REPRODUCTIVE MEDICINE & ASSISTED

RM&ART
REPRODUCTIVE TECHNIQUES SERIES

Vitrification
in Assisted
Reproduction
Second Edition
Edited by
Michael J. Tucker
Juergen Liebermann
 Vitrification
in Assisted
Reproduction
Second Edition
 Vitrification
in Assisted
Reproduction
Second Edition
Edited by
Michael J. Tucker, PhD, FIBiol, HCLD(AAB)
Director of IVF and Embryology Laboratories
Shady Grove Fertility RSC
Rockville, Maryland, USA
Juergen Liebermann, PhD, HCLD(AAB)
Director of Embryology, Andrology &
Endocrinology Laboratories
Fertility Centers of Illinois
Chicago, Illinois, USA
CRC Press
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Michael J. Tucker wishes to recognize his friends and family whose love and support helped
make this new edition happen, especially the indefatigable Juergen Liebermann, and of
course, his wonderful wife Megan and children Harry, Nick, Isabella, and Eliza, who had to
suffer through all his complaints!
Juergen Liebermann gives most sincere thanks to his wife Maike and his sons and daughter
Richard, Lennart Martin, Tobias Georg, Alexander Johannes, and Franziska Sophie for
providing unfailing support and encouragement to bring this work to reality.
Contents

Foreword ix
Preface xi
Acknowledgments xiii
Contributors xv

1 Overview of biological vitrification 1

Gregory M. Fahy

2 Vitrification of oocytes and embryos: Finally a recognized technique, but still

a source of concern and debate 23
Pierre Vanderzwalmen, Nicolas H. Zech, Fabien Ectors, Yannis Panagiotidis,
Achillae Papatheodorou, Prapas Yannis, and Barbara Wirleitner

3 Intracellular concentration of cryoprotectant during vitrification and slow-freezing

cryopreservation procedures 35
Pierre Vanderzwalmen, Fabien Ectors, Barbara Wirleitner, Astrid Stecher, Deborah Desmet,
Jacqueline Greindl, Amandine Helson, Sabine Vanderzwalmen, Mark G. Larman,
Nicolas H. Zech, and Delphine Connan

4 Importance of cooling versus warming rates in vitrification techniques 43

Shinsuke Seki

5 The movement of water and cryoprotectants in mammalian oocytes and embryos:

Membrane permeability and aquaporins 47
Keisuke Edashige and Magosaburo Kasai

6 Open versus closed systems 55

Mark G. Larman and Pierre Vanderzwalmen

7 Automatic vitrification: Development of the Gavi system 61

Tammie K. Roy, Susanna Brandi, Cara K. Bradley, and Teija T. Peura

8 Vitrification at minimum volume: From basic science to clinical application 69

Amir Arav

9 Vitrification of oocytes: General considerations and the use of the Cryotec method 77

Masashige Kuwayama

10 Safety of vitrification and cryostorage and optimization of cryopreservation protocols 87

Lodovico Parmegiani and Marco Filicori

11 Physiological aspects of oocyte vitrification 95

Mark G. Larman and David K. Gardner

vii
 contents

12 Vitrification of oocytes: Imprinting and disturbance in spindle formation and

chromosome segregation 105
Tom Trapphoff

13 Metabolic profile of day 3 embryos arising from vitrified oocytes 117

Damià Castelló and Francisco Domínguez

14 Vitrification of human oocytes for in vitro fertilization patients 125

Laura Rienzi, Benedetta Iussig, and Filippo Maria Ubaldi

15 Oocyte vitrification: Donor “egg banking” 129

Zsolt Peter Nagy, Ana Cobo, and Ching-Chien Chang

16 Fertility preservation for oncology patients 137

Kara N. Goldman, Caroline McCaffrey, and Nicole Noyes

17 Vitrification of human ovarian tissue 147

Mona Sheikhi and Outi Hovatta

18 Vitrification of cleavage-stage embryos and blastocysts and their neonatal outcomes 151

Tetsunori Mukaida and Chikahiro Oka

19 Vitrification of human blastocysts: Clinical realities and neonatal outcomes 163

Juergen Liebermann

20 Development and hatching of human blastocysts after vitrification and warming 175

Joe Conaghan and Sergio Vaccari

21 Does storage of vitrified blastocysts have an impact on implantation potential and birth rate? 185
Barbara Wirleitner, Nicolas H. Zech, and Pierre Vanderzwalmen

22 Ovarian tissue vitrification—Clinical realities and outcomes 191

Sherman Silber

23 Vitrification of human testicular tissue, spermatogonia, and spermatozoa 197

Christine Wyns, Gael Abou-Ghannam, and Jonathan Poels

24 Vitrification in pluripotent stem cell banking: Requirements and technical solutions

for large-scale biobanks 203
Julia C. Neubauer, Axel F. Beier, Frank Stracke, and Heiko Zimmermann

25 Scrying the future: The ongoing transformation of reproductive medicine through

vitrification 225
Kevin S. Richter, James R. Graham, and Michael J. Tucker

viii
Foreword

In vitro fertilization (IVF) is, in the majority of cases, the solution to all these problems, and has ushered in a new era
most direct and effective solution to the current epidemic of successful “egg freezing.” This has kickstarted the “donor
of infertility surreptitiously plaguing modern societies egg bank” industry, has enabled easy use of vitrification to
and wreaking havoc with their economies, while causing cryostore eggs if a husband is unable to provide sperm on
the grief of unwanted childlessness to ruin people’s lives. the day of an IVF egg retrieval, and, perhaps most impor-
Of course, IVF also poses a host of problems, but many of tantly and of greatest media interest, has addressed the
these can now be resolved with vitrification. “ticking biological clock” haunting the reproductive poten-
For example, most women will have more than one tial of women.
embryo, and before clinical use of vitrification, slow The major cause of the current infertility epidemic is
freezing of surplus embryos was problematic, yield- that women are putting off childbearing until they are
ing only inconsistent survival and pregnancy rates. older, because of better opportunities for career and edu-
Consequently, there was a strong temptation to transfer cation. Many women today do not consider having a baby
more than one or two fresh embryos to increase chances until their mid-thirties or even later, and, by then, over
of pregnancy—thus elevating the risk of a high order of 20% are infertile; by age 40, the vast majority are infer-
multiple pregnancies, which, as an unintended conse- tile simply because their eggs have aged significantly.
quence, has been IVF’s biggest problem. However, with However, cancer patients who have had their eggs or
the more recent introduction of vitrification technol- ovary frozen before undergoing sterilizing chemotherapy
ogy, we are able to cryopreserve extra embryos that are and radiation do not have to worry about this widespread
not transferred, and to assure patients that this process concern, because they have young eggs or ovary safely in
will neither damage their embryos nor necessarily lower the freezer. With vitrification, we can now achieve, in all
their ultimate cumulative pregnancy rate. Indeed, clini- women, that dream of safely preserving fertility in youth
cal experience is now showing that pregnancy rates from to protect them from the biological clock’s relentless
“frozen/thawed” embryos may be even higher than those progress.
from “fresh” embryos. Innovating vitrification in the 1980s, and then perfect-
The benefits of vitrification also extend beyond embryo ing it so that it would be more reliable in the early 2000s,
cryopreservation, being applicable to both oocytes and was not an easy journey for its clinical pioneers, such as
ovarian tissue freezing, and including even the whole Rall and Fahy in the mid-1980s in the United States, and
ovary. “Egg freezing” had always been somewhat of an Kuwayama in Argentina and Japan in the 1990s, and up
unfulfilled dream, whereas, at least with embryo slow to the present. IVF clinicians were reluctant to leave the
freezing (the norm for two decades), clinical outcomes comfortable space of old ideas and the use of “slow freez-
­following thawing yielded adequate survival much of the ing,” and often ridiculed vitrification; after all, it seemed
time, but still gave a distinctly lower pregnancy rate than too easy, when in fact it was very difficult, simply because it
did transfer of fresh embryos. Slow freezing of oocytes was has to be just exactly right to achieve success. It took much
never able to provide clinically reliable outcomes, and experimentation with many protocols; and having found
remained largely sidelined as a routine procedure. The the right one, it was most important not to tamper with
­reasons are severalfold: (1) the mature oocyte is the biggest it and not to deviate from the details. Cryobiology can be
cell in the body and contains a high proportion of water, a very empirical science, with much painstaking trial and
making it difficult to remove enough water during slow error (of course, based on scientifically derived postulates)
freezing to avoid ice crystal formation; (2) the mature over many years until you finally have the knowledge to get
oocyte, with chromosomes precariously aligned on the it right. It is not an easy procedure, with numerous points
metaphase II spindle, is extremely sensitive to damage in the vitrification process of just one oocyte, and if a step
from ice crystal formation and may easily be disrupted; is inadvertently not carried out perfectly, it spells disaster
(3) the oocyte is also exquisitely vulnerable to mild chill- for that oocyte.
ing, which may cause significant compromise or even The purpose of this book, therefore, is to provide read-
complete degeneration. So application of an “ultra-rapid ers with all the information they will need to perfect vit-
freeze” approach with vitrification allows a consistent rification in their own laboratories. Dr. Michael Tucker

ix
 foreword
and Dr. Juergen Liebermann include every major scien- carry out a perfect job in their own IVF lab and make the
tist who has worked on vitrification in recent times, and dream of preserving fertility a clinical reality.
describe how these individuals brought us closer to this
clinical breakthrough. In addition, readers will under- Dr. Sherman Silber
stand the controversies and pitfalls of the process, and Medical Director, Infertility Center of St. Louis,
how to resolve and avoid such issues, allowing them to St. Lukes Hospital, St. Louis, Missouri

x
Preface

Assisted reproductive technology (ART) has been evolv- effort and is nothing short of a small miracle, given that
ing for more than 30 years, and cryopreservation has it relies on such an intensive cooperation between science
become one of the keystones in such clinical infertil- and clinical application. The development of gamete and
ity treatment. However, the true “explosion” in many embryo vitrification specifically represents the trium-
embryologists’ minds in recognizing the true value of phant culmination of several decades of frustrating explo-
cryopreservation occurred with the appearance of vit- ration with “slow freeze” technology. The first edition of
rification techniques in the mid-1980s. Very gradually this book, published in 2007, was subtitled: “A user manual
since that time, vitrification has established itself as “the and trouble-shooting guide.” This second edition, being
cryopreservation technique of choice,” through consis- published more than 7 years later, has evolved into a more
tency and predictability in terms of the quality of the cell comprehensive book now containing chapters reporting
survival following the vitrified “state of suspended ani- the successful application of vitrification from research up
mation.” Using a vitrification approach, oocyte freezing to the level of its use in routine clinical therapy. We as edi-
has become a reality and routine, not to mention embryo tors of this book are excited, pleased, and grateful to have
vitrification, which in turn provides outcomes compa- chapters contributed by scientists and clinical embryolo-
rable to those achieved with fresh transferred embryos. gists who have been involved in the development of vitri-
Furthermore, vitrification has opened the doors for new fication at the very highest level. The list of contributors
areas in the field of ART such as oocyte banking, preim- demonstrates a global collaboration of professionals who
plantion diagnostics at the blastocyst stage, and notably have given their time, energy, and intellect to share their
it has helped to fuel the burgeoning adoption of elective scientific observations and clinical experiences with the
single-embryo transfers. This final area of impact is prob- future readership of this book and beyond.
ably the most important development, because it defines Our destiny working in the field of IVF every single
“quality of ART” in a new light—the delivery of a healthy day is based on strong values, and one of these is to fulfill
single baby in each IVF cycle. the dreams of our patients in creating healthy families for
The success of cryopreservation in benefiting the them; vitrification plays an increasingly essential role in
very specialized field of ART has arisen only after much achieving this goal.

xi
Acknowledgments

The editors Michael J. Tucker and Juergen Liebermann we thank them enormously for allowing us to act as coor-
would like to thank the following individuals for their dinating editors to put this book together, on a subject
efforts in bringing this publication to fruition: Robert that we think is both interesting and important. We are
Peden, who as the acquisitions editor at CRC Press, guided also grateful to all the River North IVF laboratory staff
us expertly and thoughtfully through the publication at the Fertility Centers of Illinois: Jill Matthews, Elissa
steps, and has been extremely helpful in keeping this pub- Pelts, Becky Brohammer, Sara Sanchez, Yuri Wagner,
lication on track throughout the vagaries of the process to and Ewelina Pawlowska, whose clinical skills facilitated
provide a well-focused and timely book. All contributing the clinical application of routine vitrification within that
authors are gratefully acknowledged for their commit- laboratory; likewise, all the IVF laboratory colleagues at
ment to providing current and comprehensive chapters Shady Grove Fertility, especially Jim Graham, Josh Lim,
in this area of cryobiology. Their enthusiasm and profes- Taer Han, Patricia Dhlakama, and Marc Portmann—to
sionalism in their work are evident in their writings, and all we say a very special thank you.

xiii
Contributors

Gael Abou-Ghannam Francisco Domínguez Amandine Helson

Service de Gynécologie-Andrologie Fundacion Instituto Valenciano de IVF Unit
Cliniques Universitaires Saint-Luc Infertilidad (FIVI) Centre Hospitalier Inter-Regional
Brussels, Belgium Instituto Universitario IVI (IUIVI) Edith Cavell (CHIREC)
and Braine l’Alleud, Bruxelles, Belgium
Amir Arav
INCLIVA Biomedical Research
FertileSafe Outi Hovatta
Institute
Ness Ziona, Israel Department of Clinical Science,
Valencia, Spain
Axel F. Beier Intervention, and Technology
Fraunhofer Institute for Biomedical Fabien Ectors Karolinska University Hospital
Engineering GIGA & FARAH Transgenic Platform Stockholm, Sweden
St. Ingbert, Germany University of Liège
Liège, Belgium Benedetta Iussig
and GENERA Centres for Reproductive
Hubrecht Institute and University Keisuke Edashige Medicine
Medical Center Utrecht Laboratory of Animal Science Rome, Marostica, Umbertide,
Utrecht, the Netherlands College of Agriculture and Napoli, Italy
Kochi University
Cara K. Bradley Nankoku, Kochi, Japan Magosaburo Kasai
Genea Biomedx Laboratory of Animal Science
Sydney, New South Wales, Australia Gregory M. Fahy College of Agriculture
21st Century Medicine, Inc. Kochi University
Susanna Brandi
Fontana, California Nankoku, Kochi, Japan
Genea Biomedx
Sydney, New South Wales, Australia Marco Filicori Masashige Kuwayama
Damià Castelló Reproductive Medicine Unit Repro-Support Medical Research
IVI Valencia GynePro Medical Centers Centre
Valencia, Spain Bologna, Italy Shinjuku, Tokyo, Japan

Ching-Chien Chang David K. Gardner Mark G. Larman

Reproductive Biology Associates School of BioSciences Vitrolife
Atlanta, Georgia University of Melbourne Englewood, Colorado
Melbourne, Victoria, Australia
Ana Cobo
Juergen Liebermann
IVI-Valencia Kara N. Goldman Fertility Centers of Illinois
Valencia, Spain New York University (NYU) Chicago, Illinois
Joe Conaghan Fertility Center
Pacific Fertility Center NYU Langone Medical Center Caroline McCaffrey
San Francisco, California New York, New York New York University (NYU)
Fertility Center
Delphine Connan James R. Graham NYU Langone Medical Center
GIGA & FARAH Research Centers Shady Grove Fertility Reproductive New York, New York
University of Liège Science Center
Liège, Belgium Rockville, Maryland Tetsunori Mukaida
Hiroshima HART Clinic
Deborah Desmet Jacqueline Greindl Naka-ku, Hiroshima, Japan
IVF Unit IVF Unit
Centre Hospitalier Inter-Regional Centre Hospitalier Inter-Regional Zsolt Peter Nagy
Edith Cavell (CHIREC) Edith Cavell (CHIREC) Reproductive Biology Associates
Braine l’Alleud, Bruxelles, Belgium Braine l’Alleud, Bruxelles, Belgium Atlanta, Georgia

xv
 contributors
Julia C. Neubauer Shinsuke Seki Pierre Vanderzwalmen
Fraunhofer Institute for Biomedical Division of Stem Cell Therapy IVF Centers Prof. Zech
Engineering Center for Stem Cell Biology and Bregenz, Austria
St. Ingbert, Germany Regenerative Medicine
and
Institute of Medical Science
Nicole Noyes University of Tokyo IVF Unit
New York University (NYU) Tokyo, Japan Centre Hospitalier Inter-Regional
Fertility Center Edith Cavell (CHIREC)
NYU Langone Medical Center Mona Sheikhi Braine l’Alleud, Bruxelles, Belgium
New York, New York Stockholm IVF and
Stockholm, Sweden
Chikahiro Oka GIGA & FARAH Research Centers
Tokyo HART Clinic University of Liège
Sherman Silber
Minato-ku, Tokyo, Japan Liège, Belgium
Infertility Center of St. Louis
St. Lukes Hospital
Yannis Panagiotidis St. Louis, Missouri Sabine Vanderzwalmen
IVF Centers Iakentro IVF Unit
and Centre Hospitalier Inter-Regional
Thessaloniki, Greece
Department of Obstetrics and Edith Cavell (CHIREC)
Achillae Papatheodorou Gynecology, Reproductive Braine l’Alleud, Bruxelles, Belgium
IVF Centers Iakentro Endocrinology
Thessaloniki, Greece University of Amsterdam Barbara Wirleitner
Amsterdam, the Netherlands IVF Centers Prof. Zech
Lodovico Parmegiani Bregenz, Austria
Reproductive Medicine Unit Astrid Stecher
GynePro Medical Centers IVF Centers Prof. Zech
Christine Wyns
Bologna, Italy Bregenz, Austria
Pôle de Gynécologie
Institut de Recherche Expérimentale
Teija T. Peura Frank Stracke et Clinique
Genea Biomedx Fraunhofer Institute for Biomedical Université Catholique de Louvain
Sydney, New South Wales, Australia Engineering and
St. Ingbert, Germany Service de Gynécologie-Andrologie
Jonathan Poels Cliniques Universitaires Saint-Luc
Pôle de Gynécologie Tom Trapphoff Brussels, Belgium
Institut de Recherche Expérimentale Department of Gene Technology
et Clinique and Microbiology Prapas Yannis
Université Catholique de Louvain University of Bielefeld IVF Centers Iakentro
Brussels, Belgium Bielefeld, Germany Thessaloniki, Greece

Kevin S. Richter Michael J. Tucker Nicolas H. Zech

Shady Grove Fertility Reproductive Shady Grove Fertility Reproductive IVF Centers Prof. Zech
Science Center Science Center Bregenz, Austria
Rockville, Maryland Rockville, Maryland
Heiko Zimmermann
Laura Rienzi Filippo Maria Ubaldi Fraunhofer Institute for Biomedical
GENERA Centres for Reproductive GENERA Centres for Reproductive Engineering
Medicine Medicine St. Ingbert, Germany
Rome, Marostica, Umbertide, Rome, Marostica, Umbertide,
and Napoli, Italy and Napoli, Italy and
Molecular and Cellular
Tammie K. Roy Sergio Vaccari Biotechnology/Nanotechnology
Genea Biomedx Pacific Fertility Center Saarland University
Sydney, New South Wales, Australia San Francisco, California Saarbrücken, Germany

xvi
1 Overview of biological vitrification
Gregory M. Fahy

INTRODUCTION a broader perspective, and that is the goal of the pres-

Since 1984, vitrification has been used with rapidly ent chapter. It is worth noting, however, that the general
expanding frequency to accomplish cryopreservation popularity of vitrification was originally based on its effi-
in a way that was not previously possible (Figure 1.1). cacy for preserving embryos,1 and that a major portion of
The expected number of new citations of vitrification to the continuing interest in this technique is sustained by
appear in PubMed from the time the previous edition of reproductive cryobiologists. There is now a vast literature
the present volume was published to the end of 2014 is describing the present use of vitrification for the routine
projected to be in the vicinity of 1600–1700 papers, if not cryopreservation of human2,3 and animal3 embryos, and
more. It is not possible for a short review to encompass for more experimental but increasingly routine cryo-
all of the findings in this rapidly expanding literature, or preservation of ovarian tissue,4,5 ova,6–8 and even small
even just all of the findings in the realm of reproductive whole ovaries.9
cryobiology, but it is possible to place this research into In this chapter, an overview is provided concerning
the precedent for artificial vitrification that is represented
by examples of vitrification or the potential for vitrifica-
tion in the natural world, the advantages and disadvan-
2500 tages of vitrification, the different eras of research in
1600
applied approaches to vitrification, the general physical
Citations since start of 2008

1400
principles governing vitrification and devitrification, and
1200 basic methodological and theoretical considerations for
2000 1000 maximizing biological viability while carrying out vit-
800 rification procedures. It is hoped that these perspectives
Cumulative citations

600 will assist investigators in their design of more successful

1500 400 methods based on a better understanding of the physical
200
and biological principles of vitrification.
0 VITRIFICATION UNDER NATURAL CONDITIONS
1000 2008 2010 2012 2014
Despite concerns over global warming, the record for
Year
the lowest temperature ever measured on Earth, previ-
ously standing at −89.3°C,10 was recently bested by over
500 5 degrees at −94.7°C in eastern Antarctica.11 The lowest
temperature recorded so far in Alaska, −79.8°C11, is just
below the sublimation temperature of dry ice. More com-
0 monly, the lowest environmental temperatures encoun-
1980 1990 2000 2010 tered are between −30 and −60°C.10,12 By comparison,
Year glass transition temperatures (TGs) higher than −50°C
have frequently been measured in living organisms13–17
Figure 1.1 Cumulative citations of vitrification as a and have been documented in freeze-concentrated mam-
method of cryopreservation in PubMed since 1980 malian tissue.18 Furthermore, “poikilohydric” species,
(main figure), and since the beginning of 2008 (inset). whose water content declines with declining ambient
(Modified from earlier versions Fahy GM, Rall WF. humidity, can experience apparent cytoplasmic vitrifica-
Vitrification: An overview. In Tucker MJ, Liebermann J tion in connection with partial drying at both low and
(eds.), Assisted Reproduction: A User’s Manual and high temperatures,10,17,19–23 in the most extreme cases
Troubleshooting Guide. Informa Healthcare: London, even allowing survival in nature under high-temperature
2007. p. 1–20; With kind permission from Springer conditions.24,25 In view of such observations, many have
Science+Business Media: Cryopreservation and Freeze considered it plausible that many species may overwin-
Drying Protocols, Principles of cryopreservation ter in a partly or completely vitrified state,13,14 and even
by vitrification, 2015, Fahy GM, Wowk B. Wolkers that this is a more common natural strategy for survival
WF, Oldenhof H (eds.); Fahy GM, Wowk B, Wu J. than freeze tolerance.10,23 Such a possibility derives fur-
Rejuvenation Res 2006;9:279–91.) ther support from observations in plants by investigators

1
 vitrification in assisted reproduction
such as Sakai, who has concluded 20: “the most appropri- remarkable examples involves vitrification per se, they do
ate interpretation is that completely hardy plants form both point to the desirability of avoiding ice formation
aqueous glasses intracellularly.” in organized tissues, which provides additional though
A particularly interesting and well-documented indirect support to the strategy of avoiding ice formation
example of a complex organism that is in principle able entirely by means of vitrification.
to survive vitrification in nature is that of the Alaskan
red flat bark beetle, Cucujus clavipes puniceus.12 No lar- ADVANTAGES AND DISADVANTAGES OF
vae showed exotherms (freezing events) when cooled to VITRIFICATION VERSUS FREEZING
−150°C in a differential scanning calorimeter (DSC) in Vitrification has been pursued by many for three reasons.
the laboratory. Small larvae showed TGs ranging from The first is that it can be quick and simple in comparison
−58°C to −76°C, which is consistent with the possibility to freezing, and requires less expensive and complicated
of vitrification under natural conditions, at least for those equipment. The second it that it transcends the need to
larvae with the higher TGs. Two large larvae had a sec- cool and warm at identified and rigidly controlled opti-
ondary TG at −96°C or −98°C, but still had primary TGs mal rates to maximize survival29–31 and avoids the gener-
of −76°C and −75°C, respectively. Furthermore, despite ally imperfect30,32,33 compromise between intracellular ice
technical problems, some larvae survived cooling to formation and excessive dehydration injury that is a com-
−100°C, which is below the lowest TG of even the large mon feature of freezing protocols, thus leading to both
larvae. In the field setting, about 50% of larvae failed to greater convenience and frequently improved or at least
freeze when cooled to about −60°C to −70°C, and about equal results compared to freezing. The third impetus in
50% also survived after cooling to and warming from the many cases is the ability to use vitrification to attain cool-
mean whole body TG (−71°C), suggesting that survival is ing rates that are sufficient to “outrun” chilling injury34–38
related to successful ice avoidance and even vitrification. without paying the penalty of intracellular ice formation
Of particular interest in the case of this organism is the at high cooling rates.
fact that the larvae employed large concentrations of glyc- The motivation behind the events that led to the post-
erol (up to 6.5 M in the cited study, and up to 10 M in a 1984 explosion of interest in vitrification was the advan-
separate study26), which mimics the use of high concen- tage of avoiding mechanical injury to organized tissues
trations of permeating cryoprotectants in artificial vitrifi- from ice.1,39–41 Both organized tissues42–48 and cells in
cation and makes these beetles some of the closest natural suspension30,47 can be injured from the growth of ice
counterparts to laboratory vitrification. either during cooling or as a result of recrystallization
There is a particularly striking example of convergent on warming. This concern is particularly pertinent to the
evolution that illustrates, for organized tissues in both cryopreservation of gonadal tissue, as well as to the pres-
the plant and the animal kingdoms, the basic desirabil- ervation of tissues of the reproductive tract,5,49,50 although
ity of avoiding invasion by ice. In the terrestrial frog, ice the influence of ice formation is most powerful in systems
forms first in relatively tolerant peripheral tissues, such as that require vascular support after transplantation.43,49
between the skin and the muscle, as well as in the abdom- The primary disadvantage of vitrification is the need
inal cavity.27 Because freezing takes place very slowly and for high concentrations of cryoprotectant, which compli-
with shallow thermal gradients in an animal that is rather cates the avoidance of osmotic damage and the avoidance
small in size and contains empty cavities, there is time for of cryoprotectant toxicity. Despite this disadvantage,
water to migrate from vital structures such as the brain many successful vitrification protocols have been devel-
and the heart to the growing ice crystals in these benign oped, and many methods for avoiding toxic and osmotic
locations rather than freezing in situ. The liver, heart, injury have been demonstrated. These issues are dis-
brain, intestines, and kidneys can actually be observed to cussed in more detail below.
lose volume as ice masses increase in volume, 27 very much Another disadvantage can be fracturing of the vitri-
akin to the way single cells shrink in response to growing fied medium, resulting in such lesions as cleavage of the
extracellular ice rather than freezing intracellularly, pre- zona pellucida51 or macroscopic damage to larger three-
sumably surviving as a result. A plant counterpart to this dimensional structures.52 This problem can be solved by
ice avoidance strategy is provided by the silver fir, Abies storing at temperatures that are intermediate between
sachalinensis. In the fir tree, ice forms in a tolerant area those of the glass transition and liquid nitrogen53 or by
adjacent to the apical meristem and is prevented from careful cooling and warming protocols that avoid suffi-
growing into the meristem by a diffusion barrier that cient stress development to result in fracture formation.51
allows water to diffuse through it but blocks the growth Another disadvantage of vitrification is that it usu-
of ice. This results in the movement of meristem water ally results in nucleation of unfrozen freezable water at
from the meristem to the growing ice crystal, preventing low temperatures, which may lead to the freezing of this
death of the shrinking meristem (A. Sakai, personal com- water upon warming, with attendant injury. This injury
munication). Other plants appear to have evolved essen- can in principle be avoided by sufficiently rapid warming,
tially the same mechanism.28 Although neither of these but warming may need to be more rapid than is required

2
 overview of biological vitrification
after most freezing protocols, in which there is more con- the cryoprotectants or during cryoprotectant removal.
cern over the recrystallization of previously formed ice Despite these major limitations, it is remarkable how suc-
than the formation of additional ice, much of which may cessful some of Luyet’s experiments actually were.74,75
in principle be intracellular. The partial successes achieved by Luyet, both in terms
Certain vitrification protocols entail an additional dis- of biological recovery and in terms of visual observations
advantage, at least in principle, which is contamination that suggested successful vitrification, were encourag-
resulting from the use of open containers or container- ing enough to sustain research into this approach to
less methods of vitrification.54 However, these methods cryopreservation until 1958. However, in 1954, Audrey
seem certain to be superseded by equally effective meth- Smith strongly questioned Luyet’s interpretations and
ods that employ closed containers.55 rationales, pointing out both that survival after cooling
and warming was not necessarily proof of vitrification,
THE DIFFERENT EPOCHS OF RESEARCH ON and that transparency rather than opacity after cooling or
CRYOPRESERVATION BY VITRIFICATION during warming was not necessarily proof of vitrification
The Era of Rapid Cooling with Little to No (Deliberate) and the avoidance of devitrification.61 Four years later,
Intracellular Cryoprotection Luyet and Rapatz77 and Meryman78 presented the results
Vitrification of water was originally contemplated as being of their investigations of Smith’s criticisms of Luyet’s evi-
primarily a matter of attaining the required cooling rate.56–60 dence for vitrification. The results were undoubtedly dis-
Inspired61 in part by Tammann’s observation62 that rapid appointing for Luyet.
cooling enabled the vitrification of 38% of all organic liq- The first study77 showed that thin films of gelatin gel
uids investigated and Tammann’s theory that it should be subjected to Luyet’s vitrification procedure contained
possible to vitrify any liquid through the use of sufficiently “evanescent spherulites,” ice crystals that, while thin and
high cooling rates, as well as by observations of Moran63 undetectable by the naked eye, were nonetheless quite
and Hardy64 indicating that it might be possible to vitrify real and prominent under light microscopic examination
gelatin gels, the first serious work on vitrifying living sys- through crossed polarizing filters. The second study78
tems was launched by Father Basile J. Luyet in a classic showed X-ray diffraction patterns in similar gels, another
paper published in 1937.65 As Luyet said in explaining the verification of the presence of ice. However, only two
idea of vitrification in this paper, “The essential problem of diffraction peaks were seen, which was interpreted as
the vitrification technique consists of … obtaining a cool- indicating incompletely formed crystals. Dowell et  al.79
ing velocity sufficient to prevent the formation of crystals.” later concluded that the ice formed was cubic ice, but evi-
As reviewed in more detail elsewhere,66 Luyet and his asso- dently cubic ice does not exist,80 and the patterns seen by
ciates published many papers from 1937 to 1958 in pursuit Meryman are actually consistent with ordinary, freshly
of the goal of vitrification using a variety of rapid cooling nucleated but nonrecrystallized ice.80 Therefore, the
techniques. Most of this literature was undoubtedly pub- results of both investigations were in agreement in show-
lished in Biodynamica, a journal established by Luyet in ing that Luyet’s methods could not be relied upon to cap-
October of 1934, which began as a journal dedicated to ture living systems in the vitreous state.
discovering the nature of life,67 but transformed fairly rap-
idly into the world’s first journal dedicated to cryobiology, The Era of Rapid Cooling with Minimal but Deliberate
with a heavy emphasis on vitrification. Cryoprotection
Although cryoprotection had been described even Attempted cryopreservation by vitrification ended with
before Luyet’s time,68 the use of cryoprotectants to mini- the Luyet/Meryman experiments of 1958 and did not
mize freezing injury was not generally known until 194969 resume until being re-introduced exactly 20 years later,
or generally understood until 195370 (although freeze con- but with a significant difference. Pierre Boutron was the
centration as a mechanism of cryoinjury was elucidated first to actively propose that cryoprotectants be used in
much earlier71–73). Therefore, the use of cryoprotectants higher than usual concentrations to effect vitrification,
and/or drying by Luyet and his colleagues to facilitate or at least partial vitrification at high cooling rates.81
vitrification was not initially intended to slow the rate of There was considerable irony in this, because Boutron’s
crystallization or limit the maximum possible amount proposal was based in large part on observations made
of crystallization of water as it is today, but was merely by Luyet and his colleagues after Luyet had abandoned
intended to dehydrate the system just before cooling to the idea of vitrification. The latter observations indicated
minimize the amount of water requiring vitrification.74–76 that, in high concentrations, cryoprotectant solutions
Use of cryoprotectants for dehydration by exosmosis really could be vitrified, and in the highest concentra-
rather than for intracellular uptake was presumably based tions, could even be vitrified at low cooling rates.82–88
simply on the understanding that smaller objects, and thus Presumably, Luyet, like Boutron, assumed after 1958
smaller amounts of water, are easier to vitrify than larger that high intracellular concentrations of cryoprotectants
ones. Little consideration was given to osmotic damage could never be tolerated by living cells, and so never pur-
in such procedures, either during the exposure phase to sued this approach. This is particularly ironic because

3
 vitrification in assisted reproduction
Rapatz and Luyet actually achieved the first clear-cut and red cells were not in immediate need of better cryo-
success in vitrifying living cells in 1968 by cooling red preservation at the time. In addition, Boutron’s experi-
blood cells in glycerol, and showing that their success- ments were complex, difficult to understand, and largely
ful preservation (lack of hemolysis and integrity in the theoretical. Therefore, although working at the edge of the
electron microscope) could be obtained coincident with glass-forming concentration range, as Boutron often did,
the disappearance of both intracellular and extracellular has become almost a way of life in more recent years, at
ice in freeze-fracture/freeze-etch electron microscope the time it did not inspire general interest in cryopreser-
images,89 although they never related this achievement vation by complete vitrification, a goal that, in Boutron’s
to Luyet’s original decades-long quest to accomplish the early papers, seemed to be envisioned mostly as a desir-
very same thing, but in a very different way. able dream81 that could only be attained by finding new
The difference between Luyet and Boutron was that cryoprotectant systems that permitted vitrification at low
Boutron was prepared to look for a way to circumvent concentrations (which is now seen as a largely counter-
cryoprotectant toxicity and achieve vitrification using a productive approach101).
combination of two approaches. First, he began a system- Not long after Boutron revived interest in the amor-
atic series of studies on cryoprotectant mixtures81,90–94 phous state, however, Eric James began working on ways
in the hope of finding some mixtures that enabled vit- to vitrify parasitic worms, using necessarily (due to
rification at particularly low, and therefore perhaps non- observed apparent toxicity) marginal concentrations of
toxic, concentrations. Second, he studied in great detail cryoprotectant (methanol102 or ethylene glycol [EG]103,104)
the previously unmapped kinetics of ice crystallization for vitrification in combination with high cooling rates,
as a function of the interaction between concentra- and achieving some degree of success. Although he
tion and cooling rate,81,95,96 and achieved survival of red explicitly referred to the Luyet approach, and not to
blood cells by vitrification in 1984,97 and more clearly in Boutron, he clearly recognized the need for intracellular
1988.98 Boutron pioneered the use of differential scanning uptake of the cryoprotectant to facilitate intracellular vit-
calorimetry and its marriage with x-ray crystallography rification,104 and in this respect, proceeded more in accord
as a means of verifying, quantifying, and characterizing with Boutron’s approach than with Luyet’s concepts.
vitrification and the ice that forms when vitrification is
incomplete, as well as mathematical analysis of crystal- The Era of High Concentrations and Relaxed Cooling
lization under borderline conditions and quantitative and Warming Rates
analysis of devitrification.81,99 These were all extraordi- A practical universal approach to vitrification was first pro-
nary contributions, and Boutron made additional contri- posed by Fahy in 198139,41 as a possible general solution to
butions by succeeding in his quest to find cryoprotectants the difficult problem of organ cryopreservation.1,39–41,105–108
that could vitrify water at remarkably low concentrations, The inspiration to pursue vitrification stemmed from dis-
the two outstanding examples being first propylene gly- appointing experiments with transplanted dog kidneys,109
col (PG),100 and later, 2,3-butanediol.93 which appeared to be viable after freezing and thawing
Boutron’s work, however, while describing the physics when tested in vitro (good vasoconstriction response to
of ice in great detail and truly re-introducing the idea of infused pressors after being frozen at −30°C for 1 week,
vitrification in a way that could actually be definitively confirming similar results obtained slightly earlier in rab-
achieved in practice, did not inspire much enthusiasm bits110), but passed whole blood and turned blue from
for practical application, probably for several reasons. vascular stasis shortly after transplantation, which was
First, Boutron was focused on the study of cryoprotectant interpreted as reflecting mechanical injury from ice,
solutions that are hard to recover from the vitrified state reminiscent of the injury seen by electron microscopy in
without seemingly overwhelming problems from devit- frozen-thawed kidney slices.42,111 Inspired by unpublished
rification, a nominally lethal event. Second, the meth- results of Rajotte and McGann that suggested the possibil-
ods of vitrification that he did provide were only applied ity of deep supercooling as a means of relatively short-term
successfully to red blood cells, and then only obscurely, organ preservation,109 Fahy attempted deep supercooling
as they were buried in data on the effects of freezing in but found that the supercooled state was insufficiently
198497 and 1988,98 and did not actually improve survival stable for reliable week-long storage periods.112,113 But the
in those systems over what was achieved without vitrifi- thought then occurred that perhaps this instability prob-
cation. Although the red cell papers, remarkably, showed lem could be solved simply by continuing to cool all the
that cells can survive devitrification (see the in-depth way to below the TG, and this was the genesis of the idea
analysis below), this observation was only possible using of large-system, low-cooling rate vitrification,113 which
warming rates that would be discouraging except for aca- simultaneously, of course, opened the door to the ready
demic purposes, since clinical red cell freezing involved vitrification of smaller systems as well.
volumes that were far too large to warm at rates in excess The idea of using high concentrations of cryoprotec-
of 1000°C/min. In addition, red blood cells are not nucle- tants to allow vitrification of whole organs could have and
ated, so the generality of Boutron’s methods was not clear, should have been not only proposed, but also achieved

4
 overview of biological vitrification
long before 1981. In 1965, John Farrant114 was able to of the highest cryoprotectant concentrations at reduced
recover excellent contractile function in whole guinea temperatures1,65; and restriction of concentrations of
pig uteri after cooling them to and rewarming them from cryoprotectant to the minimum levels needed to vitrify at
−79°C, using what is now called the “liquidus tracking” the cooling rates applicable to organs, as determined both
method,115 which he invented, to entirely avoid freezing. empirically and on theoretical grounds.61,63,64 By 1984, the
But he was unaware of the fact that he could have cooled his first full and generally available description of the con-
uteri to below TG and vitrified them successfully, because cept of high-concentration, low-cooling rate vitrification
the revelation that high concentrations of cryoprotectant was provided, together with a demonstration that it was
vitrify when sufficiently cooled did not begin to be pub- possible to design solutions that could vitrify at a cooling
lished until the end of 1966.116 Similarly, Huggins, who rate of only 10°C/min and yet could permit 90% recovery
came close to the same idea at the same time in attempting of the renal cortical K+/Na+ ratio.107 This paper also dem-
to explain red cell cryoprotection,117 did not contemplate onstrated that the fracturing that had dissuaded Rapatz
extending cooling to below TG. However, when Elford could be avoided, even in a whole vitrified rabbit kidney
and Walter applied Farrant’s method to smooth muscle weighing far more than a frog heart.
in 1972 and achieved similarly outstanding results,118 The final step in demonstrating the intrinsic work-
they too did not mention the possibility of using their ability of this general approach was to establish its effi-
modified method to achieve vitrification, even though the cacy for the cryopreservation of at least one convincing
last paper on the subject of high-concentration physical model system. In fact, two such proofs of principle rapidly
vitrification to appear in Biodynamica had by then been became available. The first came as a result of a collabora-
published 2 years previously.88 A case can also be made tion between Fahy and William F. Rall, and the second
for another lost opportunity to propose vitrification on was provided by an independent parallel investigation
the part of the same Gabriel L. (“Lou”) Rapatz who had, led by Tsuneo Takahashi. Both studies began at about the
with Luyet, demonstrated successful vitrification of red same time and in the same laboratory at the American
blood cells in 1968.89 In 1970, he reported119 at the annual Red Cross.
meeting of the Society for Cryobiology in Los Angeles his Rall, after being exposed to an explanation of the idea
first results indicating the possibility of recovering good of complete (intracellular and extracellular) vitrifica-
function in at least some frog hearts after cooling them to tion,106 was attracted to it as a result of his previous expe-
dry ice temperature using 11 M (68.3% w/v, a vitrifiable rience with the freezing of mouse embryos, which had
concentration) EG and a “liquidus tracking” approach led him to realize that their survival was dependent on
analogous to that of Farrant. He reported elsewhere the intracellular vitrification,125–127 and he and his colleagues
formation of ice in at least some of his hearts,120 but did had even shown, in 1984, that the unfrozen medium in
not directly suggest that the hearts that showed good which the embryos were embedded in the frozen state
recovery might have escaped ice formation, although just before rapid cooling to lower temperatures was suf-
he did try, unsuccessfully, to improve the recovery rate ficiently concentrated to vitrify.128 Rall and colleagues
by increasing the exposure time to EG.121 Interestingly, recognized that this phenomenon was not unique to
he reported from the podium, but did not record in his embryos, and concluded: “Other cryopreservation meth-
published abstract,119 that plunging the hearts into liq- ods that employ a protective additive and rapid cooling
uid nitrogen and then back into a dry ice-temperature from an intermediate subzero temperature may rely on
bath caused the hearts to “shatter,” which also suggests the ability of the residual liquid to form a metastable glass
glass-like behavior in these hearts. Later, Rapatz refined during rapid cooling.”128
his methods and reported near-normal recovery of frog These observations convinced Rall that complete vit-
hearts cooled to dry ice temperature using 10 M versus rification should be feasible, and he relocated from the
11 M EG, and showed a video of these hearts contracting United Kingdom to Bethesda, Maryland, to join forces
normally after rewarming, yet still never suggested that with Fahy and apply his expertise with embryos to prove
they might have been vitrifiable.122 the feasibility of the new method. The effort was a true
Fahy, in contrast, focused on vitrification and on collaboration in which Fahy provided the solution and
the need to obviate cryoprotectant toxicity in order to some of the cooling methods, and Rall provided the pro-
approach the goal of vitrification,62–66 in this effort com- tocol for adding and removing the vitrification solution,
bining the use of putative “toxicity neutralizers”39,123; and both worked equally hard, and interactively, on the
Boutron’s 1,2-propanediol39,100,109; the use of elevated manuscript. The result, which is well known, showed that
hydrostatic pressure39,40,109; nonpenetrating cryoprotec- mouse embryos could indeed be vitrified and rewarmed
tants to enable reduction of intracellular permeating cryo- with equal success over a range of cooling and warming
protectant concentrations107; mild osmotic dehydration rates as long as the warming was rapid enough to pre-
to facilitate intracellular vitrification, reduce toxicity, and vent devitrification.1 The published paper coined the term
facilitate cryoprotectant washout107; exponential addi- “vitrification solution,” a bit of new jargon that was origi-
tion and washout of cryoprotectants107,124; introduction nated by Rall during discussions of how to present the

5
 vitrification in assisted reproduction
data in the paper. Before long, it was confirmed that vit- very rapid chilling injury seen in this species, the only
rified mouse embryos were able to develop to term after path forward that was not doomed to failure was vitri-
transfer to female hosts,129 and the race to find even better fication at high cooling rates, with very rapid warming.
vitrification solutions was begun. Five years later, Steponkus et al. reported that using what
The second proof of principle, by Takahashi et  al.,130 he referred to as a modified version of the Rall and Fahy
has received much less attention, but is also robust. These method (which employed 8.5 M EG37 rather than the VS1
authors showed, using both DSC and freeze-fracture/ solution of Rall and Fahy) and cooling at ~400°C/s on a
freeze-etch electron microscopy, that essentially the same copper grid immersed in liquid nitrogen slush yielded
solution used for vitrification by Rall and Fahy, as well as over 18% egg development into larvae and 3% egg devel-
human monocytes embedded in it, indeed vitrified when opment into fertile adults.36 Two years after this, Mazur
cooled, although some small ice crystals, which had no et al. achieved a hatching rate of 68% and a rate of devel-
apparent effect on viability or cell function, were seen in opment to fertile adults of hatched flies of 40% using
some organelles. Rewarming allowed >90% recovery of essentially the same vitrification procedure, but modify-
both cell numbers and all measured cell functions pro- ing noncryobiological aspects of the procedure (the pre-
vided devitrification was not permitted, recovery that was cise embryo age at the time of vitrification, for example),
not different from the recovery of monocytes treated with and incidentally cooling at 100,000°C/min.37
the vitrification solution but not vitrified. Devitrification, In 1996, Martino, Songasen, and Leibo,34 taking a hint
which was prominent at warming rates of less than 80°C/ from the Drosophila work, described perhaps the first
min and minimal though still present at 80°C/min, resulted similar attempt to “outrace” chilling injury in a mamma-
in both extracellular and intracellular ice and caused major lian system (bovine oocytes) using a combination of rapid
injury to the monocytes. Avoidance of cryoprotectant tox- cooling on copper electron microscopy grids and, in this
icity required addition of the vitrification solution at 0°C case, either 5.5 M EG + 1 M sucrose or 4 M EG + 0.5 M
rather than at room temperature. sucrose. Encouraging and similar results were obtained
The era of high-concentration, relaxed-rate vitrifica- with both vitrification solutions even though the latter,
tion continues to the present day, particularly for sys- when cooled in straws, was found not to vitrify, and the
tems that cannot be cooled or warmed at high rates.131,132 authors remarked that even if extracellular ice were to
However, the success of this method opened up many form, it might not matter.
opportunities for vitrification under more marginal and The Martino et  al. paper, with one foot in the stable
ambiguous circumstances, thus leading to the next, and vitrification tradition and another in the tradition of
also still-continuing, era of vitrification research. unstable vitrification134 harkening back to the work of
James and the early work of Boutron, may have re-estab-
The Era of Marginal or Accelerated-Rate Vitrification lished a precedent for pushing the cryoprotectant con-
and Rapid Freezing centration as low as possible, thus essentially launching
Not long after the onset of the relaxed-rate era of vitrifi- the next era in vitrification research. Since Martino et al.,
cation research, much innovative work was undertaken an avalanche of studies, which continues to this day, has
to adapt the idea of complete vitrification to a variety proceeded to develop methods that differ in detail but are
of living systems. In most cases, adaptation was neces- united in seeking rapid cooling and warming rates, usu-
sary given difficulties in directly transferring the mouse ally in combination with concentrations of cryoprotec-
embryo methods to other systems. There were three main tant that are, as in the Martino et al. study, marginal in
drivers of the direction of this adaptation. First, problems their ability to support vitrification and especially to pre-
encountered with cryoprotectant toxicity suggested the vent devitrification. This work, which is of unquestioned
need to use lower concentrations and to substitute higher value and has frequently produced very good results, is
rates of cooling and warming for some of the cryoprotec- ably described in the chapters that follow, and needs no
tant, a tradeoff that was generally possible for reproduc- additional summarization here.
tive preparations such as oocytes and embryos. Second, However, there is an extreme version of this form of
lower cryoprotectant concentrations also simplified the attempted vitrification that does deserve a brief comment,
avoidance of osmotic damage and the procedures for and that is the idea that vitrification can be achieved,
adding and removing the cryoprotectants. But perhaps with recovery of viability, even in the complete absence
most definitively, and urgently, there was the necessity for of cryoprotectants, an idea that is essentially the same as
avoiding chilling injury. that of Luyet rather than being derived from the other tra-
In the same year the Rall and Fahy paper was pub- ditions described above. This idea has been championed
lished  in Nature, the National Science Foundation con- specifically for spermatozoa on the premise that their low
vened a meeting to discuss the cryopreservation of water content135,136 may make the use of permeating cryo-
Drosophila embryos and selected Peter Mazur and Peter protectants unnecessary for vitrification.136–140 However,
Steponkus as the cryobiologists to lead the project for- these claims, while plausible, at least for the intracel-
ward.133 Both laboratories soon learned that due to the lular compartment if not the extracellular one, remain

6
 overview of biological vitrification
unsubstantiated, as reviewed in detail elsewhere,134 and 20
contradictory information exists.141 Therefore, additional TM I II III IV V
0
development of this line of research is still needed before CV
definitive conclusions can be drawn. Freeze-fracture –20 CNDV
methods have been used to substantiate vitrification at TH

Temperature (°C)
–40
least since 1968,89 and there is no obvious reason that this CU
technique could not be applied to rapidly cooled sperm in –60
order to enable direct observation of either ice or vitrified –80
cytoplasm.
Although they are of practical value, it is necessary to –100
TD TG
point out that many accelerated-rate methods of vitrifi- –120
cation tend to obscure understanding of the underlying V
physical basis of the preservation attained, essentially –140 PCG
blurring the meaning of what a vitrification procedure 0 20 40 60 80 100
actually is. Rapid freezing is not the same thing as vit-
Concentration (% w/w)
rification, although the word “vitrification” is sometimes
used to describe methods that are in all likelihood really
Figure 1.2  Relationships between the melting point, TM,
a form of rapid freezing in the presence of higher-than-
the glass transition temperature, TG, the homogeneous
traditional cryoprotectant concentrations, which may
nucleation (ice self-nucleation) temperature, TH, and the
moderate the biological consequences of intracellular ice
devitrification temperature, TD, observed when small
formation. The confusion that arises from attempting to
samples of ethylene glycol–water mixtures are cooled
achieve vitrification under marginal or even beyond the
and warmed at moderate rates. Although not indicated
marginal limits of the method provides ample justifica-
in the diagram, ice nucleation is likely in Region I above
tion for the brief review of the physics of vitrification
TH due to the unavoidable presence of heterogeneous
in the next section, which may enable all of the various
nucleating agents, whereas both heterogeneous and
approaches to vitrification, “one-way” vitrification (see
homogeneous nucleation become less effective at higher
below), or rapid freezing to be placed into a clearer physi-
concentrations, permitting the glass transition to be
cal context.
observed on cooling and the conversion of the amor-
phous state to the frozen state to be observed on warm-
PHYSICAL ASPECTS OF VITRIFICATION
ing (at TD). In Region II, living systems can be vitrified,
In this section, a general framework for understanding
but are heavily nucleated. In Region III, nucleation is
the physics of cryopreservation by vitrification is pro-
much less severe because TH < TG, which enables TD
vided, including the role of cryoprotectants, the effects of
to move upward and eventually vanish. Region IV is a
the cooling rate, the effects of the warming rate, projected
concentration range in which samples can be vitrified at
rates of biological deterioration as a function of tempera-
very low cooling rates and warmed slowly without devit-
ture, and the thermodynamic necessity of vitrification.
rification, yet in which pre-existing ice, as exists during
More in-depth discussions of the physics underlying bio-
the slow freezing of a lower concentration, can continue
logical vitrification are available,66,142–145 and the reader is
to grow during slow cooling. The white points on the TM
invited to consult them to obtain a deeper understanding
line, which were associated with the TGs noted by the
of specific issues.
large points on the TG curve, represent inferred TMs of
concentrations generated by maximum freeze-concen-
Concentration Dependence of Vitrification
tration, the most extreme of which defines the boundary
The fundamental relationships between vitrification and
between Regions IV and V. CV: lowest concentration for
the use of cryoprotectants are summarized in Figure 1.2,
vitrification without homogeneous nucleation; CNDV:
using as an example a supplemented phase diagram for
lowest concentration leading to no devitrification; CU:
EG composed from data taken from many sources.85,87,88
maximum concentration attainable by freeze-concen-
The temperatures noted for the events in this figure per-
tration; PCG: partially crystallized glass. (Data from of
tain to moderate cooling and warming rates. The non-
Fahy GM et al., Cryobiology 1984;21:407–26.)
thermodynamic temperatures are cooling and warming
rate dependent, but the qualitative relationships shown
are pertinent to all but the most extreme (and in some temperatures (THs), and devitrification temperatures
cases, unattainable) cooling and warming rates, and the (TDs), all of which are a function of the concentration
level of detail shown in Figure 1.2 has been worked out of cryoprotectant and all of which behave in the same
only using relatively low cooling and warming rates. qualitative way for all major low-molecular mass cryo-
The curve labels in Figure 1.2 indicate thermodynamic protectants.107 The interplay between the events denoted
melting points (TMs), TGs, homogeneous nucleation by these curves enables the definition of different regions

7
 vitrification in assisted reproduction
of concentration dependence of vitrification-related phe- amount of growth of an astronomical number of nuclei148
nomena that have significant relevance for attempted will evolve appreciable heat.
cryopreservation by vitrification. Glasses formed at concentrations for which TH > TG
In Region I of this diagram, the dominant limiting are referred to as being “doubly unstable,”146,149 or as
factor for vitrification is homogeneous nucleation, or the being “partially crystallized glasses (PCG in Figure
self-nucleation of water, which is a very rapid process in 1.2).”145 Although vitrification is clearly incomplete in
dilute solutions and is hard to “outrun” by rapid cool- these circumstances, the nuclei formed, since they pre-
ing.146 Above TH, ice nucleation is also possible due to dominantly form at temperatures too low to enable
the presence of heterogeneous nucleating agents, which, crystal growth (temperature optima for nucleation are
at low concentrations of cryoprotectant, tend to cause very much below the temperature optima for ice crystal
freezing between TM and TH, but, as the upper concen- growth in good vitrification solutions66,144,150), tend to
tration limit of Region I is approached, become less and be submicroscopic147,151 and uniform in size,142 and will
less likely to manifest themselves. The property of being generally not be damaging to living cells unless they are
below TM without freezing is known as supercooling, able to grow to damaging sizes151,152 during warming.
and vitrification is made possible by the ability of aque- The uniformity of crystal sizes will also tend to inhibit
ous solutions, especially at concentrations above those recrystallization, another potentially favorable factor
of Region I, to supercool all the way to TG. In Region I, for survival.41 As indicated by the low temperatures of
by contrast, even rapidly cooled small systems tend to detectable devitrification, however, the warming rates
become opaque on cooling due to ice formation, and only required for successful recovery tend to be quite high, and
cooling rates on the order of 105–106°C/second58 (almost low warming rates are usually lethal.153 Even when warm-
107–108°C/min) are sufficient to suppress ice formation in ing leads to survival, however, this does not necessarily
pure water or dilute aqueous solutions, which renders at mean that devitrification has been avoided (see below),
least the lower concentration portion of Region I off lim- and for that reason, methods that employ vitrification
its for vitrification, except under the most extreme condi- in Region II are often likely to represent what has been
tions (see below). called “one-way vitrification,”112,134 in which vitrification
In Region II, the kinetics of homogeneous nucleation is enabled on cooling but the amorphous state is not fully
are significantly impeded by the presence of cryoprotec- maintained during rewarming. High survival rates after
tant, and TH becomes harder to define (gray line). In most “one-way vitrification” are feasible, in general, for systems
examples available, TH is not seen when TG is seen, but in that can be warmed at more than about 1000°C/min, the
the case of the EG–water system, remarkably, TH can be same warming rate that enables appreciable survival after
seen at 40% w/w solute, even though TG can first be detected extensive intracellular ice formation using no or low con-
at 30% w/w EG. Nevertheless, in Region II, homogeneous centrations of cryoprotectant.134,154
nucleation likely becomes distributed over a much broader The boundary between Region II and Region III is
range of temperatures as concentration rises, with the TH defined by the intersection of the TH curve with the TG
curve then defining the upper boundary, rather than the curve (circle marked with a V), which in principle allows
precise temperature of this event. As TH curve approaches vitrification without homogeneous nucleation at this
the curve for the TG, increasing viscosity undoubtedly has concentration and above, and in practice, 8-mL samples
a further significant effect on homogeneous nucleation, cooled at about 10°C/min show no visible ice crystals
but the details have not been explored to date, and other when the concentration is just sufficient for TH to coin-
subtleties may actually facilitate nucleation, as discussed cide with TG in multiple cryoprotectant solutions, and at
in more detail below. For our present purposes, it is suf- both ambient and elevated pressures.40,107 This enables the
ficient to note that the ability to cool small systems without definition, for any given circumstance, of a specific con-
massive homogeneous nucleation enables the detection centration needed to vitrify (CNV or CV).
of the TG curve for the first time in Region II. This thus It is interesting that devitrification is seen in Region III
enables vitrification of the great majority of the solution in even though homogeneous nucleation is not expected. The
Region II. However, since TG is below TH, the glass formed natural explanation for this is heterogeneous nucleation,
is undoubtedly heavily nucleated,145,147 and this is indicated and although high-temperature heterogeneous nuclea-
by the appearance of the TD curve (curve showing crys- tors are not plentiful, weak heterogeneous nucleators that
tallization upon warming, which reflects the growth of would only act at temperatures too low to enable ice growth
previously formed nuclei as well as the growth of any addi- are expected to be more plentiful.155 However, cryoprotec-
tional nuclei formed during warming, which will also form tants also tend to inactivate heterogeneous nucleators. A
mostly below the TH line), as well as by the fact that the TD different explanation for the observed devitrification is
curve can be very close to TG despite the extreme viscosity suggested by the observation of a consistent coincidence
of solutions near TG. The ability to crystallize a detectable between the onset of nucleation and the onset of the glass
amount of water at such low temperatures is a reflection of transition in two significantly different vitrification solu-
the high nucleation density, which means that even a small tions.144,150 It is therefore suggested here that nucleation is

8
 overview of biological vitrification
catalyzed by the lack of mobility of water near the glass regarded as the cooling rate sufficient to reduce x to 3.6%
transition, where local rotation and limited diffusion156 of the maximum amount of ice that can form at low
sufficient to slowly but irreversibly reorient water into an cooling rates.158 Between x = 1 (maximal ice formation)
ice embryo can still take place, without the high-velocity and x = 0 (no ice formation), x as calculated by the above
collisions between the water molecules within the nascent equation varies in a sigmoidal fashion with the log of the
cluster that would normally destabilize the growing ice cooling rate.
embryo at higher temperatures, where kinetic energy is When x is small, the above equation can be greatly
more abundant. Beyond these sources of nucleation, it is simplified. For example,158 for x = 10−6,
also possible to observe the continuation of the TH curve
below TG in some systems.157 = 33.33 k4.
v 
Despite the persistence of devitrification in Region
III, as the concentration is further raised, devitrifica- To calculate the cooling rate required to establish an
tion takes place at higher and higher temperatures until, absolute mass fraction of ice in the solution of interest,
at some concentration (the concentration permitting no rather than a mass that is a particular fraction of the total
devitrification, or CNDV in Figure 1.2), devitrification is no amount of ice that can form in that solution, Boutron’s
longer seen. The latter concentration defines the bound- equation can be simplified for the limiting case of low x
ary between Region III and Region IV. Region IV would by substituting in the definition of x, which is x = q/qmax
be ideal theoretically, since systems could be cooled and (where q represents the amount of heat evolved as a result
warmed even at low rates without any danger of ice for- of freezing and qmax is the amount of heat evolved by
mation, but given the increasingly overwhelming prob- freezing at low cooling rates).158 For example, the cooling
lems of cryoprotectant toxicity when concentrations are rate required to reduce the absolute amount of ice to 0.2%
raised to such levels, Region III is generally the preferred of the total solution mass is158,159
concentration range for vitrification when the warming
rates required for success in Region II cannot be attained.
k4
Above Region IV is Region V, in which ice cannot v= ,
(3[0.2/q max ](1/ 3) )
grow even when it is already present in the solution. The
boundary between Region IV and Region V defines the
upper limit of freeze-concentration possible after devitri- where qmax in this case is the maximum mass fraction of
fication; that is, the maximum possible concentration of the solution that can freeze at low cooling rates.
cryoprotectant that can be generated by freezing84,150 (the Boutron and Baudot et  al., using the latter definition
“unfreezable” concentration, or CU in Figure 1.2). of the critical cooling rate, tabulated the critical cooling
rates of several cryoprotectant–water solutions at several
The Effect of Cooling Rate on the Concentration of different concentrations. Most of these results are sum-
Cryoprotectant Needed for Vitrification marized in Figure 1.3, and are related to Bruggeller and
Between TM and either TH or TG, the state of the system Mayer’s58 estimates for the cooling rates needed to vitrify
is determined primarily by the competing effects of the pure water. The unity of the results, and their agreement
rates of nucleation and crystal growth on the one hand, with projections113,160 as to the critical cooling rates of
and the effects of the cooling rate (and warming rate) solutions intermediate in concentration between those of
on the other. The rates of ice nucleation and growth are pure water and those tabulated by Boutron, Baudot, and
also dependent upon both the solute in question and its colleagues, allows the “boundary conditions” shown in
concentration. Figure 1.3 to be used to evaluate the difficulty of vitrifying
The kinetics of ice formation under conditions of lower-concentration solutions, as well as claims of success
­interest to the cryobiologist were partly mapped by Luyet with the latter endeavor. However, the effects of carrier
and his colleagues, but it was not until Boutron that solutions, nonpenetrating cryoprotectants (npCPAs), and
mathematical generalizations of the net rate of ice devel- the osmolytes normally present within cells are not taken
opment as a function of cooling rate were made avail- into account in this diagram. Mapping the influence of
able.81 Boutron’s final equation95 these additional solutes remains an important task for
future research.

− ln(1 − x1/3 ) + 0.5 ln(1 + x1/3 + x 2/3 ) The Warming Rate Needed After Vitrification
⎛ √3 x ⎞ 1/3
k4 As mentioned above, survival after “one-way” vitrifi-
+ √3 arctg ⎜ = cation, especially in Region II of the phase diagram of
⎝ 2 + x ⎟⎠ | v |
1/3
Figure 1.2, indicates that some degree of devitrification,
or even complete devitrification, followed by incomplete
related the fraction of crystallizable water frozen, x, to recrystallization can be tolerated by living cells. Extensive
the cooling rate (v) and a constant, k4, which can be research has been devoted to defining the warming rate

9
 vitrification in assisted reproduction

109 8
108
G EG
Critical cooling rate (°C/min)

1,3-BD
107
6
106 2,3-BD

Log(WRcrit/CRcrit)
105
D
104 4
103 PG
102 G 2
101 E
P D
100 2,3BD
10–1 0
0 10 20 30 40 50 60 0 100 200 300 400 500 600
Concentration (% w/w) Critical cooling rate (°C/min)

Figure 1.3  Cooling rates required to vitrify all but 0.2% Figure 1.4 Relationship between the critical warming
of the mass of 0%–50% w/w cryoprotectant–water solu- rate (WRcrit) and the critical cooling rate (CRcrit) for six
tions. Data on cryoprotectant solutions are from Baudot different cryoprotectant–water systems (EG  = ethyl-
and Odagescu 229 and Baudot et al.159 The heavy vertical ene glycol; G = glycerol; 1,3-BD = 1,3-butanediol; 2,3-
line at 0% cryoprotectant represents Bruggeller and BD = levo-2,3-butanediol; PG = propylene glycol; and
Mayer’s58 estimated cooling rate range for the vitrifica- D = dimethylsulfoxide). The log of the ratio of the WRcrit
tion of pure water (105 –10 6°C/second). The cooling rates to the CRcrit rises exponentially to an apparent maxi-
projected by Toner,160 as presented by Fahy and Rall,113 mum, as CRcrit rises to greater than 200–500°C/min or
to be required for concentrations between 0% and the so. The function used for curve fitting is log([WRcrit]/
concentrations tabulated by Baudot and colleagues CRcrit]) = y0 + a(1 – e−bx), where x is CRcrit. The param-
all fall within the zone between the dashed lines.  The eters found are given in Table 1.1. A separate fit for D was
curve labels designate glycerol (G), ethylene glycol not attempted, but the fit for EG appeared to fit the data
(E), dimethylsulfoxide (D), propylene glycol (P), and for D adequately. There were insufficient data (two data
2,3-butanediol (2,3BD). (Based on Fahy GM, Rall WF. points) to fit the G results separately, but G and 1,3-BD
Vitrification: An overview. In Tucker MJ, Liebermann appeared to behave in the same fashion at overlapping
J (eds.), Assisted Reproduction: A User’s Manual and rates, so the two G data points and two nearby 1,3-BD
Troubleshooting Guide. Informa Healthcare: London, data points were pooled to allow a lumped fit through
2007. p. 1–20.) the four a­vailable grouped data points. (Data from
Boutron P. Cryobiology 1993;30:86–97; Baudot A, Alger
and temperature dependence99 (but, to a far lesser degree, L, Boutron  P. Cryobiology 2000;40:151–8; Baudot  A,
the actual extent161,162) of devitrification under a variety Odagescu V. Cryobiology 2004;48:283–94.)
of conditions, but almost no research has been done to
define the extent of devitrification that is sufficient to
limit survival. Table 1.1  Parameters Found in Figure 1.4
To begin to place this issue into perspective, Figure 1.4
plots the log of the ratio between the critical warming rate Cryoprotectants y0 a b
(the warming rate needed to prevent ice formation during EG and D 1.4659 5.8246 0.0107
warming; parameters found are listed in Table 1.1) and G and 1,3-BD 1.3668 4.9799 0.0088189
the critical cooling rate as defined in Figure 1.3. As the
2,3-BD 1.2023 4.8140 0.0085981
critical cooling rate increases, the critical warming rate
PG −0.1792 3.8417 0.0331
increases far faster, but seems to reach a maximum ratio
to the critical cooling rate that can range from just under
104 to just over 107 times the critical cooling rate, depend-
ing on the chosen cryoprotectant. with the absence of ice on warming. A few examples
Using this relationship, the critical warming rate can of such relationships are available and are a­ nalyzed in
be calculated for any known critical cooling rate that was Figure 1.5.
associated with the use of a particular cryoprotectant for
a particular vitrification experiment in which the vitri- Cells vitrified in PG
fied living system survived after warming, thus allow- Boutron and Arnaud97 cooled red cells in 30% PG at
ing a comparison to be made between the warming rate 4000°C/min, which exceeds the 1200°C/min critical
associated with survival and the warming rate associated cooling rate for this solution, and rewarmed them at

10
 overview of biological vitrification

100 and implied survival of the red cells upon rapid warming.
Assuming the critical cooling rate was the same as for 45%
10–1
w/w glycerol and that a warming rate of 105°C/min was
PG survival
10–2 achieved, the achieved warming rate would still be 4 logs
WRsurvival/WRcrit

10–3 short of the critical warming rate. Finally, Lehn-Jensen

Glycerol
and Rall127 showed survival of cattle embryos frozen in
10–4 survival
glycerol to a solute concentration of about 51.4% w/w,154
10–5 and then plunged to lower temperatures and rewarmed at
10–6
250°C/min. If one assumes that, based on a critical cooling
1,3-BD survival rate for 50% w/w glycerol of 70°C/min, the critical cooling
10–7 rate of the Lehn-Jensen and Rall solution was 35°C/min,
10–8 then survival was obtained after warming at less than 2%
101 102 103 104 105 of the rate needed to suppress devitrification.
Critical cooling rate (°C/min)
Cells vitrified in other cryoprotectants
Figure 1.5  Warming rates yielding survival after vitri- Mehl and Boutron98 vitrified red blood cells in 30% and
fication, expressed as a fraction of the critical warming 35% w/w 1,3-butanediol and obtained survival after
rates (WRcrit) for the same concentrations of pure cryo- warming at 5000°C/min, even though the critical warm-
protectant in water. Line labels refer to the cryoprotec- ing rates for these solutions are estimated to be over
tants discussed in the text. The critical cooling rate for 100,000 times higher, even for the 35% w/w solution. Rall
30% 1,3-butanediol (1,3-BD) is not known, so for the et al.,128 in experiments involving the freezing of mouse
purposes of graphical representation, the results for embryos in a dimethylsulfoxide solution along lines
this solution are plotted at 1000°C/min. Since the WRcrit similar to those discussed above, also obtained survival,
could not be calculated from a known critical cooling suggesting innocuous devitrification.125,154 By this time,
rate for this solution, the WRcrit value was conservatively there are undoubtedly vast numbers of other examples
assigned the same value as the WRcrit for the 35% w/w that could be analyzed, including for cells of reproductive
1,3-BD solution for graphical representation purposes. interest,153 but the point is clear that survival after vitrifi-
The survival data do not represent a boundary between cation is not dependent, in most systems, on the complete
survival and nonsurvival, but simply provide specific avoidance of devitrification.
illustrative examples available from the literature. The explanation for survival after devitrification is
(PG = propylene glycol.) undoubtedly multifaceted. Studies of the survival of intra-
cellular ice formation have linked death to the attainment
5000°C/min and obtained survival. Red cells cooled in of intracellular ice crystal sizes of 400 nm or above,151,152
35% PG at rates above the 240°C/min critical cooling which requires finite time to occur.161,164 In addition, the
rate for this solution, and then rewarmed at 5000°C/ higher the warming rate, the less the total amount of ice
min, also survived. Using the PG curve fit of Figure developed,161,162,165 even though devitrification is not pre-
1.4, the warming rates giving survival were 9 × 10 −4 and vented. Another important factor comes from the fact that
4.5 × 10 −3 times as high as the critical warming rates for vitrification solutions used for living cells contain more
these solutions. than cryoprotectant and water, and the contribution of
carrier solution solutes to both vitrification166,167 and the
Cells vitrified in glycerol suppression of devitrification112,113,167 is considerable. The
Nei163 found that, according to his electron microscopic inclusion of polymers is particularly effective at slowing
observations, 30% (presumably v/v or ~35% w/w) glyc- devitrification,168 which suggests that natural intracellular
erol cooled at 105°C/min, but not at 104°C/min, appeared polymers will have a similar effect, consistent with evi-
to be ice-free, and warming red cells vitrified under these dence that erythrocyte cytoplasm is more resistant to ice
conditions at unspecified warming rates allowed survival. formation than the extracellular milieu.89,163 Unfortunately,
If we generously assume that the critical cooling rate is the magnitude of these effects and systematic ways of fac-
only 2 × 104°C/min, and that the warming rate was as high toring them into predicted results remain to be adequately
as 106°C/min, however, the warming rate allowing sur- elucidated, although it appears that, as a rule of thumb, a
vival was still ~5 logs less than the critical warming rate. given percentage by weight sugar is roughly equivalent to
Rapatz and Luyet89 mixed one volume of 8.6 M glycerol the same weight percentage of cryoprotectant for suppress-
with one volume of packed red cells, producing perhaps a ing devitrification.112
5.3 M final glycerol concentration154 (or 44.3% w/w) prior A negative contributor to survival of devitrification
to cooling at a rate that was not specified but was suffi- has  been observed in slowly frozen embryos that are
cient to avoid both intracellular and extracellular ice for- killed during slow warming by an invisible event that
mation according to electron microscopic observations, ­follows devitrification, but precedes recrystallization.125

11
 vitrification in assisted reproduction
In this case and many others, protection at lower tem- We can relate viscosity to the time required for a given
peratures and susceptibility to injury at higher temper- amount of diffusion by using the Stokes–Einstein equa-
atures has been attributed to the conversion of cubic to tion, which, for temperatures above TG, relates diffusion
hexagonal ice on warming.169 Even though cubic ice is not coefficients to viscosity by
currently considered to exist,80 the correlation might still
have some validity, since the X-ray pattern suggestive of
kT
cubic ice still represents an incomplete hexagonal ice lat- D=
6πη(T)
tice,80 whose perfection upon warming might then enable
more destructive events to take place. Alternatively, an
invisible change could simply include increased cryopro- where η(T) is the viscosity at temperature T, D is the dif-
tectant toxicity in the frozen state,170 driven by freeze- fusion coefficient of the substance, and k is a constant.
concentration following devitrification. Since the time t required for a substance to diffuse a dis-
In summary, there is still too little understanding of tance d is equivalent to d 2/6D, we can combine the above
the boundary conditions for survival versus nonsur- relationships to obtain
vival of living cells during devitrification, and this is an
area deserving of much further study, for both basic and
⎛T⎞ ⎛ ⎡ 1 1 ⎤⎞
applied reasons. Beyond this, the tolerability of devitrifi- t = t1 ⎜ 1 ⎟ exp ⎜ B ⎢ − ,
cation in organized tissues introduces additional issues ⎝ T⎠ ⎝ ⎣ (T − T0 ) (T1 − T0 ) ⎥⎦⎟⎠

of great applied and theoretical interest, which are as yet
barely addressed.131,171 where t is the time required to diffuse distance d at tem-
perature T and t1 is the time required to diffuse the same
Safe Storage Times in the Vitreous or distance at reference temperature T1.
Near-Vitreous State Values of B and T0 have been determined for a vari-
It would be of great interest to know how biological dete- ety of biologically relevant solutions, including the M22
rioration rates scale with temperature in nonfreezing vitrification solution172,173 used to vitrify and transplant
systems near TG, in part to enable a rational choice of a rabbit kidney, with subsequent permanent survival of
storage temperature. Because glasses are unable to relieve the rabbit after excision of the nonvitrified contralat-
developed thermal stress and are fragile particularly dur- eral kidney,171 based on measured viscosity data and the
ing warming, they are at risk of fracturing, particularly assumption that the viscosity at TG equals 1013 poise.174
when cooled and warmed at higher rates, and this risk Using these parameters and the assumption that t1 is 1
increases as temperature decreases below TG. In addition, ­minute at −22°C (a minimal time to accumulate renal
liquid nitrogen poses safety risks of various kinds, both to injury during M22 perfusion at −22°C), storage times
the stored samples and to banking personnel, and vapor equivalent to t1 at different temperatures have been cal-
storage would be a desirable alternative provided safe culated, and are given in Table 1.2. Although the results
vapor storage temperatures could be defined and main- at ­specific temperatures differ, remarkably, by four orders
tained. Limited empirical data suggest that storage just of magnitude between the different solutions (driven in
below TG is safe,53 but a better theoretical understanding part by a combination of different assumed TGs as well as
of rates of change near TG would be reassuring, particu- different i­nitial viscosities at −22°C), they suggest that,
larly given that nucleation is known to be able to continue even in the worst case, storage of living systems not far
even below TG.142,144,150,157 below TG should be safe.
It was suggested by Fahy and Rall113 that rates of bio- The times calculated in Table 1.2 assume instanta-
logical change ought to scale with molecular mobility, neous cooling to the tabulated temperatures, whereas it
which can be related to the viscosity of the solution. The will be necessary to integrate the time- and temperature-
viscosity of a glass-forming liquid can be fit, for example, dependent total amount of diffusion during cooling to
to the Vogel–Tammann–Fulcher (VTF) equation various temperatures, as a function of the cooling rates
actually contemplated, in order to correct the estimated
safe storage times. The fact that many systems survive vit-
⎛ B ⎞ rification well, however, including embryos cooled at only
η(T2 ) = η(T1 )exp ⎜
⎝ [T2 − T0 ] ⎠⎟ 20°C/min, suggests that, in most cases, damage accumu-

lation during cooling to and warming from TG will not be
where η(T1) is a known reference viscosity at temperature a significant factor for permissible storage times below TG.
T1, η(T2) is the viscosity at temperature T2, and B and T0 The effect of nucleation near TG on safe storage times will
are constants. The VTF equation can be used down to also require additional analysis. For the time being, the
approximately TG, below which viscosity shows Arrhenius predictions of Table 1.2 and similar calculations remain
behavior rather than super-exponential behavior. entirely theoretical, and await experimental testing.

12
 overview of biological vitrification
Table 1.2  Projected Safe Storage Times in the Amorphous (a complete loss of liquid-like properties, as required to
Statea avoid Kauzmann’s paradox).
Solution
BIOLOGICAL CONSIDERATIONS FOR
Constants M22 b DAP10c DMSOc SUCCESSFUL VITRIFICATION
B 1112 1187.1 906.9 What Is Cryoprotectant Toxicity?
T0 −155.4 −154.5 −159.3 New insights are beginning to develop into the nature of
TG −123.3 −124 −135 cryoprotectant toxicity under conditions of cryoprotec-
tant exposure that are relevant to actual cryopreservation
Temperature Time Equivalents Time Units protocols. One of the more interesting developments is
−22°C 1 1 1 Min an expression profiling study involving precision-cut rat
−80°C 13.2 23.2 2.7 Hours liver slices,177 one interpretation of which is that a signifi-
−90°C 5.5 12.0 0.62 Days cant driver of injury may be protein denaturation.134,178
−100°C 18.0 53.3 0.85 Weeks This overall conclusion is consistent with evidence that
−105°C 31.2 114 0.83 Months the toxic effects of vitrification solutions are correlated
with the strength of interaction between the permeating
−110°C 30.5 145 0.39 Years
cryoprotectants and water,101 with stronger interactions
−115°C 652 4379 3.18 Years
potentially causing injury by affecting protein hydration
−120°C 32,837 352,276 44.4 Years
and hence stability. Interactions between cryoprotectants
−121°C 82,374 990,388 81.7 Years and water could account for changes in temperature-
a Temperatures expressed in degrees Celsius, but T1/T calculated dependent actin and microtubule polymerization and
from degrees Kelvin. aggregation, which has been a factor in the destabiliza-
b Data source: Mullen SF, Fahy GM. Fundamental aspects of vit- tion of the meiotic spindle.179 Although individual cryo-
rification as a method of reproductive cell, tissue, and organ protectants show toxic effects that occur at concentrations
cryopreservation. In Donnez J, Kim S (eds.), Principles & lower than those required to denature proteins,180,181 vit-
Practice of Fertility Preservation. Cambridge University Press: rification solutions consisting of mixtures of individual
Cambridge, 2011. p. 145–63. cryoprotectants exhibit toxicity at much higher total
Data source: Fahy GM, Rall WF. Vitrification: An overview. In
c
concentrations, suggesting that the use of mixtures over-
Tucker MJ, Liebermann J (eds.), Assisted Reproduction: A User’s comes toxic mechanisms that are specific to individual
Manual and Troubleshooting Guide. Informa Healthcare: agents and replaces them with denaturation associated
London, 2007. p. 1–20, with error in decimal place for the B with high aggregate concentrations.134,178 If denaturation
value for DAP10 corrected; DAP10 refers to 40% w/v DAP10
is indeed a significant factor in vitrification solution tox-
(10% w/v 1,2-propanediol plus 30% w/v “DA”, a 1:1 mole ratio
of dimethylsulfoxide and acetamide) + 6% PEG (polyethylene
icity, there are likely to be several effective interventions
glycol, Mr 6000); DMSO refers to 50% DMSO in a carrier con- to lessen its effects.
taining 2.7% w/v dextran and 2.25% glucose.
Methods of Preventing Injury from
The Thermodynamic Necessity of Vitrification Cryoprotectant Exposure
During the cooling of vitrifiable liquids above TG, their It is now possible to list at least 14 different methods for
greater molecular mobility in comparison to frozen sys- controlling apparent cryoprotectant toxicity,134 which
tems causes them to lose entropy more rapidly than fro- are recapitulated here with additional notes about appli-
zen systems. Since the vitrifiable liquid does not freeze, it cability to manual pipetting methods for oocytes or
was noticed by Kauzmann175 that a physically impossible embryos:
situation (Kauzmann’s paradox) would arise if the liq-
uid did not become immobilized with cooling: namely, 1. Avoid osmotic injury.
its entropy would eventually, at what is now called the 2. Use mixtures of permeating cryoprotectants
Kauzmann temperature (TK), become less than the (pCPAs; mutual dilution effect).
entropy of the corresponding frozen system. This obser- 3. Include npCPAs to enable the use of less pCPA.107
vation can be taken to indicate that the energy needed to 4. Keep temperatures as low as possible without exac-
drive liquid-like motions is effectively removed by cool- erbating Method 1 above or inducing intolerable
ing prior to reaching TK, thus providing a thermody- chilling injury, and be aware of and avoid transient
namic explanation for the kinetic phenomenon known warming during procedures such as pipetting.
as the glass transition. In fact, TK is quantitatively almost 5. Select an appropriate carrier solution.
identical176 to the T0 of the VTF equation (see above), 6. In the case of oocytes exposed to 1,2-propanediol,
which, according to the VTF equation, would be the use lower concentrations of calcium in the carrier
temperature at which viscosity approaches infinity solution.182

13
 vitrification in assisted reproduction
7. Keep exposure time to the cryoprotectant as low as is available elsewhere,134 but by using appropriate com-
possible, and do not allow differences in pipetting puter modelling,196,197,222–227 limited concentration steps,
times to create exposure time differences between optimized temperatures (which may be higher at lower
different specimens in the same batch. concentrations than at higher ones1), and osmotic buffers,
8. Employ cryoprotectant toxicity neutralization.181 osmotic injury can generally be avoided. Although the
9. Do not use more cryoprotectant than necessary (use importance of osmosis may seem obvious, it is frequently
care in defining the CV), but be aware of and cor- overlooked.228
rect for the fact that pipetting from one solution to
another contaminates the new solution with the old. SUMMARY AND CONCLUSIONS
10. Employ ice blockers5,183,184 to assist with Method 9, The cryobiologist is beginning to catch up to nature in
including emerging novel ice blockers185–194 as they the ability to preserve increasingly complex systems at
become available. low temperatures without ice crystal damage. The mod-
11. Employ 3-methoxy-1,2-propanediol.134,195 ern success of vitrification as a method of cryopreserva-
12. Preferentially use weakly glass-forming pCPAs.101 tion for living systems in general, and for cells and tissues
13. Minimize the “cost function” of cryoprotectant related to reproductive functions in particular, is the result
exposure.196,197 of a decades-long process of gradually improving under-
14. For perfused organs, use newly emerging perfusion standing of both the physical and biological requirements
techniques.131 of this approach to cryopreservation. Despite many basic
misunderstandings, false starts, and failures along the
way, the intrinsic feasibility of cryopreservation by vitri-
What Is Chilling Injury? fication is now clear, and applications are still continuing
A recent DNA microarray study of chilling injury in explosive growth three decades after the first modern wave
rat liver slices177 suggests134,178 that chilling injury in the of enthusiasm for vitrification was launched. A variety of
presence of vitrification solutions can be an extension of new methods for avoiding injury, increasing safety, and
cryoprotectant toxicity, with many changes being similar improving ease of use continues to be established, and
to but intensified versions of changes seen with cryopro- this can be expected to continue. A hopeful new develop-
tectant exposure alone.177 A suggested possibility134,178 is ment for even more forward momentum for the field is
that protein destabilization by cryoprotectants may sum- just beginning to dawn in the form of new examinations
mate with protein destabilization by cooling,198–200 lead- of the molecular biological effects of vitrification solutions
ing to an increase in injury associated with cooling per and chilling injury, an enterprise that seems likely to bring
se. This is a departure from, but is not incompatible with, with it new remedies for old problems and the ability to
prior observations linking chilling injury, especially at address new problems that were previously beyond reach.
higher temperatures, to membrane lipid phase transi- At the same time, large areas of very basic investigation
tions,201–208 and is supported by the finding that chill- have hardly even begun in, for example, defining the stor-
ing stimulates heat shock protein production,209 which age stability of vitrified or near-vitrified biological systems
appears to be protective,209 as well as “cold shock” pro- near TG, the contribution of intracellular biomolecules
teins.210,211 Protein changes are certainly associated with and surfaces to vitrification tendency, the limits of use of
meiotic spindle ­disassembly and mis-reassembly, which npCPAs for vitrification, the nature, rate, and extent of
contribute to chilling injury in OOCYTES,179,212 and may nucleation in concentrated solutions both above and below
be exacerbated by cryoprotectants.213 TG, the limits of tolerance of living systems to devitrifica-
tion, the mechanisms by which devitrification kills cells
Methods of Preventing Chilling Injury and damages intercellular structures, and “cost function”
Chilling injury has been blocked by changing the mem- approaches to reducing cryoprotectant toxicity in many
brane lipid composition,202,214–216 by using antifreeze pro- systems. In conclusion, vitrification remains a dynamic
teins to modify membrane lipid phase behavior217,218 and and productive area of research in cryobiology, and is
permeability,217 by prior heat shock,209 to some extent by likely to remain so for the foreseeable future.
lipid segregation or removal methods,207,219 by tonicity
adjustment,172,220 and by cooling more rapidly than chill- ACKNOWLEDGMENTS
ing injury can induce damage.35–37 I thank 21st Century Medicine, Inc., for enabling the
writing of this contribution and the conduct of the origi-
Methods of Preventing Osmotic Injury nal research mentioned in this chapter.
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2 Vitrification of oocytes and embryos: Finally a recognized technique,
but still a source of concern and debate
Pierre Vanderzwalmen, Nicolas H. Zech, Fabien Ectors, Yannis Panagiotidis,
Achillae Papatheodorou, Prapas Yannis, and Barbara Wirleitner

Even though vitrification was initiated as a viable cryo- embryo transfer of a warmed embryo in a subsequent
preservation technique 30 years ago,1,2 we have had to cycle.16,17 There have been reports of improved implanta-
wait more than 20 years before a shift away from con- tion and pregnancy rates with frozen embryo transfer as
ventional controlled-rate slow freezing (SF) toward vit- compared to fresh autologous embryo transfer, suggest-
rification occurred. It is now recognized worldwide that ing superior endometrial receptivity in the absence of
vitrification is more effective than the standard SF tech- ovarian stimulation.17,18 Clinical outcomes of vitrifica-
nique for cryopreservation of oocytes and embryos at dif- tion/warming cycles support the expectation that trans-
ferent stages of development,3–11 resulting in a doubling fer of an embryo into a more “physiologic environment”
of pregnancy rates.3,4 The implementation of this efficient will result in improved pregnancy rates.19,20 In line with
technique gives us the opportunity to change the strat- this, recent data also support a potential decrease of
egy for embryo transfer and to extend the indications for both maternal and perinatal morbidity in cryo-cycles.21
cryopreservation of oocytes and embryos. Moreover, vitrification cycles are a valuable option when
In the past, oocyte cryopreservation (SF or vitrifica- blastocysts originate from in vitro maturation cycles.12,22
tion) was the only viable option for preserving the fertility With the final objective of increasing pregnancy
of  women who can be predicted to undergo premature rates, an efficient, standardized, reliable, and harmless
ovarian failure due to pathological or iatrogenic alterations. vitrification protocol is mandatory. Vitrification pro-
In addition, to overcome legal, ethical, and religious restric- cedures consist of several steps. Some of them generate
tions, or in cases of absence or delayed semen production, debate. In this review, several phases of the procedure
oocyte cryopreservation has been utilized; and also for that have aroused controversy are discussed. For exam-
medical reasons (e.g., polycystic ovarian syndrome and ple: (a) which blastocysts should be selected before and
ovarian hyper-stimulation syndrome), oocyte cryopreser- after vitrification? (b) Is it necessary to collapse the blas-
vation may be the only alternative. However, for other indi- tocoele? (c) Are concerns about the use of high concen-
cations such as oocyte banking or family planning, it was trations of cryoprotectant solutions (CPsol) justified? (d)
not considered reasonable to propose cryopreservation of Is it possible to vitrify using reduced cooling conditions
oocytes. The patients were informed that cryopreservation and achieve aseptic vitrification ? (e) Is the warming
was still in an area of active research and that one could not process more important than the cooling one? (f) And
guarantee the success of the procedure for the long-term finally, there are concerns about the stability of vitrified
future. Owing to the remarkable improvement in the effi- biological materials over time.
ciency of vitrification techniques as an alternative to the
classical SF procedure, the proportion of births following PRINCIPLE, DESCRIPTION, AND QUESTIONS
the transfer of cryopreserved oocytes has risen substan- THAT ARISE DURING THE DIFFERENT PHASES
tially. Vitrification is no longer recognized as an experi- OF A VITRIFICATION PROCEDURE
mental method, and the proportion of vitrification cycles Vitrification and warming of embryos (blastocysts) con-
for banking purposes as a replacement for surplus embryo sist of several steps (Figure 2.1):
storage and for social reasons is on the increase.
The same tendency is also true for embryos. If we look Step 1: Selection of blastocysts before vitrification with or
back 15 years, a patient had to undergo several oocyte without the artificial blastocoele collapsing.
retrievals to achieve one pregnancy. As a result of our Step 2: Exposure of blastocysts to the CPsol.
accomplishments regarding a single blastocyst transfer Step 3: Loading on the carrier device and plunging it in
in combination with the very efficient aseptic vitrifica- liquid nitrogen (LN2).
tion technique on day 5 or 6,12–15 we are now able to maxi- Step 4: Storage in LN2 containers.
mize cumulative pregnancy rates.16 From a single oocyte Step 5: The warming process.
retrieval, a woman may conceive several babies after the Step 6: Rehydration and removal of the intracellular cryo-
transfer of one vitrified blastocyst at a time. protectant (CP).
At present, there is a growing trend toward a “vitrify- Step 7: Selection of warmed blastocysts before embryo
all” strategy after in vitro fertilization (IVF) with a single transfer.

23
 vitrification in assisted reproduction

37°C
RT 2 6 7
1 A
1. Selection of the biological material
Blastocyst: Artificial blastocoele collapsing

2. Exposure to nonvitrifying and vitrifying solutions in

minimal two steps

A. Loading on carrier devices

3 5
3. Plunge in LN2 after loading the biological material on
an “open” carrier or “closed” carrier device

4. Storage in LN2 or vapor

5. Fast warming (>20.000°C/min)

–150°C 6. Dilution of CP in steps and ET

–196°C
4
7. Selection of warmed blastocysts before embryo transfer

Figure 2.1  Chronology of the steps in a vitrification/warming protocol. (CP = cryoprotectant; ET = embryo trans-
fer; LN2 = liquid nitrogen; RT = room temperature.)

Even though the basic principle for vitrifying biologi- According to our experience, we suggest that, in prin-
cal material is quite similar according to different proto- ciple, one will be mainly presented with four groups of
cols, some questions are still a matter of debate. We will blastocysts on day 5:
present our opinions about several of the major questions
involved, with special reference to the vitrification of 1. Good-quality blastocysts defined by ICM and TE
blastocysts. classified as A and/or B, accompanied by a degree
STEP 1: SELECTION OF BLASTOCYSTS BEFORE of expansion of 6 (Figure 2.2a), 5 (Figure 2.2b), 4
VITRIFICATION WITH OR WITHOUT THE (Figure 2.2c,d), 3 (Figure 2.2e) or 2 according to
ARTIFICIAL BLASTOCOELE COLLAPSING the morphological scoring criteria of Gardner and
Selection of Blastocysts before Vitrification: Should colleagues.28
We Discard Poor-Quality Embryos? 2. The second group is composed of embryos with an
A pivotal question, especially in autologous cycles, is the ICM and TE classified as B and C, with a degree of
selection of blastocysts before vitrification. Which ones expansion of 5, 4, 3, or 2 (Figure 2.3a,b,c).
should be vitrified and which ones discarded? This raises 3. The third group encompasses early blastocysts of
the question of how to define a good-quality blastocyst: good and nontop quality (Figure 2.3d,e).
the one that looks morphologically normal or the one that 4. The fourth group includes embryos with delayed
actually implants? blastulation as defined by no signs of cavitation on
Grading of blastocyst morphology incorporates day 5, plus embryos that show an arrested develop-
­assessment of the degree of expansion of the blastocyst ment on day 3 (Figure 2.3f).
and the quality of the inner cell mass (ICM) and troph-
ectoderm (TE).23,24 Several studies have shown that the Interestingly, according to the vast majority of data
TE is determined to be significantly related to the rate published on cryo-cycles, predominantly blastocysts
of ongoing pregnancies and miscarriages. By contrast, that fulfill the criteria of the first group are selected for
neither the ICM nor blastocyst expansion was statisti- vitrification, and more rarely embryos from the second
cally significantly related.25,26 In a more recent article, group. Those that fall into the third and fourth groups
Ahlström et al.27 analyzed which pre-freeze morphologi- are commonly not selected. Thus, clinical results always
cal parameters can be used to predict live birth outcomes appear outstanding at first glance. However, if all of the
after vitrified/warmed blastocyst transfer cycles. They patients who had no embryo transfer and those where no
stated that blastocoele expansion and TE grade were surplus embryos were cryopreserved due to poor mor-
identified as the most significant pre-freeze morphologi- phology would be included in the data, then the overall
cal predictors of live birth. success rates would look rather more disappointing. But

24
 vitrification of oocytes and embryos

(a) (b) (c)

(d) (e)

Figure 2.2 Good-quality blastocysts with optimal inner cell masses and trophectoderm formation and a high
degree of expansion.

(a) (b) (c)

Bl 4CA Bl 3BB Bl 2BB

(d) (e) (f)

Fbl top Fbl non top

Figure 2.3  Poor-quality blastocysts with poor inner cell masses and trophectoderm formation as well as a low grade
of expansion.

25
 vitrification in assisted reproduction
Table 2.1  Clinical Outcomes after Vitrification/Warming of Blastocysts according to the Morphological Quality
Blastocyst quality before vitrification Figure Cycles Survival rate (%) Birth rate (%)
i AA, AB, BA Figure 2.2c 311 87 30
ii BB Figure 2.3b 237 85 32
ii BC, AC, CA, CB Figure 2.3a 88 88 35
i AA, AB, BA Figure 2.2e 125 89 29
ii BB Figure 2.3b,c 203 90 31
ii BC, AC, CA, CB 66 92 23
iii Top-grading Figure 2.3d 44 86 25
iii Nontop-grading Figure 2.3e 34 77 22
iv Blastocysts delayed in development Figure 2.3f 29 98 37

do we have to present only excellent results, knowing that many of these poor-morphology embryos in order to
if we took the patients’ point of view, their advice might “beautify” the implantation rates.
be totally different? It is worth mentioning that with current technology,
According to our experience (Table 2.1), we still it is difficult to select the right embryos even in a rou-
observe birth rates of 22%–25% when early blastocysts tine IVF laboratory. Of course, when good-morphology
are vitrified and transferred in a cryo-cycle. For those embryos are present, they will be selected for transfer
from the fourth group, an extra culture period to day 6 or for cryopreservation. However, there is still no clear
is a valuable option, instead of discarding them accord- method or algorithm that will improve this selection. But
ing to tangible criteria. Interestingly, we observe a higher the more difficult task at present is how to select from a
birth rate after vitrification of poor-quality embryos on cohort of poor-morphology embryos the one that may
day 5 or 6 when compared to fresh embryo transfers of implant after fresh ET or cryopreservation. Again, what
similar quality. should we do: keep everything, or deselect all?
In the case of slow-blastulating embryos on day 5 Following the results of our data, it should be man-
(Figure 2.3f), there is a benefit to vitrifying them instead datory to cryopreserve all supernumerary blastocysts
of performing fresh embryo transfer. A total of 488 fresh of moderate to good quality in order to increase the
transfers with slow-blastulating embryos on day 5 were cumulative pregnancy rate. With regard to this, it is very
analyzed. Clinical outcomes were compared: (i) with important to achieve a reproducible outcome, especially
82 cryo-cycles, after day 5 vitrification and warming in terms of survival after warming independent of the
24 hours before cryo-embryo transfer; and (ii) after pro- quality, to allow high success rates after vitrified/warmed
longed culture of slow-blastulating embryos for 24 hours blastocyst transfer.
that resulted in 58 cycles with day 6 blastocyst vitrifica-
tion. Birth rates after a day 5 fresh single embryo transfer Is It Necessary to Collapse the Blastocoele?
with top early blastocysts (mean female age: 37.5 years) With respect to blastocyst quality and survival, the ques-
were 26.1%, with nontop early blastocysts (mean age: tion arises as to whether artificial shrinkage or collaps-
37.1 years) at 6.5%. In cryo-cycles (mean age: 38.2 years) ing should be mandatory (Table 2.2). Since the size of
with vitrified day 5 early blastocysts/morulae, warmed the blastocoele corresponds to the amount of water in
24 hours prior to embryo transfer (ET), birth rates were this cavity, larger blastocysts might show reduced cryo-
18.3%. When vitrification was done on day 6, the birth rate survival potential due to ice crystal formation during
with top-grade blastocysts derived from slow embryos on the cooling step and ice recrystallization (devitrification)
day 5 was 42.9%. Nontop-grading blastocysts vitrified on during the warming step.
day 6 showed birth rates of 18.3%. Better synchronization In order to reduce the likelihood of ice crystal forma-
between the embryo stage and endometrium is suggested tion, artificial shrinkage or collapsing of expanded blas-
to be mainly responsible for this outcome. tocysts has been suggested using either micropipettes30 or
Acceptable results were also obtained after vitrifica- laser pulses.31
tion of blastocysts that originated from embryos that However, with use of vitrification solutions containing
were not selected for fresh ET and underwent cryopreser- higher sucrose concentrations of 0.75 M instead of 0.5 M,
vation on day 3 because of their poor quality. Fifty blasto- we observed that artificial shrinkage of the blastocoelic
cyst warming cycles resulted in a 76% survival rate, 44% cavity is less necessary for preventing injury from intra-
clinical pregnancy rate, and 39% implantation rate.29 cellular ice, because of sufficient dehydration during the
So, is it really an option to reject those blastocysts for exposure to the vitrification solutions. As another strat-
transfer or vitrification? It may be that we discard too egy, Zech et al.32 showed the benefit of opening the zona

26
 vitrification of oocytes and embryos
Table 2.2  Impact of Artificial Collapsing before Concentration of CP and Cooling/Warming Speed:
Vitrification Using Different Types of Carrier Devices Two Parameters to Control in Order to Achieve a
and Vitrification Media Vitrified State
When the temperature decreases, liquid water can be
Concentration Concentration Carrier Artificial Survival
converted either to a solid crystal or to a solid amorphous
nVS Sucrose VS devices collapsing (%)
glass when the supercooled water is dropped instanta-
20% 40% 0.3 M Open No 20–30 neously below the glass transition temperature (TG). It is
3 min 30 s Closed Yes 78 simply the ability of being able to prevent ice crystals from
10%–20% 40% 0.75 M Open No 92 forming inside the cell (which can happen during the
4–5 min 60 s Closed Yes 90 cooling as well as the warming process) that will deter-
15% 30% 0.5 M Open No 82 mine the viability of the embryos after cryopreservation.
8–12 min 60–90 s Closed No 64 The physical process by which a superviscous liquid solu-
Yes 88 tion remains supercooled during transition through the
crystalline phase and reaches the solid glassy state when
Note:
it is cooled below its TG is called vitrification.1 According
nVS, nonvitrification solutions:
20%: 10%–10% EG/DMSO;
to this definition, with the application of the vitrification
15%: 7.5%–7.5% EG/DMSO. process, ice crystal formation is still possible either in
VS, vitrification solutions: the intracellular or in the extracellular spaces, but solidi-
40%: 20%–20% EG/DMSO; fication of a superviscous liquid solution that remains
30%: 15%–15% EG/DMSO. supercooled during the cooling process will obviate this
possibility (Figure 2.4).
pellucida some hours before the vitrification process. The fundamental issue in all vitrification methods is
They observed that blastocysts with a larger blastocoelic achieving and maintaining conditions within the cells
cavity survived vitrification better when they were par- that guarantee an amorphous state throughout the cool-
tially or completely hatched, even with a short exposure ing, as well as during the warming process. Independent
to CP solutions. of the carrier device that determines the cooling and/or

Cooling– CPs
warming concentration
rates

T° P=
Liquid H2O
Solution
TM volume
0°C
Heterogenous Nucle
ation
TH area
~–40°C

TM, Melting T°
Solid crystalline H2O
TH, Homogenous nucleation
–100°C TG, Glass transition T°
TG

–120°C Solid glass–vitreous-H2O

A B

Concentration of solute (salt–CP)

Figure 2.4  Phase diagram presenting the different phases of water according to the temperature. The equation rep-
resents the factors that influence the probability of achieving the vitrified state. The arrows represent the cooling
speed and the size of ice crystal formation that are associated. Arrow A represents a less fast cooling than arrow B.
(CP = cryoprotectant.)

27
 vitrification in assisted reproduction
the warming rate, the key to success, in order to achieve a Consequently, we may state that although SF has been
“glass-like” state, depends on an optimal balance between the standard cryopreservation method for more than
the speed of cooling, rewarming (time and temperature), 25 years, few practitioners were aware that cell survival
and the optimal cell dehydration and penetration of CP was principally a result of the presence of an intracellu-
when the cells are exposed to concentrated hypertonic lar vitrified state. This vitrified state is a consequence of
solutions.33 the prolonged effect of the solution during cooling, and is
a reflection of a very high concentration of CP. Thus, we
STEP 2: EXPOSURE OF BLASTOCYSTS TO HIGH may suggest that a drop in the survival after SF is prob-
CONCENTRATIONS OF CP SOLUTIONS—WHAT ably more related to osmotic shock after warming than to
IS THE FINAL INTRACELLULAR mechanical injuries due to the formation of intracellular
CONCENTRATION OF CP? ice crystals. To conclude, we may state that SF is nothing
There is no doubt that all CPs are toxic and that each CP more than another way of performing vitrification.
has its own intrinsic toxicity that induces biophysical and
biochemical interaction with the cellular structures. This STEP 3: LOADING THE CARRIER DEVICE AND
toxicity is related to the type of CP, the incubation tem- PLUNGING INTO LN2: CAN WE FORESEE AN END
peratures, and the concentrations. During the SF process TO THE “OPEN” SYSTEM?
using 1–2 propanediol, an extracellular and intracellular A summary of the open and closed vitrification devices
accumulation of 1–2 M propanediol is generated by long with their specific volume and cooling/warming rates
exposure times before freezing and by the formation of will be discussed in Chapter 6.
extracellular ice crystals. Could this explain why Pinborg
et  al.34 reported an increased birth weight of children Open Systems
born after SF embryo transfer using 1–2 propanediol It has been postulated that ultra-rapid cooling and warm-
as the CP? It is reported that 1–2 propanediol displays ing rates (as high as 20,000–30,000°C/min) are obligatory
higher genotoxicity and induces DNA damage in vitro, during the vitrification process in order to reduce the risk
leading to chromosomal mutations.35,36 In addition, cyto- of intracellular crystal formation and the concomitant
toxic effects should not be underestimated.37 damage to the cell structures.40 In order to achieve ultra-
In the majority of vitrification solutions, ethylene rapid cooling rates, very small volumes of vitrification
glycol is associated with dimethyl sulfoxide (DMSO). solution of less than 1 µL are deposited on open carrier
DMSO was shown to alter DNA methylation, changing devices (e.g., Cryotop, Vitriplug, Cryoloop, and copper
the epigenetic profile of cells.38 There are, in fact, plenty electron microscopy grids), which are directly plunged
of reports with controversial conclusions related to the into LN2.41–43
toxicity of CPs. Therefore, the use of CPs cannot be easily The advantage of such an approach is that blastocysts
dismissed as a nonissue. What is clear is that the use of are exposed in two steps to increasing concentrations of
very high concentrations of CPs may be a strong argu- CP. This is only for a short period of time, but nevertheless
ment against the increasing use of vitrification. it is long enough to permit the extraction of the intracel-
The total concentrations required to obtain a vitreous lular water while limiting the amount of CP permeating
state are usually between 6 and 8 molal and are very near into cells, thus limiting osmotic stress.
the tolerable limits for cells. As a consequence, fear about One drawback of the “open” carrier devices is that
exposing gametes and embryos to high amounts of CP the blastocysts are directly exposed to LN2 during the
solutions that exceed three- to four-fold of those applied cooling process, as well as during the whole storage
in SF has been the central part of a debate initiated by time. This is not in concordance with medical direc-
advocates of the SF procedure, where lower concentra- tives for the storage of biological tissues that are put in
tions of penetrating CPs are used. This point of the debate place to minimize the risk of cross-contamination from
is extensively described in Chapter 3. infected material, and also from the LN2 itself, which
Concisely, we have found that only a small percent- is not sterile. In spite of the existing theoretical risk of
age of the CP present in the vitrification solutions enters cross-­contamination by bacteria, viruses, or fungi dur-
through the cell membrane. We have found that the intra- ing cooling as well as during storage in LN2 , the clini-
cellular concentration of cryoprotectant (ICCP) in mouse cal significance is widely debated.44,45 Retrospective
zygotes during the vitrification procedure prior to plung- investigations in which commercial LN2 cryotanks were
ing them in LN2 is approximately equal to 2.14 M, and examined after 35 continuous years of service revealed
is three-fold below the concentration in the vitrification various bacterial and fungal contaminations in the
solution that surrounds the cell.39 Furthermore, contrary LN2 detritus, but reassuringly, many of the identified
to common belief, it was observed that the intracellular bacteria isolated were universal environmental micro-
concentration of CP in vitrified zygotes is even lower than organisms and were rare opportunistic pathogens of
after SF, despite working with higher amounts of CPs in low significance to producing disease in humans or ani-
the solutions. mals.46 In addition, the potential probability of a toxic

28
 vitrification of oocytes and embryos
effect with the reactive chemical compounds present in fluorescence and confocal laser scanning microscopy.
LN2 raises safety concerns.47 Even though there was a significantly higher incidence of
Various methods for sterilizing LN2 have been pro- abnormal spindles in the vitrified group compared with
posed or are under development, including ceramic fil- the fresh group, the high survival rate following warming
ters48 or ultraviolet light, with subsequent hermetically and the large proportion of normal spindle/chromosome
sealed cryostorage49,50; or even by using LN2 vapor for configurations suggest that aseptic vitrification of blasto-
storage.51 cysts on day 5 in carrier tools sealed into a container does
Although the probability of an impairment of cellular not adversely affect the development of human embryos
structures by contact with LN2 is still being discussed, this and the ability of spindles to form and continue normal
potential risk is important, and the ongoing discussion cell division.
indicates that the storage system, especially long term, There is no direct evidence of cryopreserved human
should be revised. Even the standard storage conditions oocytes/embryos becoming contaminated during cooling
with refilling of the tanks pose a hazard, when oxygen and cryostorage.58 However, given the available informa-
from the surrounding air condenses and mixes with LN2 tion from bovine embryo studies and potential concerns
during the regular opening of the dewar tank for routine with long-term storage in open devices, it would appear
refilling, or indeed whenever straws are added or with- prudent to avoid such direct contact with LN2 during vit-
drawn. Although it is generally assumed that thermally rification and subsequent cryo-storage, by using closed
driven reactions do not occur in cells at −196°C, it has devices.
been reported that in the case of irradiation of an LN2/
oxygen mixture, a synthesis of oxygen radicals resulting STEP 4: STORAGE IN LN2 CONTAINERS:
from ozone formation and decomposition cannot be dis- FOR HOW LONG IS THIS POSSIBLE?
counted; and this may even be enhanced by the catalytic Since the goal of cryopreservation is to preserve cells and
effect of nitrogen. Mouse oocytes have shown impaired tissue in a solid state for long periods, the stability of vitri-
survival, fertilization rates, and embryonic development fied biological materials over time is an important issue,
after prolonged contact with LN2.47 and certainly is still a matter of debate when consider-
ing the thermodynamic aspect. The instability phenom-
Closed Systems enon under the TG is of particular interest, because the
The European Directives (2004/23/EC)52 and the Food and considerable molecular mobility that persists near and
Drug Administration (FDA) directives on tissue and cell under the TG makes the stability of such storage tempera-
storage53 dictate adherence to certain safety regulations, tures unclear. In fact, crystalline solids are more stable
ensuring that gametes and embryos are protected from any because there is usually the energy of crystallization that
possible contamination with pathogens and attempting to is released upon crystallization with interactions between
prevent them from any harmful physical conditions during molecules. By contrast, the conversion to a glass-like state
cryo-storage. To comply with the EU directive, a valuable during vitrification is not accompanied by exothermic
option consists of switching from an open vitrification car- heat of fusion, but is accompanied by changes in the
rier device to a protocol that entails complete isolation of heat capacity of the sample.59 The glass transition can be
the biological samples from LN2 during both the cooling understood on a molecular level as a loss of translational
process and storage by hermetically isolating the embryos and rotational degrees of freedom over a particular mea-
from LN2 in the dewar tanks. surement timescale, but still leaves bond vibration within
A huge difference in cooling/warming rates exists a fixed molecular structure.60
between vitrification protocols with open (>25,000°C/ Although diffusion is practically nonexistent below the
min) or closed (<2000°C/min) carrier devices. This TG, small local movements of molecules related to relax-
reduction in the cooling rate is responsible for the ongo- ation have consequences for cryobiology.61 This is a result
ing debate, as the cooling rate is widely believed to be an that occurs during the forming of amorphous solids when
important factor in the success of vitrification protocols. substances are cooled so quickly that their molecules move
However, several studies have shown that vitrification of too slowly to orient themselves. It is commonly admitted in
oocytes,54 zygotes,19 and blastocysts in closed carriers can the field of thermodynamic cryobiology that once the glass
achieve good results in clinical studies.12,13,15,55,56 Taking state is formed, it can be aged. Aged amorphous materials
into account that the reported survival rates after asep- show decreased physical and chemical reactivity compared
tic vitrification correspond with data observed in open to un-aged materials.
devices, the functional aspect of embryos/gametes frozen Although many thousands of children have already
with the different devices remains to be investigated. been born after blastocyst vitrification, many aspects of
In a recent study, Chatzimeletiou et  al.57 investigated this technique remain to be elucidated. New applications,
the effects of aseptic vitrification on the cytoskeleton and such as fertility preservation, lead to long storage times of
development of human blastocysts by analyzing survival vitrified gametes or embryos. However, it remains to be
rates and spindle and chromosome configurations by determined whether these vitrified gametes and embryos

29
 vitrification in assisted reproduction
are stable over time. In the assisted reproductive tech- STEP 5: IS THE WARMING PROCESS MORE
nology field, little is known about the risks of prolonged IMPORTANT THAN THE COOLING ONE?
storage of cryopreserved cells, given that vitrification is Since the rate of cooling engendered a hot debate, it is
the solidification of a fluid without the formation of crys- surprising that little emphasis was put on the warming
talline structures—a physically disorganized and there- procedure. However, it has become clear that the warm-
fore potentially unstable system. This raises the question ing rate might play a greater role in establishing cryo-
that if this state changes over time, will this significantly survival after vitrification than the cooling rate. Seki and
impair the cryo-survival and implantation potential of Mazur63,64 have shown that mouse MII oocytes survived
vitrified gametes and embryos? Subsequently, any poten- well in reduced cooling conditions, as long as they were
tial impact on the health of the newborn remains largely warmed as rapidly as possible.
unknown. According to the theoretical definition of vitrifica-
A study by Wirleitner et al.62 included the transfer of tion and the probability of obtaining a vitreous state with
blastocysts that had been vitrified aseptically with the the increasing concentration of CP, we may expect com-
Vitrisafe® device, and showed that the storage of vitrified plete intracellularly vitrified states (Figure 2.5, situation
blastocysts in aseptic conditions did not impair blasto- cell 3). Even without the presence of small nuclei of ice
cyst viability. The survival rate after warming during the crystals, the warming rate is crucial in order to achieve
first year of storage was 83.0%, compared with 83.1% after high survival rates. When absolute intracellular vitrifica-
5–6 years of storage (no significant difference); there were tion is completed (Figure 2.5: situation cell 3), a slow rise
also no decreased pregnancy rates, as the clinical preg- in the temperature above the TG will induce first a direct
nancy rate after 1 year of storage was 40.0% versus 38.5% devitrification (Figure 2.5A and B), with the formation of
after 6 years (no significant difference). In addition, no plenty of small intracellular crystals that will reorganize
increase in the neonatal malformation rate over time was (Figure 2.5C and D) with a further rise in temperature.65
observed. In contrast to what happens during the cooling process,

E T°
Liquid H2O
TM, Melting T°
TM
0°C TH, Homogenous nucleation

D TG, Glass transition T°

TH
~–40°C

Solid crystalline H2O

C

–100°C

B
TG Solid glass–vitreous-H2O
–120°C
A B C
A

Concentration of solute (salt–CP)

Figure 2.5  Devitrification and ice re-crystallization during warming procedures that are too slow. (A) Vitrified—
maintain a fully amorphous intracellular state. (B) Devitrification may occur when the temperature crosses
the TG. (C) Freezing—crystallization step. (D) Re-crystallization to a damaging size. (E) Thawing and cell lysis.
(CP = cryoprotectant.)

30
 vitrification of oocytes and embryos
in which the sample is in a supercooled condition after STEP 7: SELECTION OF WARMED BLASTOCYSTS
crossing the melting temperature (TM), the sample above BEFORE ET: WHICH ONE TO SELECT?
the TG will start rapidly to develop ice crystals. Though some post-thaw morphological predictors have
We have shown that the intracellular concentration of been investigated in SF of blastocysts (e.g., immedi-
CP is substantially lower than that present in the vitrifi- ate re-expansion68,69 or 24-hour survival), no such data
cation solutions.39 So the question is—even though good have yet been published for vitrified blastocysts. It has
survival rates are obtained after vitrification, are fully vit- been suggested that as a result of the presence and size
rified states obtained intracellularly? It has been demon- of the blastocoelic cavity, vitrified warmed blastocysts
strated that the faster the cooling rate, the smaller the size experience several morphologic changes and become col-
of the ice crystals that will be formed66 (Figure 2.4: arrow lapsed during cryopreservation. Thus, it is more difficult
B vs A). According to the previous study, Seki and Mazur to score a vitrified blastocyst after warming than a fresh
believe that the crystal size determines the rate of warm- one.69 Several factors (unrelated to the vitrification pro-
ing, and consequently the survival rate.65 Therefore, the cess) are known to directly influence the fate of a cryo-
primary cause of injury or cell death in the vitrification preserved blastocyst after thawing/warming and transfer.
procedure is ice re-crystallization (devitrification) during It is important to realize that the survival rates reported
warming, and not failure to vitrify during cooling.63,64 in the literature are not easily comparable, due to the fact
If very small intracellular ice crystals are present in the that some embryologists focus on immediate cryo-sur-
intracellular compartment, they will be innocuous dur- vival, while others suggest an additional waiting period
ing the cooling process (Figure 2.5: situation cells 1 and of 24 hours, for example, to facilitate control of growth.
2), but they may become lethal according to the warm- Differences in implantation rates may also be attributed to
ing rate. In the case of warming being too slow, there is the fact that not all working groups apply assisted hatching
sufficient time for the aggregation of many small-sized to the thawed blastocysts (whilst shrunk), though this has
crystals into larger ones65 (Figure 2.5B–D). The oocytes been found to improve outcomes.40 Re-expansion of the
or embryos will then not survive the re-crystallization blastocoele (and consequently the blastocyst) after thaw-
that occurs during the warming process, as the growing ing is expected within 24 hours.68,69 However, immediate
ice crystals will reach a lethal size. Therefore, to compen- re-expansion (e.g., within the first 2 hours after warming)
sate for the greater driving force, one needs to warm more has not been used for prognostic purposes for vitrified
rapidly to avoid re-crystallization. blastocysts. Since in SF, approximately half of the frozen
It is well known that for any given concentration of CP, blastocysts re-expanded immediately after 2–4 hours in
the critical warming rate is much higher than the criti- culture,69 it has been reported that when using vitrifica-
cal cooling rate.67 Consequently, the minimal concen- tion, a higher rate of re-expansion is observed.70
tration of CP to prevent crystallization during warming Even if it can be assumed that all viable blastocysts will
must be higher than during cooling. This means that it re-expand after several more hours, any delay in this pro-
might be easier to maintain a vitrified state during the cess could be the manifestation of altered osmotic and/or
cooling p ­ rocess than during the warming process for metabolic conditions. This is comparable to the situation
the same concentration of CP. If the warming rate is found during blastocoele development, when water enters
reduced by using a device that isolates the drop contain- the blastocoelic cavity via tight junctions either by dif-
ing the embryos from the LN2, then higher intracellular fusing passively or being pumped actively. To conclude,
concentrations of CP are needed in order to reduce the then, a fast re-expansion post-warming is the best indica-
likelihood of re-crystallization. However, these higher tor of blastocyst viability.
concentrations of CP might be toxic to the cells.
Hence, in order not to increase the concentration of CONCLUSIONS
CP above the toxic level, the biological material has to be It is now proven that aseptic vitrification with the closed
warmed extremely rapidly. Consequently, most open or devices such as the Vitrisafe® carrier can be effective for
closed devices that are used produce very high warming oocytes, zygotes, and blastocysts at different stages of devel-
rates exceeding 20,000°C/min. Mechanistically, this has opment and quality. Vitrification has to be adapted accord-
resulted in the confounding of whether the cooling rate ing to the cooling/warming conditions. Higher levels of
or the warming rate is relatively more important. intra- and extra-cellular CPs are needed to keep the solu-
tion in a vitrified state in the event that there is a reduced
STEP 6: WHY IS DILUTION OF THE CP cooling as well as warming rate. However, the fear about
REQUIRED? exposure of biological material to higher concentrations of
During warming, water re-enters the cells and CPs are CPs does not appear justified. Even though a lower cooling
washed out. This has to be performed in a controlled way rate exists with aseptic vitrification, it remains an effective
in order to avoid cellular damage. The influx of water procedure if higher warming rates are achieved. It is our
being too rapid is circumvented by a stepwise exposure to advice that this strategy of “better safe than sorry” has no
solutions containing reducing sucrose concentrations.39 negative impact on the viability of vitrified blastocysts over

31
 vitrification in assisted reproduction
a period of 6 years. Critical warming rates are typically two 13. Stachecki J, Garrisi J, Sabino S et al. A new safe, sim-
or more orders of magnitude greater than critical cooling ple and successful vitrification method for bovine
rates. Collapsing of the blastocoele is not really necessary and human blastocysts. Reprod Biomed Online 2008;​
and has to be adapted to the rate of dehydration that occurs 17:360–7.
in the vitrification solution. Finally, the speed of re-expan- 14. Van Landuyt L, Stoop D, Verheyen G et al. Outcome of
sion after warming is a good sign of viability. closed blastocyst vitrification in relation to blastocyst
quality: Evaluation of 759 warming cycles in a single-
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effects of cryoprotectants as single-component and gen vapour as a risk factor in pathogen transfer.
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115–22. 52. European Parliament Directive (EU Tissues and Cells
38. Iwatani M, Ikegami K, Kremenska Y et al. Dimethyl Directive 2004/23/EC). URL: http://eur-lex.europa.
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39. Vanderzwalmen P, Connan D, Grobet L et al. Lower 53. Food and Drug Administration (FDA). Human Cells,
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to higher concentration of cryoprotectant solutions. URL: http://www.gpo.gov/fdsys/pkg/FR-2007-06-19/
Hum Reprod 2013;28:2101–10. pdf/E7-11795.pdf.

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 vitrification in assisted reproduction
54. Papatheodorou A, Vanderzwalmen P, Panagiotidis 63. Seki S, Mazur P. The dominance of warming rate over
Y et  al. Open versus closed oocyte vitrification sys- cooling rate in the survival of mouse oocytes sub-
tem: A prospective randomized sibling-oocyte study. jected to a vitrification procedure. Cryobiology 2009;​
Reprod Biomed Online 2013;26:595–602. 59:75–82.
55. Kuwayama M, Vajta G, Leda S et al. Comparison of 64. Seki S, Mazur P. Effect of warming rate on the survival
open and closed methods for vitrification of human of vitrified mouse oocytes and on the recrystallization
embryos and the elimination of potential contamina- of intracellular ice. Biol Reprod 2008;79:727–37.
tion. Reprod Biomed Online 2005;11:608–14. 65. Mazur P, Seki S. Survival of mouse oocytes after being
56. Liebermann J. Vitrification of human blastocysts: An cooled in a vitrification solution to –196°C at 95° to
update. Reprod Biomed Online 2009;19(Suppl. 4):4328. 70,000°C/min and warmed at 610° to 118,000°C/min:
57. Chatzimeletiou K, Morrison EE, Panagiotidis Y et al. A new paradigm for cryopreservation by vitrification.
Cytoskeletal analysis of human blastocysts by confo- Cryobiology 2011;62:1–7.
cal laser scanning microscopy following vitrification. 66. Van Venrooij V., Aersten A, Hax W et  al. Freeze-
Hum Reprod 2012;27:106–13. etching: Freezing velocity and crystal size at different
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study of amorphous CaAl2Si2O8. Geochim Cosmochim 69. Shu Y, Watt J, Gebhardt J et  al. The value of fast
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34
3 Intracellular concentration of cryoprotectant during vitrification
and slow-freezing cryopreservation procedures
Pierre Vanderzwalmen, Fabien Ectors, Barbara Wirleitner, Astrid Stecher,
Deborah Desmet, Jacqueline Greindl, Amandine Helson, Sabine
Vanderzwalmen, Mark G. Larman, Nicolas H. Zech, and Delphine Connan

INTRODUCTION publications were reassuring in terms of obstetric and

To reduce the likelihood of intracellular ice crystal for- perinatal outcomes,16–20 further studies were necessary
mation, which has detrimental effects on cell organelles to prove the safety of the procedure and to exclude any
and membranes, vitrification (VIT) was first introduced negative impacts on the development of vitrified warmed
as an alternative to slow freezing (SLF) to cryopreserve embryos.
embryos and gametes 30 years ago.
It is now well documented that compared to SLF, VIT is EXPOSURE OF OOCYTES OR EMBRYOS
a very effective procedure to cryopreserve MII oocytes,1–6 TO HIGHER CONCENTRATIONS OF CPS:
zygotes,7,8 and embryos at different stages of development.9–14 WHY AND WHEN?
Following the reports of an efficient VIT technique, a change The central part of this debate concerns first the use of
in both the strategy for embryo transfer and an expansion solutions containing high concentrations of CP, and fur-
of indications for cryopreservation of oocytes and embryos thermore the reasons why in specific situations we have to
is occurring. For example, the “freeze-all” strategy, in com- increase the time of exposure to penetrating CPsol.
bination with single-embryo transfer and preimplantation
genetic screening (PGS) on day 5 or 6 will likely become the Why Use Such High Concentrations of CPs in the Last
standard procedure to increase the cumulative birth rate. Solution before Plunging in Liquid Nitrogen?
Furthermore, at present, in addition to fertility preservation, General principle
to avoid legal, ethical, or religious restrictions, oocyte cryo- Before the oocytes or embryos are immersed in liquid
preservation is now commonly applied for oocyte banking nitrogen (LN2), they are exposed to CPsol in order to
and for women who want to preserve their oocytes for social increase the intra- and extra-cellular viscosity to a level
reasons. such that the liquid water molecules will solidify so
quickly that they will not have time to rearrange them-
VIT: REASONS FOR LACK OF ENTHUSIASM selves into a crystalline structure when they have to cross
A DECADE AGO the crystalline phase. This means that before plunging the
In spite of the first announcement of successful VIT in biological material in LN2, the intracellular compartment
the mammalian model in 1985,15 we had to wait for more has to be conditioned to allow the emergence and mainte-
than two decades of SLF use before the full acceptance of nance of a vitreous state.21
VIT in the assisted reproduction technology field. To achieve this objective, in nearly all VIT methods,
How can this unwillingness to move to VIT be the biological materials are exposed to a minimum of
explained, despite the up-to-date publications being reas- two steps of gradually increasing concentrations of non-
suring in terms of obstetric and perinatal outcomes? One vitrifying solution (nVS) and vitrifying solution (VS).8,11
part of the answer is directly related to the methodology Usually, oocytes or embryos are first exposed to two nVS
of VIT that is totally different when compared to SLF in (nVS1 and nVS2) containing cell-penetrating CPs (2.3–
term of cryoprotectant (CP) concentration. 3.2 M).1,8,11 In the last step, just before being plunged in
A fear about exposing gametes and embryos to such LN2, they are exposed for a short time (45–90 seconds) to
high amounts of CPs that exceed by three- to four-fold a VS containing very high concentrations of penetrating
those found in SLF (CP solutions [CPsol] of 1.5 M) was (4.8–6.4 M)1,8,11 and nonpenetrating (0.5–0.75 M) CPs.
the central part of a debate initiated by advocates of the
SLF procedure in which lower concentrations of penetrat- The objectives of exposure to the nVS and VS
ing CPs are used. The objectives of exposing the biological material to the
It is well accepted that all CPs are potentially toxic. The VS that contains nonpermeable CPs (sucrose [SUC] and
impact of the CPs used on the safety of VIT has always trehalose) with low or high molecular weights (Ficoll) are
been a major scientific concern. Although the up-to-date two-fold.

35
 vitrification in assisted reproduction
The first is to create an intracellular vitrifiable state • The ICCP at the end of the exposure steps to the
due to the dehydration of the cells in contact with the CPsol.
SUC that concentrates the intracellular solutions of salts, • Which of the techniques—SLF or VIT—result in
(glyco)proteins, and also CPs that have previously pen- the highest ICCP.
etrated the cell in the course of exposure to the nVS. The
cytoplasm contains various intrinsic macromolecules Determination of the ICCP by Cellular Volume
and salts that are concentrated during the final dehydra- Monitoring
tion step, so favoring the amorphous state. This generates Strategy and method for ascertaining the ICCP
an intracellular environment that promotes the mainte- The ICCP was determined at the end of the exposure steps
nance of a vitreous state when cells are rapidly plunged to nVS1, nVS2, and VS using cell volume measurements.
into LN2 or warmed. Cells adapt their intracellular compartment, by vol-
The second aim of exposing the cells to such high con- ume modification, in reaction to osmotic modifications in
centrations of CPsol is to create a vitrifying extracellular their environment. Exploiting this mechanism, we deter-
sheath to avoid ice crystal formation, which may have mined the final ICCP after the incubation steps in the
detrimental effects. In fact, the extracellular vitrifiable VIT solutions by exposing the cells to defined molarities
state is obtained by the high concentration of CPs in the of SUC solutions. No changes in cell volume, and there-
VS that encapsulates the embryo in a vitrifying sheath. fore no movement of water through the cell membrane,
indicates equivalent intra- and extra-cellular osmotic
In Which Situation Do We Have to Increase the pressures on both sides of the membrane. Knowing the
Exposure Time to the CPsol? osmolarity of the SUC solution consequently allows
VIT is the conversion of a superviscous, supercooled liq- determination of the ICCP. The ICCP was then compared
uid into a glassy state when it is cooled below its glass tran- with the one in the VS.
sition temperature (TG).21 The cooling and warming rates, Mouse zygotes were exposed to nVS1, nVS2, and
the volume of the drop containing the biological mate- VS (Figure 3.1b) before placing them in solutions with
rial, and the exposure of the biological material to high various  SUC molarities. The observations were per-
concentrations of CPsol are conditions supporting the formed under an inverted microscope (IX51; Olympus)
maintenance of an intra- and extra-cellular environment equipped with a camera (Olympus DP50), and software
during the transition from a liquid to a solid glass-like that records the pictures (AnalySIS version 3.2; Olympus)
state, and in reverse when warming back to a liquid state. (Figure 3.1a). In order to maintain the zygote at the same
During the steps of exposure to the different nVS, focal plane during the transit from one CPsol to the
a certain amount of CP enters the cells. This may take next, a holding pipette fixed on a micromanipulator was
3–15 minutes according to the type of CP used and the installed (Figure 3.1b).
cooling rate, which in turn depends on the carrier device.
The time of exposure to the nVS at a defined temperature Results and arguments for reduced ICCP
is of utmost importance and determines the amount of Using a cinematographic recording system, we estimated
intracellular CP. the degree of dehydration and penetration of CPs inside
The duration of exposure to the permeable CPs is the cells during the exposure to nVS1, nVS2, and VS
determined by several biophysical factors, such as the (Figure 3.1c).
membrane properties (cellular permeability to water and The cells in our VIT protocol were exposed for short
CP), the type and concentration of CP, the surface/vol- periods to nVS1 and nVS2, and did not reach complete
ume ratio of the cells, and the rate of cooling and warm- equilibration. Hence, cells were exposed for a short period
ing.22–25 The nVS is exclusively composed of permeable to the VS containing SUC, and an intracellular vitrify-
CPs (e.g., dimethyl sulfoxide [DMSO], ethylene glycol, ing state was obtained after dehydration and exposure to
1,2-propanediol [PROH], and glycerol). concentrations of the previously entered nVS1 and nVS2
(Figure 3.1c, steps v and vi).
ASSESSMENT OF THE INTRACELLULAR The cinematographic analysis also allowed us to deter-
CONCENTRATION OF CP mine the SUC concentration in which no modification of
The questions regarding the use of high concentrations the cell volume was observed, allowing inference of the
of CP, or increasing the intracellular concentration in ICCP.
relation to the type of device used, were the origins of a After the steps in the nVS and VS, zygotes were exposed
debate. In this context, the question was: how high is the to increased solutions of SUC of increasing molarity
intracellular concentration of CP (ICCP) during the VIT (Figure 3.2). We have observed that the volume variations
and SLF cryopreservation procedures? decreased when increasing the SUC molarity. This decrease
In order to find an answer for those who are concerned in amplitude of swelling is the result of reduced inflow of
about the high concentration of CPs during the VIT pro- water. Movements of water across cell membranes occur
cess, we determined: easily and rapidly, and continue until intracellular and

36
 intracellular concentration of cryoprotectant

Record the pictures

(a)
Camera Microscope Computer

Image analysis software

(b)
Holding pipete

CM nVS1 nVS2 VS

(c)
nVS1 nVS2 VS
100
Relative zygote volume (%)

i
80 iii v ICCP
?
60
ii iv vi
40 nVS1: 5% DMSO–5% EG

20 nVS2: 10% DMSO–10% EG

0 VS: 20% DMSO–20% EG–
0 20 40 60 80 100 120
Time (s) Ficoll–SUC 0.5 M
(d) i CM iii nVS1 v nVS2

ii iv vi

ICCP
?
nVS1 nVS2 VS

Figure 3.1  Quantitative assessment of the ICCP by cinematographic analysis. (a) Technical design for the record-
ing and measurement of the zygote. (b) Method used to monitor the change in volume of the zygote during expo-
sure to nVS1, nVS2, VS, and various molarities of SUC (holding pipette used in order to maintain the zygote in
the same focal plane from one droplet to the next). (c) Evolution of the relative volume of one zygote through the
various CP solutions. (d) Pictures of the zygotes at different time points: (i) the culture medium; (ii) at the end
of the dehydration step in nVS1; (iii) before transferring in nVS2; (iv) at the end of the dehydration step in nVS2;
(v) before transferring in VS; (vi) arrow indicates the time point at which the ICCP was evaluated. (CM = culture
medium; DMSO = dimethyl sulfoxide; EG = ethylene glycol, ICCP = intracellular concentration of cryoprotectant;
nVS = Non-vitrifying solution; SUC = sucrose; VS = vitrifying solution.)

extracellular tonicities are identical. When incubated in the cell due to the fast entrance of water. Based on this
2.14 M SUC solution, only minimal changes in cell volume assumption, two protocols were designed allowing com-
occurred, indicating an iso-osmotic situation between the parison of the survival rates of mouse zygotes after SLF
intracellular and extracellular compartments. During on the one hand, and VIT on the other, with (i) various
VIT, therefore, this intracellular osmolarity approaches warming rates; and (ii) various concentrations of SUC in
2.14 M before plunging in LN2. the warming dilution medium (Figure 3.3).

Comparison of the ICCP after SLF or VIT Comparison of survival rates after VIT or SLF relative
In a second study, we evaluated in which of the cryo- to the warming rate
preservation techniques (VIT versus SLF) the final ICCP
was higher (Figure 3.3). • Principle. It is well known that the higher the
It is accepted that the higher the ICCP, the lower the ICCP, the lower the probability of recrystalliza-
probability of (re)crystallization during the warming tion and cell lysis when reduced warming rates are
step, and the higher the probability of overswelling of applied.

37
 vitrification in assisted reproduction
220

200

180

160 0M
Relative zygote volume (%)

140 Hypo‐osmotic
120

100 0.45 M
0.82 M
VS 80
60 Iso‐osmotic
f 1.82 M
40

20 Hyper‐osmotic
2.14 M
0
400 420 440 460 480 500 520 540 560 580 600 620 640 660 680
Time (s)

Figure 3.2  Evolution of the relative zygote volume with exposure to solutions of different concentrations of sucrose
(0, 0.45, 0.82, 1.82, and 2.14 M), following the exposure steps to nonvitrifying solution 1, nonvitrifying solution 2,
and VS. The volume of the picture at 2.14 M is almost the same as the volume of the picture in (f), indicating an iso-
osmotic situation between the intracellular and extracellular compartments. (VS = vitrifying solution.)

Slow freezing Vitrification

20
Liquid state

–7 ICCP ?

–30
0.3°C/min
Temperature (°C/min)

–40
Crystal state
ICCP ?

Comparison
of the ICCP
TG
LN2
Vitrous state LN2

Solute concentration (salt + CP) Solute concentration (salt + CP)

Survival rates in relation to:

Low and fast warming rates
Dilution in different SUC solutions

Figure 3.3  Experimental design for the comparison of the ICCP when VIT and SLF cryopreservation procedures
were performed. (ICCP ? = time point where the ICCPs were compared; CP = cryoprotectant; ICCP = intracellular
concentration of cryoprotectant; LN2 = liquid nitrogen; SLF = slow freezing; SUC = sucrose; TG = glass transition
temperature; VIT = vitrification.)

38
 intracellular concentration of cryoprotectant
• Design of the experiment.  According to this assump­ Comparison of survival rates after VIT or SLF in relation
tion, an experiment was conducted comparing the to dilution in various SUC concentrations
rate of zygote survival after fast and slow warm-
ing following either SLF or VIT. The design of the • Principle.  The higher the ICCP, the higher the SUC
experiment is explained in Figure 3.4a. concentration necessary to minimize overswelling
• Results and arguments for low ICCP after VIT ver- of the cell due to the fast inflow of water through
sus SLF.  We observed that the survival rates after the cell membrane.
SLF is poorly affected by the warming rate, suggest- • Design of the experiment.  In this experiment, the
ing that the high ICCP prevents intracellular crys- percentages of lysed zygotes were evaluated in vari-
tallization (devitrification). ous SUC-containing warming solutions. This was
performed either after VIT or SLF. The design of
After VIT, however, only 10% of the zygotes survived the experiment is described in Figure 3.5a.
when the warming process took place in air (slow warm- • Results and arguments for low ICCP after VIT ver-
ing). By contrast, a high warming speed (higher than sus SLF.  A significantly higher rate of lysed zygotes
20,000°C/min) after VIT prevented cell lysis (Figure 3.4b). was observed after SLF than after VIT.
This is a physical argument indicating that the ICCP is
higher after SLF than VIT (Figure 3.4c). This is due to the Immediately after warming, SLF embryos presented
fact that in the VIT process, the ICCP is too low to prevent obvious signs of overswelling when dilution was per-
recrystallization during slow warming when the tempera- formed in culture medium or in solutions with low con-
ture exceeds the TG (~−120°C). If the warming speed is not centrations of SUC (Figure 3.5b).
fast enough, the supercooled liquid is rapidly transformed The higher the ICCP, the higher must be the SUC con-
into ice crystals in the interval between the TG and the centration in the warming medium to minimize the over-
melting temperature. swelling of the cells.

(a)
Slow freezing Vitrification
‐ 15 min PROH 1.5 M Suc 0.1 M ‐ DMSO – EG 1.6 M 5 min
‐ 0.3°C/min 0.25 mL straw 3.2 M 4 min
‐ at 36°C 6.4 M 60 s
‐ Plunge in LN2 ‐ Plunge in LN2

Thawing–warming rates

Water bath 37°C 2280°C/min Fast Plunge in SUC 1 M 21,000°C/min

In air 680°C/min Slow In air 1140°C/min

(b) (c)
Slow warming *
SLF VIT
100 Fast warming Warming Slow Fast Slow Fast
80 Crystallization Extracellular YES YES NO NO
Survival rates (%)

during
cooling Intracellular NO NO NO NO
60
*P < 0.001 Crystallization Extracellular YES YES NO NO
40 during
warming Intracellular NO NO YES NO
20

0 High survival: Low survival: (devitrification)

SLF VIT Intracellular vitrification Intracellular crystallization
during warming: ICCP HIGH during slow warming: ICCP lOW

Figure 3.4  ​Comparison of survival rates after VIT or SLF in relation to the warming rate. (a) Design of the experi-
ment. (b) Results. (c) Arguments for low ICCP after VIT. (ICCP = intracellular concentration of cryoprotectant;
SLF = slow freezing; VIT = vitrification.)

39
 vitrification in assisted reproduction
(a) Design of the experiment

Slow freezing Vitrification

‐ 15 min PROH 1.5 M SUC 0.1 M ‐ DMSO – EG 1.6 M 5 min
‐ 0.3°C/min 0.25 mL straw 3.2 M 4 min
‐ at 36°C 6.4 M 60 s
‐ Plunge in LN2 ‐ Plunge in LN2

Thawing–warming
Direct dilution in:

PBS ‐ 0.125 M ‐ 0.25 M ‐ 0.5 M

(b) Results (c) Arguments for low ICCP after VIT

100
SLF VIT
80 Dilution in SUC 0M 0.25 0.5 0M 0.25 0.5
Survival rates (%)

Water
60
SLF entrance and +++ ++ + ++ + /
osmotic shock
40 VIT

20
LOW survival = HIGH survival =
0 Important osmotic shock reduce osmotic shock
0 0.25 0.5 High ICCP Low ICCP
SUC concentration

Figure 3.5  Comparison of survival rates after VIT or SLF in relation to dilution in various SUC concentrations after
the warming step. (a) Design of the experiment. (b) Results. (c) Arguments for low ICCP after VIT. (ICCP = intra-
cellular concentration of cryoprotectant; PBS = phosphate buffer saline; SLF = slow freezing; VIT = vitrification.)

This higher rate of lysed zygotes in low concentrations 2.14 M. This deduced low-level ICCP explains why VIT
of SUC can be explained by the too rapid and finally lethal gives better results than SLF; although high extracellu-
inflow of water into the cells. This is a tangible argument lar concentrations of CPs are needed to allow VIT, only
for the presence of a higher intracellular osmolarity in a fraction of them enter the cell, thereby reducing their
SLF, when compared to that present after VIT of cells toxicity. It is probable that the problems linked to SLF are
(Figure 3.5c). more likely to be due to osmotic shock during the warm-
ing–dilution process than due to ice crystal formation, as
GENERAL CONCLUSIONS it was previously thought.
Is it justified to be anxious about the use of high concen- It has become obvious that warming speeds play an
trations of CPs and to favor SLF? even more important role in cell survival after VIT than
From two studies using mouse zygotes in conjunc- the cooling rates.27–29 As critical warming rates are higher
tion with cinematographic analysis, we can conclude that than critical cooling rates, the minimal concentration
the ICCP during VIT is a lot lower (2.14 M) than the CP of CPs for preventing recrystallization during warming
concentrations present in the VS solution (>6 M).26 We must be higher than during cooling. This means that it
have clearly shown that the ICCPs in the vitrified zygotes might be easier to maintain a vitrified state during cool-
are, in contrast to common belief, even lower than those ing than during the warming process, given the same
observed after a SLF procedure. From all of these experi- concentration of CPs.
ments, we can conclude that with our VIT procedure, the During SLF, when cooling is applied at a rate of 0.3°C/
ICCP that is reached after VIT of mice zygotes is not as min, cells are equilibrated with a low concentration of a
high as it was previously thought. As a consequence, we penetrating CP whose concentration increases through-
can demystify the problem of the high level of CPs inside out the cooling process, as a consequence of the increas-
the cell. ing formation of extracellular ice crystals.
The CPs are only part of the intracellular agents inhib- Knowing that a cell survives a cryopreservation
iting crystallization, so the true ICCP is even lower than procedure only if the development of intracellular ice

40
 intracellular concentration of cryoprotectant
crystals is avoided, survival after SLF ref lects the pres- an oocyte-donation programme: A prospective ran-
ence of an intracellular vitrified state. SLF has been the domized study. Reprod Biomed Online 2013;26:470–6.
standard cryopreservation method for more than 25 13. Cobo A, de los Santos MJ, Castellò D, Gámiz P,
years, without us being fully aware of the presence of Campos P, Remohí J. Outcomes of vitrified early
a vitrified intracellular state obtained with a very high cleavage-stage and blastocyst-stage embryos in a cryo-
ICCP. preservation program: Evaluation of 3.150 warming
cycles. Fertil Steril 2012;98:1138–46.
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approach to overcome an impaired uterine environ- and utility of vitrification solutions. Cryobiology
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9. Isachenko V, Katkov II, Yakovenko S et al. Vitrification 22. Kasai M, Edashige K. Movement of water and cryo-
of human laser treated blastocysts within cut standard protectants in mouse oocytes and embryos at different
straws (CSS): Novel aseptic packaging and reduced stages: Relevance to cryopreservation. In Ri-Cheng C,
concentrations of cryoprotectants. Cryobiology Quinn P (eds.), Fertility Cryopreservation. New York:
2007;3:305–9. Cambridge University Press, 2010. p. 16–23.
10. Balaban B, Urman B, Ata B et al. A randomized con- 23. Leibo S. Water permeability and its activation energy
trolled study of human day 3 embryo cryopreserva- of fertilized and unfertilized mouse ova. J Membrane
tion by slow freezing or vitrification: Vitrification is Biol 1980;53:179–88.
associated with higher survival, metabolism and blas- 24. Leibo SP, Pool TB. The principal variables of cryo-
tocyst formation. Hum Reprod 2008;9:1976–82. preservation: Solutions, temperatures, and rate
11. Vanderzwalmen P, Ectors F, Grobet L et  al. Aseptic changes. Fertil Steril 2011;96:269–76.
vitrification of blastocysts from infertile patients, 25. Vanderzwalmen P, Ectors F, Grobet L et al. Adaptation
egg donors and after IVM. Reprod Biomed Online of a universal procedure for cryopreservation of dif-
2009;5:700–7. ferent developmental stages: Is it conceivable? In
12. Panagiotidis Y, Vanderzwalmen P, Prapas Y et  al. Varghese AC, Sjöblom P, Jayaprakasan K (eds.),
Open versus closed vitrification of blastocysts from Practical Guide to Setting Up an IVF Lab, Embryo

41
 vitrification in assisted reproduction
Culture Systems and Running the Unit. Jaypee Brothers 28. Seki S, Mazur P. The dominance of warming rate
Medical Publishers (P) Ltd, New Delhi, 2013, p. 118– over cooling rate in the survival of mouse oocytes
31 and 2101–10. subjected to a vitrification procedure. Cryobiology
26. Vanderzwalmen P, Connan D, Grobet L et al. Lower 2009;1:75–82.
intracellular concentration of cryoprotectants after 29. Mazur P, Seki S. Survival of mouse oocytes after being
vitrification than after slow freezing despite exposure cooled in a vitrification solution to −196°C at 95° to
to higher concentration of cryoprotectant solutions. 70,000°C/min and warmed at 610° to 118,000°C/min:
Hum Reprod 2013;28:2101–10. A new paradigm for cryopreservation by vitrification.
27. Seki S, Mazur P. Effect of warming rate on the survival Cryobiology 2011;62:1–7.
of vitrified mouse oocytes and on the recrystallization
of intracellular ice. Biol Reprod 2008;79:727–37.

42
4 Importance of cooling versus warming rates in vitrification techniques
Shinsuke Seki

INTRODUCTION cooling and a high concentration of cryoprotectants. In

In the cryopreservation of oocytes/embryos, it is known this chapter, I will describe the results that support my
that the formation of more than a trace amount of ice in hypothesis.
the cell is lethal. Strategies developed to prevent intracel-
lular ice formation (IIF) are slow freezing1 and vitrifica- OBSERVATION OF IIF IN MOUSE OOCYTES
tion.2 More recently, because of its simplicity and high Since IIF is lethal, the most important factor determin-
survival, vitrification has become used more widely. ing the success of cryopreservation is whether intracel-
When cells are vitrified with a minimum degree of lular ice forms or not during cryopreservation. The only
supercooling (i.e., in near equilibrium), no ice should way to avoid IIF is to vitrify cells; that is, to convert cell
form in the cell. In most cases, however, the degree of water into a glass rather than ice. Despite the importance
supercooling is not minimal, thus invisible minute ice of IIF, the mechanism by which IIF is induced was not
crystals will form in cells during vitrification. The min- understood in detail. At first, I would like to show how
ute crystals are innocuous as long as they remain minute. IIF is induced.3,10,11
During warming, however, they can recrystallize into Mouse oocytes at metaphase II (oocytes) were sus-
larger lethal crystals, as a result of differences in surface pended in 1.5 M ethylene glycol/phosphate buffered saline
free energy.3 In the present article, intracellular ice means (PBS), placed on linkam BCS196 cryostat, and cooled to
recrystallized lethal ice. −25°C at 50°C/min. At this temperature, extracellular ice
With a few exceptions,4,5 the literature on vitrifica- had been induced, but intracellular ice was not formed.
tion has almost wholly emphasized the role of the cool- Samples were held there for 0, 5, 10, 20, 30, or 40 minutes,
ing rate on preventing IIF. As a strategy to increase the then cooled to −70°C at 50°C/min, and warmed to 20°C
cooling rate, ultra-rapid vitrification was developed, in moderately (10°C/min). The temperature of the cryostage
which small tools, such as electron microscope grids,6 was controlled with a programmable freezer. By changing
open pulled straws,7 Cryoloops,8 and Cryotops,9 are used. the holding time at −25°C, the water content of the cells was
By using these devices, it is possible to achieve very high varied, because oocytes are dehydrated very slowly (water
cooling rates (>10,000°C/min) by manipulating oocytes/ permeability, Lp being 1.82 × 10−3 μm/min/atm) at such a
embryos with a very small volume (~0.1 μL) of vitrifica- low temperature (−25°C). The morphological appearance
tion solution. Given the higher cooling/warming rates, of oocytes during cooling and warming was observed by
cells are able to survive during this greater supercool- taking pictures at fixed times, and IIF was evidenced by
ing. Ultra-rapid vitrification was first developed for the blackening of the cells.
cryopreservation of bovine oocytes to circumvent chill- When the holding time was 0–10 minutes, the water
ing injury,6 but now it is mainly used to prevent IIF in content was more than 50%, and IIF was induced at below
less permeable cells like oocytes and blastocysts; the −40°C.11 This shows that IIF is induced during cooling
plasma membrane of blastocysts is quite permeable, but when the water content is high.
the whole embryo is less permeable because blastocysts When the holding time was 10–30 minutes, the water
have different compartments, as described by Edashige content was 20%–50% and IIF was not induced during
and Kasai in this book (Chapter 5). cooling. During warming, however, IIF was induced at
The literature has also emphasized the role of the con- between −56°C and −46°C.11 This shows that IIF is more
centration of vitrification solution; that is, the vitrifica- likely to form during warming than during cooling in
tion solution in which cells are suspended must have a cells with 20%–50% water.
high concentration of cryoprotective solutes, especially When the holding time was 40 minutes, the water
cell-permeating cryoprotectants. Therefore, it is believed content was less than 20% and IIF was induced neither
that vitrification requires both the cooling rate and the during cooling nor warming, even when both cooling
concentration of cell-permeating cryoprotectant(s) to be and warming rates were low.11 This shows that IIF is
quite high for prevention of IIF. not induced during cooling/warming if cells are dehy-
However, my findings with the vitrification of mouse drated sufficiently. Recently, Jin et  al.12 and Mochida
oocytes are not in accordance with this belief. My hypoth- et  al.13 reported near-equilibrium vitrification, in
esis is that high survival of vitrified mouse oocytes/ which vitrified mouse embryos actually survived slow
embryos is derived more from rapid warming than rapid warming.

43
 vitrification in assisted reproduction
HOW CAN INTRACELLULAR ICE BE PREVENTED was assessed morphologically, or functionally by observa-
FROM FORMING IN MOUSE OOCYTES? tion of cleavage to the two-cell stage after in vitro fertiliza-
The results of the experiments described above show tion (IVF).
that IIF is not induced during cooling to −70°C when With full-strength (1.0×) EAFS10/10, morphologi-
the water content is less than 50%. As it is not difficult to cal survival rates were more than 65%, regardless of the
dehydrate cells to less than 50% in vitrification, I assumed cooling rate, if the warming rate was 2950°C/min, but the
that  the cooling rate would not need to be increased to rates dropped gradually to less than 56% and 45% when
avoid IIF if cells were dehydrated enough, whereas the the tonicity of EAFS10/10 was decreased to 0.75× and
warming rate would need to be rapid to avoid IIF by 0.5×, respectively.
recrystallization. Firstly, I examined the effect of the If the warming rate was decreased only slightly
cooling rate and warming rate on the survival of oocytes (2170°C/min), the drop in the survival with diluted
vitrified with a solution containing a lower concentration EAFS10/10 became exaggerated, such that with 0.5×
of cryoprotectants,14 in which ice is more likely to form. EAFS10/10, the survival rate approached 0%. When
Mouse oocytes were suspended in a vitrification solu- oocytes were warmed at the highest rate (117,500°C/min),
tion containing 10% ethylene glycol, 10% acetamide, 24% on the other hand, morphological survival rates were
Ficoll, and 0.4 M sucrose (EAFS10/10),15 cooled to −196°C 80%–90%, regardless of the tonicity of EAFS10/10 (1.0×,
with plastic cryostraws at rates ranging from 37°C/min 0.75×, or 0.5×) and the cooling rate (95°C/min, 880°C/
to 1827°C/min, and warmed at rates ranging from 137°C/ min, or 69,000°C/min). These results show that the warm-
min to 2950°C/min between −70°C and −35°C. Survival ing rate definitely has a more dominant effect over both
was assessed morphologically by osmotic responsiveness. the concentration of cryoprotectants and the cooling rate.
I used EAFS10/10 for the experiments, but this vitrifica- Even when oocytes were cooled and warmed at the
tion solution contains acetamide, which would be quite highest cooling rates, functional survival rates decreased
toxic to oocytes for practical use. slightly from 81% to 67% by decreasing the tonicity of
When vitrified oocytes were warmed at the lowest rate EAFS from 1.0× to 0.5×. When cooled at 880°C/min,
(137°C/min), the survival rate was close to 0%, regardless functional survival rates were 54%–73%; while with the
of the cooling rate. When they were warmed at a higher slowest cooling rate (95°C/min), the rates decreased yet
rate (2950°C/min), however, the survival rate was more further (29%–49%). Therefore, the cooling rate affects the
than 80% over a wide range of cooling rates (187–1827°C/ functional survival of the oocytes, although the effect is
min), showing that the warming rate is a more dominant smaller than that of the warming rate.
effect over the cooling rate. Furthermore, a similar experiment was conducted
Then, mouse oocytes were vitrified with Cryotops, with embryos: eight-cell mouse embryos were vitri-
to apply higher rates of cooling/warming.16 When fied using Cryotops with 1.0×, 0.75×, 0.5×, or 0.33×
oocytes were cooled at 95, 880, and 69,000°C/min, then EAFS10/10.18 They were cooled at various rates ranging
warmed at the highest rate attainable (117,500°C/min), from 95°C/min to 69,000°C/min, and warmed rapidly
similar results to those with cryostraws were obtained. (117,500°C/min). Survival was assessed by the ability to
Morphological survival exceeded 80%, even when the develop to expanded blastocysts in culture. The survival
prior cooling rate was as low as 880°C/min. These results rates of embryos vitrified with 1.0× to 0.5× EAFS10/10
also show that the warming rate is a more dominant effect were more than 80%, even if the cooling rate was mod-
over the cooling rate, probably by eliminating time for erate (880°C/min). Even when the tonicity of EAFS was
the growth of minute intracellular ice crystals through reduced to 0.33×, 40% of the embryos survived.
recrystallization. In conclusion, rapid warming has a more dominant
Cryobiologists think that if cells could be cooled and effect over rapid cooling and a high concentration of
warmed extremely rapidly, thus eliminating the time for cryoprotectants in the vitrification of mouse oocytes. The
IIF, then the cells would survive even if the cryopreser- results obtained in oocytes should probably be applicable
vation medium did not contain cryoprotectant, at least to embryos as well.
theoretically. When the cooling and warming rates are
high, therefore, cells should survive after cryopreserva- APPLICATION OF THESE STUDIES TO THE ART
tion, even when using a lower concentration of cryopro- The importance of the warming rate should have practi-
tectants. Therefore, I examined the effect of decreasing cal implications for successful cryopreservation of oocytes/
cryoprotectant concentration on the survival of vitrified embryos. Given that the cooling rate does not need to be
oocytes.17 very high, it is therefore not necessary to apply high cooling
Mouse oocytes were suspended in various tonici- rates by direct contact of cells with liquid nitrogen, which
ties (1.0×, 0.75×, or 0.5×) of EAFS10/10, loaded in cryo- may not be aseptic. As a consequence, oocytes/embryos
straws or on Cryotops, cooled at rates ranging from 37°C may possibly be cryopreserved within a closed carrier (e.g.,
to 69,000°C/min, vitrified at −196°C, and warmed rapidly cryostraws); and yet vitrified oocytes/embryos do appear
(2170°C/min, 2950°C/min, or 117,500°C/min). Survival to need to be warmed rapidly.

44
 importance of cooling versus warming rates in vitrification techniques
When a solution is vitrified, the liquid phase turns to 7. Vajta G, Booth PJ, Holm P et al. Successful vitrifica-
solid during cooling, and the solid phase turns to liquid tion of early stage bovine in vitro produced embryos
during warming at the glass transition temperature (TG), with the open pulled straw (OPS) method. Cryo Lett
which is at around −130°C. During the phase transi- 1997;18:191–5.
tion, the volume slightly changes. If a solution is cooled/ 8. Lane M, Schoolcraft WB, Gardner DK. Vitrification
warmed rapidly, the solution contains both liquid and of mouse and human blastocysts using a novel cry-
solid compartments for a moment, and fracture planes oloop container-less technique. Fertil Steril 1999;72:​
form between the two phases. If cells are situated in the 1073–8.
fracture plane, they are physically dissected.19 During 9. Kuwayama M, Vajta G, Kato O et al. Highly efficient
cooling and warming, therefore, it is necessary to pass vitrification method for cryopreservation of human
through the TG moderately to prevent fracture damage, oocytes. Reprod Biomed Online 2005;11:300–8.
unless the sample volume is small enough (0.1 μL) to 10. Mazur P, Seki S, Pinn IL et  al. Extra- and intracel-
form fracture planes. My results suggest that moderate lular ice formation in mouse oocytes. Cryobiology
cooling can be applied at any temperature, but moderate 2005;51:29–53.
warming should be applied only at around −130°C (below 11. Mazur P, Pinn I, Kleinhans FW. Intracellular ice for-
−80°C). mation in mouse oocytes subjected to interrupted
In addition, vitrification with a lower concentration of rapid cooling. Cryobiology 2007;55:158–66.
cryoprotectants may well be possible to reduce the toxic- 12. Jin B, Mochida K, Ogura A et al. Equilibrium vitrifica-
ity of the vitrification solution used. tion of mouse embryos. Biol Reprod 2010;82:444–50.
13. Mochida K, Hasegawa A, Li MW et al. High osmo-
ACKNOWLEDGMENT lality vitrification: A new method for the simple and
I acknowledge Dr. Peter Mazur because this sequential temperature-permissive cryopreservation of mouse
research was conducted in his laboratory under his super- embryos. PLoS One 2013;8:e49316.
vision. I thank Dr. Magasaburo Kasai for pre-submission 14. Seki S, Mazur P. The dominance of warming rate
review and comments. over cooling rate in the survival of mouse oocytes
subjected to a vitrification procedure. Cryobiology
REFERENCES 2009;59:75–82.
1. Whittingham DG, Leibo SP, Mazur P. Survival of 15. Pedro P, Yokoyama E, Zhu S et  al. Permeability of
mouse embryos frozen to −196 and −269°C. Science mouse oocytes and embryos at various develop-
1972;178:411–4. mental stages to five cryoprotectants. J Reprod Dev
2. Rall WF, Fahy GM. Ice-free cryopreservation of 1997;51:235–46.
mouse embryos at −196°C by vitrification. Nature 16. Mazur P, Seki S. Survival of mouse oocytes after being
1985;313:573–5. cooled in a vitrification solution to −196°C at 95° to
3. Seki S, Mazur P. Kinetics and activation energy of 70,000°C/min and warmed at 610° to 118,000°C/min:
recrystallization of intracellular ice in mouse oocytes A new paradigm for cryopreservation by vitrification.
subjected to interrupted rapid cooling. Cryobiology Cryobiology 2011;62:1–7.
2008;56:171–80. 17. Seki S, Mazur P. Ultra-rapid warming yields high
4. Boutron P, Mehl P. Theoretical prediction of devitri- survival of mouse oocytes cooled to −196°C in dilu-
fication tendency: Determination of critical warming tions of a standard vitrification solution. PLoS One
rates without using finite expansions. Cryobiology 2012;7:e36058.
1990;27:359–77. 18. Seki S, Jin B, Mazur P. Extreme rapid warming yields
5. Fahy GM. Biological effects of vitrification and high functional survivals of vitrified 8-cell mouse
devitrification. In Pegg DE, Karow AM Jr (eds.), The embryos even when suspended in a half-strength
Biophysics of Organ Cryopreservation. Plenum Press: vitrification solution and cooled at moderate rates to
New York, 1987. p. 265–97. −196°C. Cryobiology 2014;68:71–8.
6. Martino A, Songsasen N, Leibo SP. Development into 19. Kasai M, Zhu SE, Pedro PB et al. Fracture damage of
blastocysts of bovine oocytes cryopreserved by ultra- embryos and its prevention during vitrification and
rapid cooling. Biol Reprod 1996;54:1059–69. warming. Cryobiology 1996;33:459–64.

45
5 The movement of water and cryoprotectants in mammalian oocytes
and embryos: Membrane permeability and aquaporins
Keisuke Edashige and Magosaburo Kasai

INTRODUCTION permeability is suggestive of the movement of water by

During the cryopreservation of cells, intracellular ice simple diffusion—that is, via a channel-independent
formation is the most common cause of cell death. To ­process—whereas higher permeability to water with a
prevent this in oocytes/embryos, cytoplasm must be lower E a value is suggestive of the movement of water
vitrified using cell-permeating cryoprotectants, which by facilitated diffusion via channel processes. Verkman
can cause other major types of cell injury; for example, et  al.3 suggested that low permeability to water with an
chemical toxicity of cryoprotectant during loading and E a value higher than 10 kcal/mol is suggestive of water
osmotic swelling during removal of permeated cryopro- movement principally by simple diffusion, whereas per-
tectant.1 Especially in vitrification, the potential toxic- meability higher than 4.5 μm/min/atm with an E a value
ity of the final vitrification solution is high, because of lower than 6 kcal/mol is suggestive of water movement
the high concentration of cryoprotectants needed to principally via channels. The existence of water channels
achieve the vitrified state. Therefore, the exposure time in embryos can be inferred by Verkman et al.’s criteria, by
of oocytes/embryos to the vitrification solution has to be the presence of mRNA for water channels, by detection of
limited. However, short exposure times can cause insuf- channel proteins, and also by the decreased water perme-
ficient permeation by the cryoprotectant and insufficient ability in embryos when the expression of such channel
dehydration/concentration of the cytoplasm, resulting in proteins is suppressed.
intracellular ice formation. To prevent these major inju- In mouse oocytes, the permeability to water in a
ries during vitrification, rapid movement of water and sucrose solution is low at 20–25°C (0.4–1.0 μm/min/atm),
cryoprotectants is preferable. and its E a value is higher (11–15 kcal/mol) than 10 kcal/
Here, we summarize the movement of water and cryo- mol (Figure 5.1). 4–11 Applying Verkman et al.’s criteria to
protectants at 20–25°C across the plasma membrane of mouse oocytes, this implies that water moves slowly by
metaphase II stage oocytes and embryos at various devel- simple diffusion, and that channels do not play a role in
opmental stages in the mouse, bovine, pig, and human the movement of water (Figure 5.2a).12
systems. In mouse embryos at the early (one- to four-cell) stages,
the permeability to water is low (0.4–0.7 μm/min/atm)
THE MOVEMENT OF WATER ACROSS THE and the Ea value is high (12–13 kcal/mol),4,9,11 similar to
PLASMA MEMBRANE OF MAMMALIAN oocytes, although these cells do express a small amount
OOCYTES AND EMBRYOS IN A HYPERTONIC of mRNAs for aquaporins and aquaporin proteins.13–15 In
SUCROSE SOLUTION one- to four-cell embryos, therefore, water seems to move
In most types of cells, water moves slowly across the across the plasma membrane principally by simple diffu-
plasma membrane by simple diffusion via the lipid sion, and perhaps minimally by channel processes.
bilayer. However, the plasma membrane of some cells— In mouse morulae and blastocysts, on the other hand,
for example, human red blood cells and cells in renal water permeability is high (3.1–4.5 μm/min/atm) and the
proximal tubules—is extremely permeable to water. In E a value is low (5.1–6.3 kcal/mol) (Figure 5.1),11 suggest-
the 1990s, small intrinsic membrane proteins that act as ing that the movement of water is principally dependent
water channels, called aquaporins, were discovered and on facilitated diffusion via channel processes. Among
characterized.2 Therefore, water is able to move across the aquaporin families, aquaporin 316 is expressed abun-
plasma membrane of oocytes/embryos slowly by simple dantly in mouse morulae11,14 and blastocysts.14 In mouse
diffusion, or rapidly by facilitated diffusion through blastocysts, mRNA of aquaporin 3 is much more abun-
aquaporins. dantly expressed than the mRNA of other aquaporins.17
The pathway for the movement of water across the Furthermore, by suppressing the expression of aquaporin
plasma membrane can be deduced from the permeabil- 3 in mouse morulae/blastocysts, the high permeability to
ity to water, and its temperature dependency expressed water markedly decreases.18 In mouse morulae/blasto-
as Arrhenius activation energy (E a). In general, lower cysts, therefore, water moves rapidly principally through
permeability to water with a higher E a value for the aquaporin 3 (Figure 5.2b).12

47
 vitrification in assisted reproduction
(μm/min/atm) oocytes is significantly higher than that of bovine mor-
7 ulae when the expression of aquaporin 3 is suppressed
15°C (0.6 μm/min/atm). In bovine embryos at the 16-cell
6 (6)
25°C stage, similar results to those as with bovine oocytes
Hydraulic conductivity

5 are reported.19 In bovine oocytes and 16-cell embryos,

therefore, water moves across the plasma membrane
4 principally by simple diffusion, although the channel
pathway via aquaporins can also partially be involved
3
in water movement.
2 In bovine morulae and blastocysts, on the other
(12) hand, the permeability to water is high (3 μm/min/
1 atm), although slightly lower when compared to the
0 mouse (4.5 μm/min/atm), and the E a value for the per-
Oocyte Morula meability is much lower (3 kcal/mol) than 6 kcal/mol.19
Furthermore, the suppression of the expression of aqua-
Figure 5.1 Permeability to water (hydraulic conduc- porin 3 in bovine morulae markedly decreases the per-
tivity, LP) of mouse oocytes and morulae in a hyper- meability to water. Thus, in bovine morulae/blastocysts,
tonic solution containing sucrose at 15 and 25°C. LP water moves across the plasma membrane principally via
values were determined from the measurement of the the channel pathway through aquaporin 3, as in mouse
shrinkage of oocytes and morulae in a modified phos- morulae/blastocysts.
phate-buffered saline containing 0.5 Osm/kg sucrose In pig oocytes, the permeability to water is low (1.0 μm/
(0.8 Osm/kg in total) for 5 minutes at 15 and 25°C. min/atm) and the E a value is quite high (19 kcal/mol), as
Values in parentheses are the activation energies for the in mouse oocytes.20 In pig morulae, the permeability to
permeability (kcal/mol). (Data from Edashige K et  al. water remains low, unlike in mouse and bovine morulae,
Biol Reprod 2006;74:625–32.) probably because pig embryos become compacted at the
four-cell stage. In pig oocytes and morulae, therefore,
water seems to move across the plasma membrane princi-
pally by simple diffusion.
In bovine oocytes, the permeability to water is low
In pig blastocysts, the permeability to water increases
(1.8 μm/min/atm),19 although higher than that of mouse
at the unexpanded stage, and further increases at the
oocytes (0.4–1.0 μm/min/atm), and the E a value for the
expanded stage (3.4 μm/min/atm).20 The Ea value for the
permeability is high (9 kcal/mol), although slightly
permeability to water in expanded blastocysts (7 kcal/mol)
lower than the 10 kcal/mol level for mouse oocytes.
is close to 6 kcal/mol, as seen in the mouse. Therefore,
Furthermore, the permeability to water of bovine
water seems to move across the plasma membrane of pig
expanded blastocysts principally via channel pathways.
Since pig expanded blastocysts express mRNA of aqua-
porin 3 abundantly,20 aquaporin 3 seems to be responsible
(a) (b) AQP3
for the high permeability to water of pig expanded blasto-
cysts, as in mouse and bovine morulae/blastocysts. In pig
embryos, therefore, the expression of aquaporin 3 appar-
ently increases at a slightly later stage.
In human oocytes, the permeability to water in hyper-
Water Water tonic solutions containing a nonpermeating solute is also
low (0.4–1.0 mm/min/atm),5,21,22 and the E a value is high
Figure 5.2  Schematic representation of the pathway for (9–11 kcal/mol).5,22 In human oocytes, therefore, water
the movement of water across the plasma membrane of seems to move across the plasma membrane slowly prin-
mouse oocytes and morulae in a hypertonic solution cipally by simple diffusion, as in animal oocytes.
containing sucrose. Solid lines indicate the movement In human embryos, the permeability to water has not
of water across the plasma membrane by facilitated been reported, but it would be reasonable to presume
diffusion via channel pathways, whereas dotted lines that permeability is essentially similar to that in embryos
indicate the movement of water across the plasma from other experimental models.
membrane by simple diffusion. (a) The movement of In mammalian oocytes and embryos at early stages,
water in oocytes. (b) The movement of water in moru- therefore, water moves across the plasma membrane
lae. (AQP3 = aquaporin 3.) (Modified from Kasai  M, slowly principally by simple diffusion. In embryos at later
Edashige K. Fertility Cryopreservation. Cambridge stages, on the other hand, water moves rapidly principally
University Press: Cambridge, 2010. p. 16–23.) by facilitated diffusion through aquaporin 3, although

48
 the movement of water and cryoprotectants in mammalian oocytes and embryos
the developmental stage at which the pathway changes (×10–3 cm/min)
may shift slightly in some species. (9)
14

Cryoprotectant permeability
12
THE MOVEMENT OF CELL-PERMEATING
Oocyte at 15°C
CRYOPROTECTANTS ACROSS THE PLASMA 10
Oocyte at 25°C
MEMBRANE OF MAMMALIAN OOCYTES Morula at 15°C
8
AND EMBRYOS (10) Morula at 25°C
Water channels occur in two groups: one is highly selec- 6
(12) (20)
tive for water, and the other transports not only water
4 (12)
but also neutral solutes with a smaller molecular weight, (20)
such as cell-permeating cryoprotectants.2 Therefore, cell- 2
(17) (23) (18)
permeating cryoprotectants will move across the plasma (42)
0
membrane either slowly by simple diffusion, or rapidly by Gly EG AA DMSO PG
facilitated diffusion through aquaporins. Cryoprotectant
Although no report is available for quantitative evalua-
tion of the movement of cell-permeating cryoprotectants,
Figure 5.3 The permeability to cell-permeating cryo-
it would be reasonable to deduce the pathway of their
protectants (PS) of mouse oocytes and morulae and the
movement from the permeability and its E a value, based
activation energy for the permeability. PS values were
on the case of permeability to water; low permeability to
determined from the measurement of the shrinkage and
cryoprotectant with a relatively high E a value is suggestive
swelling of oocytes and morulae in a modified phos-
of the movement of cryoprotectant principally by simple
phate-buffered saline containing 10% v/v glycerol (Gly),
diffusion, whereas an increase in permeability with a
8% v/v ethylene glycol (EG), 1.5 M acetamide (AA), 9.5%
decrease in the E a value for the permeability is sugges-
v/v dimethyl sulfoxide (DMSO), or 10% v/v propylene
tive of movement principally by facilitated diffusion via
glycol (PG) for 5–20 minutes at 15 and 25°C. Values in
channels. Among the major permeating cryoprotectants
parentheses indicate the values of the activation energies
are glycerol, ethylene glycol, dimethyl sulfoxide (DMSO),
for the permeability (kcal/mol). (Data from Edashige K
acetamide, and propylene glycol.
et al. Biol Reprod 2007;77:365–75.)
Glycerol
In mouse oocytes and embryos at early developmental (0.6 × 10 −3 cm/min), similar to the permeability to
stages, the permeability to glycerol is quite low (0.01– water. 20 The E a value for the permeability to glycerol of
0.02 × 10−3 cm/min),11,23–25 and the E a value is remarkably expanded pig blastocysts is much lower (10 kcal/mol)
high (42 kcal/mol) (Figure 5.3).11,18 In mouse oocytes and than that of oocytes (27 kcal/mol). Furthermore, pig
embryos at early stages, therefore, glycerol would move expanded blastocysts express mRNA of aquaporin 3
slowly across the plasma membrane by simple diffusion. abundantly. 20 Thus, in expanded pig blastocysts, glyc-
In mouse morulae, on the other hand, the permeability erol seems to move rapidly principally by facilitated dif-
to glycerol is markedly higher (4–5 × 10−3 cm/min) than fusion through aquaporin 3.
that of mouse oocytes (0.01–0.02 × 10−3 cm/min), and the In human oocytes, no data are available for the perme-
Ea value for the permeability (10 kcal/mol) is lower than ability to glycerol; the permeability of oocytes to glycerol
that of mouse oocytes (42 kcal/mol).11 Furthermore, the (MW 92) would be too low as a permeating cryoprotec-
high permeability is remarkably decreased by suppressing tant for practical use.
the expression of aquaporin 3.18 In mouse morulae (and
probably blastocysts), therefore, glycerol moves rapidly Ethylene Glycol
across the plasma membrane by facilitated diffusion via In mouse oocytes, the permeability to ethylene glycol is
the channel pathway through aquaporin 3 (Figure 5.4a).12 low (0.6 × 10−3 cm/min) and the E a value is high (17 kcal/
In bovine oocytes/embryos, essentially similar results mol) (Figure 5.3),18 similar to the permeability to glyc-
to those in mouse oocytes/embryos have been reported.19 erol. Therefore, ethylene glycol will move through mouse
In pig oocytes, the permeability to glycerol is low oocytes (and probably embryos at early stages of develop-
(0.02 × 10−3 cm/min), and the E a value is high (27 kcal/ ment) principally by simple diffusion.
mol), as with mouse oocytes.20 In pig morulae, the per- In mouse morulae, on the other hand, the permeability
meability to glycerol remains low (0.05 × 10−3 cm/min),20 to ethylene glycol is extremely high (10 × 10−3 cm/min),
unlike in mouse morulae. So in pig oocytes and morulae, and the E a value is lower (9 kcal/mol) than that in mouse
glycerol seems to move slowly by simple diffusion. oocytes (17 kcal/mol).18 Furthermore, the suppression of
In pig blastocysts, the permeability to glycerol the expression of aquaporin 3 in mouse morulae mark-
increases at the unexpanded stage (0.3  × 10 −3 cm/ edly decreases the high permeability to ethylene glycol,
min), then further increases at the expanded stage while exogenous expression of aquaporin 3 in mouse

49
 vitrification in assisted reproduction
(a) AQP3 (b) AQP3 the permeability to ethylene glycol does not increase at
Gly EG the morula stage but at the blastocyst stage,20 as with the
permeability to glycerol.
In human oocytes, the permeability to ethylene glycol
(1.2×10−3 cm/min) is low and the E a value is high (15 kcal/
mol),22 as in mouse oocytes, suggesting that ethylene gly-
Water Water col moves slowly principally by simple diffusion.

(c) AQP3 Other (d) AQP3 Dimethyl Sulfoxide

DMSO channels PG In mouse oocytes, the permeability to DMSO is low
(1.0 × 10−3cm/min), and its E a value is high (18 kcal/mol)18
(Figure 5.3), suggesting that DMSO moves slowly by sim-
ple diffusion.
In mouse morulae, on the other hand, the perme-
Water Water
ability is higher (3.0 × 10−3 cm/min) than that in oocytes
(1.0 × 10−3 cm/min), and the Ea value is lower (12 kcal/
mol) than that in mouse oocytes (18 kcal/mol), suggesting
Figure 5.4 Schematic representation of the pathway
that  DMSO principally moves via channels.18 However,
for the movement of cell-permeating cryoprotectants
the suppression of aquaporin 3 in mouse morulae does
and water in the presence of cryoprotectants across
not decrease the permeability to DMSO, and the exog-
the plasma membrane of mouse oocytes and morulae.
enous expression of aquaporin 3 in mouse oocytes does not
Solid lines indicate the movement of water and cryo-
increase the permeability to DMSO.18 These results show
protectants across the plasma membrane by facilitated
that aquaporin 3 is not involved in the channel pathway
diffusion via channel pathways, whereas dotted lines
for the movement of DMSO in mouse morulae. Therefore,
indicate the movement of water and cryoprotectants
DMSO must move through mouse morulae principally
by simple diffusion. (a)  The movement of water and
via channels other than aquaporin 3 (Figure 5.4c).12
glycerol (Gly). (b) The movement of water and ethylene
In bovine oocytes, the permeability to DMSO is rel-
glycol (EG). (c)  The movement of water and dimethyl
atively low (1.5 × 10−3 cm/min) and the E a value is high
sulfoxide (DMSO). (d) The movement of water and pro-
(13 kcal/mol), suggesting that DMSO moves slowly by
pylene glycol (PG). “Other channels” refers to DMSO-
simple diffusion.19
permeable channels. Open circles represent water
In bovine morulae, the permeability to DMSO remains
molecules; shaded circles represent cryoprotectant mol-
low (1.7 × 10−3 cm/min),19 while the Ea value is high
ecules. (AQP3 = aquaporin 3.) (Modified from Kasai M,
(21 kcal/mol), which is actually higher than that in bovine
Edashige K. Fertility Cryopreservation. Cambridge
oocytes (13 kcal/mol). In bovine blastocysts, no reports on
University Press: Cambridge, 2010. p. 16–23.)
the movement of DMSO are available, but it would be rea-
sonable to assume that the movement of DMSO is essen-
oocytes (which do not express aquaporin 3) increases tially similar to that in bovine morulae.
their permeability to ethylene glycol.18 In mouse moru- In bovine oocytes/embryos, therefore, DMSO seems to
lae (and probably blastocysts), therefore, ethylene glycol move across the plasma membrane principally by simple
moves principally via the channel pathway through aqua- diffusion regardless of the developmental stage.
porin 3 (Figure 5.4b).12 In pig oocytes/embryos, similar results to those in
In bovine oocytes, the permeability to ethylene glycol mouse oocytes/embryos are reported.20 However, the per-
is essentially similar to that in mouse oocytes.19 However, meability to DMSO increases marginally at the expanded
the permeability is higher (3.5 × 10−3 cm/min) than that blastocyst.
in mouse oocytes (0.6 × 10−3 cm/min), and the E a value In human oocytes, the permeability to DMSO is low
is slightly lower (14 kcal/mol) than that in mouse oocytes (1.6 × 10−3 cm/min), as in mouse oocytes (1.0 × 10−3 cm/
(17 kcal/mol). Since bovine oocytes express a small min),22 suggesting that DMSO moves slowly principally
amount of water channels on the plasma membrane by simple diffusion.
as described above for the permeability to water in the
presence of sucrose, ethylene glycol would move through Acetamide
bovine oocytes mainly by simple diffusion, and partially In mouse oocytes/embryos, the movement of acetamide
via channel processes through water channels. is essentially similar to that of DMSO (Figure 5.3).18
In bovine morulae, the permeability to ethylene glycol Therefore, acetamide would also move through mouse
is essentially similar to that in mouse morulae.19 oocytes by simple diffusion and through mouse morulae
In pig oocytes/embryos, essentially similar results to by facilitated diffusion via channels other than aquapo-
those in mouse oocytes/embryos are reported, although rin 3 (Figure 5.4c).

50
 the movement of water and cryoprotectants in mammalian oocytes and embryos
In bovine, pig, and human oocytes/embryos, no data developmental stages, glycerol and ethylene glycol move
are available for the movement of acetamide; in any event, principally by facilitated diffusion through aquaporin 3,
acetamide may be too toxic for practical use. DMSO and acetamide move principally via channels
other than aquaporin 3 in many species, and propylene
Propylene Glycol glycol moves by simple diffusion but relatively rapidly.
In mouse oocytes, the permeability to propylene glycol
is relatively low (1.7 × 10−3 cm/min), and the E a value is THE MOVEMENT OF WATER ACROSS THE
high (20 kcal/mol) (Figure 5.3),18 suggesting that propyl- PLASMA MEMBRANE OF MAMMALIAN
ene glycol also moves through mouse oocytes by simple OOCYTES AND EMBRYOS IN THE PRESENCE OF
diffusion. However, the permeability of mouse oocytes A CELL-PERMEATING CRYOPROTECTANT
to propylene glycol is higher (1.7 × 10−3 cm/min) than the The permeability to water in oocytes/embryos can be
permeabilities to other cryoprotectants (0.6–1.0 × 10−3 assessed in a hypertonic solution containing a nonper-
cm/min). This would be due to the molecular configu- meating solute; for example, sucrose, as described above.
ration of propylene glycol; propylene glycol is a more In cryopreservation, however, oocytes/embryos are sus-
hydrophobic molecule than other permeating cryopro- pended in a solution also containing a cell-permeating
tectants. Therefore, propylene glycol would move across cryoprotectant(s). In this case, the movement of water
the plasma membrane by simple diffusion more rapidly. is not always the same as that in a solution containing
In mouse morulae, on the other hand, the permeability just a nonpermeating solute, because water will interact
to propylene glycol is more than twice as high (3.8 × 10−3 with the permeating cryoprotectant. The pathway for
cm/min) as that in mouse oocytes (1.7 × 10−3 cm/min),18 the movement of water in the presence of a permeating
although the reason for this is not clear. The permeability cryoprotectant may be deduced from the permeability
to propylene glycol (3.8 × 10−3 cm/min) is similar to the to water and its E a value, as with the movement of the
permeabilities to glycerol (4–5 × 10−3 cm/min) and DMSO cryoprotectant.
(3.0 × 10−3 cm/min) in mouse morulae, in which water
moves principally via the channel pathway. However, the In the Presence of Glycerol
E a value for the permeability to propylene glycol remains In mouse oocytes, the permeability to water in the pres-
high (20 kcal/mol), unlike the E a values for the perme- ence of glycerol is low (0.5–0.6 μm/min/atm) and the E a
ability to glycerol and DMSO. Furthermore, the suppres- value is high (14 kcal/mol) (Figure 5.5),18 suggesting that
sion of aquaporin 3 in mouse morulae and the exogenous water moves across the plasma membrane slowly by sim-
expression of aquaporin 3 in mouse oocytes do not affect ple diffusion.
the permeability to propylene glycol.18 These results sug- In mouse morulae, on the other hand, the permeability
gest that the relatively high permeability to propylene gly- to water in the presence of glycerol is higher (2.2 μm/min/
col in mouse morulae does not rely on channel processes, atm) than that in oocytes (0.5–0.6 μm/min/atm), and the
but on simple diffusion (Figure 5.4d).12 From these results, E a value for the permeability is lower (9 kcal/mol) than
it is speculated that in mouse morulae, the movement of that in oocytes (14 kcal/mol).11,18 Furthermore, the sup-
propylene glycol interacts with water, which hinders each pression of the expression of aquaporin 3 in mouse mor-
other’s movement through aquaporin, as described below ulae significantly decreases the permeability to water.18
for the permeability to water in the presence of propylene In mouse morulae, therefore, water in the presence of
glycol. glycerol moves rapidly across the plasma membrane
In bovine oocytes/embryos and pig oocytes/embryos, principally via the channel pathway through aquaporin 3
similar results to those as in mouse oocytes/embryos are (Figure 5.4a).12 This movement is essentially similar to the
reported.19,20 movement of water in the presence of sucrose.
In human oocytes, the permeability to propylene In bovine oocytes/embryos, the movement of water
glycol (2.2 × 10−3 cm/min)22 is similar to that in mouse in the presence of glycerol is essentially similar to that in
oocytes (1.7 × 10−3 cm/min), suggesting that propylene mouse oocytes/embryos.19
glycol moves slowly principally by simple diffusion. In pig oocytes, the permeability to water in the pres-
In mammalian oocytes/embryos, therefore, propylene ence of glycerol is low (0.7 μm/min/atm) and the E a value
glycol seems to move across the plasma membrane princi- is high (17 kcal/mol).20 In pig morulae, the permeability
pally by simple diffusion but relatively rapidly, regardless of to water in the presence of glycerol remains low (0.5 μm/
the expression of water/cryoprotectant channels. min/atm), unlike in mouse morulae. In pig oocytes/­
morulae, therefore, water seems to move across the
SUMMARY plasma membrane slowly by simple diffusion.
In mammalian oocytes and embryos at early develop- In pig blastocysts, the permeability to water in the pres-
mental stages, cell-permeating cryoprotectants move ence of glycerol increases at the unexpanded stage and
across the plasma membrane principally by simple dif- further increases at the expanded stage (1.2–2.0 μm/min/
fusion, like water in sucrose solution. In embryos at later atm),20 as with the permeability to water in the presence

51
 vitrification in assisted reproduction
(μm/min/atm) by simple diffusion, regardless of the expression of aqua-
3 (9) (9) porin 3. Therefore, it is speculated that the movement
(7)
2.5 of water is hindered by the movement of ethylene glycol
Hydraulic conductivity

Oocyte at 15°C
through aquaporin 3, as a result of strong interactions
Oocyte at 25°C
2 Morula at 15°C between water and ethylene glycol (Figure 5.4b).12
Morula at 25°C In bovine oocytes/embryos and in pig oocytes/
1.5
(15)
embryos, the movement of water in the presence of ethyl-
1 (14) ene glycol is essentially similar to that in mouse oocytes/
(14) (12) (13)
(10) (15) embryos,19,20 although the permeability does not increase
0.5
at the morula stage, but at the blastocyst stage in pig
0 embryos.20
Gly EG AA DMSO PG
In human oocytes, the permeability to water in the
Cryoprotectant
presence of ethylene glycol is low (0.8 μm/min/atm) and
the E a value is high (11 kcal/mol),22 as in mouse oocytes,
Figure 5.5 The permeability to water (hydraulic con- suggesting that water in the presence of ethylene glycol
ductivity [LP]) of mouse oocytes and morulae in the moves across the plasma membrane slowly principally by
presence of a cryoprotectant, and the activation energy simple diffusion.
for the permeability. LP values were obtained when the
permeability to cell-permeating cryoprotectant values In the Presence of DMSO
were estimated in Figure 5.3. Values in parentheses In mouse oocytes, the permeability to water in the pres-
are the activation energies for the permeability (kcal/ ence of DMSO is low (0.5 × 10−3 μm/min/atm) and the E a
mol). (AA = acetamide; DMSO = dimethyl sulfoxide; value is high (12 kcal/mol) (Figure 5.5),18 suggesting that
EG = ethylene glycol; Gly = glycerol; PG = propylene water moves slowly by simple diffusion.
glycol.) In mouse morulae, on the other hand, the permeabil-
ity is higher (2.3 × 10−3 μm/min/atm) than that in oocytes
(0.5 × 10−3 μm/min/atm), and the E a value for the perme-
of sucrose. The E a value for the permeability in expanded ability is lower (7 kcal/mol) than that in oocytes (12 kcal/
blastocysts is much lower (5 kcal/mol) than that in pig mol).18 Furthermore, the suppression of the expression
oocytes (17 kcal/mol). Furthermore, pig expanded blas- of aquaporin 3 in mouse morulae decreases the perme-
tocysts express the mRNA of aquaporin 3 abundantly.20 ability to water, and exogenous expression of aquaporin
In pig expanded blastocysts, therefore, water in the pres- 3 in mouse oocytes increases the permeability to water.18
ence of glycerol seems to move principally via the channel In mouse morulae, therefore, water in the presence of
pathway through aquaporin 3 rapidly. DMSO moves via the channel pathway through aquapo-
In human oocytes, the permeability to water in the rin 3.
presence of glycerol has not been reported, but it would be In mouse oocytes and morulae, therefore, the move-
reasonable to presume that the permeability is essentially ment of water in the presence of DMSO is essentially
similar to that in animal oocytes. similar to that in the presence of sucrose (Figure 5.4c).18
In mammalian oocytes/embryos, therefore, the pres- In bovine oocytes, the permeability to water in the
ence of glycerol does not affect the movement of water presence of DMSO is relatively low (1.2 μm/min/atm) and
markedly. the E a value is high (11 kcal/mol), suggesting that water
principally moves slowly by simple diffusion.19
In the Presence of Ethylene Glycol In bovine morulae, the permeability remains low
In mouse oocytes, the permeability to water in the pres- (1.4 × 10−3 μm/min/atm), although the E a value is mark-
ence of ethylene glycol is low (0.4 μm/min/atm) and the edly lower (2 kcal/mol) than that in bovine oocytes
E a value is high (10 kcal/mol) (Figure 5.5),18 suggesting (11  kcal/mol). Furthermore, the suppression of the
that water moves across the plasma membrane slowly by expression of aquaporin 3 in bovine morulae significantly
simple diffusion. decreases the permeability to water to a value lower than
In mouse morulae, on the other hand, the movement that in intact bovine oocytes, and exogenous expres-
of water in the presence of ethylene glycol is different sion of bovine aquaporin 3 in mouse oocytes increases
from that in the presence of sucrose; the permeability the permeability to water, although the permeability to
is low (0.5 μm/min/atm) and the E a value is quite high DMSO does not increase.19 In bovine morulae, therefore,
(14 kcal/mol),18 regardless of marked expression of aqua- water in the presence of DMSO seems to move principally
porin 3 at this stage. Furthermore, exogenous expression via the channel pathway through aquaporin 3, although
of aquaporin 3 in mouse oocytes increases the permeabil- the increase in the permeability is marginal.
ity to ethylene glycol, but not to water.18 Therefore, water In pig oocytes/embryos, essentially similar results to
in the presence of ethylene glycol seems to move slowly those in mouse oocytes/embryos are reported, although

52
 the movement of water and cryoprotectants in mammalian oocytes and embryos
the increase in the permeability to water in the presence MEMBRANE PERMEABILITY AND SUITABLE
of DMSO is observed not at the morula stage, but at the CONDITIONS FOR VITRIFICATION OF
expanded blastocyst stage.20 OOCYTES/EMBRYOS
In human oocytes, the permeability to water in the In vitrification, the time and temperature for exposure of
presence of DMSO is low (0.6 μm/min/atm), as with oocytes/embryos to the vitrification solution are impor-
mouse oocytes,22 suggesting that water in the presence of tant, because oocytes/embryos could easily be injured by
DMSO moves across the plasma membrane slowly prin- the toxicity of a high concentration of the cryoprotectant.
cipally by simple diffusion. Exposure of embryos to cryoprotectant solutions at a
In mammalian oocytes/embryos, therefore, water in high temperature needs to be avoided, because cryopro-
the presence of DMSO moves through oocytes slowly by tectants are more toxic at higher temperatures.
simple diffusion, and moves through morulae by facili- In order to design protocols suitable for the vitrifica-
tated diffusion through aquaporin 3 rapidly in mouse tion of mammalian oocytes/embryos, it would be valu-
morulae and pig expanded blastocysts, or relatively able to consider the pathway for the movement of water
slowly in bovine morulae. and cell-permeating cryoprotectants for each stage of
development. Our assessment of the movement of water
In the Presence of Acetamide and cell-permeating cryoprotectants in oocytes/embryos
The movement of water in the presence of acetamide shows that the pattern of the movement is rather more
(Figure 5.4c) is essentially similar to that in the presence stage specific than species specific, although some spe-
of DMSO in the mouse.18 Therefore, water in the presence cies-specific cases exist. Therefore, cryopreservation
of acetamide moves through oocytes slowly by simple dif- protocols developed for oocytes/embryos in one species
fusion, and through morulae/blastocysts rapidly by facili- should be applicable to protocols of various species at
tated diffusion through aquaporin 3. similar stages in terms of permeability.
In oocytes and embryos at early cleavage stages (includ-
In the Presence of Propylene Glycol ing pig morulae), water and cryoprotectants principally
In mouse oocytes, the permeability to water in the pres- move across the plasma membrane slowly by simple diffu-
ence of propylene glycol is low (0.5 μm/min/atm) and the sion. To prevent injury from the toxicity of cryoprotectants
E a value is high (13 kcal/mol) (Figure 5.5),18 suggesting in vitrification, a stepwise treatment of oocytes/embryos
that water moves principally by simple diffusion. is effective; oocytes and embryos at early stages are sus-
In mouse morulae, the permeability to water in the pended first in a solution containing a lower concentration
presence of propylene glycol is low (1.0 μm/min/atm), of cryoprotectant for permeation, and then in a vitrifica-
although higher than that in oocytes (0.5 μm/min/atm), tion solution for a short time to maximize the cellular con-
and the Ea value for the permeability is quite high (15 kcal/ centration by rapid dehydration. In oocytes and embryos
mol) (Figure 5.5).18 Furthermore, the suppression of the at early cleavage stages, it is preferable to handle them at
expression of aquaporin 3 in mouse morulae and the exog- a moderate temperature (room temperature) to optimize
enous expression of aquaporin 3 in mouse oocytes do not the movement of water and cryoprotectant during loading
affect the permeability to water. In mouse morulae, there- and during removal of cryoprotectant, because they move
fore, water in the presence of propylene glycol presumably slowly by simple diffusion, and their movement is greatly
moves across the plasma membrane principally by simple affected by temperature.
diffusion, regardless of the abundant expression of aqua- In morulae (in many species), water and cryopro-
porin 3, unlike water in the presence of other cryoprotec- tectants move across the plasma membrane rapidly by
tants. From these results, it is speculated that water and facilitated diffusion via channels. It is known that the
propylene glycol interact and hinder each other’s move- expression of aquaporin 3 (and DMSO/acetamide chan-
ment through aquaporin 3 (Figure 5.4d).12 nels in some species) markedly increases at later stages
In bovine oocytes/embryos and in pig oocytes/ of embryonic development. Facilitated diffusion not only
embryos, the movement of water in the presence of increases membrane permeability, but also decreases the
propylene glycol is essentially similar to that in mouse temperature dependency of the permeability. Therefore,
oocytes/embryos.19,20 morulae can be handled at a lower temperature (e.g.,
In human oocytes, the permeability to water in the 20°C) without a large decrease in the permeability.
presence of propylene glycol is low (1.1 μm/min/atm),22 In blastocysts, water and cryoprotectants also move
as with mouse oocytes, suggesting that water in the pres- across the plasma membrane via channels rapidly, but
ence of propylene glycol moves across the plasma mem- optimal diffusion into the inner cell mass and trophec-
brane slowly principally by simple diffusion. toderm can be problematic, and so affect the most suit-
In the presence of propylene glycol, therefore, water able conditions for vitrification. Notably, the inner cell
appears to move through mammalian oocytes/embryos mass faces not the outside of the blastocyst, but abuts the
slowly principally by simple diffusion, regardless of the trophectoderm or the blastocoele. Therefore, water and
expression of water/cryoprotectant channels. cryoprotectants do not move across the plasma membrane

53
 vitrification in assisted reproduction
of the inner cell mass directly from/to the solution in 12. Kasai M, Edashige K. Movement of water and cryo-
which blastocysts are suspended, but indirectly through protectants in mouse oocytes and embryos at differ-
the trophectodermal cells or the blastocoelic fluid. ent stages: Relevance to cryopreservation. In: Chian
Furthermore, in blastocysts with a larger amount of water, R-C, Quinn P (eds.), Fertility Cryopreservation.
most of the water in the blastocoele has to be removed Cambridge University Press: Cambridge, 2010.
before the cryopreservation process. At the same time, p. 16–23.
the cryoprotectant has to permeate the blastocoele via the 13. Offenberg H, Barcroft LC, Caveney A et al. mRNAs
trophectodermal cells. To minimize the toxic effect of the encoding aquaporins are present during murine
cell-permeating cryoprotectant whilst promoting perme- preimplantation development. Mol Reprod Dev
ation and dehydration, stepwise treatment is more effec- 2000;57:323–30.
tive, as with oocytes, for preventing ice formation in the 14. Barcroft LC, Offenberg H, Thomsen P et al. Aquaporin
blastocoele. Consequently, puncturing the blastocoele to proteins in murine trophectoderm mediate transepi-
collapse it is the most effective strategy for promoting both thelial water movements during cavitation. Dev Biol
permeation of cryoprotectant and dehydration. 2003;256:342–54.
15. Edashige K, Sakamoto M, Kasai M. Expression of
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11. Edashige K, Tanaka M, Ichimaru N et  al. Channel- 25. Jackowski S, Leibo SP, Mazur P. Glycerol permeability
dependent permeation of water and glycerol in mouse of fertilized and unfertilized mouse ova. J Exp Zool
morulae. Biol Reprod 2006;74:625–32. 1980;212:329–41.

54
6 Open versus closed systems
Mark G. Larman and Pierre Vanderzwalmen

INTRODUCTION from infected women.7 No viral sequences were detected,

Vitrification is now a part of nearly every in vitro fertil- suggesting the risk of cross-contamination is low. Safer
ization (IVF) cycle. Gametes and embryos are, therefore, cryopreservation methods would, however, avoid any of
stored in liquid nitrogen (LN2) for a number of years and the possible contaminations mentioned above.
possibly decades, especially when considering donor
cryo-banks and gamete freezing for social reasons or fer- MINIMIZING CONTAMINATION
tility preservation. Many cryopreserved samples within DURING LN2 STORAGE
IVF are stored in containers that are not hermetically There are several steps that can be taken to mini-
sealed, which is not in concordance with medical direc- mize the risk of contamination during cryo-storage.8
tives for the storage of biological tissues. Such directives Cryopreserved germplasm should be quarantined until
have been put in place to minimize the risk of cross-con- it is deemed free of infectious entities, which will mini-
tamination from infected material and also the LN2 itself, mize any risk of cross-contamination. Semen is par-
which is not sterile. ticularly susceptible to a high microbial load. Therefore,
This chapter will discuss the potential concerns with semen samples should always be stored separately from
the storage of gametes and embryos in LN2 and how those oocytes and embryos, although washing has been shown
risks can be minimized. The most obvious solution is to to significantly reduce or remove viruses and bacteria.9
prevent contact with LN2 completely via the use of closed The zona pellucida does offer some protection to oocytes
vitrification devices. Therefore, different closed devices and embryos. As with semen, multiple washing is very
and their clinical comparison with open devices will be effective in removing microbial and viral pathogens.10
discussed. However, it is now very common for the zona pellucida to
be breached because of intracytoplasmic sperm injection
LN2 AND CONCERNS OF CONTAMINATION (ICSI), biopsy, and assisted hatching, which may make the
The risk of contamination during storage in LN2 has been oocytes and embryos more susceptible to contamination.
of concern for a number of years. In a 1976 study, glass It is difficult to sterilize large quantities of LN2 and also
vials containing vesicular stomatitis were breached dur- impossible to maintain that sterility in IVF clinics. Cryo-
ing storage causing contamination of the LN2.1 The risk of tanks and dry shippers should undergo periodic decon-
actual cross-contamination during cryo-storage of bio- tamination with a solution that does not react with the
logical material was first clinically reported following the lining, and then rinsed with sterile water.8 Even though
transmission of hepatitis B from cryo-stored bone mar- LN2 is not generally provided sterile to the clinic, most
row.2 Microbial transmission should also be considered, contamination is probably introduced during opening,
given that ice sediments from LN2 tanks were found to distribution to storage vessels, and contaminated samples
contain both bacterial and fungal contaminations that being stored in open containers. Sterile filtration of LN2
are capable of causing illness.3 Bacterial and fungal spe- at the outlet is feasible using a 0.22 µm filter.11 Ultraviolet
cies were also found in LN2 used to store bovine embryos (UV) radiation is a further alternative for LN2 steriliza-
and semen.4 tion. Microbial sterilization of small volumes (500 mL)
As there is the potential, during cryo-storage, to con- of LN2 is possible with UV radiation.12 UV-sterilized LN2
taminate germplasm, it is not too surprising that viral can also be used to wash devices to remove any external
and microbial transmissions have been investigated. microbial contamination.13 Filtration and UV irradia-
Cross-contamination with bacteria between samples was tion of LN2 can therefore offer protection against bacte-
first demonstrated with semen pellets, and it occurred rial and fungal contamination. Another option consists
within 2 hours.5 Spiking LN2 with different viruses and of storing the carrier containing the biological material
microbes demonstrated that bovine embryos could also in LN2 vapor.14,15 It is, however, still unclear whether LN2
be contaminated.4,6 As expected, using sealed containers vapor completely eliminates the potential for contamina-
prevented contamination, suggesting that this should be tion.16,17 Furthermore, one of the drawbacks of this form
routine and mandatory. One study, albeit with relatively of storage is the need for careful monitoring of tempera-
limited numbers, performed viral screening on spent cul- tures in different parts of the container and ensuring they
ture media and LN2 used to vitrify oocytes and embryos remain below the glass transition temperature (–130°C).18

55
 vitrification in assisted reproduction
In summary, even though it is possible to sterilize LN2, for a continued debate, as it is widely believed to be the
the methods do not afford complete viral elimination, most important factor for achieving successful vitrifica-
may be impractical or prohibitively expensive, and most tion.8 This now appears not necessarily to be the case.
importantly do not prevent subsequent cross-contamina- Peter Mazur’s group has determined the functional
tion during LN2 storage. It must also be noted that the relationship between cooling and warming rates and
IVF laboratory is not a sterile environment, so the ste- survival of mouse oocytes.22–24 It was found that mouse
rility of the LN2 cannot be guaranteed over time. There oocyte survival was more negatively affected by slower
is no direct evidence of cryopreserved human oocytes/ warming rather than slower cooling rates. The rationale
embryos becoming contaminated during cryo-storage, behind the critical importance of the warming rate may
and subsequently transmitting disease or causing infec- be that, although small ice nucleation events might occur
tion. Given the available information from bovine embryo with slower cooling rates, the warming rate must be fast
studies, however, and potential concerns with long-term enough to prevent them from aggregating and forming
storage in open devices, it would appear prudent to avoid the larger, damaging ice crystals.
such direct contact with LN2 during vitrification and sub- Despite the slower cooling rates, closed devices allow
sequent storage by using closed devices. The remainder equivalent warming rates to open devices, and there
of this chapter will discuss such closed devices and their is now growing evidence that closed systems can be as
efficacies with regard to clinical comparisons with open effective as their open counterparts for the vitrification
systems. of both human oocytes25–29 and embryos.26,30–40 A sum-
mary of the open and closed vitrification devices with
CLINICAL COMPARISONS OF OPEN AND their approximate volumes and cooling/warming rates is
CLOSED VITRIFICATION DEVICES: CAN WE presented in Table 6.1.
FORESEE THE END OF THE “OPEN” SYSTEM? When the Cryotip was compared to the Cryotop, the
The principal of using a closed system, which would elim- survival and pregnancy rates with human blastocysts
inate the risk of contamination, is not new. Kuleshova were comparable between the two devices.31 One study,
and Shaw sealed an open pulled straw in an outer straw however, has reported unacceptably low recovery with
to provide a closed storage system.19 This device was the Cryotip,41 and another showed more ultra-structural
successful at vitrifying mouse embryos19,20 and human damage of human oocytes with the Cryotip compared to
pronuclear oocytes.21 There is, however, a significant dif- the Cryotop.42 The Cryotip as well as the Cryopette are
ference in cooling rate between vitrification protocols very fine straws based on the principle of the open pulled
with open (>25,000°C/min) and closed (<2000°C/min) straw. It is true that different users of such devices observe
devices. This difference in cooling rates is responsible problems in terms of recovery. Furthermore, with such

Table 6.1  A Review of the Main Open and Closed Vitrification Devices
Device Volume (µL) Cooling rate (°C/min) Warming rate (°C/min)

Open devices
Cryotop Flat strip <0.1 23,000 >25,000
Hemi-straw Small gutter 0.3 >20,000 >25,000
Cryoloop Nylon loop <0.1 >20,000 >25,000
Cryoleaf Flat strip 1 23,000 >25,000
Cryolock Flat strip 1 >20,000 >25,000
Vitri-inga Hole 1 20,000 >25,000
Open pulled straw Mini-straw 1 16,700 <20,000
Fiber plug Hook >1 10,000 >25,000

Closed devices
0.25-mL straw Straw 25–100 <2500 ~1300
Vitrisafe Small gutter 0.3 1300 >25,000
HSV Small gutter 0.5 2000 >25,000
Rapid-i Hole 0.05 1200 >25,000
Cryotip Mini-straw 1 12,000 <20,000
Cryopette Mini-straw 1 23,700 <20,000
Ultravit Quartz glass microcapillary 0.5 Unpublished Unpublished

56
 open versus closed systems
devices, the warming rate is lower compared to devices in Table 6.3  Clinical Results Following Blastocyst
which the vitrification medium (containing the oocytes/ Vitrification Using Either the Rapid-i or Cryotop
embryos) comes in direct contact with the warming
Rapid-i Cryotop
solution. These lower warming rates could explain the
(n = 100) (n = 163)
observed negative effect on the cytoskeleton after using
the Cryotip.42 Implantation rate (%) 54 53
The Rapid-i uses supercooled air and is the most Ongoing pregnancy rate (%) 45 47
tested closed vitrification device. It was developed using
Source: Modified from Hashimoto S et al. J Assist Reprod Genet
mouse embryos and is capable of vitrifying mouse pronu- 2013;30:371–6.
clear oocytes with a 100% survival rate. The subsequent
embryo development, cell number, and embryo viability
following embryo transfer were not affected when com- pregnancy rates were similar for the Rapid-i and the
pared to sibling nonvitrified embryos.43 The Rapid-i has Cryotop (Table 6.3).
been compared to two open systems for human embryo As with embryos, it appears that human oocytes can
vitrification. The results of vitrifying day 3 embryos and also be vitrified with the Rapid-i. Sibling in vitro-matured
blastocysts using the Rapid-i were compared to an open human oocytes were vitrified using either the Rapid-i
system (Cryoloop).36 For day 3 embryos, the survival rates or the Cryotop.28 The survival rates were 92% and 90%,
for the Rapid-i and Cryoloop were 99%. The implantation respectively. Currently, the Rapid-i is being evaluated in
rates were 37% and 35% and the clinical pregnancy rates a clinical trial for donor oocyte vitrification. Over 500
were 47% and 49%, respectively. For blastocysts, the sur- oocytes have been vitrified and warmed with a survival
vival rates for the Rapid-i and Cryoloop were 97% and rate of 94%. Following ICSI, the fertilization rate was
91%, respectively. The Rapid-i supported a trend to higher 76%. Blastocyst transfer resulted in a 49% ongoing preg-
implantation (49%) and clinical pregnancy (59%) rates nancy rate,29 with 40 healthy live births now recorded.
than the Cryoloop (38% and 46%, respectively). A prospective, randomized study was performed by
The Rapid-i has also been compared to the Cryotop.35 Papatheodorou et  al.25 to validate the effectiveness of
The first comparison in this study used zygotes previously using the Vitrisafe device32 in both an open and closed
cryopreserved at the pronuclear stage. After rewarming, method for oocyte cryopreservation. Sibling oocytes
embryo development was assessed. There were no dif- donated from the same donor were randomly and equally
ferences between the Rapid-i and the Cryotop in terms assigned to closed or open vitrification groups. A total of
of survival (100% and 97%, respectively), blastulation, 75 vitrification–warming cycles were performed in each
good blastocyst rates, or mean cell numbers (Table 6.2). group. Apart from the survival rate (82.9% versus 91.0%,
To investigate the influence of the vitrification method P < 0.05) in favor of the open system, no statistically sig-
on apoptosis, blastocysts were vitrified–warmed and nificant differences were observed in clinical pregnancy
compared to nonvitrified blastocysts. There was no dif- (36.0% and 28.0%) and live birth (36.0% and 24.0%) rates
ference in the proportion of dead cells between the three between the closed and open groups, respectively.
treatments. Lastly, the two devices were compared clini- In another prospective randomized study, Panagi­
cally. A total of 263 high-grade blastocysts were ran- otidis et al. analyzed the outcome of vitrified blastocysts
domly assigned to vitrification using either the Rapid-i being randomly allocated to either an open hemi-straw or
or the Cryotop. The survival rates for both devices were closed Vitrisafe device.37 There were no statistically sig-
the same (97%). Single-blastocyst transfer was per- nificant differences in the parameters measured: embryo
formed after warming, and the implantation and ongoing survival rate (84.1% and 82.1%, respectively), clinical
pregnancy rate (45.9% and 42.4%, respectively), implan-
tation rate (25.6% and 24.5%, respectively), cycle cancel-
Table 6.2  Two-to Four-Cell Embryos Vitrified with the lation rate (6.7% and 8.4%, respectively), and live birth
Rapid-i or Cryotop rate (41.2% and 40.9%, respectively).
In a recent study, Chatzimeletiou et al. investigated the
Rapid-i Cryotop
(n = 34) (n = 32)
effects of aseptic vitrification with Vitrisafe on the cytoskel-
eton, chromosome alignment, and development of human
Blastulation rate at 120 h (%) 68 56 blastocysts.44 Even though there was a significantly higher
Good blastocysts (%) 47 41 incidence of abnormal spindles in the vitrified group com-
Mean cell number 137 ± 14 138 ± 18 pared with the fresh group, the high survival rate following
Source: Modified from Hashimoto S et al. J Assist Reprod Genet
warming and the large proportion of normal spindle/chro-
2013;30:371–6. mosome configurations suggests that aseptic vitrification
Note: The subsequent blastulation rate and percentage of good- at the blastocyst stage does not adversely affect the develop-
quality blastocysts are shown with the mean cell number ment of human embryos and the ability of spindles to form
in each blastocyst. and continue normal cell divisions.

57
 vitrification in assisted reproduction
It also appears that long-term storage of embryos in samples of oocytes and embryos from hepatitis B, hep-
closed systems is not an issue. Mouse zygotes stored for atitis C, and human immunodeficiency virus chroni-
2 years with the Rapid-i have equivalent embryo devel- cally infected women undergoing in vitro fertilization
opment and viability compared to nonvitrified zygotes.43 cycles. Fertil Steril 2012;97:74–8.
The study of Wirleitner et al. included the transfer of blas- 8. Bielanski A, Vajta G. Risk of contamination of germ-
tocysts that had been vitrified for different time periods.45 plasm during cryopreservation and cryobanking in
The data demonstrated that long-term storage of vitrified IVF units. Hum Reprod 2009;24:2457–67.
blastocysts using the Vitrisafe device did not impair blas- 9. Maertens A, Bourlet T, Plotton N, Pozzetto B, Levy
tocyst viability. The survival rate after warming during R. Validation of safety procedures for the cryopreser-
the first year of storage was 83.0% compared with 83.1% vation of semen contaminated with hepatitis C virus
after 5–6 years of storage. The clinical pregnancy rate in assisted reproductive technology. Hum Reprod
after 1 year of storage was 40.0% versus 38.5% after 6 2004;19:1554–7.
years. Furthermore, no increase in the malformation rate 10. Stringfellow DA, Seidel S. Manual of the International
over time was observed. Embryo Transfer Society, 3rd edn. IETS: Savoy, IL,
1998. p. 4–6.
CONCLUSIONS 11. McBurnie LD, Bardo B. Validation of sterile filtration
There are concerns with regard to contamination from of liquid nitrogen. Pharm Technol 2002;26:74–82.
LN2 when using open systems that use direct contact 12. Parmegiani L, Accorsi A, Cognigni GE, Bernardi
with LN2 during vitrification and subsequent long- S, Troilo E, Filicori M. Sterilization of liquid nitro-
term storage. A European Parliament directive (EU gen with ultraviolet irradiation for safe vitrifica-
Tissues and Cells Directive 2004/23/EC), revised in 2006 tion of human oocytes or embryos. Fertil Steril
(Commission Directive 2006/86/EC), has defined medi- 2010;94:1525–8.
cal safety requirements and the use of closed systems for 13. Parmegiani L, Accorsi A, Bernardi S, Arnone A,
the cryopreservation of human cells.46,47 There is now evi- Cognigni GE, Filicori M. A reliable procedure for
dence that closed vitrification devices are as efficacious as decontamination before thawing of human specimens
open devices. Therefore, to prevent any contact with LN2 cryostored in liquid nitrogen: Three washes with ster-
during the procedure and subsequent storage, the most ile liquid nitrogen (SLN2). Ferti Steril 2012;98:870–5.
straightforward solution is to employ a closed device. 14. Eum JH, Park JK, Lee WS, Cha KR, Yoon TK, Lee DR.
Long-term liquid nitrogen vapor storage of mouse
CONFLICT OF INTERESTS embryos cryopreserved using vitrification or slow
Mark G. Larman is an employee of Vitrolife AB. cooling. Fertil Steril 2009;91:1928–32.
15. Cobo A, Romero JL, Perez S, de Los Santos MJ,
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2. Tedder RS, Zuckerman MA, Goldstone AH et al. gen vapour as a risk factor in pathogen transfer.
Hepatitis B transmission from contaminated cryo- Theriogenology 2009;71:1079–82.
preservation tank. Lancet 1995;346:137–40. 17. Criado E, Moalli F, Polentarutti N et al. Experimental
3. Morris GJ. The origin, ultrastructure, and microbiol- contamination assessment of a novel closed ultravit-
ogy of the sediment accumulating in liquid nitrogen rification device. Fertil Steril 2011;95:1777–9.
storage vessels. Cryobiology 2005;50:231–8. 18. Clarke GN. Sperm cryopreservation: Is there a sig-
4. Bielanski A, Bergeron H, Lau PC, Devenish J. nificant risk of cross-contamination? Hum Reprod
Microbial contamination of embryos and semen dur- 1999;14:2941–3.
ing long term banking in liquid nitrogen. Cryobiology 19. Kuleshova LL, Shaw JM. A strategy for rapid cooling
2003;46:146–52. of mouse embryos within a double straw to eliminate
5. Piasecka-Serafin M. The effect of sediment accumu- the risk of contamination during storage in liquid
lated in containers under experimental conditions nitrogen. Hum Reprod 2000;15:2604–9.
on the infection of semen stored directly in liquid 20. Isachenko V, Montag M, Isachenko E, van der Ven H.
nitrogen (–196 degree C). Bull Acad Pol Sci Biol Vitrification of mouse pronuclear embryos after polar
1972;20:263–7. body biopsy without direct contact with liquid nitro-
6. Bielanski A, Nadin-Davis S, Sapp T, Lutze-Wallace C. gen. Fertil Steril 2005;84:1011–6.
Viral contamination of embryos cryopreserved in liq- 21. Isachenko V, Montag M, Isachenko E et  al. Aseptic
uid nitrogen. Cryobiology 2000;40:110–6. technology of vitrification of human pronuclear
7. Cobo A, Bellver J, de los Santos MJ, Remohí J. Viral oocytes using open-pulled straws. Hum Reprod
screening of spent culture media and liquid nitrogen 2005;20:492–6.

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22. Seki S, Mazur P. Effect of warming rate on the survival approach to overcome an impaired uterine environ-
of vitrified mouse oocytes and on the recrystallization ment. Reprod Biomed Online 2012;25:591–9.
of intracellular ice. Biol Reprod 2008;79:727–37. 35. Hashimoto S, Amo A, Hama S, Ohsumi K, Nakaoka
23. Seki S, Mazur P. The dominance of warming rate Y, Morimoto Y. A closed system supports the develop-
over cooling rate in the survival of mouse oocytes mental competence of human embryos after vitrifica-
subjected to a vitrification procedure. Cryobiology tion. J Assist Reprod Genet 2013;30:371–6.
2009;59:75–82. 36. Desai NN, Goldberg JM, Austin C, Falcone T. The
24. Mazur P, Seki S. Survival of mouse oocytes after being new Rapid-i carrier is an effective system for human
cooled in a vitrification solution to –196°C at 95° to embryo vitrification at both the blastocyst and cleav-
70,000°C/min and warmed at 610° to 118,000°C/min: age stage. Reprod Biol Endocrinol 2013;11:41.
A new paradigm for cryopreservation by vitrification. 37. Panagiotidis Y, Vanderzwalmen P, Prapas Y et  al.
Cryobiology 2011;62:1–7. Open versus closed vitrification of blastocysts from
25. Papatheodorou A, Vanderzwalmen P, Panagiotidis an oocyte-donation programme: A prospective ran-
Y et  al. Open versus closed oocyte vitrification sys- domized study. Reprod Biomed Online 2013;26:470–6.
tem: A prospective randomized sibling-oocyte study. 38. Schiewe M. MicroSecure Vitrification (μS-VTF) pro-
Reprod Biomed Online 2013;26:595–602. cedure: Optimum simplicity, security, cost-savings
26. De Munck N, Verheyen G, Van Landuyt L, Stoop and effectiveness combining FDA-approved prod-
D, Van de Velde H. Survival and post-warming in ucts. J Clin Embryol 2010;13:33–51.
vitro competence of human oocytes after high secu- 39. Isachenko V, Katkov, II, Yakovenko S et al. Vitrification
rity closed system vitrification. J Assist Reprod Genet of human laser treated blastocysts within cut standard
2013;30:361–9. straws (CSS): Novel aseptic packaging and reduced
27. Stoop D, De Munck N, Jansen E et al. Clinical vali- concentrations of cryoprotectants. Cryobiology 2007;​
dation of a closed vitrification system in an oocyte- 54:305–9.
donation programme. Reprod Biomed Online 40. Liebermann J. Vitrification of human blastocysts:
2012;24:180–5. An update. Reprod Biomed Online 2009;19(Suppl.
28. Pinasco M, Hickman T, Russell H, Rashiv B. Oocyte 4):4328.
vitrification freeze/thaw survival rates using an open 41. AbdelHafez FF, Desai N, Abou-Setta AM, Falcone T,
versus a closed system. Fertil Steril 2012;97(Suppl. Goldfarb J. Slow freezing, vitrification and ultra-rapid
3):S18. freezing of human embryos: A systematic review and
29. Machac S, Hubrinka V, Larman M, Koudelka M. A meta-analysis. Reprod Biomed Online 2011;20:209–22.
novel method for human oocyte vitrification with 42. Bonetti A, Cervi M, Tomei F, Marchini M, Ortolani
a closed device using super-cooled air. Fertil Steril F, Manno M. Ultrastructural evaluation of human
2013;100(Suppl. 3):S108. metaphase II oocytes after vitrification: Closed versus
30. Stachecki J, Garrisi J, Sabino S et al. A new safe, sim- open devices. Fertil Steril 2011;95:928–35.
ple and successful vitrification method for bovine 43. Larman MG, Gardner DK. Vitrification of mouse
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2008;17:360–7. 1462–6.
31. Kuwayama M, Vajta G, Ieda S, Kato O. Comparison of 44. Chatzimeletiou K, Morrison EE, Panagiotidis Y et al.
open and closed methods for vitrification of human Cytoskeletal analysis of human blastocysts by confo-
embryos and the elimination of potential contamina- cal laser scanning microscopy following vitrification.
tion. Reprod Biomed Online 2005;11:608–14. Hum Reprod 2012;27:106–13.
32. Vanderzwalmen P, Ectors F, Grobet L et  al. Aseptic 45. Wirleitner B, Vanderzwalmen P, Bach M et  al. The
vitrification of blastocysts from infertile patients, egg time aspect in storing vitrified blastocysts: Its impact
donors and after IVM. Reprod Biomed Online 2009;​ on survival rate, implantation potential and babies
19:700–7. born. Hum Reprod 2013;28:2950–7.
33. Van Landuyt L, Stoop D, Verheyen G et al. Outcome 46. European parliament directive (EU Tissues and Cells
of closed blastocyst vitrification in relation to blas- Directive 2004/23/EC). URL: http://eur-lex.europa.
tocyst quality: Evaluation of 759 warming cycles eu/Lex-UriServ/LexUriServ.do?uri=OJ:L:2004:102:0
in a single-embryo transfer policy. Hum Reprod 048:0058:en:PDF.
2011;26:527–34. 47. Directive 2006/17/CE de la Commission du 8 février
34. Vanderzwalmen P, Zech NH, Ectors F et al. Blastocyst 2006. URL: http://www.agence-biomedecine.fr/IMG/
transfer after aseptic vitrification of zygotes: An pdf/directive-2006_17_ce.pdf.

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7 Automatic vitrification: Development of the Gavi system
Tammie K. Roy, Susanna Brandi, Cara K. Bradley, and Teija T. Peura

THE CHALLENGES OF VITRIFICATION from a single ovarian stimulation cycle. By the mid-2000s,
AND IN VITRO FERTILIZATION clinical evidence of improved ART outcomes from embryos
(IVF) AUTOMATION cryopreserved by vitrification as compared with the tradi-
The cryopreservation of embryos during assisted repro- tional method of slow freezing was accumulating.8,9 Genea
duction technologies (ARTs) is an essential practice for (previously known as Sydney IVF), a leading Australian
clinics to maximize cumulative pregnancy rates. The most IVF clinic,2 also performed testing of the two methods, with
effective method of cryopreserving embryos is vitrifica- embryo recovery and pregnancy outcomes clearly favoring
tion, which typically uses high concentrations of cryo- vitrification (unpublished data). As a result, vitrification
protectants and ultra-rapid cooling to preserve cells in a became the sole method for embryo cryopreservation in
glass-like (vitreous) state without detrimental ice crystal Genea clinics during 2006. While the clinical benefits of
formation.1 When successfully executed, vitrification pro- embryo vitrification for our patients were clearly apparent,2
duces extremely high embryo recovery rates, in excess of the labor-intensive and high-skill nature of the procedure
90%, and pregnancy, live birth, and neonatal outcomes provided challenges for our clinic and made it unattractive
equivalent to (or better than) those from fresh transfers.2–4 or not feasible for many other ART clinics.
However, vitrification is a manual, time-consuming, In 2009, Genea decided to address the drawbacks
and high-skill procedure, and outcomes can vary greatly of vitrification through automation of the procedure.
between operators and ART clinics.5 Multiple vitrifica- However, we understood that our strength lay in our fer-
tion devices and protocols are currently in use, which vary tility and vitrification expertise, and that to be success-
greatly in the constitution of cryoprotectants and condi- ful in this endeavor, we would need to collaborate with
tions of embryo exposure, the rate of cooling and subse- additional skill sets. Therefore, Genea established a part-
quent warming, and whether or not the embryo comes nership with Planet Innovation (Melbourne, Australia),
into direct contact with liquid nitrogen.6,7 The numerous a technology innovation and development company with
vitrification variables involved, including actual tempera- commercially focused product developers. Teams of
tures during the process, the exact duration of embryo embryologists, scientists, and engineers, located at both
exposure to solutions, and the diffusion gradient of solu- Genea and Planet Innovation primary sites, were formed
tions at the embryo level, make it very difficult to standard- to collaboratively develop and optimize various aspects of
ize the procedure when performed manually. an automated vitrification system.
The recognized need for standardization, as well as
the high-labor and high-skill demands of vitrification, The Requirements of an Automated
could potentially be addressed by automation. However, Vitrification System
automation of processes involving fluid exchange and Prior to attempting to automate vitrification, in-depth
embryos presents many challenges. The most significant “voice-of-customer” studies to understand ART clinics’
of these is how to secure small and fragile embryos, with requirements for an automated vitrification system were
highly variable physical characteristics depending on performed. Several key needs were identified, with one of
developmental stage, during automated fluid movement. the most important being that the recovery and survival
Here, we describe our approach to this problem and its rates of embryos processed using an automated system
application in an automated vitrification instrument, had to be comparable to current commercially available
which forms the basis of the Gavi system. devices. Another key requirement was that the system had
to be closed to prevent the embryo and vitrification solu-
DEVELOPMENT OF AN AUTOMATED tion coming into contact with liquid nitrogen. This avoids
VITRIFICATION SYSTEM potential contamination with pathogens that have been
The Beginning: A Partnership between Embryologists shown to survive in liquid nitrogen,10 and is essential for
and Engineers ART clinics in countries of the European Union to con-
In 1984, the first human baby was born from a frozen- form with their regulatory requirements.11 At Genea, the
thawed embryo cryopreserved by slow freezing. For the vitrification device used was the Cryotop®, an open sys-
next 20 years, this method was used by ART clinics world- tem that is arguably the gold-standard for embryo vitrifi-
wide, and this greatly improved cumulative pregnancy rates cation, and therefore, the decision was made to develop a

61
 vitrification in assisted reproduction
closed system with outcomes comparable to the Cryotop® to fully enclose the embryo within the Pod. Following
method. Furthermore, the system had to be single use, investigation of a number of options, it was decided to
simple to use, and vitrify multiple embryos simultane- use a heat-sealing technique to apply the lid. In order to
ously, ideally all blastocysts from a given patient in a sin- prevent transfer of heat to the embryo during this pro-
gle run. Additionally, the system would ideally be able to cess, various engineering concepts were introduced into
process oocytes, cleavage-stage embryos, and blastocysts. the Pod to minimize energy transfer from the seal loca-
tion (top of the Pod) to the embryo located in the channel
The Greatest Challenge and the Pod Solution divot. As such, the embryo does not experience tempera-
The first and greatest hurdle in developing an automated tures beyond physiological levels after sealing if trans-
vitrification system was to solve the problem of how to ferred to liquid nitrogen within the recommended time.
secure embryos during automated fluid exchange. An The Pod evolved significantly over the development
additional challenge was that the device used to hold process to address the many requirements for function-
the embryo during fluid exchange ideally would be ality and usability, and to date, there have been over 15
used as the vitrification and embryo storage device, thus main (over 40 total) Pod prototypes. The first “test bed”
negating the need for embryo handling after automated Pods, designed for proof of principle of the fluid dynamic
equilibration. Numerous concepts were brainstormed, channel, were made by manually molding pre-existing
including a mesh, a restricted tube, and an open micro- thin concave plastic. Subsequently, numerous concept
fluidic channel (Figure 7.1). Subsequently, nine different demonstrator prototypes were manufactured by injec-
“concept demonstrator prototypes” were developed by tion molding using a single-cavity tool. Design modifica-
rapid prototyping and their potential evaluated using a tions were incorporated in later alpha and beta models,
“fail fast” approach. Most prototypes were rapidly elimi- including the refinement of the microfluidics channel and
nated including the initial favorites, the mesh, which the addition of a handle and label area for embryo iden-
resulted in embryo disintegration, and the restricted tification Pods (Figure 7.2). Additionally, a metallic metal
tube, from which the embryo could not be consistently insert was added to the beta Pod to allow easy and secure
retrieved. Instead, a most unexpected candidate became placement into the cassette, as well as removal from the
the most promising concept: an open microfluidic chan- cassette both in and out of liquid nitrogen. Here, consid-
nel. Subsequently renamed the Pod, this approach holds erable effort went into injection molding technology to
the embryo in place during fluid exchange not by using achieve the thin wall section and co-molding the plastic
a physical barrier, but by fluid dynamics12; the embryo with an inert metallic insert.
resides in a microfluidic channel that restricts the move-
ment of the embryo but allows the passage of solutions The Gavi System
(for visualization of fluid dynamics).13 The Gavi system performs automatic equilibration for
To fulfill the requirements of a closed system, the Pod closed-system vitrification on up to four embryos at
was designed for a “lid” to be applied after equilibration a time. This occurs in Pods using the benchtop Gavi

The Road to Market: The Product Development Process

The project resulting in the Gavi instrument and consum- were undertaken by Planet Innovation, and all science
ables was executed following a tightly structured product aspects by Genea Biomedx. From the science aspects, Gavi
development process using international standards for the vitrification outcomes were always benchmarked against the
design and development of medical devices. The process Cryotop® system in regard to equivalent recovery of embryos,
consisted of stage-gated project phases, with the first phase initial survival, and further in vitro embryonic development.
defining the initial customer needs and product require- Engineering aspects of the Gavi system included a variety of
ments, development of product concepts, and their test- variables, such as testing of electronic systems, instrument
ing. The second phase contained further refinement of the operating software, fluidic exchange protocol software, pre-
core technology and feasibility testing of the overall system cision pipetting and liquid handler unit functions, tempera-
utilizing concept demonstrator prototype units. The third ture control, movable platform and sealing unit operations,
phase locked in the final design and verified that all prod- and many others. Furthermore, a significant amount of
uct requirements were met utilizing more refined pre-veri- science-related testing was undertaken to ensure the safety
fication prototype units (alpha and beta), while the fourth and efficacy of the system and its parts. This included sta-
phase (currently in progress) undertakes the final valida- bility testing of Gavi consumables such as Pods and pipette
tion of the complete system to ascertain whether the prod- tips using accelerated (heat and light induced) and real-time
uct functions as intended, utilizing pre-production units aging, as well as the effect of sterilization by irradiation and
and production units. simulated transportation. Biocompatibility and sterility
Both science and engineering aspects were addressed testing were part of all development phases, as well as final
and tested at all phases. All engineering aspects of the work product verification and validation.

62
 automatic vitrification
Mesh Restricted tube Microfluidics channel
Fluidic Fluidic
movement movement
Fluidic
movement
Embryo

Embryo
Divot
Embryo
Pressure Pressure

Figure 7.1  Examples of concepts investigated to secure the embryo during automated fluid exchange.

Handle and allow the media to equilibrate to the platform tempera-

label area Cross-section through Pod channel ture. Next, Pods are placed into the “cassette,” followed
A
by a single embryo being loaded into each Pod. The cas-
B A sette is then loaded into the appropriate position of the

Divot Pipette tip

B Pod channel (loading site) well Medium
cartridge Tip and seal Pod
Figure 7.2  Gavi Pod. Computer-aided design drawings
showing Pod (left) and a cross-section through the Pod
microfluidic channel (right).
1 2 4
Cassette
instrument, which contains high-precision liquid han-
6 5
dling pipettes and sealing units with vertical move- Operating
ment capabilities. At the base of the instrument is the tray
carriage, located on a horizontal moving platform. The 3
carriage has fine temperature control with heating and Blastocyst
cooling capabilities that ensure very little temperature Gavi instrument
variation during the protocol execution. Within the car-
riage are allocated sites for required consumables for 7
automated vitrification, including Pods and vitrification
solutions. The instrument contains an inbuilt computer 8
with sophisticated software that controls all aspects of the
instrument, including spatial and temporal movements. Automated processing LN2 bucket
The various steps of the equilibration protocol are exe-
cuted by coordination of the platform movement along Figure 7.3  Procedure to vitrify embryos using the Gavi
the horizontal axis with the high-precision pipettes, and system. Steps to operate the Gavi system: (1) load the
sealing units along the vertical axis. “medium cartridge” into the “operating tray”; (2) load
The procedure to operate the Gavi system is simple and the “tip and seal” into the operating tray; (3) load the
the key skill set required is embryo handling (Figure 7.3). operating tray into the Gavi instrument; (4) load the Pod
Preparation of the Gavi instrument involves placing sin- into the “cassette”; (5) load a single embryo into the Pod;
gle-use consumables “medium cartridge” (containing the (6) load the cassette into the operating tray in the Gavi
vitrification solutions) and “tip and seal” (containing a instrument; (7) start the protocol; (8) upon completion
sterile pipette tip and lid seal) on the operating tray, which of the protocol, immerse the cassette in the LN2 bucket.
is then loaded into the Gavi instrument on the carriage to (LN2 = liquid nitrogen.)

63
 vitrification in assisted reproduction
operating tray located within the instrument. The num- The main requirements considered to ensure good vitrifi-
ber of Pods to be processed is indicated via the software cation outcomes using the Pod were
interface before the “start” button is pressed. Automated
equilibration of the embryos then commences, running • Securing the embryo during fluid exchange with-
an approximately 18-minute (human) blastocyst proto- out mechanical stress or toxicity
col. Once the protocol is complete, the cassette is removed • That the embryo would at all times be covered by
from the operating tray and manually dunked in the “liq- solution
uid nitrogen bucket,” also located on the Gavi instru- • A high rate of cooling and warming
ment. The liquid nitrogen bucket is then used to transport • Good optics for embryo visualization
the embryos, now contained in fully closed Pods, to long- • Ease of handling
term liquid nitrogen storage. • Consistent successful sealing (and subsequently
opening) to create a closed system
Optimization of Gavi Protocols • Withstanding pressures of greater than 1 bar, a
Simultaneous to the development of the engineering requirement due to extreme temperature changes
aspects of the Gavi system, there has been a large amount from vitrification and warming
of research into protocol optimization. This involved in-
depth experimentation with the many different variables in Often, these functional requirements conflicted with
the equilibration process, including the temperature, time, each other. For example, the width of the Pod plastic had
volume, media concentration, and aspiration/dispense to be thin enough to ensure high cooling and warming
speed of the equilibrations steps; an example of this can rates, but thick enough to ensure Pod endurance under
be seen in Table 7.1, which revealed that an exposure time pressure. The material used to manufacture the Pod also
of 60 seconds, but not 90 seconds, in vitrification solution provided challenges; for example, one plastic we trialed
2 for mouse zygotes produced equivalent in vitro develop- had excellent optical properties but was difficult to seal
ment outcomes post-warming compared to the manual reliably, while another plastic was ideal for both but was
Cryotop® method. In many cases, modification of a single extremely hydrophobic and irreparably disrupted the
variable elicited the need for further refinement and opti- fluid dynamics.
mization of other variables. Through this lengthy and time- As a consequence of conflicting functional require-
consuming process, we have generated a standard protocol ments, many acceptable compromises had to be navi-
for all blastocyst types, including fully hatched blastocysts gated. This included Pod cooling and warming rates being
(see Roy et al., 2014, Table III and Table IV13). The fact that approximately 14,100 and 11,200°C/min, respectively,
we can achieve equivalent in vitro cryopreservation out- using the final alpha Pod prototype,13 and approximately
comes to that of Cryotop® controlled for both mouse (non- 11,400 and 8600°C/min, respectively, (unpublished data)
hatched) blastocysts and fully hatched blastocysts using the using the final beta Pod prototype. The reason for this
same protocol on the Gavi system challenges the notion decrease in Pod cooling and warming rates in successive
that vitrification needs to be personalized depending on an prototypes reflects minor revisions to the Pod design,
embryo’s (visual) response to vitrification solutions. materials, and manufacture, which were essential for
ensuring reliable and consistent vitrification outcomes.
Getting the Pod Just Right Critically, extensive in vitro testing using mouse embryos
During development, extensive testing of multiple Pod with beta Pods revealed that these changes did not com-
prototypes for vitrification and engineering outcomes was promise the vitrification outcomes achieved with the
performed to ensure optimal design and functionality. system (unpublished data), and in fact, our cooling rate

Table 7.1  Example of Optimization of the Gavi System

≥Expanding Fully hatched
blastocyst blastocyst

Treatment Number Recovered Survived Day 5 Day 7

Cryotop® 68 68 (100%) 67 (98.5%)* 54 (79.4%)* 39 (57.4%)
Gavi (V2 60 s) 60 58 (96.7%) 51 (87.9%)* 44 (75.9%) 33 (56.9%)
Gavi (V2 90 s) 64 62 (96.9%) 54 (87.1%)* 37 (59.7%)* 29 (46.8%)
Note: The effect of time spent in vitrification solution 2 (V2) on mouse F1 C57Bl/6J × CBA zygotes processed using
the alpha Gavi system. Embryos were incubated in V2 for either 60 or 90 seconds and the results were com-
pared with the manual Cryotop® method. Embryos were considered to have survived if all cells were intact at
the completion of warming.
* Significant difference (p < 0.05) between test groups.

64
 automatic vitrification
far exceeds that of other commercially available closed- no difference in in vitro embryo development at 24 hours
system vitrification devices.14,15 (day 6) and 48 hours (day 7) after warming between Gavi-
and Cryotop® -processed embryos. This includes develop-
OUTCOMES OF THE GAVI SYSTEM ment to fully hatched blastocysts by day 6 being 31.6% for
Vitrification of Mouse Blastocysts embryos processed with the Gavi system as compared
As reported previously,13 using the alpha model Gavi with 30.8% for embryos processed using the manual
system, we achieved in vitro vitrification outcomes for Cryotop® method.
mouse zygotes, cleavage-stage embryos, and blastocysts
(including fully hatched blastocysts) that were compa- Vitrification of Human Blastocysts
rable to Cryotop® controls. Using Gavi pre-production Encouraged by the positive outcomes for mouse embryos
units, we have processed a total of 2876 mouse blasto- processed using Gavi pre-production units, we have com-
cysts, of which we have recovered 99.9% of embryos after menced testing of Gavi production units using human
vitrification–warming. Embryo survival after warming embryos excess to reproductive needs and donated for
was 94.9% for blastocysts processed with the most recent research. These embryos are blastocysts that have either
Gavi protocol, and was comparable to the 96.0% obtained been cryopreserved by slow freezing or vitrification, and
using the Cryotop® method (Table 7.2). There were fewer are warmed with appropriate protocols for the cryopreser-
mouse embryos considered to be re-expanded 2 hours vation method, before being vitrified using the Gavi sys-
after warming (defined as occupying 90% or greater of tem or the Cryotop® method. To date, the recovery of 18
its original volume) using the Gavi system as compared human embryos processed with Gavi production units is
with Cryotop® controls. However, the lower re-expan- 100% (Table 7.3). The proportion of embryos considered to
sion rate for embryos processed with the Gavi system have survived after warming was 94.4%, which is compa-
was not indicative of poor survival, as the developmen- rable to the 86.4% achieved with the Cryotop® system. The
tal potential of the embryos remained intact; there was proportion of embryos considered to have re-expanded

Table 7.2  Evaluation of the Gavi System Using Mouse Blastocysts

Fully hatched blastocyst

Treatment Number Recovered Survived Re-expanded Day 6 Day 7

Cryotop® 273 273 (100%) 262 (96.0%) 211 (77.3%)* 84 (30.8%) 112 (41.0%)
Gavi 336 335 (99.7%) 318 (94.9%) 225 (67.2%)* 121 (36.1%) 160 (47.8%)

Note: Outcomes for Quackenbush Swiss mouse blastocysts vitrified after automated processing with Gavi pre-production
units as compared with the manual Cryotop® method. Embryos were considered to have survived if 75% or more
cells were intact at the completion of warming. Embryos were considered to have re-expanded if occupying 90% or
greater of their original volume 2 hours after warming.
* Significant difference (p < 0.05) between test groups.

Table 7.3  Evaluation of the Gavi System Using Human Blastocysts

24 h

Fully hatched Hatching/expanding

Treatment Number Recovered Survived Re-expanded blastocyst blastocyst
Cryotop® 22 22 (100%) 19 (86.4%) 13/19 (68.4%)* 2 (9.1%) 15 (68.2%)
Gavi 18 18 (100%) 17 (94.4%) 9 (50.0%) 5 (27.8%) 11 (61.1%)

Note: Outcomes for human blastocysts vitrified after automated processing with Gavi production units as compared with
the manual Cryotop® method. Embryos were donated for research via an informed consent process (National Health
and Medical Research Council [NHMRC] license 309718) and were either slow frozen (50%) or vitrified blastocysts
(50%) that the patients had deemed as excess to their reproductive needs. Only embryos that were grade I or grade
II2 prior to Gavi (50% expanding blastocysts or greater) or Cryotop® (59% expanding blastocysts or greater) vitrifica-
tion were used. Embryos were considered to have survived Gavi or Cryotop® vitrification if 75% or more cells were
intact at the completion of warming. Embryos were considered to have re-expanded if occupying 90% or greater of
their original volume 2 hours after warming. Embryo classifications at 24 hours were restricted to grade I and
grade II2 embryos.
* Not all embryos were assessed for re-expansion.

65
 vitrification in assisted reproduction

Prior to After Gavi vitrification-warming

Gavi vitrification (Immediately) (24 h)

Embryo 1
Embryo 2
Embryo 3

Figure 7.4  Examples of human blastocysts vitrified using the Gavi system. Embryos were donated for research via
an informed consent process (NHMRC license 309718) and were either slow frozen (embryo 2) or vitrified blasto-
cysts (embryo 1 and embryo 3) that the patients had deemed as excess to their reproductive needs. Embryo 1 was
a pre-implantation genetic diagnosis (PGD)-biopsied hatching blastocyst. Embryo 2 and embryo 3 were expanded
blastocysts. All images were taken at 200× magnification, with the exception of the images of embryo 1 and embryo
3 at 24 hours after Gavi vitrification–warming, which were taken at 100× magnification.

2 hours after warming was slightly lower for the Gavi even very junior technicians achieved similar outcomes
system as compared with Cryotop® controls (50.0% and to senior embryologists (unpublished data). In fact, the
68.4%, respectively), and is consistent with the mouse greatest challenge when training new staff for this project
blastocyst data. Furthermore, in vitro embryo develop- has been mastering the manual Cryotop® method, and
ment after vitrification–warming was comparable between given that this is our vitrification benchmark, stringent
Gavi- and Cryotop®-processed embryos; at 24 hours post- certification criteria were necessary before considering
warming, 88.9% of Gavi-processed embryos were grade I new users’ Cryotop® results. However, we do not believe
or grade II2 expanding blastocysts or greater, as compared that the Gavi system reduces the need for skilled embry-
with 77.3% for Cryotop® controls (Figure 7.4). ologists in the clinic, although it does provide valuable
time to embryologists for other work; when consider-
THE BENEFITS OF THE GAVI SYSTEM ing the “hands-on” time to vitrify four human blasto-
The Gavi system provides many benefits to vitrifica- cysts, the Gavi system takes an estimated 36 minutes less
tion users compared with manual vitrification. A major than the manual Cryotop® method.13
advantage of the system is the ability to control and Another advantage of the Gavi system for ART clin-
standardize vitrification variables, a very difficult feat ics struggling with the challenges of manual vitrification,
if performed manually, thus allowing the procedure to and even more so for clinics currently performing slow
be performed the same way each time. The Gavi system freezing, consists of the in vitro vitrification outcomes
also reduces the constant need for monitoring individual achieved with the system. The recovery rate alone, being
operator outcomes, something that is critical even after 99.9% using mouse blastocysts with Gavi pre-production
a user achieves training requirements. The key skill set units, is outstanding, particularly given it being a closed
required for operation of the Gavi system is basic embryo system. It is difficult to know how this compares with
handling, and during the development of the system, other vitrification devices in a clinical setting, given that

66
 automatic vitrification
embryo recovery rates are usually not stated and can may provide further improvements in assisted reproduc-
vary greatly depending on the device used and operator tion outcomes.
skill. Furthermore, the Gavi system achieves equivalent
embryo survival and subsequent in vitro development to CONCLUSION
that of the gold-standard Cryotop® system, with which Vitrification is an essential practice in ART clinics for
we have proven expertise.2 ensuring optimal patient outcomes. However, vitrifica-
tion is labor intensive and time consuming, and outcomes
THE FUTURE OF THE GAVI SYSTEM vary between operators and ART clinics. The Gavi system
Toward Clinical Use automates the equilibration of embryos prior to vitrifica-
Despite the extremely promising in vitro results using tion using a novel closed-system device. This allows pre-
the Gavi system on mouse embryos and donated human cise control of the many vitrification variables—a very
blastocysts reported here and elsewhere,13 the ultimate difficult feat if performed manually. Testing of Gavi sys-
test for determining its success will be in the clinic. When tem prototypes—the alpha system reported previously13
the Gavi system passes the requirements for scientific and and the pre-production and production units reported
engineering validation, as well as a number of other strict here—has shown equivalent in  vitro outcomes to those
criteria, including toxicity studies on the plastic consum- of the gold-standard Cryotop ® system. Further in vitro
ables and stability testing of the vitrification solutions, testing of production units is currently underway, and it
the system will be used to vitrify human blastocysts in a is expected that usage in a clinical setting will begin in
number of Genea ART clinics. This clinical usage, which 2015. The Gavi system has the potential to revolutionize
will include analysis of neonatal outcomes, is expected to and standardize vitrification, and opens up the possibil-
commence in 2015, and will be monitored very closely to ity of automating other complicated IVF procedures.
ensure patient outcomes are not compromised in any way.
The Gavi system (with the blastocyst protocol) is UPDATE
expected to gain regulatory clearance and consequently Since the submission of this chapter the Gavi verifica-
should be commercially available to ART clinics in late tion and validation and clinical evaluations has been
2015. The long time frame to develop the Gavi system completed and the instrument has been CE marked.
(7 years) reflects the challenging nature of developing
an automated vitrification system, the very stringent ACKNOWLEDGMENTS
criteria set for success, and the large amount of testing The authors wish to thank the staff of Genea Biomedx
required before clinical usage and regulatory clearance. and Planet Innovation involved in the development and
Furthermore, research and development of the Gavi sys- testing of the Gavi system, particularly Genea Biomedx
tem will be ongoing and will include optimization of pro- embryologists Ms. Naomi Tappe for investigation of the
tocols for cleavage-stage embryos and oocytes. These will effect of time spent in vitrification solution 2 on mouse
be incorporated into the Gavi system by software updates. zygotes and Ms. Breanna Evenden for validation of Gavi
Additionally, it is anticipated that future versions of the pre-production units using mouse blastocysts, and Planet
Gavi system may automatically place equilibrated sealed Innovation engineers Mr. Eduardo Vom and Mr. Simon
Pods into liquid nitrogen, thus removing the current Hobbs for engineering aspects of the Gavi system.
requirement for manual transfer.
REFERENCES
Potential Application to Other IVF Processes 1. Kuwayama M. Highly efficient vitrification for cryo-
While the Gavi system has the potential to revolutionize preservation of human oocytes and embryos: The
and standardize embryo cryopreservation, the system Cryotop method. Theriogenology 2007;67:73–80.
may have applications for assisted conception procedures 2. Roy TK, Bradley CK, Bowman MC et al. Single-
beyond that of vitrification. As the Pod solves the most embryo transfer of vitrified-warmed blastocysts
difficult issue of how to hold the embryo in place dur- yields equivalent live-birth rates and improved neo-
ing automated fluid exchange, it may be possible to use natal outcomes compared with fresh transfers. Fertil
this new technology in other areas to minimize embryo Steril 2014;101:1294–301.
handling and thus stress and the potential for embryo 3. Kato O, Kawasaki N, Bodri D et al. Neonatal outcome
loss. We are also applying our knowledge and experience and birth defects in 6623 singletons born following
from developing the Gavi system to finding solutions to minimal ovarian stimulation and vitrified versus
other challenges and inefficiencies in the ART clinic. This fresh single embryo transfer. Eur J Obstet Gynecol
includes combining advanced embryo incubator technol- Reprod Biol 2012;161:46–50.
ogy with time-lapse monitoring of embryo development, 4. Takahashi K, Mukaida T, Goto T et al. Perinatal out-
which may assist in the identification of embryos with the come of blastocyst transfer with vitrification using
highest pregnancy potential.16 These technologies will cryoloop: A 4-year follow-up study. Fertil Steril
provide another step toward ART standardization and 2005;84:88–92.

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 vitrification in assisted reproduction
5. Gosden R. Cryopreservation: A cold look at technol- 2004 on setting standards of quality and safety for
ogy for fertility preservation. Fertil Steril 2011;96:​ the donation, procurement, testing, processing,
264–8. preservation, storage and distribution of human tis-
6. Alpha Scientists in Reproductive Medicine. The Alpha sues and cells. Official Journal of the European Union
consensus meeting on cryopreservation key perfor- 2004;102:48–58.
mance indicators and benchmarks: Proceedings of an 12. Henderson CJ, Lewis CM, Roy TK et al. Improved
expert meeting. Reprod Biomed Online 2012;25:​146–67. micromanipulation and storage apparatus and meth-
7. Kader AA, Choi A, Orief Y et al. Factors affecting the ods patent WO2011146998A1. Dec 1, 2011.
outcome of human blastocyst vitrification. Reprod 13. Roy TK, Brandi S, Tappe NM et al. Embryo vitrifi-
Biol Endocrinol 2009;7:99. cation using a novel semi-automated closed sys-
8. Kuwayama M, Vajta G, Ieda S et al. Comparison of tem yields in vitro outcomes equivalent to manual
open and closed methods for vitrification of human Cryotop method. Hum Reprod 2014; 29(11):2431–8.
embryos and the elimination of potential contamina- 14. Desai NN, Goldberg JM, Austin C et al. The new
tion. Reprod Biomed Online 2005;11:608–14. Rapid-i carrier is an effective system for human
9. Stehlik E, Stehlik J, Katayama KP et al. Vitrification embryo vitrification at both the blastocyst and cleav-
demonstrates significant improvement versus slow age stage. Reprod Biol Endocrinol 2013;11:41.
freezing of human blastocysts. Reprod Biomed Online 15. Vanderzwalmen P, Ectors F, Grobet L et al. Aseptic
2005;11:53–7. vitrification of blastocysts from infertile patients,
10. Bielanski A. A review of the risk of contamination egg donors and after IVM. Reprod Biomed Online
of semen and embryos during cryopreservation and 2009;19:700–7.
measures to limit cross-contamination during bank- 16. Meseguer M, Rubio I, Cruz M et al. Embryo incuba-
ing to prevent disease transmission in ET practices. tion and selection in a time-lapse monitoring system
Theriogenology 2012;77:467–82. improves pregnancy outcome compared with a stan-
11. European Union. Directive 2004/23/EC of the dard incubator: A retrospective cohort study. Fertil
European Parliament and of the Council of 31 March Steril 2012;98:1481–9.

68
8 Vitrification at minimum volume: From basic science
to clinical application
Amir Arav

WHAT IS VITRIFICATION? it in the liquid or gas state and cool it rapidly so as to skip
Vitrification is the process by which a liquid is super- over the zone of crystallization temperatures in less time
cooled to a temperature at which the viscosity is so high than is necessary for the material to freeze. Therefore, one
that the liquid can be defined as being at an amorphous should consider the crystallization velocity. It is evident
glassy solid state, having no ice crystals. The under- that when the crystals grow faster one must traverse the
standing of the vitrification process has deepened over crystallization zone more rapidly if one wants to avoid
the years and has been applied for cryopreservation. crystallization.”1
Currently, it is the method of choice for oocyte and
embryo cryopreservation. THE FIRST CELLS SURVIVING AFTER
VITRIFICATION
HISTORY OF VITRIFICATION In 1938, Basil J. Luyet and Eugene L. Hodapp published
Basil Luyet stated in 1940: “Some of the oldest investi- the first successful vitrification of sperm.5 They demon-
gations on subcooling were made by Gay Lussac (1836) strated for the first time the successful cryopreservation
who observed that water can be subcooled to –12°C of frog sperm by vitrification using a 2 M sucrose solu-
when it is enclosed in small tubes.”1 Supercooling is cool- tion in small drops. Luyet started his research with col-
ing under the freezing point without crystallization, loids (i.e., gelatin, milk, or agar) and found that their
and it had already been thought of at the beginning of water content determines the possibility or impossibil-
the nineteenth century. It was done by the great French ity of vitrification. In general, solutions containing 50%
chemist and physicist Joseph Louis Gay Lussac. He is gelatin could vitrify successfully with layers of 0.3 mm in
known mostly for his two laws of gases and for his work thickness (by the method of immersion in liquid nitrogen
on alcohol–water mixtures. Gay Lussac found that water [LN]), while solutions containing 90% water could vit-
can be cooled to −12°C when enclosed in small tubes rify only with smears of a few microns in thickness.1 We
without freezing, finding with this discovery the basis of recently repeated Luyet’s experiment using human sperm
vitrification.2 and obtained a very high survival rate of sperm “vitri-
In 1804, Gay Lussac ascended in a hot-air balloon fied” in liquid air (−190°C) using a solution that does not
and noticed that the drops in the clouds were not frozen contain intracellular cryoprotectants but is based only on
despite the subzero temperatures. He later published the sugars and proteins.6
discovery of the effect of small volumes of water droplets In 1949, Polge et  al.,7 trying to repeat Luyet’s results,
on supercooling; indeed, the size of water drops in clouds discovered the cryoprotective property of glycerol and so
is about 8–10 µm, which maintains them at a liquid state opened the field to slow freezing.
at the subfreezing temperature of −5°C. In 1898, Gustav Currently, there are two methods for gamete cryo-
Heinrich Johann Apollon Tammann pointed out that a preservation: slow freezing and vitrification. Slow freez-
large number of substances can be obtained as glasses ing has the advantage of using low concentrations of
and suggested that this property might be universal.3 cryoprotectants (CPs), which are associated with chemi-
In 1858, Johann Rudolf Albert Mousson sprayed drop- cal toxicity and osmotic shock. Vitrification is a rapid
lets of water less than 0.5 mm in diameter onto a dry sur- method that reduces chilling sensitivity and crystalliza-
face and observed that the smaller the drops, the longer tion damage caused to cells.
they stayed subcooled.4 However, the volume is not the For many years, slow freezing and not vitrification has
only important factor for achieving supercooling; among been the method of choice, since vitrification was not
other factors that might have an influence on inducing achieved easily due to the need for high CP concentrations
crystallization, as were mentioned by Luyet, are the cool- and relatively high-volume samples to be cryo-stored.
ing velocity and the concentration of the supercooled or
supersaturated solutions. Luyet wrote: “To avoid freezing, THE MINIMUM DROP SIZE TECHNIQUE:
the temperature should drop at a rate of some hundred MY PERSONAL STORY
degrees per second, within the objects themselves,” and The first successful vitrification of mouse embryos using
also: “The only method of vitrifying a substance is to take a relatively large-volume sample was done in 1985.8 These

69
 vitrification in assisted reproduction
researchers vitrified mouse embryos with 6.5 M of glyc- and William F. Rall published in 2007 the critical cool-
erol in a large volume inside a 0.25-mL straw plunged ing rates needed to vitrify aqueous solutions contain-
into LN. At that time, I was a veterinary student at the ing different concentrations of CPs15; from this, it was
University of Bologna, Italy, and I met Bill Rall, who told extrapolated that for pure water, a cooling rate greater
me about the exciting work he did on mouse embryos. than 100 million °C/min is needed to form a glass state
Two years later, I started to work with Boris Rubinsky without crystallization (Figure 8.3). Also, it is interesting
on the cryomicroscopy of oocytes and embryos, and in to note that for 15% (v/v) of most CPs, a cooling rate of
our laboratory, we used to prepare oocytes for histology almost 1  million °C/min is needed, which is extremely
evaluation by fixing them with a small drop over a micro- difficult to achieve. We showed that 15% (v/v) of CPs can
scopic slide. This gave me the idea for using the same vitrify at a relatively slow cooling rate when the volume
technique for vitrification in a small drop, which I later of the drop is 0.07 µL.10 Therefore, it is more feasible and
called the “minimum drop size” (MDS) (Figure 8.1).9–12 easier to achieve vitrification by lowering the sample vol-
The volume we used for vitrification was in the range of ume than by increasing the cooling rate.
0.07–0.1 µL, and the concentration of the vitrification James H. Walton and Roy C. Judd measured the veloc-
solution (VS) was about 50% lower than of the VS used ity of ice crystal growth and found that it is in the range
for large-volume vitrification (Figures 8.1 and 8.2).10 of 65 mm/s.16 This means that if we wish to avoid crys-
We called it the MDS as this was the minimal size that tallization in a drop having a diameter of 0.01 mm, we
allowed us to keep oocytes or embryos without damage will need a velocity of 1/6500 mm/s, which is 0.0001 s.
due to desiccation. Vitrification of embryos, on the other If we cool from room temperature to −180°C, this means
hand, although initially attempted in the late 1980s, has we need to drop 200°C at a rate of 0.1 microseconds, or
not been clinically applied until recently. Vitrification is at 120 × 106 °C/min. This is actually very similar to the
currently producing very satisfactory outcomes by means cooling rate that was estimated by Balds and Bruggeler
of different methodologies using a minimum volume (Figure 8.3).1,15 However, since achieving this cooling rate
principle both for oocytes and embryos.13,14 is virtually impossible, the best possible way to achieve
vitrification of pure water is in a small drop (diameter of
FACTORS AFFECTING VITRIFICATION 10 µm) at relatively slow cooling rates. This indicates that
The velocity of cooling, which is a major factor in the vit- volume has an independent effect on the probability of
rification process, depends on the thermal mass of the vitrification.
sample and on its surface area. To achieve rapid cooling, Later attempts at vitrifying pure water have been
we should use materials that have the lowest heat mass made by a few investigators; L. Hawkes17 published an
and maximum surface-to-volume ratio. Gregory M. Fahy experiment in which a drop of solid amorphous water

Solution toxicity

40%
CP concentration (w/v)

Drop volume (μL)

MDS
20% 1.6 × 10–2

0 5.2 × 10–7
103 104 >105

Cooling rate (K/min)

Possibility of vitrification: CP solution

Possibility of vitrification: 1/2 CP solution
Cryomicroscope
Possibility of vitrification: pure water

Figure 8.1 The effect of volume, cooling rate, and CP concentration on vitrification. (CP = cryoprotectant;
MDS = minimum drop size.)

70
 vitrification at minimum volume

0.2
0.18
0.16
0.14
Volume (μL)
0.12
0.1
0.08
0.06 FCS + glycerol
0.04 + sucrose
FCS + sucrose
0.02 FCS
0 PBS
17.5 20 26 35
PG concentration (% v/v)

Figure 8.2  The probability of vitrification as it depends on cooling rate, volume, and composition of the solution.
As is noted in Figure 1 and 2 taken from Arav (1989),9 the probability of vitrification increases as the volume of the
sample decreases. At 0.02 µL, it is possible to achieve vitrification even with only 17.5% propylene glycol (PG) + 2.5%
glycerol + 20% fetal calf serum (FCS) in phosphate-buffered saline (PBS). However, the minimum drop size was
designed in the range of 0.035 µL, which requires about 30% cryoprotective agent (26% PG and 2.5% glycerol and
sucrose) and is very similar to the concentration levels used currently (Figure 8.3).

Cooling rate Drop on the container, the volume, thermal conductiv-

80 × 106 diameter ity, solution composition, etc.19 To achieve LN slush,
e
°C/min
lum (mm)/ the LN needs to be cooled close to its freezing point
Vo Volume (−210°C). The VitMaster is a device that generates LN
0.5/ slush (IMT Ltd, Ness Ziona, Israel), which reduces the
20,000 MDS C
oo 0.035 μL temperature of LN to between −205°C and −210°C by
lin
gr
ate
applying negative pressure. When LN slush is formed,
0.1/0.2 nL the cooling rate is dramatically increased. The cooling
0.01/ rate is especially enhanced in the first stage of cooling,
0.0002 nL when cooling down from room temperature to 0°C.
0% 15% 30% 40%
Concentration of CPs The cooling rate is enhanced two to six times more
than plunging into LN (−196°C) with 0.25-mL straws,
Figure 8.3 The probability of a vitrification solution or any other device such as open-pulled straws (OPS)
containing different concentrations of CPs (the volume or electron microscope (EM) grids.11 It was shown for
is calculated for a half sphere). (CP = cryoprotectant; oocytes and embryos that increasing the cooling rate
MDS = minimum drop size.) improves survival rates by up to 37%.20 In Figure 8.2,
we can see that cooling at 80 million °C/min is needed
to vitrify pure water, while cooling at 20,000 million
was obtained during rapid cooling. Burton and Oliver °C/min is needed to vitrify 30% of CPs using a small
obtained, from steam, some solid water in which X-ray volume in the range of 0.1 µL (the MDS technique),
analysis did not reveal any crystalline structures.18 As we and only 15% of CPs is needed if we reduce the diame-
can see, these achievements were mainly due to the small ter of the drop to 100 µm.21 This means that it is easier
sample volume, and not the velocity of cooling. to achieve vitrification by reducing the volume rather
than increasing the cooling rate. It was recently dem-
IMPORTANT FACTORS THAT SHOULD BE onstrated by Seki and Mazur that the warming rate
CONSIDERED is dominant over the cooling rate for mouse oocyte
1. Cooling and warming rate—high cooling rates can be vitrification22; however, to reduce chilling damage,
achieved with LN or LN slush, and a warm water bath which occurs during cooling and warming, we should
for warming. When using LN, the sample is plunged keep the cooling rate as rapid as possible.
into LN, resulting in cooling rates of hundreds to tens 2. Viscosity of the medium in which the embryos are
of thousands of degrees Celsius per minute, depending suspended, or the glass transition capability of the

71
 vitrification in assisted reproduction
solution at low temperatures, is defined by the con- OOCYTE VITRIFICATION
centration and behavior of various CPs and other Oocytes are very different from sperm or embryos with
additives during vitrification. The higher the con- respect to cryopreservation. The volume of the mamma-
centration of CPs, the higher the glass transition lian oocyte is in the range of three to four orders of magni-
temperature (TG), thus lowering the chance of ice tude larger than that of the spermatozoa, thus substantially
nucleation and crystallization. Different CPs and decreasing the surface-to-volume ratio. However, this is
other additives have different toxicities, penetra- not the reason why the oocytes are sensitive to low tem-
tion rates, and TGs. A combination of different CPs peratures and to slow freezing; that is, mature oocytes are
is often used to increase viscosity, increase TG, and very sensitive to slow freezing, and even after fertilization,
reduce the level of toxicity. So in the cattle industry, the volume of the oocytes remains the same and their sen-
to avoid handling of the post-warmed embryos and sitivity is reduced to a minimum. In fact, this is the best
allow direct transfer, ethylene glycol (EG) is often stage for freezing human oocytes (2 pro-nuclei [2PN]
used as the permeating CP because of its high pen- freezing).50 The reason why oocytes are nonfreezable is due
etration rate23 and its high glass transition ability.24 to their chilling sensitivity, which occurs at different cel-
3. Volume—the smaller the volume, the higher the lular levels: the zona pellucida, plasma membrane, meiotic
probability of vitrification.9–12 Smaller volumes allow spindle, cytoskeleton, etc.
better heat transfer, thus facilitating higher cooling The plasma membrane of oocytes at the metaphase II
rates. Although small volume has an independent (MII) stage has a low permeability coefficient, thus mak-
effect on the probability of nucleation, as was dis- ing the movement of CPs and water slower.51 In addi-
covered 200 years ago by Guy Lussac, only recently tion, the freeze–thaw process causes premature cortical
have techniques been developed to reduce sample granule exocytosis, leading to zona pellucida hardening,
volume, with an explosion of methods appearing thus making sperm penetration and fertilization impos-
in the literature during the last decade. These tech- sible.52–54 These later consequences can be overcome
niques can generally be divided into two categories: by the use of intracytoplasmic sperm injection (ICSI).
surface techniques and tubing techniques.12 The Oocytes also have a high cytoplasmic lipid content that
surface techniques include the EM grid,25 MDS,9,11,12 increases chilling sensitivity.51 They have less sub-mem-
Cryotop,26 Cryoloop,27,28 Hemi-straw,29 solid sur- branous actin microtubules,55 making their membranes
face,30 nylon mesh,31 Cryoleaf,32 direct cover vitrifi- less robust. Cryopreservation can cause cytoskeleton
cation,33 fiber plug,34 vitrification spatula,35 Cryo-E,36 disorganization and chromosome and DNA abnormali-
plastic blade,37 and Vitri-Inga.38 To the tubing tech- ties.56 The meiotic spindle, which has been formed at the
niques belong the plastic straw,8 OPS,39,40 closed- MII stage, is very sensitive to chilling and may be com-
pulled straw,41 Flexipet-denuding pipette,42 superfine promised as well.57 It does, however, tend to recover to
OPS,43 CryoTip,44 pipette tip,45 high-security vitrifi- some extent after thawing or warming and during in vitro
cation device,46 sealed pulled straw,47 Cryopette,48 culture; this recovery is faster following vitrification than
Rapid-i,49 and JY Straw (RC Chian, personal com- following slow freezing.57 Oocytes are also more suscep-
munication). Each of these two groups has its spe- tible to the damaging effects of reactive oxygen species.58
cific advantages. In the surface methods, the size of Many of these parameters change after fertilization,
the drop (0.1 µL) can be controlled, a high cooling making embryos less chilling sensitive and easier to
rate is achieved because these systems are open, and cryopreserve.55,59,60
high warming rates are achieved by direct exposure Vitrification requires the presence of high concentra-
to the warming solution. The tubing systems have tions of CPs. The presence of CP in the VS decreases the
the advantage of achieving high cooling rates in probability of intracellular crystallization, which is con-
closed systems, thus making them safer and easier to sidered to cause the most damage when very rapid cool-
handle. Decreasing the vitrified volume and increas- ing takes place, but the higher concentrations of the CPs
ing the cooling rate allows a moderate decrease in are toxic and cause osmotic injury to the oocytes even
CP concentration, so as to minimize their toxic and without cooling. It is therefore important to minimize
osmotic hazardous effects.47 Combining these three the damage caused to cells by the osmotic stress and/or
factors can result in the following general equation chemical toxicity. No ideal CPs that meet the require-
for the probability of vitrification: ments of all different species and developmental embry-
onic stages have been found; vitrification studies should
Arav “equation”: therefore be preceded by osmotic and cytotoxic studies.
Different methods have been used to reduce this “solu-
Cooling/Warming rate tion effect”: (i) short time of exposure to CPs24,61; (ii) use
of low-toxicity CPs62 or mixtures of them63; (iii) addition
× Viscosity of nonpermeating CPs24; (iv) reduction of the CP concen-
Probability of vitrification =
Volume tration62; and (v) exposure at low temperatures.62 Among

72
 vitrification at minimum volume
these methods, the use of nonpermeating CPs is very use- 1. Increasing the cooling rate (CR) will increase the
ful, either because the shrinkage of the oocyte and con- probability of vitrification; however, it will also
sequently the amount of water inside the cell that may increase the probability of fractures.
crystallize during rapid cooling and warming is lower,62 2. Increasing the viscosity (μ) will also increase
or because of the reduction of the amount of the CP the probability of vitrification because the TG
that penetrates the cell, thus reducing the possible toxic will increase,19 thus increasing the probability of
effects.64 In addition, the carbohydrates used as nonper- fractures.
meating CPs have a stabilizing effect on membranes.65 In 3. The only parameter that will increase the probabil-
a study reported by ourselves,66 trehalose was less harm- ity of vitrification, and at the same time decrease
ful than sucrose. Determination of the Boyle–Van’t Hoff the probability of fractures, is to reduce the volume
relationship for both sucrose and trehalose produced the (V) to the value of the MDS.
same regression line, so it is possible that this beneficial
effect could be a consequence of its interaction with the The reason for the increasing probability of fractures in
membrane polar lipid groups by the trehalose.65 We also high concentrations of VS is thought to be related to the
showed that only 10 minutes of exposure are required for TG. We know that fractures can form only at temperatures
equilibration in propylene glycol and dimethyl sulfoxide below that at which the liquid turns into glass (the TG) and
(DMSO) solutions or mixtures of them,66 as the mem- above the LN temperature (−196°C). We also know that a
brane is very permeable to both of them. The results of solution with higher CP concentrations will have a higher
the vitrification provide evidence that propylene glycol TG. Therefore, if the temperature gradient increases, as in
can be used successfully. Indeed, the in vitro fertiliza- the case of higher TG, then the probability of fracturing
tion (IVF) rate of the bovine oocytes vitrified in a solu- will also increase. Finally, the results of the vitrification of
tion containing 40% (w/v) propylene glycol was 37%, bovine oocytes at the MII or germinal vesicle (GV) stage,
and is not different from the results obtained using a with a concentration of 75% v/v of VS/phosphate-buffered
slow-freezing protocol.67 The viability of vitrified mouse saline, have been reported.71 We achieved 72% and 38%
embryos was successfully increased by reducing the con- cleavage and blastocyst formation rates, respectively, for
centration of the CP.68 However, concentrated solutions the vitrified MII oocytes, and 27% and 14% cleavage and
of permeating CPs are required for successful cryopreser- blastocyst formation rates, respectively, for oocytes vit-
vation of oocytes when rapid cooling and warming rates rified at the GV stage. We conclude that the new vitrifi-
are used. In earlier reports on immature pig oocytes, we cation procedure, which features small volumes, direct
showed that when lower concentrations of CPs are used, contact with supercooled LN and low concentrations of
despite apparent vitrification, membrane destruction was VS, reduces chilling injury and provides a high probability
unavoidable.69 In 1990, M. Kasai was the first to describe of vitrification in the absence of glass fractures.
the use of EG for mouse embryo vitrification.70 Today, the
most frequent solutions for oocytes vitrification are based WHY VITRIFICATION WITH OPEN SYSTEMS
on a mixture of DMSO and EG.13,44 WORKS BETTER FOR OOCYTES
Open versus Closed Systems
Small Volumes Can Resolve Many Problems that Most of the methods that work very well use direct expo-
Occur During Vitrification sure to LN, which introduces a potential risk of contami-
Three major problems are associated with vitrification: nation and cross-contamination72; in fact, contamination
(1)  crystallization (during cooling); (2) devitrification of bovine embryos by viral pathogens during storage in
(crystallization during storage or during warming); and LN has already been reported.72,73 Most likely, volume is
(3) fractures of the glassy solution that cause devitrification the important factor for successful vitrification in open
due to the release of energy by the fracture. Surprisingly, systems, as it was demonstrated that open systems give
at 1 µL, fractures appeared only when the concentration better results than closed systems, and these were exclu-
of the VS was high (100% VS = 38% EG, 0.5 M trehalose sively for surface systems.74–76 If we analyze the differences
and 4% bovine serum albumin in tissue culture medium between the two systems, we can see that the warming
[TCM]), but not at lower concentrations.12 This means that rate is not the reason for these differences, as it is the same
the probability of fractures increases with the increasing in both systems (we open the container in LN and plunge
of the TG or the viscosity of the VS. At low concentration it directly into a warm dilution medium in a closed sys-
of VS (50% VS), fractures were observed only at very high tem as well). The VS is also not very much different for
cooling rates. We suggest here a simple explanation of this both systems, and the cooling rate in both systems is sim-
phenomenon, based on the following equations: ilar (actually, in small tubes like Cryopette, the cooling
rate is very high). Therefore, the only factor that differs
Probability of fracturing = CR × μ × V between the two systems is the volume of the drop; while
in the open system the drop volume is reduced to a mini-
Since probability of vitrification is = CR × μ × 1/V mum (in the range of 0.1 µL or less), the volume in the

73
 vitrification in assisted reproduction
closed system is in the range of 0.5 µL or more in order 3. Tammann G. Ueber die abhangkeit der Kernr, welche
to avoid rehydration and desiccation damage during the sich in verschiedenen flussigkeiten bilden, von der
introduction to the container and heat sealing. temperature. Z Phys Chem 1898;25:441–79.
4. Mousson A. Einige Tatsachen betreffend das
CLAir SYSTEM Schmelzen und Gefrieren des Wassers, Annalen Der
CLAir is an automatic bench-top device for producing Physik Und Chemie 1858;181(10):161–74.
sterile liquid air. The device employs LN and produces 5. Luyet BJ, Hoddap A. Revival of frog’s spermatozoa
clean liquid air, which is supplied into a specially designed vitrified in liquid air. In Proceedings of the Meeting of
sterile cup. Liquid air is at the same temperature range of Society for Experimental Biology, 1938, p. 433–4.
LN (between −190°C and −196°C) and thus can be used 6. Arav A, Patrizio P, Pabon D, Cobo A, De Los Santos
for cryopreservation. CLAir comes in two sizes (CLAir MJ, Barak Y. “Vintage” in sperm cryopreserva-
and CLAir XL, FertileSafe, Israel), which are able to pro- tion: Droplets in liquefied air. Reproductive Biomed
duce 70 mL and 350 mL of clean liquid air every 10 min- 2014;28(S1):S5 (Online).
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higher than LN (−190°C) due to the presence of 20% liq- zoa after vitrification and dehydration at low temper-
uid oxygen with a temperature of −180°C. However, the atures. Nature 1949;164:666.
cooling rate is very similar in LN or clean liquid air. We 8. Rall WF, Fahy GM. Ice-free cryopreservation of
have vitrified bovine GV and MII oocytes and human mouse embryos at −196 degrees C by vitrification.
oocytes by direct immersion into sterile Styrofoam cups Nature 1985;313(6003):573–5.
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 vitrification in assisted reproduction
49. Larman MG, Gardner DK. Vitrifying mouse oocytes 63. Massip A, Vanderzwalmen P, Ectors F. Recent progress
and embryos with super-cooled air. Hum Reprod in cryopreservation of cattle embryos. Theriogenology
2010;25:i265. 1987;27:69–79.
50. Testart J, Lassalle B, Belaisch–Allart J. High preg- 64. Széll A, Shelton JN. Osmotic and cryoprotective
nancy rate after early human embryo freezing. Fertil effects of glycerol-sucrose solutions on day-3 mouse
Steril 1986;46:268–72. embryos. J Reprod Fertil 1987;80(1):309–16.
51. Ruffing NA, Steponkus PL, Pitt RE, Parks JE. 65. Crowe JH, Crowe LM, Mouradian R. Stabilization
Osmometric behavior, hydraulic conductivity, and of biological membranes at low water activities.
incidence of intracellular ice formation in bovine Cryobiology 1983;20(3):346–56.
oocytes at different developmental stages. Cryobiology 66. Arav A, Shehu D, Mattioli M. Osmotic and cytotoxic
1993;30:562–80. study of vitrification of immature bovine oocyte.
52. Carroll J, Depypere H, Matthews CD. Freeze–thaw- J Reprod Fertil 1993;99:353–8.
induced changes of the zona pellucida explains 67. Schellander K, Brackett BG, Fuhrer F, Schleger W.
decreased rates of fertilization in frozen–thawed In vitro fertilization of frozen–thawed cattle oocytes.
mouse oocytes. J Reprod Fertil 1990;90:547–53. In Proceedings of the 11th Congress on Animal
53. Mavrides A, Morroll D. Bypassing the effect of zona Reproduction and Artificial Insemination, 1988.
pellucida changes on embryo formation following p. 26–30.
cryopreservation of bovine oocytes. Eur J Obstet 68. Rall WF, Meyer TK. Zona fracture damage and its
Gynecol Reprod Biol 2005;118:66–70. avoidance during the cryopreservation of mamma-
54. Bogliolo L, Ledda S, Innocenzi P et al. Raman micro- lian embryos. Theriogenology 1989;31:683–92.
spectroscopy as a non-invasive tool to assess the 69. Rubinsky B, Arav A, DeVries AL. Cryopreservation of
vitrification-induced changes of ovine oocyte zona oocyte using directional cooling and antifreeze pro-
pellucida. Cryobiology 2012;64(3):267–72. teins. Cryo Lett 1991;12:93–106.
55. Gook DA, Osborn SM, Johnston WIH. 70. Kasai M, Komi JH, Takakamo A, Tsudera H, Sakurai T,
Cryopreservation of mouse and human oocytes using Machida T. A simple method for mouse embryo cryo-
1,2-propanediol and the configuration of the meiotic preservation in a low toxicity vitrification solution,
spindle. Hum Reprod 1993;8:1101–9. without appreciable loss of viability. J Reprod Fertil
56. Luvoni GC. Current progress on assisted reproduc- 1990;89:90–7.
tion in dogs and cats: In vitro embryo production. 71. Arav A, Pearl M, Zeron Y. Does lipid profile explain
Reprod Nutr Dev 2000;40:505–12. chilling sensitivity and membrane lipid phase
57. Ciotti PM, Porcu E, Notarangelo L, Magrini O, transition of spermatozoa and oocytes? Cryo Lett
Bazzocchi A, Venturoli S. Meiotic spindle recovery is 2000;21:179–86.
faster in vitrification of human oocytes compared to 72. Bielanski A, Bergeron H, Lau PC, Devenish J.
slow freezing. Fertil Steril 2009;91:2399–407. Microbial contamination of embryos and semen dur-
58. Gupta MK, Uhm SJ, Lee HT. Effect of vitrification ing long term banking in liquid nitrogen. Cryobiology
and betamercaptoethanol on reactive oxygen species 2003;46(2):146–52.
activity and in vitro development of oocytes vitri- 73. Bielanski A, Nadin-Davis S, Sapp T, Lutze-Wallace C.
fied before or after in vitro fertilization. Fertil Steril Viral contamination of embryos cryopreserved in liq-
2010;93:2602–7. uid nitrogen. Cryobiology 2000;40(2):110–6.
59. Fabbri R, Porcu E, Marsella T et al. Technical aspects 74. Paffoni A, Guarneri C, Ferrari S et al. Effects of two
of oocyte cryopreservation. Mol Cell Endocrinol vitrification protocols on the developmental poten-
2000;169:39–42. tial of human mature oocytes. Reprod Biomed Online
60. Ghetler Y, Yavin S, Shalgi R, Arav A. The effect of 2011;22(3):292–8.
chilling on membrane lipid phase transition in human 75. Bonetti A, Cervi M, Tomei F, Marchini M, Ortolani
oocytes and zygotes. Hum Reprod 2005;20:3385–9. F, Manno M. Ultrastructural evaluation of human
61. Arav A, Gianaroli L, Suriano P. Titration of vitrifi- metaphase II oocytes after vitrification: Closed versus
cation solution in mouse embryo cryopreservation. open devices. Fertil Steril. 2011;95(3):928–35.
Crybiology 1988;6:567. 76. Papatheodorou A, Vanderzwalmen P, Panagiotidis Y
62. Rall WF, Wood MJ, Kirby C, Whittingham DG. et  al. Open versus closed oocyte vitrification sys-
Development of mouse embryos cryopreserved by tem: A prospective randomized sibling-oocyte study.
vitrification. J Reprod Fertil 1987;80(2):499–504. Reprod Biomed Online 2013;26(6):595–602.

76
9 Vitrification of oocytes: General considerations and the use
of the Cryotec method
Masashige Kuwayama

WHY DO WE NEED TO CRYOPRESERVE of humankind to alleviate their handicapped situation in

HUMAN OOCYTES? the field of reproduction.
In most papers dealing with this topic, a long list of sim- Unfortunately, although not at all according to their
ple and obvious reasons to answer this question can be original intentions, modern reproductive technologies in
found. These answers will also be listed below. However, the human have made the situation even worse. With the
let this review start with a more general and rarely men- application of intracytoplasmic sperm injection (ICSI),
tioned argument: the handicapped situation of women one gamete from both genders may be enough to pro-
from the standpoint of reproduction. duce an offspring. Compared to the enormous number of
Although gender-based discrimination is less and sperm available, the ten-, sometimes twenty-fold artificial
less acceptable in most human societies, nature still pre- increase in number of available oocytes (as the result of an
serves the right to make a seemingly unfair but strong expensive, painful, and sometimes risky medical inter-
distinction between males and females in certain areas vention) does not fully compensate for this imbalance. It
including—obviously—reproduction. In most mamma- is even more embarrassing that with ICSI, incompletely
lian species, females bear most of the weight of reproduc- capacitated or matured, handicapped, immotile, or dying
tion, including discomfort related to reproductive cycles spermatozoa can still be successfully used for fertiliza-
and pregnancy, pain of labor, and nursing of babies. In tion, while the slightest deviation in morphology, func-
humans, even with the best intentions, male partners tion of oocytes (including those caused by the supportive
cannot share most of these sacrifices, and most women medical intervention itself), or the smallest inaccuracy in
accept them as inevitable parts of their full life, which the stage of maturation may seriously compromise their
are at least partially compensated by the most intimate further developmental competence.
relationship with their babies. However, there are addi- Finally, when we consider storage possibilities of the
tional, even more frustrating differences between males male and female gametes of mammals, the difference is
and females regarding the possibility of distributing and even more frustrating. In many domestic species, large
preserving genetic material. A healthy male can produce commercial networks are dealing with the collection,
many millions of sperm cells every day, while the number deep-freezing, and distribution of the sperm of valuable
of oocytes is highly restricted in many mammals, includ- animals, by using highly standardized procedures, and
ing humans, to one or two per month. Moreover, males with an efficiency that almost completely eliminates the
normally preserve their reproductive ability for their need for natural mating or artificial insemination with
whole life, while the time for women is rather limited. fresh semen. The establishment of the “cryoprotectant-
Theoretically, they may become pregnant and have babies free vitrification” method1 for spermatozoa also proves
before their menopause—which also means almost halv- the extreme tolerance of male germ cells toward cryoinju-
ing the available time compared to males—but in prac- ries, although the term itself may raise some concerns. In
tice, even this period is drastically shortened by the fact the mouse, a technology has also been developed enabling
that the quality of oocytes decreases sharply after the age sperm transportation in a sealed envelope by ordinary
of 35, restricting the real freedom to reproduce without mail. On the other hand, the recovery, laboratory han-
concern and fear to approximately 15 years, coinciding dling, and especially cryopreservation of mammalian
exactly with the time that is the most critical to establish- oocytes from live animals is still regarded as a challenge
ing a professional career for a lifetime. and restricted (with a few exceptions) to the experimen-
We may be referring to the inevitable order or laws of tal field. The situation is no better in humans: sperm can
nature, but aging and loss of teeth or hair may also be be easily collected, frozen, stored, and utilized in small
referred to as such, not to mention blindness, deafness, aliquots, creating the commercial distribution of this
or other serious handicaps. We make considerable and supposedly valuable male genetic “stuff” as a prosperous
fully justified efforts to eliminate these unfair differ- business in some countries. In contrast, the storage and
ences created by life and nature and we institute legisla- use of the female gamete is seriously restricted by the
tive and financial help to ensure equal opportunities in above-mentioned biological, technical, and psychological
many fields. We probably should focus more on this half problems related to collection, as well as by legal measures

77
 vitrification in assisted reproduction
restricting experimental use and/or donations in many cell cultures can be cryopreserved with high efficiency
countries, and by the poor and inefficient technologies and without any sophisticated approach by using sim-
available for their cryopreservation. Until recently, due to ple media, a refrigerator, and a deep freezer or liquid
the cumulative effect of these factors, the efficiency of the nitrogen. For the even smaller bacteria and viruses, we
whole procedure was so low that practically every baby meet the frustrating evidence every day: they are pres-
born after oocyte cryopreservation deserved a scientific ent in almost every liquid nitrogen tank, and preserve
publication. their viability without any protection and in spite of
Establishment and widespread application of an effi- our best intentions (although apart from the size, some
cient and safe cryopreservation method for oocytes other factors—for example, their simple structure—may
would not eliminate differences in reproductive flexibility also play roles in this resistance). In reproductive biol-
between females and males, but may mean a solution to ogy, we just referred to the above-mentioned differences
many specific problems and eventually reverse the actual between cryotolerance of spermatozoa and oocytes that
trend of this unacceptable artificial widening of the gap can be at least partially attributed to the differences in
between the two genders. Accordingly, this area deserves volume. Quite controversially, the cumulative mass of
special attention, and should be regarded as more than cells decreases exponentially during the first week of
just a subject of scientific ambition of a few, accidentally embryo development, and at the expanded blastocyst
selected scientists, making this an area that is benignly stage, it may become as low as 1/10–1/100 of that of the
and respectfully disregarded by the vast majority of oocyte, with obvious similar decreases of the water con-
reproductive specialists. tent. Although the solution accumulated in the blasto-
The need for change in the general attitude toward coele may mean a potential source of damage either by
oocyte cryopreservation is even more justified by the ice crystal formation or through the accumulation and
recent rapid advancement in technology now outlining slow dilution of toxic cryoprotectants, 8–10 these mecha-
the perspective of a real breakthrough, and offering a def- nisms obviously cause more harm when they occur
inite solution right now in many important fields, includ- intracellularly in the oocyte.
ing the following: Apart from the size, the shape of the oocyte is also
most unfortunate. The almost perfect sphere slows down
1. Malignant diseases where systemic anticancer the formation of an equal distribution of any substance,
treatment is required 2 including permeable cryoprotectants coming from out-
2. Surgical procedures resulting in loss of ovarian side or released from the oocyte. Accordingly, for a rela-
function3 tively long period of time, a continuous concentration
3. Treatment of patients with polycystic ovarian gradient from the periphery to the center or vice versa
syndrome4,5 exists, resulting in toxic damage in one part while provid-
4. Patients with ovary hyperstimulation syndrome3 ing less than optimal protection in the other. From this
5. Poor responders to ovarian stimulation3 point of view, the change in shape caused by the osmotic
6. Patients at risk of ovarian function loss through effect at equilibration may offer some kind of benefit, but
premature menopause3 it may also contribute to the damage to the cytoskeleton
7. In cases of male factor infertility or problems asso- (see later in this chapter).
ciated with difficulty of sperm collection, inad- The third major factor is the lowest possible cell num-
equate seminal samples, or nonviable spermatozoa ber. From this point of view, the oocyte resembles a gam-
at the time of oocyte retrieval3 bler who puts all of their money on the very first bet: all or
8. To overcome ethical concerns and legal restric- nothing. Multicellular embryos can survive and compen-
tions in several countries associated with embryo sate for as much as 50% loss of their cells (and supposedly
cryopreservation6 also some level of injury in the remaining ones), as dem-
9. Cryobanking oocytes for young women who wish onstrated by biopsies, bisection of embryos, or just the
to delay motherhood for various reasons (career, less-than-optimal culture conditions, quite apart from
lack of appropriate partner, etc.) the cryopreservation experience. The oocyte has only one
10. Cryobanking oocytes for egg donation programs or chance, and there is no backup to regenerate from a seri-
for research purposes7 ous injury. We have to use an extremely careful approach
to get out of the game as winners!
WHY IS CRYOPRESERVATION OF OOCYTES Unfortunately, apart from the factors listed above,
DIFFICULT? there are still many other factors that contribute to the
Some of the reasons for this, including the size, shape, sensitivity of oocytes to cryoinjuries. Chilling injury,
and cell number, are quite obvious. which occurs at relatively high temperatures and induces
It is well known that oocytes are the largest cells irreversible damage of the cytoplasmic lipid droplets,
of the human body. In cryobiology, the size, or rather lipid-rich cell membranes, and microtubules, mostly
the mass, is a decisive factor. Suspensions of somatic affects the latter two structures in the human oocyte

78
 vitrification of oocytes
(in  contrast, for example, to pigs), as in humans, cyto- justified by practice, although it should be confessed that
plasmic lipid droplets are less abundant. On the other the sequence of events was (as usual) inverted: the empiri-
hand, the membranes are extremely sensitive and rap- cally established methods were retrospectively supported
idly undergo a transition from the liquid state to the gel by the subsequent detailed theoretical analyses of events.
state, an irreversible process that is detrimental for future Firstly, we need a method that minimizes chilling
development. For unknown reasons, just a step ahead, injury. So far, in mammalian embryos and oocytes, two
after fertilization, the membranes of zygotes are much approaches have been successfully applied for this pur-
less sensitive to this type of injury.11 pose: the removal of the lipid droplets (by high-speed
The depolymerization of microtubules, misalignment centrifugation and micromanipulation, although the
of the chromosomes, and the possible increased risks latter step is not required with the use of some recent
of aneuploidy are frequently emphasized and have wide techniques)17 and by radically increasing the cooling and
experimental backgrounds,12 although in the human, warming rate to minimize the duration of exposure to
comparative examinations may not entirely confirm the dangerous temperatures. As human oocytes contain rela-
seriousness of this problem,13 and the supposed beneficial tively low amounts of lipids, centrifugation does not sig-
effects of some agents (cytoskeleton relaxants or stabiliz- nificantly improve survival chances. On the other hand,
ers) are not fully proven. Similar to somatic cell nuclear all forms of traditional slow-rate freezing are obviously
transfer, spindle reorganization may occur surprisingly less appropriate for the purposes of oocyte cryopreserva-
efficiently, and the number of chromosomal abnormali- tion than high-rate cooling vitrification strategies.
ties in children born after oocyte vitrification does not The large cell mass and spherical shape of the oocyte
seem to show a significant increase. necessitates the use of highly permeable cryoprotectants
A strange and not completely understood phenom- with low toxicity. As in many areas of vitrification in
enon is the change in cryosensitivity of oocytes during mammalian embryology, ethylene glycol is the candidate
the maturation process. Although there is only a mini- of choice for this purpose. According to earlier investi-
mal difference between their size and shape, immature gations in rabbits,18 the permeability of ethylene glycol is
oocytes are usually more sensitive to cryopreservation facilitated by dimethyl sulfoxide (DMSO). Further stud-
than mature (Meiosis II [MII] phase) oocytes.11,15 The ies have also demonstrated that DMSO may have a ben-
contrary might be supposed based on the known sensi- eficial effect on spindle polymerization, and consequently
tivity of the meiotic spindle to chilling. More research is a protective effect on oocyte vitrification.3 Although
needed to understand the reasons for this difference, and various proportions of DMSO and ethylene glycol were
the alteration of the sensitivity of membranes may be one extensively tested for the vitrification of bovine oocytes
of the possible explanations. and embryos, the best results were always achieved with
The osmotic shock at equilibration may result in a 1:1 mixture (G. Vajta, unpublished data). To facilitate
shrinking and misshaping of the oocytes, supposedly dehydration, thus decreasing the chances of intracellular
damaging the cytoskeleton. However, the effect of other ice formation, the addition of nonpermeable cryoprotec-
agents (e.g., pronase digestion of the zona pellucida) tants is also required. Various substances, including poly-
induces much more serious deformation, followed by mers with low toxicity, were suggested for the purpose;
surprisingly rapid recovery and maintenance of devel- however, the traditionally used sugars (i.e., sucrose or tre-
opmental competence. On the other hand, the osmotic halose) seem to be more appropriate. Curiously, although
shock that may occur during dilution may result in exten- trehalose has been reported many times to be superior, in
sive swelling, rupture of the membrane, lysis, and imme- the past few years, it has gradually disappeared from the
diate death of the oocytes. list of frequently used cryoprotectants.
Hardening of the zona pellucida, attributed by some In the past few years, two basically different strategies
authors to premature cortical granule release, may cause of equilibration before cooling have been applied.19 It was
decreased rates of fertilization.13 proposed that dehydration may even be more important
Fracture is a common consequence of all cryopreser- than cryoprotectant concentration for the prevention of
vation procedures16 and does not seem to occur more fre- ice crystal formation, suggesting extremely short equili-
quently in oocytes than in embryos. However, while the bration times both for the diluted and concentrated cryo-
consequences for zona fracture may be similar for both, protectant solutions.
embryos may survive some level of cell membrane dam- This strategy was successfully applied subsequently by
age, while for the oocyte, any injury at this level is evi- many others for domestic animal oocytes and embryos.
dently fatal. However, more recently, another approach has received
more attention and seems to be more efficient for mamma-
WHAT IS THE BEST APPROACH? lian oocytes: an extended equilibration in a rather diluted
Based on the points listed above, the principles of a first cryoprotectant solution, followed by a short, but
successful cryopreservation strategy can be outlined. slightly prolonged incubation in a second, relatively con-
Although infrequent in biological study, theory is mostly centrated vitrification solution containing, in addition, a

79
 vitrification in assisted reproduction
nonpermeable cryoprotectant.20–22 Although the time of eliminated by the application of the ultra-rapid open vit-
the exposure is significantly increased, the cumulative rification systems.
toxic effect (as a result of the lower concentration) may be Finally, the problem of zona hardening and subsequent
the same or even lower, and the prolonged equilibration low levels of fertilization has been eliminated entirely
may ensure proper penetration of cryoprotectant, provid- with the discovery and subsequent widespread applica-
ing appropriate protection to the entire oocyte. The other tion of ICSI. Although not included in the original goals,
way to minimize the toxic and osmotic effects of cryopro- the application of ICSI after cryopreservation has contrib-
tectants is to decrease the required concentration while uted much to increases of efficiency, and opened the gate
maintaining the ice-free solidification pattern. Currently, to widespread application of oocyte cryopreservation.
the only practical way to achieve this goal is with an
extreme increase in cooling rates. Among the various THE MOST RECENTLY DEVELOPED CLINICAL
tools applied for this purpose, electron microscopic grids, VITRIFICATION PROTOCOL: THE CRYOTEC
Cryoloop, and Cryotop seem to be the most appropriate METHOD
choices, although recently, similar results were achieved The first consistently successful vitrification protocol of
with the open pulled straw technique.23 Either directly by human oocytes was reported by Kuwayama et al., 24 incor-
the higher rate of cooling or indirectly by the decreased porating the Cryotop as the vitrification carrier. In his
toxic and osmotic effect, the Cryotop and Cryoloop vitri- experiments at the Kato Ladies Clinic (the largest in vitro
fication approaches with a mixture of relatively low con- fertilization [IVF] center in the world), almost 95% of
centrations of DMSO and ethylene glycol do not seem to oocytes survived vitrification and ICSI, and cleavage
cause serious anomalies in the spindle structure, and may rates did not differ from those of controls in our labora-
ensure relatively high developmental rates. tory. When blastocyst transfer was applied, 45% of vitri-
As mentioned, fracture damage is not specific to fied oocytes developed to healthy babies (Figures 9.1 and
oocyte cryopreservation, although the consequences may 9.2). Other groups began applying this Cryotop approach
be more detrimental. Fortunately, the open vitrification for oocyte vitrification with good success3: 89.2% sur-
systems have drastically reduced the occurrence of this vival rates have been reported after Cryotop vitrifica-
type of damage. Retrospectively, it may be supposed that tion of oocytes, as well as a 56.5% pregnancy rate (13 of
in a closed system, the extreme pressure changes caused 23 patients) with an average of 4.63 embryos transferred
by rapidly cooling or warming air bubbles induce dis- to patients. This Colombian group also achieved the first
locations in the partially solidified solution, and with a baby born after oocyte vitrification in South America
scissor-like effect cut the zona pellucida or the cell mem- (E. Lucena, unpublished data). From Mexico, 401/445
branes. In the open systems, such mechanical forces are (90.1%) survival and 34.1% pregnancy rates were reported
almost completely avoided. The extremely small volume after Cryotop vitrification.25 In Valencia, Spain,26 a total
of solutions used also minimizes the chance of frac- of 225 MII oocytes were vitrified, of which 217 (96.5%)
tures. Accordingly, this type of damage is almost entirely survived cryopreservation and 165 (76.0%) were normally

Before vitrification Immediately after thawing 2 hours after culture

PN stage (day 1) Four-cell stage (day 2) Blastocyst stage (day 5)

Figure 9.1  Inverted microscopic pictures of human oocytes before and after vitrification, intracytoplasmic sperm
injection and in vitro culture (on days 1, 2, and 5). PN = pronucleus.

80
 vitrification of oocytes
100 Here, we provide some technical details required for
90 96 successful cryopreservation of MII-phase human oocytes.
94
90 92 Day 2 ET The commercially available vitrification kit (Repro-
86
80 Day 5 ET Support Medical Research Centre, Tokyo, Japan) contains
70 73 the Cryotec vitrification container, a filmstrip attached
to a plastic handle also equipped with a cap to cover the
60
filmstrip for safe handling and storage (Figure  9.3), and
50 all media required for washing, equilibration, vitrifica-
50
40
45 45 tion, warming, and dilution. These solutions are based
39 on minimal essential medium (MEM) supplemented
30 with hydroxypropyl cellulose and xanthan gum as serum
28
20 replacement, and containing ethylene glycol, DMSO, and
endotoxin-free trehalose as permeating and nonpermeat-
10
ing cryoprotectants. All media and manipulations should
0 be performed at 25–27°C, except for warming, in which
Surv 2PN 2cell BL Preg Deliv
the medium should be warmed to 37°C. A pulled, fire-
polished glass pipette with a 140–150-μm inner diam-
Figure 9.2 Results of oocyte cryopreservation per-
eter is suggested for all of the manipulations for oocytes
formed at Kato Ladies Clinic with Cryotop vitrifica-
vitrification.
tion. Columns refer to percentages of vitrified oocytes
Oocytes can be vitrified 1–6 hours after the ovum pick-
surviving vitrification (Surv), developing to the two
up, immediately after denudation. Oocytes are first placed
pronucleate embryo stage (2PN), proceeding to cleavage
on the surface of the equilibration solution that contains
(2cell), developing to the blastocyst stage (BL), result-
the permeating cryoprotectants. Oocytes immediately
ing in pregnancy (Preg), and resulting in the delivery
shrink to approximately 50% volume of their original size,
of healthy babies (Deliv). The total numbers of oocytes
and then start to recover. When oocyte volume has recov-
used for day 2 and day 5 embryo transfer were 86 and
ered to the original size, it is time to end this step. This
25, respectively. Finally, 18 and 11 embryos were trans-
normally takes 12–15 minutes for oocytes.
ferred on days 2 and 5, respectively (2.9 and 1 embryos
After the completion of the equilibration step, oocytes
per recipient).
are then placed in the bottom of vitrification solution,
which is a solution that has higher concentrations of cryo-
fertilized after ICSI, and this was not different from the protectants. Oocytes start to rise to the surface because of
controls. A total of 93.9% of zygotes underwent cleav- the different specific gravities between the equilibration
age on day 2, and the blastocyst per fertilized oocyte and vitrification solutions, and the purpose of this step is
rate (22.4%) did not differ from the control. Twenty-one to complete the exchange of the solutions from the equili-
embryo transfers were performed with embryos from vit- bration to the vitrification solution, which is completed
rified oocytes, resulting in 13 pregnancies (61.9% preg- automatically (Figure 9.4a).
nancy and 37.2% implantation rates). This is followed by oocyte loading onto the Cryotec
The Cryotop method has been in widespread use in (filmstrip), a carrier device set on the vitrification plate
more than 40 countries during the last decade, and a total (Figure 9.4b), and then directly plunged into liquid nitro-
of 70,000 healthy babies have been obtained from vitrified gen (Figure 9.4c). The Cryotec is then covered with the
oocytes, and all the while the protocol has been improved cap while still under liquid nitrogen to avoid mechanical
several times for better survival and easier handling to damage during transfer to the container and storage.
reduce the potential for human error by embryologists at At warming, the film part of the Cryotec should
all stages of oocytes and embryos. This completed non- be submerged quickly into the 37°C warming solu-
invasive clinical vitrification method has been called the tion in a warming plate (Figure 9.4d) to achieve the
Cryotec method. Now, the Cryotec method is being rap- required 42,000°C/min warming rate. After 10 seconds,
idly adopted in many countries and successfully applied, the oocytes are gently removed from the surface of the
so that the Cryotec method is routinely providing almost Cryotec by themselves, and start to float in the warming
100% survival for both human oocytes and embryos. solution. After 1 minute, the dilution should be continued

(a)
(b)

Figure 9.3  Cryotec vitrification container (a) without and (b) with cover cap.

81
 vitrification in assisted reproduction

(b)
(a)

(c) (d)

Figure 9.4 Cryotec vitrification protocol: (a) equilibration of equilibration solution/vitrification solution/

vitrification solution (ES/VS/VS), (b) loading of oocyte, (c) ultra-rapid cooling, and (d) ultra-rapid warming.

in dilution solution, washing solutions 1 and 2 for 3, 5, those achieved previously with conventional vitrification
and 1 minutes, respectively. Oocytes should be cultured and traditional freezing.
for an additional 2 hours before the ICSI. Morphologies
before and after vitrification, ICSI, and embryo culturing CONCLUSION
are shown in Figure 9.1. The high number of healthy babies born (more than
70,000 babies) in over 40 countries after Cryotop and
INITIAL OOCYTE VITRIFICATION RESULTS Cryotec vitrification worldwide over 10 years clearly
ACHIEVED WITH THE CRYOTEC METHOD proves that oocytes cryopreservation by this successful
Gandhi et  al.27 introduced the protocol details of vitrification approach is emerging as a very useful tool
the Cryotec method as one of the latest versions of in the human reproduction field across many applica-
Dr.  Kuwayama’s various novel vitrification protocols, tions. All data obtained from many different laboratories
and they introduced their clinical results in which they (including survival, fertilization, embryo development,
reported the same rates of fertilization (83.0% versus and pregnancy rates) suggest that oocytes vitrified with
80.9%), cleavage (96.5% versus 94.4%), pregnancy (56.3% this technology are highly viable, and their developmen-
versus 54.9%), implantation (28.0% versus 31.1%), and live tal competence is comparable with that of fresh oocytes.
births (45.0% versus 45.1%) between fresh and vitrified The increasing evidence proves that Cryotec vitrification
oocytes when using the Cryotec method. may offer solutions for women with various fertility prob-
Kagalwala et al.28 reported a direct comparison of the lems, and may participate in the compensation for the
Cryotop and the Cryotec methods: from 611 vitrified handicap of women from the standpoint of reproduction.
oocytes, survival rates, fertilization rates, and cleavage
rates of the Cryotec and Cryotop methods were 97.2% and REFERENCES
95.1%, 90.7% and 86.2%, and 96.9% and 91.9%, respec- 1. Isachenko V, Isachenko E, Montag M, Zaeva V,
tively. After embryo transfer (ET), pregnancy rates of the Krivokharchenko I, Nawroth F, Dessole S, Katkov
Cryotec and Cryotop methods were 54.8% and 40.6%. II, van der Ven H. Clean technique for cryoprotec-
Furthermore, when oocytes vitrified by the Cryotop tant-free vitrification of human spermatozoa. Reprod
method were warmed by the Cryotec method, higher Biomed Online 2005;10:350–4.
survival (92.5% versus 87.8%) and higher pregnancy rates 2. Meirow D. Reproduction post-chemotherapy
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Saito H, Ishida GM, Kaneko T et al. Application of vitri- Walker D, Tummon IS, Hammit DG et  al. Vitrification
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10 Safety of vitrification and cryostorage and optimization
of cryopreservation protocols
Lodovico Parmegiani and Marco Filicori

INTRODUCTION Single-straw closed carriers

As vitrification is a cryopreservation technique increas- As an alternative to straw-in-straw, other types of closed
ingly applied in clinical practice for reproductive cells systems, such as CryoTip20 or Cryopette,21 allow faster
and tissue, the main focus of this chapter is to analyze rates of cooling. These closed carriers consist of a very
the risks related to this technique and to propose pos- thin straw specifically designed to load cells with the
sible solutions. Since there may be concerns regarding the minimum volume of cryoprotectant solution, and to be
safety of vitrification procedures and cryostorage, due hermetically sealed; in this way, direct contact between
to the contact of cell/tissue/carrier with liquid nitrogen cells and LN2 is avoided. Unfortunately, because of their
(LN2), these aspects are comprehensively treated in the design, these systems would not avoid the transmission
“Safety of vitrification and cryostorage” section. of microorganisms into the culture medium during the
Furthermore, since today different cryopreserva- warming procedure, arising from accidental contamina-
tion protocols with various cryoprotectant formulations tion of the LN2; this is due to the direct contact between
can be used both for vitrification and slow freezing, the the LN2 and the external surface of the carrier.22,23 In
“Optimization of cryopreservation protocols” section practice, the contamination of cells occurs at 37°C, when
of this chapter will describe the possibility of using a any cryopreserved microorganism found in the LN2
single “universal warming protocol” for any frozen cell, reactivates after thawing in the culture medium. Even
irrespective of the freezing protocol and cryoprotectant though in vitro fertilization (IVF) culture media are sup-
cocktail used at freezing. plemented with antibiotics, some micro-organisms may
resist the antibiotic and infect the culture. In these cir-
SAFETY OF VITRIFICATION AND CRYOSTORAGE cumstances, the bacterial or viral particles released into
Vitrification Carriers the culture medium may attach themselves to the oocyte/
Open carriers embryo zona pellucida, if this is cracked.14,24 Another pro-
During vitrification, cells and tissue need to be cooled and cedure to decontaminate the straw is to quickly wipe the
warmed at an extremely rapid rate.1 This can be achieved carriers with 70% ethanol for disinfection at warming.20
by using specific “open carriers” such as the open pulled However, the deactivation of all microorganisms can be
straw,2 Cryoloop, 3 hemi-straw,4 Cryotop,5 Cryoleaf,6 obtained only by a 5-­minute contact between ethanol and
Cryolock,7 Vitri-inga,8 etc.; these “open carriers” are gen- carrier25; this prolonged contact time can damage human
erally preferred for oocytes.9–13 However, these systems cells, which remain inside the carrier in the warmed vit-
cannot avoid the hypothetical risk of microorganism rification solution, rich in potentially toxic cryoprotec-
contamination during the vitrification procedure, if the tants.26 For these reasons, sterilization of the LN2 before
LN2 is accidentally contaminated.14,15 The sterilization of the vitrification procedure is recommended for the safe
LN2 before vitrification procedures is thus recommended clinical application of these systems.
to ensure the safety of clinical application.
Nitrogen vapors/supercooled air vitrification
Straw-in-straw closed carriers It has been demonstrated that it is possible to vitrify
Another option for vitrification is the closed carrier based reproductive cells by exposure to nitrogen vapors.27,28
on the “straw-in-straw” mode (high-security vitrifica- Other authors have proposed inserting the carrier con-
tion), designed to insulate the inner carrier containing taining the cells into the supercooled air of a straw for
the cells/tissue against LN2 during vitrification by using instantaneous vitrification, and then to seal the open
a sealed external straw.16,17 This system avoids direct end of the straw (post-sealing method) to avoid direct
contact between specimens and LN2 and also any hypo- contact with LN2.28 It is important to point out that the
thetical risk of contamination and leads to good results supercooled air inside the straw is basically composed
with zygotes, cleaved embryos, blastocyst and ovarian of nitrogen vapor owing to nitrogen’s rapid evaporation
tissue.17–19 However, the “straw-in-straw” system causes a and molecular weight.29 For this reason, any microorgan-
reduction in the rate of cooling, and is not routinely used ism accidentally present in the LN2 can also pass to the
in the clinical cryopreservation of oocytes. nitrogen vapor phase,30 and thus lead to the hypothetical

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 vitrification in assisted reproduction
contamination of the oocyte/embryo and the inner car-
rier. This means that contamination issues associated
with LN2 cannot be entirely avoided by using supercooled
air, and the sterilization of LN2 before evaporation is also
recommended with this method.29

Solid surface vitrification

With these systems, vitrification is performed on the solid
surface of a chilled metal block partially submerged in LN2
(CryoLogic Vitrification Method—http://www.­cryologic.
com/cvm.htm). Following vitrification, specimens are
inserted into a sleeve that is then fully heat sealed. Since
the air around the block is composed of nitrogen vapor,31
these systems require LN2 sterilization as well.

Cryostorage
Currently, human cells and tissues are mainly cryostored
in LN2 or in nitrogen vapor (NV); such cryostorage is
potentially hazardous because many pathogens can sur-
vive at the low temperature of LN2/NV,14,15,30,32–35 and they
may contaminate the frozen cells or their carriers/con-
tainer surface inside the cryobanks.14,15,24,30,31,34–36
To date, there have been no reported cases of dis-
ease transmission by transferred cryopreserved human Figure 10.1  Hermetical goblet for the cryostorage of vit-
embryos35,37–39; however, we have no specific studies rification carriers. A hermetical goblet made using some
regarding possible negative effects of LN2/NV infectious disposables that are commonly marketed for human
agent contamination on the final outcome of IVF frozen cell cryostorage: Cryoflex, a polyethylene tube specifi-
cycles, although it is generally known that some of these cally designed for cryostorage in liquid nitrogen (Nunc,
microorganisms negatively affect gametes and embryonic Roskilde, Denmark); a plastic Visotube for cryostorage
development at warming.15,40–42 In addition, vitrification is (Cryo Bio System, L’Aigle, France); plus a stainless steel
increasingly used for human cells, and this cryo-­procedure weight. The hermetical goblet must be prepared in a
appears to be riskier than slow freezing due to the direct sterile environment (e.g., an IVF flow-hood).
contact between cells/tissue and LN2 required for “open
systems.” The hypothetical risk of culture contamination
at warming cannot be excluded even when using some absorbs large quantities of heat, making it very effective
“closed vitrification systems.”22,29,31 as a coolant. Although LN2 should have a low microbial
Some precautions may be routinely used in IVF labo- content (in nature, very few microorganisms are able to
ratories to minimize the risk of cross-contamination survive at temperatures around −195°C), nevertheless
during cryopreservation. For example, cryostorage in the need to guarantee the absolute sterility of nitrogen is
hermetically sealed containers and the use of a second- at present still felt in view of its critical applications in
ary sleeve (straw-in-straw) is recommended for human cryobiology. Many methods are used for obtaining clean
specimens for both vitrification and slow freezing LN2 at the time of production, and companies that sup-
(Figure 10.1).2,31,35,43,44 Cryostorage contamination may be ply LN2 can certify the level of purity of the LN2 sup-
avoided by storing the vitrified embryos in LN2 vapor45,46: plied. Owing to the particular composition of this liquid,
Cobo et  al. have demonstrated that vitrified human however, it is not possible to seal the containers used to
oocytes can also be safely cryostored in LN2 vapor.47 transport it, and consequently it is not possible to guar-
Periodic cleaning and refilling of cryo-dewars with antee or certify the sterility of the LN2 before it is actually
sterile LN2 (SLN2) are additional precautions for mini- used. Very often, between the time of leaving the manu-
mizing the potential risk of cross-contamination; today, facturer and the time of reaching the end user, the LN2
certified SLN2 can be easily obtained through ultraviolet passes through several hands, exposing it to risks of con-
(UV) irradiation (Figure 10.2).43,48 tamination. Furthermore, incorrect sanitizing or safety
procedures during the handling of potentially infected
Sterilization of LN2 for vitrification via UV radiation biological material in the hospital laboratory or medical
LN2 is obtained by compressing gaseous nitrogen (N2), center where the cryogenic container is located may also
which has a very low boiling point (−195.82°C). In its lead to LN2 contamination. It is therefore possible for the
practical applications, LN2 evaporates when released and contaminated LN2 to infect a biological sample through

88
 safety of vitrification and cryostorage and optimization of cryopreservation protocols
N2, the absence of a dipole moment permits electronic
absorption only in the far UV spectral region (wavelength
<145 nm). Hence, N2 is largely transparent to infrared and
visible radiation. The radiations from the most common
commercial UV sources, usually working at wavelengths
between 185 and 366 nm, are not involved in the N2
absorption process. Since in the case of small molecules
such as N2 no significant spectral difference transpires
from the absorption spectra obtained in liquid and gas
phases,51–53 it can be hypothesized that direct UV steriliza-
tion is applicable to LN2. However, even though UV steril-
ization is used for liquids similar in composition to water,
this method is difficult to apply to LN2, which evaporates
rapidly. The decontamination of LN2 via UV irradiation
comprises calculating the minimum irradiation time nec-
essary to kill the microorganisms that are resistant in LN2.
This calculation takes into account that the rate at which
the microorganisms are deactivated is related to the quan-
tity of LN2 to be sterilized, and depends on the physical
properties of the container in which the LN2 is located; the
LN2 released in a container absorbs large quantities of heat
and evaporates, and it is desirable to calculate irradiation
times in such a way as to complete irradiation before the
Figure 10.2  Specifically designed device for ultraviolet nitrogen evaporates completely. Since the rate at which the
liquid nitrogen sterilization (Nterilizer—www.nteril- microorganisms are deactivated depends on the efficiency
izer.com). of the UV irradiation system, which is in turn inversely
proportional to the temperature at which the irradiation
system operates, it is important to check the efficiency of
direct contact with it; for example, during specific cryo- the system by measuring the temperature of the bulb wall
preservation procedures, or in the case of samples that are of the lamp so as to calculate its relative UV output, even
cryogenically preserved, in improperly sealed or damaged in the presence of the highly refrigerating effect induced
devices. It has also been demonstrated that LN2 may be a by the evaporation of the LN2. The relative UV output
potential source of infection for cryostored gametes and is required to calculate the time of irradiation (T = UV
embryos.14 Defects in sealing materials may cause contact dose/I; T stands for residence time and I for UV inten-
of cryopreserved cells and tissues with LN2.15 Oocyte and sity). The lamp output, at various bulb wall temperatures,
embryo contamination by LN2 may occur, for example, if is usually described by the manufacturer of the UV ger-
any bacterial or viral particles in the LN2 make contact micidal lamp.
with the exposed cryoprotectant and then, after thawing, In a 2010 study,54 a method for sterilizing LN2 was pro-
attach to a cracked zona pellucida.14,24 posed based on emitting the minimum dose of UV radia-
It has been hypothesized that filtration and UV radia- tion necessary to kill microorganisms that can survive at
tion of LN2 may protect against contamination during the the boiling point of nitrogen (−195.82°C), and which is
vitrification procedure.46 The use of UV radiation to ster- irradiated in a temperature-controlled regimen, within a
ilize surgical material, work surfaces, and water or other short time interval, before the LN2 completely evaporates.
liquids is widespread. UV radiation is sub-divided into This study described an experimental microbial con-
UV-A (wavelength 315–400 nm), UV-B (280–315 nm), tamination and disinfection via UV irradiation of LN2,
and UV-C (40–280 nm). UV-C is the most energetic and with the irradiation being suspended after the adminis-
dangerous portion of UV radiation. It can cause electronic tration of the minimum UV dose needed to deactivate
excitation, leading to the breaking of chemical bonds and the most resistant microorganism inoculated in the
the formation of unpaired electron species, known as open dewar (Aspergillus niger: 330,000 UV dose). This
radicals.49 These chemical species are highly reactive and study confirmed that the microorganisms used in this
can damage DNA and cell replication, in this way deac- experiment were able to survive in LN2, but showed that
tivating the growth of all kinds of microorganisms, from decontamination of LN2 via UV irradiation is feasible
viruses to fungi.50 With some liquids, depending on their and simple. Considering that UV radiation deactivates
specific physical and chemical composition, the UV radia- the growth of all kinds of microorganisms, from viruses
tion may be absorbed before it can reach the microorgan- such as hepatitis (which requires 8000 UV dose) to fungi
ism to deactivate it. For homonuclear molecules such as like Aspergillus niger (330,000 UV dose),50 this technique

89
 vitrification in assisted reproduction
allows UV–SLN2 to be easily obtained for every use, and can be hypothesized that the same warming protocol can
in particular for a safe vitrification procedure.54 be potentially used regardless of the freezing protocol.59
Furthermore, in any vitrification protocol or any “vitrifi-
Three-wash procedure in sterile-certified LN2 cation–warming ready-to-use” kit, the concentration of
A reliable procedure exists to decontaminate frozen the nonpermeating cryoprotectant in the first warming
human specimens before warming.55 This procedure con- solution is 1.0 M, irrespective of the concentration and
sists of washing the specimens with SLN2, and this has types of cryoprotectants used.60 This suggests that a single
been shown to efficiently decontaminate vitrification car- “­universal warming protocol” via vitrification warming
riers in extreme experimental conditions. This procedure solutions can potentially be used to warm any reproductive
could be routinely performed in IVF laboratories for the cell/tissue, thereby simplifying the laboratory protocol.
safe thawing of human specimens that are cryostored in
“non-hermetical” cryocontainers, particularly in the case “Universal warming protocol” via
of “open” or “single-straw closed” vitrification systems. vitrification–warming solution
It is possible to warm slow-frozen cells by using the same
Regulations and quality assurance warming solutions as used in vitrification protocols,
Hypothetical cell/tissue contamination by LN2/NV which may also increase their survival rates. This has
requires us to guarantee the sterility of vitrification been demonstrated in a study on sibling oocytes random-
procedures, particularly in Europe, due to the direc- ized for conventional rapid thawing, or rapid warming
tives on tissue manipulation (European Union Tissues via vitrification–warming solution.59 The study assessed
and Cells Directive EUTCD: 2004/23/EC, 2006/17/EC, the survival at 2 hours, and also examined some of the
and 2006/86/EC). These directives have been issued by surviving oocytes for parthenogenesis. The survival
the European Parliament in order to increase the safety rate was significantly higher (90.2%) in rapidly warmed
and quality of tissues—including reproductive cells— oocytes than in rapidly thawed oocytes (74.6%), and rap-
processed for human re-implantation, through the con- idly warmed parthenotes grew faster.
trol of equipment, devices, and environment. Similar An interesting observation of this study was that, dur-
regulations may be introduced by the Food and Drug ing warming, slow-frozen oocytes in the rapidly warmed
Administration at some point in the future. Such require- group displayed behavior typical of vitrified oocytes; as
ments will be applicable to all assisted reproductive soon as the oocytes were expelled from the straw, they
centers in the United States.37 Thus, both in Europe and tended to float and appear “vitreous” in 1.0 M sucrose
potentially in the United States, human reproductive cells solution. They subsequently shrank, and then progres-
are to be treated in the same way as other nonreproduc- sively recovered their original shape (see Figures 10.3
tive tissues. For this reason, even though Pomeroy et al. and 10.4).60 For this reason, it seems critical to expel the
considered the cross-contamination of infectious agents oocytes into the first warming solution at 37°C as soon as
to be a negligible risk,37 and the majority of cryobiologists the external ice around the straw has melted, in order to
and embryologists maintain that vitrification with open
systems using nonsterile LN2 is a safe practice, interna-
tional regulations and quality assurance require specific
procedures in embryo/oocyte/ovarian tissue cryopreser-
vation in order to avoid any hypothetical contamination
of human cells through direct contact with accidentally
contaminated LN2.

OPTIMIZATION OF CRYOPRESERVATION
Intracellular “Glassy State”
The final goal of cell cryopreservation protocols is to
convert the cell to a “glassy state” without ice crystal
formation, to avoid cryoinjury.56,57 This occurs despite
the behavior of different cryopreservation media when
cooled below the solidification point; in fact, the medium
used for vitrification appears “glassy,” whereas the slow-
freezing medium is “iced” due to the presence of ice crys-
tals. As Katkov puts it, “Slow freezing is just a method
of intracellular vitrification with ice being present in the Figure 10.3  ​Slow-frozen oocyte in 1 M sucrose. During
extracellular compartment.”58 the rapid warming procedure, the oocyte is expelled
For this reason, since both vitrified and slow-frozen into 1 M sucrose at 37°C still shrunken; its rehydration
reproductive cells/tissues have a vitrified cytoplasm, it occurs within 1 minute.

90
 safety of vitrification and cryostorage and optimization of cryopreservation protocols
may be useful not only for reproductive cells/tissue but,
in the future, also for other human specimens, even
including whole organs. In current directives worldwide,
there are no specific indications against direct contact
between human specimens and LN2/NV; for this reason,
vitrification “open systems” can comply with any existing
directive, as long as aseptic procedures during vitrifica-
tion–cryostorage–warming are established.31,44,45
Despite the increase of vitrification worldwide, to date,
many slow-frozen oocytes/embryos/ovarian tissue speci-
mens have already been stored in IVF cryobanks. Existing
studies of a “universal warming procedure” confirm that
this increases the efficiency of slow freezing and enables
survival rates comparable with vitrification, while opti-
mizing costs and simplifying laboratory routines by
using the same single “universal warming protocol.”
Figure 10.4 Slow-frozen oocyte in washing solution
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11 Physiological aspects of oocyte vitrification
Mark G. Larman and David K. Gardner

INTRODUCTION antibodies, and subsequent visualization through con-

It has been nearly 30 years since the birth of the first child focal microscopy. Although facilitating high-­resolution
conceived through the fertilization of a frozen human images, this technique does not allow monitoring of the
metaphase II (MII) oocyte.1 Within the last decade, suc- dynamic process within the same oocyte.
cess rates and implementation of oocyte vitrification have Polarized light microscopy in combination with imag-
increased dramatically. There remains, however, room for ing software algorithms has made it possible to image the
improvement. For example, why do oocytes from 10% to meiotic spindle of the living and unfixed metaphase  II
20% of donors in a given donation cycle fail to tolerate oocyte continually, without compromising viability.6
the cryopreservation procedure? What negative impact Rienzi and colleagues7 used this technique to demon-
does vitrification have on oocyte physiology that results strate that the spindle disappears during the thawing
in a much sharper age-related decline in clinical outcome stages following slow freezing, but reforms in the major-
compared to fresh oocytes? ity of oocytes within 3 hours. This is not unsurprising
For the most part, methods for oocyte cryopreserva- considering exposures to the freeze and thaw solutions
tion have been derived by empirical approaches using during slow freezing are carried out at room tempera-
survival, fertilization, and in some cases embryo devel- ture. Not every study has demonstrated that the spindle is
opment as the criteria to assess efficacy. This approach has highly sensitive to temperatures lower than 37°C. Tamura
certainly helped improve cryopreservation techniques, et al. demonstrated that mouse oocytes maintained their
but provides minimal information on the physiology spindle for up to 2 hours at room temperature, which is
of the oocyte and how cryoprotectants and cryopreser- in contrast to many other studies.8 Exposing the oocytes
vation procedures affect cell function. Analyses of the to the vitrification solutions at room temperature, how-
effects of cryopreservation on oocyte physiology provide ever, caused depolymerization, and a loss of microtubule-
the opportunity to create a greater understanding about organizing center proteins. Furthermore, this effect was
the impacts of these techniques, and to subsequently independent of the cryoprotectants used. As the oocytes
improve the results. were warmed, it appears that they underwent excessive
polymerization initially, but 2 hours after warming, the
MEIOTIC SPINDLE meiotic spindle had normalized in terms of shape and
The maternal chromosomes are held suspended close size. It seems that this recovery period is critical for sub-
to the cortex of the MII arrested oocyte by a network sequent survival following parthenogenetic activation, as
of microtubules, called the meiotic spindle. A correctly oocytes activated before 2 hours had significantly lower
formed spindle and aligned chromosomes are required survival rates.
for the physical segregation of the chromosomes during When human oocytes were vitrified using a protocol
second polar body extrusion. An intact spindle is also that involves exposure to the vitrification (and warming)
the site for the spatial downregulation of the M-phase solutions at 37°C, the meiotic spindle remained intact
promoting factor following fertilization.2 Therefore, any (Figure 11.1).9 This means that intracytoplasmic sperm
disruption to the spindle and its function can also affect injection (ICSI) can be carried out soon after warm-
completion of meiosis, subsequent development, and via- ing, avoiding a long spindle recovery time that has been
bility, including the induction of aneuploidy. observed with slow freezing,10 ensuring that fertiliza-
Interestingly, the spindle is not a steady-state structure. tion occurs in a timely manner, and eliminating issues
Rather, tubulin monomers are in dynamic equilibrium, associated with oocyte aging. The spindle is maintained
being constantly added and lost to each individual tubule. or recovers more quickly with vitrification compared to
Cooling, cryoprotectants, osmotic stress, and cryopreser- slow freezing.9 It does appear, however, that the faster
vation itself have all been shown to induce microtubule reduction in cell volume with vitrification disrupts the
depolymerization,3–5 with the most likely explanation position of the polar body in relationship to the spindle.11
being that they affect the balance of tubulin turnover. Although it might be difficult to disrupt the spindle dur-
Many studies have investigated the effects on the meiotic ing microinjection with ICSI, care should be taken, as
spindle of MII oocytes through fixation, labeling of the the polar body might not indicate the position of the
spindle and chromosomes with fluorescent-conjugated meiotic spindle following vitrification.

95
 vitrification in assisted reproduction
Before 0h 2h

Vitrification
Slow freezing

Figure 11.1  The effect of cryopreservation on the meiotic spindle of human MII oocytes. Polarized light micros-
copy (Oosight™) was used to image the meiotic spindle before vitrification/freezing, immediately after warming/
thawing, and after recovering for a further 2 hours. The vitrification and warming protocol was performed at 37°C,
which probably explains why the spindle remained intact. Following slow freezing, it took up to 2 hours for the
meiotic spindle to reform.

It is still unclear how effectively the chromosomes spermatozoon with the oocyte, a sperm-specific phos-
maintain or reform their proper alignment following pholipase C (PLCζ) is introduced into the cytosol of the
vitrification. A study of oocytes from donor patients oocyte, triggering a series of calcium oscillations.27,28 In
(mean age 25.1 years) reported that vitrified oocytes had all mammalian oocytes, the protein triggers an initial
bipolar spindles, and equatorially aligned chromosomes transient intracellular calcium rise, followed by a series
2–3 hours after warming,12 whereas Coticchio and col- of shorter-duration calcium oscillations until pronuclei
leagues13 reported a significantly lower rate when com- form. In human oocytes, the initial increase in calcium
pared to fresh oocytes within 1 hour of warming in older lasts approximately 3–5 minutes, with shorter-duration
age patients (mean age 38.5 years). The differences in oscillations (2–3 minutes) occurring every 15–30 min-
patient age and time after warming make it difficult to utes.26 It has been known for many years that the spa-
draw solid conclusions, but perhaps this is one possible tio-temporal profile of these calcium oscillations affects
explanation for the decrease in results following vitrifica- mammalian embryo development and viability.29–34 The
tion of oocytes from older patients.14 There are some reas- downstream events are still not fully elucidated, but it is
suring data, however, that show that oocyte vitrification clear that the calcium increases are integral in initiating
does not increase the risk of aneuploidy.15 and controlling a number of signaling pathways.35–38
The initial calcium increase causes cortical gran-
INTRACELLULAR CALCIUM INCREASE AND ules to fuse to the plasmalemma, release their contents
“ZONA HARDENING” DURING OOCYTE into the adjacent perivitelline space, and therefore alter
CRYOPRESERVATION the zona pellucida. The proteolytic enzymes from the
Intracellular calcium is a ubiquitous second messenger.16 cortical granules target sperm binding proteins, pre-
It is not only involved in many somatic cell signaling path- venting further sperm from binding, and thus blocking
ways, but is pivotal in the fertilization and early embryo polyspermy. This process is referred to as zona harden-
development of many species, including mammals.17–21 ing. The subsequent calcium increases are necessary for
Studies on human oocytes have been somewhat limited embryo development, triggering the resumption of meio-
and, in most part, used in vitro-matured oocytes.22–25 To sis by regulating cell cycle proteins. Embryo activation
the authors’ knowledge, there is only one study measur- and embryo development can even be triggered without
ing intracellular calcium in fresh MII human oocytes.26 sperm by mimicking the intracellular calcium increases,
In  these studies, it did not matter whether in vitro fer- demonstrating that they are necessary and sufficient to
tilization (IVF) or ICSI was performed, as both lead to initiate embryo development. Interestingly, the calcium
increases in intracellular calcium. Upon fusion of the oscillation profile in cryopreserved human oocytes is

96
 physiological aspects of oocyte vitrification
different compared to nonvitrified oocytes.26 Both vitri- to hardening of the zona pellucida, a small hole was cre-
fied and slow-frozen human oocytes exhibit decreased ated using a laser. By circumventing the zona pellucida,
frequency and increased duration. A change in the cal- thus permitting direct access for the sperm, fertilization
cium oscillation pattern could be explained by a number rates were restored.
of mechanisms. For example, in porcine oocytes, vitri- In rat oocytes, it has been shown directly using con-
fication causes a decrease in the calcium release chan- focal microscopy that exposure to vitrification media
nel (inositol 1,4,5-trisphospahte receptor), which would containing EG and DMSO caused an increase in corti-
negatively affect the calcium oscillations following subse- cal granule release, and that even more release occurred
quent fertilization.39 following vitrification.47 Calcium-free vitrification media
As with fertilization, it appears that zona hardening, significantly decreased cortical release, and subsequently
which occurs during cryopreservation, is induced by increased the fertilization rate in rat oocytes. Similarly,
an increase in calcium. It has been determined that it an increase in fertilization was observed by the same
is the cryoprotectants, and not the physical aspects of group when calcium-free vitrification media were used
cryopreservation, which cause the calcium increase.40–44 for mouse oocytes.48 Although the majority of stud-
Vincent and colleagues were the first to demonstrate ies have been performed on other mammalian species,
that dimethyl sulfoxide (DMSO) causes zona harden- it appears that the human oocyte is also affected in the
ing and consequently reduces fertilization in mouse same way. There are a number of reports demonstrating
oocytes. This was not a direct effect on the zona pellu- that there is a decrease in cortical granules in cryopre-
cida, as the presence of the oocyte itself was required.40,41 served human oocytes.49–57 As with rodent oocytes, it
Takahashi et  al. demonstrated ethylene glycol (EG) appears that the release of cortical granules can also be
causes an increase in intracellular calcium of mouse triggered by cryoprotectants.49,58
oocytes.43 Reducing the increase in calcium with an ICSI provides a means to circumvent issues with zona
intracellular calcium chelator decreased the cytotoxic hardening during oocyte cryopreservation. Assisted fer-
effects of EG during long-term exposure. Larman and tilization does not, however, eliminate the fact that the
colleagues went on to demonstrate that EG, DMSO, and oocyte has seen a large increase in intracellular calcium
1,2-propanediol (PrOH) all induce increases in intra- and has initiated activation signaling pathways before the
cellular calcium.44,45 By removing extracellular calcium sperm has entered the oocyte. Could this explain some of
from the medium, it was possible to reduce the amount the issues observed with oocyte cryopreservation? Such
of calcium released by EG and PrOH. The response to a study would be quite difficult to perform, but the lit-
DMSO was not affected by removing extracellular cal- erature certainly indicates that cryoprotectant-induced
cium from the medium, suggesting that it enters the calcium increases occur in oocytes from different mam-
oocyte and releases calcium most likely by disrupting malian species.
the membranes of intracellular calcium-containing An investigation into the concentration of calcium
organelles, such as the endoplasmic reticulum and mito- within the vitrification media was performed with ovine
chondria. This direct intracellular effect of DMSO on oocytes.59 Calcium- and magnesium-free media with fetal
calcium makes it stand out from other cryoprotectants, calf serum significantly increased survival and embryo
and raises concerns about its ability to alter intracellular development rates when compared to a calcium-con-
signaling pathways. taining medium. Calcium-free media also significantly
It has been shown that the calcium increase caused reduced parthenogenetic activation following exposure
by cryoprotectants during cryopreservation procedures to the vitrification solutions. The negative impact of
is enough to induce zona hardening.44,46 Using proteo- cryoprotectant-induced calcium increases are further
lytic dissolution of the zona pellucida, it was possible to highlighted by the fact that exposure of mouse oocytes
measure the level of hardening. Figure 11.2a shows that to 1.5 mM PrOH for 4 or 10 minutes caused almost 40%
the zona pellucida of vitrified mouse oocytes takes much and 60% of the oocytes to degenerate, respectively. In the
longer to undergo dissolution by chymotrypsin than absence of calcium, however, survival was 100%.44
nonvitrified oocytes. If the vitrification is performed in
calcium-free media, there is a significant decrease in the ANALYSIS OF GENE EXPRESSION
time it takes to dissolve the zona pellucida. The increase Relatively little work has been performed on the effects of
in intracellular calcium induced by the cryoprotectants is cryopreservation on the stability of mRNA and/or gene
not only enough to cause zona hardening, but is also suf- expression in oocytes. Studies comparing the efficacy
ficient to significantly reduce fertilization (Figure 11.2b). of DMSO with EG have revealed that DMSO appears
Vitrified mouse oocytes have a lower fertilization rate to have a greater detrimental effect on gene expression.
compared to nonvitrified oocytes. Performing the vit- For example, in mouse MII oocytes, DMSO was found
rification in calcium-free media significantly increases to have a greater effect on the upregulation of aquapo-
the percentage of fertilized oocytes. To confirm that the rin 7 expression than either EG or sucrose.60 It was pro-
reduction in fertilization of the vitrified oocytes was due posed that rather than affecting gene expression as a

97
 vitrification in assisted reproduction
(a) (b) 100
Control
90
1000 With calcium
Control 80 *
900 Without calcium
With calcium

Eggs reaching 2-cell (%)

800 70
c Without calcium With calcium and laser
Time for dissolution (s)

700 60
600 50 *
500 b 40
400
30
300
200 20
a
100 10
0 0

Figure 11.2  The effect of vitrification on zona hardening and fertilization. (a) Vitrification causes hardening of the
zona pellucida (ZP) of mouse MII oocytes. To assess the degree of zona hardening, oocytes were exposed to a 1%
chymotrypsin solution to determine how long it would take to dissolve the ZP. The more cortical granule release the
oocyte had experienced, the more resistant the ZP would become to enzymatic digestion. The time taken for the ZP
to disappear was recorded for each treatment. Vitrification of the oocytes in calcium-containing solutions signifi-
cantly increased the time taken for the ZP to disappear compared to the control (nonvitrified) oocytes. Vitrifying
the oocytes in solutions that did not contain calcium significantly reduced the time for the ZP to disappear com-
pared to vitrification with calcium-containing solutions. Different letters indicate a significant difference between
treatments (p < 0.01). (b) The extent of zona hardening can also be examined by observing the degree of fertiliza-
tion. Those oocytes with increased zona hardening will have reduced rates of fertilization. The ability of the sperm
to bind, penetrate, and fertilize the oocyte was assessed by recording the number of two-cell stage embryos that were
present 24 hours after insemination. Fertilization of oocytes vitrified with calcium-containing solutions was sig-
nificantly reduced compared to control (nonvitrified) oocytes. A significant increase in fertilization was observed
with oocytes that were vitrified with solutions that did not contain calcium (p = 0.001). To demonstrate that the
reduction in fertilization was due to hardening, oocytes were vitrified with calcium-containing solutions and then
the ZP was breached with a laser to allow direct access for the sperm.

result of increased osmolarity, DMSO acted directly to on gene expression, similar to its action on calcium-stor-
induce changes in gene expression. Consistent with these ing organelles, reflect its high degree of solubility and
observations, when immature bovine oocytes were vit- penetration.
rified with EG together with either DMSO or PrOH, it In a study by Di Pietro and colleagues, using 25
was found that the inclusion of DMSO had a significant donated human MII oocytes and a total of 30% cryo-
negative impact upon the ability of oocytes to mature in protectant, it was determined that vitrification did not
vitro.61 Analysis of mRNA transcript abundance in sheep affect the molecular profile, nor was it associated with
oocytes revealed that when high concentrations of cryo- the degradation of mRNA.64 In a subsequent analysis of
protectants were employed (20% EG together with 20% human MII oocytes that had failed fertilization through
DMSO), there was a significant decrease in gene expres- ICSI, Monzo and colleagues compared the impact of
sion and oocyte competence.62 The significance of this slow freezing or vitrification on gene expression signa-
work was emphasized in a subsequent study by Habibi tures.65 Both forms of cryopreservation were associated
and colleagues on the mouse MII oocyte.63 It was deter- with altered gene expression compared with the non-
mined that the expression of Sod1 was affected by vitri- cryopreserved controls. While slow freezing induced
fication. What is of interest is that using a total of 30% a downregulation of genes connected to chromosomal
cryoprotectants (15% EG plus 15% DMSO) in the vitrifi- structure maintenance and cell cycle regulation, vitrifi-
cation solutions had a much greater negative effect com- cation induced the downregulation of genes associated
pared to the use of a total of 15% cryoprotectants. It was with the ubiquitination pathway, comprising genes of
not determined whether there were differences between ubiquitin-specific peptidases and subunits of the 26S
the two cryoprotectants used, but evidently, concentra- proteasome. The latter was hypothesized to result in
tion of cryoprotectant influences the gene expression of stabilization of the proteins required for oocyte func-
the oocyte, indicating that it would be prudent to work tion and viability, and may therefore go some way to
at the lowest possible effective concentrations, and plau- explaining the greater efficacy of vitrification. Similarly,
sibly avoid DMSO. The proposed direct actions of DMSO Chamayo and coworkers examined the impact of slow

98
 physiological aspects of oocyte vitrification
freezing and vitrification on donated MII human that vitrification imparts less stress on the cell.75 Salvetti
oocytes, and determined that the freezing process was and coworkers determined that although vitrification of
associated with a significant decrease in mRNA content, rabbit oocytes resulted in a loss of intracellular ATP, if
with only 39.4% of the mRNA being preserved. In con- oocytes underwent slow freezing, then the loss of ATP
trast, after vitrification, 63.3% of the mRNA remained, was even more significant.76 It is therefore feasible that
indicating that vitrification imparts less molecular using metabolic parameters will assist in identifying
injury than slow freezing.66 those treatments that have the least impact on such a cen-
In summary, vitrification appears to have less of an tral cellular function of the oocyte. As adequate energy
effect on gene expression in the mammalian oocyte than metabolism is fundamental to numerous cell processes,
slow freezing, and molecular trauma is related to the con- it would not be prudent to select a treatment that had a
centration of cryoprotectants used. negative impact on it.
One of the main negative effects of cryopreserva-
METABOLISM AND ENZYME LEAKAGE tion is damage to the plasma membrane of the cell. By
The metabolism of embryos is linked to their development quantifying the appearance of oocyte-derived proteins
in culture and subsequent viability post-transfer.67–69 in the medium, it would be possible to indirectly assess
Stress upon the embryo usually results in compromised membrane damage. As lactate dehydrogenase (LDH)
metabolic function, which in turn affects developmen- comprises a significant amount of the total protein of
tal potential.69,70 Typically, when considering environ- the oocyte (5%),77 quantification of LDH in the medium
mental stress, one thinks of pH changes, oxidative stress surrounding the oocyte makes an excellent marker of
through the use of atmospheric oxygen in the culture sys- membrane integrity. It has been revealed that while slow
tem, temperature drifts, and suboptimal media formu- freezing of mouse two-cell embryos results in the release
lations.71 However, a further source of stress to oocytes of significant amounts of LDH into the medium, vitrifica-
is cryopreservation, where exposure to cryoprotectants, tion has almost no impact on this parameter, indicating
together with shifts in temperature, can impair metabolic that the damage to cell membranes is greater with slow
function. Of note, all studies to date have determined freezing than with vitrification. Together with the data on
that the negative effects of cryopreservation are greater nutrient utilization, it is tempting to speculate that slow
for slow freezing than for vitrification. freezing has the capacity to directly affect membranes,
The majority of metabolic studies have been per- which are particularly sensitive to environmental per-
formed on embryos, but it is likely that similar paral- turbations. The exact aspect of the slow-freezing process
lels can be drawn for the oocyte. The negative impact of that is responsible for the observed effects on oocyte and
cryopreservation on embryo metabolism was initially embryo metabolism has yet to be determined.
demonstrated in bovine embryos. The metabolic rate
of blastocysts (measured by glucose consumption) fol- PROFILING THE OOCYTE PROTEOME USING
lowing slow freezing using glycerol and sucrose was SURFACE-ENHANCED LASER DESORPTION/
significantly decreased compared to levels before cryo- IONIZATION TIME-OF-FLIGHT MASS
preservation.72 Subsequently, Lane et al.73 demonstrated SPECTROMETRY
that slow freezing of two-cell mouse embryos induced a Proteomic analysis of oocytes and embryos has previ-
greater decrease in oxidative metabolism than vitrifica- ously been hindered by the large sample sizes required
tion, indicating potential damage to the mitochondria, to perform standard protein analyses, such as 2D poly-
plausibly through alterations in membrane integrity and acrylamide gel electrophoresis. Furthermore, proteins
structure. In a subsequent study on human cleavage-stage with high or low molecular masses, or those that are
embryos, Balaban and colleagues revealed that not only either acidic or hydrophobic, can be under-represented
was vitrification associated with higher embryo survival, on such gels. Recent developments in the field of mass
but that vitrification also had significantly less of an effect spectrometry have facilitated the analysis of specific
on metabolic activity than slow freezing.74 Day 3 human protein expression patterns that reflect different biologi-
embryos that underwent slow freezing had a significantly cal states.78,79 Subsequently, it has been possible to use
lower pyruvate uptake than those embryos that had surface-enhanced laser desorption/ionization time-of-
undergone vitrification, again reflecting possible distur- flight mass spectrometry (SELDI-TOF-MS) to determine
bances to mitochondrial function. the proteome of small groups (n = 5) of oocytes and
Similarly, with regard to the oocyte, it has been shown embryos.44,80,81 Consequently, it is now possible to obtain,
that slow freezing of mouse oocytes resulted in a signifi- through the use of different protein chip types, a com-
cant decrease in the uptake of the central nutrient pyru- prehensive profile of proteins in oocytes under different
vate.75 Although vitrification was also associated with a conditions, and relate this to the proteome of an in vivo-
decrease in nutrient utilization by the oocyte compared to ovulated oocyte or in vitro-generated embryo.
noncryopreserved controls, the decrease was significantly Analysis of mouse MII oocyte protein profiles through
smaller than that induced by slow freezing, indicating SELDI-TOF MS following cryopreservation using either

99
 vitrification in assisted reproduction
4000 4500 5000 Da outcomes. For example, does the hormone stimulation
protocol affect the ability of oocytes to withstand vitrifi-
Control cation? Investigating the effect of cryopreservation on the
meiotic spindle has demonstrated that 2 hours of recov-
Vitrified ery after warming is adequate to ensure that the spindle is
intact. A recovery time might not actually be required if
a protocol is used in which both vitrification and warm-
Slow frozen ing are performed at 37°C, which appears to maintain the
spindle. Using the same analysis, it has been shown that
the polar body can move during vitrification, so there
Figure 11.3 Protein expression (3500–5500  Da) in could be an advantage in using polarized light micros-
mouse MII oocytes following cryopreservation. Surface- copy to visualize the location of the spindle during ICSI,
enhanced laser desorption/ionization time-of-flight or even determine whether a meiotic spindle is present,
mass spectrometry was used to investigate the effect of and therefore if the oocyte is suitable for vitrification
cryopreservation procedures on the proteome of mouse at that time. Further, it has been revealed in a number
oocytes. The line plot shows that the expression level of of mammalian species that performing vitrification in
one protein remained unchanged following cryopreser- calcium-free media improves results. The ability to per-
vation (solid box). It was found that slow freezing affected form ICSI with human oocytes does circumvent the zona
the protein expression profile more than vitrification. In hardening, but perhaps the precocious increase in intra-
this example, slow freezing caused up- and downregula- cellular calcium caused by the cryoprotectants is detri-
tion of proteins (dashed boxes). (Modified from Gardner mental to some oocytes, and should not be discounted.
DK et al. Theriogenology 2007;67:64–72.) Many of the published studies are on donor oocytes, so
there is less known about the efficacy of oocyte vitrifica-
slow freezing (with PrOH) or vitrification (using EG and tion with older patients. The clinical outcomes following
DMSO) determined major alterations when oocytes were oocyte vitrification decline more rapidly with patient age
cryopreserved using conventional freezing techniques. than with the known age-related decline observed with
Figure 11.3 shows line plots from the SELDI-TOF MS; fresh oocytes. Are oocytes from older patients more sus-
whereas vitrified oocytes appear to be similar to noncryo- ceptible to the negative effects of cryoprotectant-induced
preserved control oocytes, oocytes that underwent slow calcium increases? Physiological studies are difficult to
freezing exhibit markedly different protein profiles. A sub- perform with human oocytes because of the paucity of
sequent analysis of the slow-freezing procedure identified material, but by conducting such analyses, it is envisaged
the dehydration of the oocyte, prior to seeding, as the time that protocols will continue to improve and minimize the
when alterations in the proteome are manifest. Further aberrations induced by cryopreservation.
analysis revealed that rather than cooling from room tem-
perature to –7°C, it is the chronic exposure to PrOH that is ACKNOWLEDGMENTS
responsible for the aberrations in the proteome.44 DKG would like to thank Vitrolife AB for their support.
Such observations are concordant with similar stud-
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2003;71:135–41. cryoprotectants, calcium and cumulus cells. J Reprod
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tion of meiosis in fertilizing mouse eggs. Curr Biol fate of cortical granules. Fertil Steril 2006;86:210–6.
2002;12:746–50. 50. Gualtieri R, Iaccarino M, Mollo V, Prisco M,
37. Marangos P, Carroll J. Fertilization and InsP3- Iaccarino S, Talevi R. Slow cooling of human oocytes:
induced Ca2+ release stimulate a persistent increase Ultrastructural injuries and apoptotic status. Fertil
in the rate of degradation of cyclin B1 specifically in Steril 2009;91:1023–34.
mature mouse oocytes. Dev Biol 2004;272:26–38. 51. Gualtieri R, Mollo V, Barbato V, Fiorentino I, Iaccarino
38. Ducibella T, Fissore R. The roles of Ca2+, downstream M, Talevi R. Ultrastructure and intracellular calcium
protein kinases, and oscillatory signaling in regulat- response during activation in vitrified and slow-fro-
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Dev Biol 2008;315:257–79. 52. Coticchio G, Borini A, Distratis V et  al. Qualitative
39. Hirose M, Kamoshita M, Fujiwara K et al. Vitrification and morphometric analysis of the ultrastructure
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receptor expression, resulting in low fertility of pig tive slow cooling protocols. J Assist Reprod Genet
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40. Vincent C, Pickering SJ, Johnson MH. The harden- 53. Bonetti A, Cervi M, Tomei F, Marchini M, Ortolani F,
ing effect of dimethylsulphoxide on the mouse zona Manno M. Ultrastructural evaluation of human
pellucida requires the presence of an oocyte and is metaphase II oocytes after vitrification: Closed versus
associated with a reduction in the number of cortical open devices. Fertil Steril 2011;95:928–35.
granules present. J Reprod Fertil 1990;89:253–9. 54. Bianchi V, Macchiarelli G, Borini A et al. Fine mor-
41. Vincent C, Turner K, Pickering SJ, Johnson MH. Zona phological assessment of quality of human mature
pellucida modifications in the mouse in the absence oocytes after slow freezing or vitrification with a
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42. Johnson J, Bagley J, Skaznik-Wikiel M et  al. Oocyte 55. Nottola SA, Macchiarelli G, Coticchio G et  al.
generation in adult mammalian ovaries by putative Ultrastructure of human mature oocytes after slow
germ cells in bone marrow and peripheral blood. Cell cooling cryopreservation using different sucrose con-
2005;122:303–15. centrations. Hum Reprod 2007;22:1123–33.
43. Takahashi T, Igarashi H, Doshida M et al. Lowering 56. Nottola SA, Coticchio G, De Santis L et  al.
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fication solution in unfertilized mouse oocytes. Mol Biomed Online 2008;17:368–77.
Reprod Dev 2004;68:250–8. 57. Nottola SA, Coticchio G, Sciajno R et  al.
44. Larman MG, Katz-Jaffe MG, Sheehan CB, Gardner Ultrastructural markers of quality in human mature
DK. 1,2-propanediol and the type of cryopreservation oocytes vitrified using Cryoleaf and Cryoloop. Reprod
procedure adversely affect mouse oocyte physiology. Biomed Online 2009;19(Suppl. 3):17–27.
Hum Reprod 2007;22:250–9. 58. Schalkoff ME, Oskowitz SP, Powers RD.
45. Larman MG, Sheehan CB, Gardner DK. Calcium-free Ultrastructural observations of human and mouse
vitrification reduces cryoprotectant-induced zona oocytes treated with cryopreservatives. Biol Reprod
pellucida hardening and increases fertilization rates 1989;40:379–93.
in mouse oocytes. Reproduction 2006;131:53–61. 59. Succu S, Berlinguer F, Leoni GG et al. Calcium con-
46. Larman MG, Sheehan CB, Gardner DK. Vitrification centration in vitrification medium affects the devel-
of mouse pronuclear oocytes with no direct liq- opmental competence of in vitro matured ovine
uid nitrogen contact. Reprod Biomed Online oocytes. Theriogenology 2011;75:715–21.
2006;12:58–61. 60. Tan YJ, Xiong Y, Ding GL et al. Cryoprotectants up-
47. Fujiwara K, Sano D, Seita Y, Inomata T, Ito J, regulate expression of mouse oocyte AQP7, which
Kashiwazaki N. Ethylene glycol-supplemented facilitates water diffusion during cryopreservation.
calcium-free media improve zona penetration of Fertil Steril 2013;99:1428–35.
vitrified rat oocytes by sperm cells. J Reprod Dev 61. Faheem MS, Baron E, Carvalhais I, Chaveiro A,
2010;56:169–75. Pavani K, da Silva FM. The effect of vitrification of
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developmental rates of mouse oocytes cryopreserved development and gene expression. Zygote 2014:1–10.

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62. Succu S, Bebbere D, Bogliolo L et al. Vitrification of 73. Lane M, Maybach JM, Gardner DK. Addition of
in vitro matured ovine oocytes affects in vitro pre- ascorbate during cryopreservation stimulates subse-
implantation development and mRNA abundance. quent embryo development. Hum Reprod 2002;17:​
Mol Reprod Dev 2008;75:538–46. 2686–93.
63. Habibi A, Farrokhi N, Moreira da Silva F et  al. The 74. Balaban B, Urman B, Ata B et al. A randomized con-
effects of vitrification on gene expression in mature trolled study of human day 3 embryo cryopreserva-
mouse oocytes by nested quantitative PCR. J Assist tion by slow freezing or vitrification: Vitrification is
Reprod Genet 2010;27:599–604. associated with higher survival, metabolism and blas-
64. Di Pietro C, Vento M, Guglielmino MR et  al. tocyst formation. Hum Reprod 2008;23:1976–82.
Molecular profiling of human oocytes after vitrifica- 75. Lane M, Gardner DK. Vitrification of mouse oocytes
tion strongly suggests that they are biologically com- using a nylon loop. Mol Reprod Dev 2001;58:342–7.
parable with freshly isolated gametes. Fertil Steril 76. Salvetti P, Buff S, Afanassieff M, Daniel N, Guerin P,
2010;94:2804–7. Joly T. Structural, metabolic and developmental
65. Monzo C, Haouzi D, Roman K, Assou S, Dechaud H, evaluation of ovulated rabbit oocytes before and after
Hamamah S. Slow freezing and vitrification differen- cryopreservation by vitrification and slow freezing.
tially modify the gene expression profile of human Theriogenology 2010;74:847–55.
metaphase II oocytes. Hum Reprod 2012;​27:2160–8. 77. Brinster RL. Lactate dehydrogenase activity in the
66. Chamayou S, Bonaventura G, Alecci C et  al. preimplanted mouse embryo. Biochim Biophys Acta
Consequences of metaphase II oocyte cryopreserva- 1965;110:439–41.
tion on mRNA content. Cryobiology 2011;62:130–4. 78. Shau H, Chandler GS, Whitelegge JP, Gornbein JA,
67. Gardner DK, Lane M, Stevens J, Schoolcraft WB. Faull KF, Chang HR. Proteomic profiling of can-
Noninvasive assessment of human embryo nutrient cer biomarkers. Brief Funct Genomic Proteomic
consumption as a measure of developmental poten- 2003;2:147–58.
tial. Fertil Steril 2001;76:1175–80. 79. Rocken C, Ebert MP, Roessner A. Proteomics in
68. Gardner DK, Wale PL, Collins R, Lane M. Glucose pathology, research and practice. Pathol Res Pract
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embryos is predictive of embryo sex and live birth 80. Katz-Jaffe MG, Linck DW, Schoolcraft WB, Gardner
outcome. Human Reproduction 2011;26:1981–6. DK. A proteomic analysis of mammalian preim-
69. Gardner DK, Wale PL. Analysis of metabolism to plantation embryonic development. Reproduction
select viable human embryos for transfer. Fertil Steril 2005;130:899–905.
2013;99:1062–72. 81. Katz-Jaffe MG, Gardner DK, Schoolcraft WB.
70. Lane M, Gardner DK. Understanding cellular disrup- Proteomic analysis of individual human embryos to
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viability and fetal development. Reprod Fertil Dev ity. Fertil Steril 2006;85:101–7.
2005;17:371–8. 82. Sheehan CB, Lane M, Gardner DK. The CryoLoop
71. Balaban B, Sakkas D, Gardner DK. Laboratory proce- facilitates re-vitrification of embryos at four succes-
dures for human in vitro fertilization. Semin Reprod sive stages of development without impairing embryo
Med 2014;32:272–82. growth. Hum Reprod 2006;21:2978–84.
72. Gardner DK, Pawelczynski M, Trounson AO. 83. Gardner DK, Sheehan CB, Rienzi L, Katz-Jaffe
Nutrient uptake and utilization can be used to select M, Larman MG. Analysis of oocyte physiology to
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Mol Reprod Dev 1996;44:472–5. 2007;67:64–72.

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12 Vitrification of oocytes: Imprinting and disturbance
in spindle formation and chromosome segregation
Tom Trapphoff

THE MEIOTIC SPINDLE intracellularly labeled structures, including the meiotic

Metaphase II (MII) spindles appear to be particularly spindle. Three-dimensional image reconstruction of dis-
vulnerable to environmental changes. Susceptibility tinct optical sections enables assessment of chromosome
of microtubules to cryoprotective agents (CPAs) has alignment at the equatorial plate, which is infeasible by
been shown, as well as to thermal, oxidative, and redox noninvasive PLM. A general drawback of fluorescence
stress.1–3 Albeit transient, cryopreservation interferes microscopy is the need for fixation of the oocytes, which
without doubt with the intracellular and extracellu- eliminates time-lapse assessment before and after vitrifi-
lar homeostasis and therefore poses a risk for inducing cation, fertilization, and embryonic development.
spindle abnormalities, chromatid nondisjunction, fer- Invasive techniques are powerful tools for basic
tilization errors, and abnormal mitotic divisions during research, although they cannot be adapted to assisted
embryonic development. reproductive therapy (ART) laboratories as an analyzing
The meiotic spindle is a multi-subunit complex crucial tool to select oocytes that are suitable for further clini-
for aligning and separating chromosomes during meio- cal applications. PLM is limited to assessing the spindle
sis I and II.4 Although the spindle apparatus consists of quantity, but it provides a useful option for verifying the
more than 100 different proteins, it is mainly composed presence of a meiotic spindle prior to in vitro fertilization
of microtubules of α- and β-tubulin dimers. Microtubule (IVF) or intracytoplasmic sperm injection (ICSI) in clini-
polymerization originates at microtubule organizing cal daily routine.
centers (MTOCs) rich in γ-tubulin and pericentrio-
lar material located at both spindle poles to anchor the Vitrification of MII Oocytes
chromosomes at the equatorial plate. Specific subcellu- Several studies focused on spindle integrity and chromo-
lar compartments containing accumulated transcripts some alignment after cryopreservation in animal mod-
and proteins necessary for spindle formation, epigenetic els and, to a lesser extent, also in humans. However, the
control, and chromosome alignment are also mandatory results were not always consistent, supposedly due to
for proper spindle functionality. Prior to and subsequent multifactorial parameters (e.g., patient age, vitrification
to the onset of meiosis, these factors are recruited in a protocol, and recovery time following vitrification). Thus,
stage-specific manner to ensure chromosome segregation the question whether and how the meiotic spindle and
at oocyte to embryo transition. Thus, besides direct sus- chromosome alignment is subjected to cryodamage has
ceptibility of microtubules to cryodamage, alterations of been discussed controversially during recent years and
crucial cytoplasmic components may also be induced by cannot be resolved by a simple “yes” or “no.”
cryopreservation. Studies concerning spindle integrity and chromosome
alignment after oocyte vitrification were initially per-
Visualization Techniques formed in animal models.5–7 Although the microtubu-
Polarization light microscopy (PLM) is based on the lar system and cytoplasmic composition in non-human
detection of anisotropic structures, as incident light models differs from that of human oocytes, early studies
with a defined plane of vibration becomes twisted when revealed several important findings. Using either slow-
it passes through an anisotropic/birefringent structure. rate freezing (SRF) or vitrification methods, spindle integ-
Because the meiotic spindle is birefringent while the sur- rity appears to be altered directly after thawing/warming
rounding cytoplasm exhibits isotropic characteristics, due to microtubule depolymerization and disorganiza-
PLM provides the opportunity to study spindles non- tion, but repolymerization and reorganization occurs later
invasively before and after vitrification without staining. dependent on time, species, and applied protocols (Figure
Commercially available polarization light microscopes 12.1).5,8–10 Chen and colleagues showed that two-thirds of
utilize liquid crystals and optimized image processing open-pulled straw (OPS)-vitrified mouse MII oocytes had
algorithms, increasing image resolution and providing the a disrupted spindle morphology immediately after warm-
possibility to quantify microtubule density as retardance. ing, while the spindle was nearly restored after 1 hour of
Epifluorescence and confocal laser scanning micros- recovery (vitrification solution [VS]: 5.5 mol/L ethylene
copy (CLSM) can be used to obtain detailed images of glycol [EG] and 1.0 mol/L sucrose).5 These findings were

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 vitrification in assisted reproduction
Prior to vitrification Directly after vitrification 2 hours after vitrification
(a) (b) (c)

Polarization microscopy

20 μm

(d) α-tubulin DNA (e) α-tubulin DNA (f) α-tubulin DNA

Confocal microscopy

10 μm

Figure 12.1  (See color insert.) Spindle dynamics after CryoTop vitrification of murine metaphase II (MII) oocytes
analyzed by noninvasive polarization light microscopy (a–c) or by confocal laser scanning microscopy (d–f)
(Trapphoff T., ­unpublished data). Spindle characteristics prior to vitrification (a and d), directly after vitrification
(b and e), and after recovery for 2 hours at 37°C (c and f). The kind of information obtained on spindle integrity
differs between polarization light and fluorescence microscopy directly after vitrification and warming (b and e).
Arrowhead: slightly aberrant spindle, arrow: displaced chromosome; both phenotypes occurred in about a third of
in vivo-ovulated and vitrified murine MII oocytes directly after warming (unpublished data).

confirmed in other model systems with different vitrifica- cryodamage is passed much faster, therefore reducing the
tion protocols and time intervals after warming.6,10,11 potential damage.
Additionally, it was demonstrated that spindle integ- Studies of human MII oocytes analyzing spindle
rity depends on the applied vitrification protocol and the morphology and chromosome alignment after vitrifica-
temperature during pre- and post-treatment. Exposure tion still remain limited. Analysis of spindle retardance
to VSs at 37°C had a profound effect on the develop- using noninvasive PLM after cryopreservation either by
ment competence and a protective influence on the CryoLoop vitrification (VS: 16% EG, 16% propanediol
spindle structure in mice, bovines, and humans (but [PROH], 0.65 mol/L sucrose, and 10 mg/mL Ficoll) or
this increased CPA toxicity).12–14 With respect to oocyte SRF (1.5 mol/L PROH and 0.1 mol/L sucrose) of human
survival, spindle integrity, chromosome alignment, and MII-stage oocytes demonstrated that vitrification
developmental potential, minimal-volume carrier devices affects the meiotic spindle only to a minor degree, while
and equilibration in mixtures of CPAs at medium molar- it was not preserved in slow-frozen oocytes.12 Although
ity, lowering the toxicity of each CPA without weaken- in the study by Martínez-Burgos et al.18 CryoTop vitrifi-
ing their protective characteristics, affected the success of cation, but not SRF, of human MII-stage oocytes tended
vitrification significantly.15–17 For instance, grids, closed to alter the angle between the meiotic spindle and the
pulled straws, and OPSs preserved spindle integrity bet- microfilament meshwork, superiority of vitrification
ter than conventional straws due to faster cooling and compared to SRF regarding spindle morphology and
warming rates (15,000–30,000°C/min versus 2500°C/ recovery dynamics was suggested using noninvasive
min) in murines, while CryoTop vitrification appeared microscopy.13,18
superior compared to OPS vitrification in bovines (VS: PLM is restricted to analysis of spindle quantity, but
20% EG, 20% dimethyl sulfoxide [DMSO], and 0.5 mol/L not quality, and it was demonstrated that morphometric
sucrose).6,17 Current vitrification protocols rely commonly spindle evaluation is not consistent between polarization
on mixtures of CPAs to lower their cellular toxicity, and light and fluorescence microscopy (Figure 12.1).19 Using
also on small-volume vitrification methods to increase CLSM, 27/43 (63%) of Cryoleaf (VS: 15% EG, 15% DMSO,
cooling and warming rates. Thus, the critical tempera- and 0.5 mol/L sucrose)-vitrified human MII-stage oocytes
ture window between 15°C and −15°C responsible for exhibited a normal bipolar spindle with focused poles after

106
 vitrification of oocytes
warming for 1 hour, compared to 22/22 (100%) from non- phenotypes) with caution. Nevertheless, normal spindle
vitrified controls.20 However, normal chromosome align- integrity and chromosome alignment was reported in a
ment at the equatorial plate and a bipolar spindle was large subset of studies using different vitrification pro-
found only in 14/43 (32%) vitrified MII oocytes, compared tocols, after an incubation period of 2–3 hours in both
to 13/22 (59%) from fresh controls. This was also accompa- non-human and human oocytes. This strongly implies a
nied by an increased pole-to-pole distance.20 negligible influence of vitrification on spindle integrity
Studies using a comparable experimental setup showed and morphology.
that the proportion of morphologically normal spindles It is noteworthy that cryopreservation poses not only
and well-aligned MII chromosomes was statistically a risk for altering microtubule stability, but also inter-
not distinguishable in vitrified/warmed oocytes, com- feres with proteins and mRNAs involved in cell cycle
pared to controls, after a recovery time of 2–3 hours.21,22 control and maintenance of the spindle. For instance,
Interestingly, the percentage of normal barrel-shaped vitrification of murine MII oocytes caused a transient
spindles and well-aligned chromosomes was much disappearance of the MTOC components pericentrin,
higher in the vitrification group (51/62, 82%; VS: 15% EG, NEDD1 and γ-tubulin directly after warming.10 High-
15% PROH, and 0.5 M sucrose; Cryoleaf), compared to throughput analysis of gene expression profiles of vitri-
oocytes cryopreserved by SRF using 1.5 M PROH and fied human MII-stage oocytes suggested, moreover, that
0.3 M sucrose (39/64, 60%) in the study by Cao et  al.,22 vitrification compromises gene expression and mRNA
while there was no significant difference between vitri- levels of cell cycle- and spindle-associated factors com-
fication (13/17, 76%) and SRF (19/23, 82%) in the study pared to fresh oocytes, although to a lesser degree than
by Cobo.21 Therein, chromosome alignment and the SRF.25,26 Alterations at the molecular level may there-
proportion of morphologically normal spindles was not fore induce unknown (long-lasting) downstream effects
distinguishable, compared to fresh controls either after that potentially interfere with intracellular checkpoints,
vitrification (VS: 15% EG, 15% DMSO, and 0.5 mol/L resulting in bypass of the spindle assembly checkpoint
sucrose; Cryotip) or after SRF using three different proto- while not possessing an intact genome at later embry-
cols (1.5 mol/L PROH supplemented with 0.2 or 0.3 mol/L onic stages.
sucrose, or with 0.3 mol/L sucrose and 137 mmol/L cho-
line chloride). In accordance with the studies of Cobo and Chromosomal Constitution in Early Embryos
Cao, chromosome alignment (15/25, 60%) and spindle and Offspring
morphology (13/25, 52%) were not statistically different Despite only transient changes (or intermediate pheno-
in in vitro-matured (IVM) and vitrified human MII-stage types) occurring, lasting alterations during early embry-
oocytes, compared to IVM controls (20/31chrom, 64%; onic development and in the offspring are still possible,
17/31spind, 54%) in a study by Lei et al. (VS: 15% EG, 15% especially when considering that early studies in mice
DMSO, and 0.5 mol/L sucrose; Cryoleaf).23 using straw vitrification revealed an approximately three-
However, the post-warming time prior to spindle analy- fold higher incidence of zygotes displaying aneuploidy,
sis and patient age (25 years,21 28 years,23 and 38 years20) and an increased amount of malformed fetuses derived
differed in the referenced studies. Oocytes from older from vitrified oocytes.27 In the limited number of stud-
patient groups exhibited a reduced recovery potential of ies on humans, no evidence for abnormal karyotypes in
the meiotic spindle compared to younger ones after cryo- children born after vitrification of mature human oocytes
preservation, as demonstrated for instance after SRF.9 As has so far been found. Chen et al. reported one child with
shown in animal models and after SRF of human MII- a normal 46,XY karyotype28; Song et  al. reported four
stage oocytes, spindle de- and re-polymerization exhibits female and five male infants with normal karyotypes29; and
dynamic characteristics, and the time point for spindle and Grifo et  al., using array comparative genomic hybridiza-
chromosome analysis is crucial.9,10 Although it should be tion, reported one healthy, euploid male infant.30 Similar
noted that microtubule repolymerization is more effective results were reported in a follow-up study of 12 children
in mouse oocytes exposed to cold stress than in human born after SRF.31 Additionally, animal models provide evi-
oocytes3,24; the cellular alterations that were reported after dence for normal karyotypes in visually normal neona-
1 hour of recovery may reflect only transient anomalies tal offspring after vitrification of mature oocytes.32 Thus,
that may be restored after a prolonged recovery time, or results regarding chromosomal constitution in children
may depend on patient cohorts that differ in age. born after vitrification of mature oocytes are reassuring,
Overall, it is rather difficult to compare spindle qual- but abnormalities in early embryos leading to aborted
ity among studies using different methodologies/param- pregnancies cannot be excluded. Pre-implantation genetic
eters. Minor protocol modifications or different storage diagnosis using fluorescence in situ hybridization (FISH)
devices may have unpredictable downstream effects on revealed euploidy for chromosomes 13, 14, 15, 16, 18, 21,
spindle integrity and chromosome alignment. Therefore, 22, and X in early cleavage-stage embryos obtained after
it is necessary to test each cryopreservation protocol indi- OPS vitrification of human MII oocytes.33 Trophectoderm
vidually, and to interpret results (especially intermediate biopsy using single-nucleotide polymorphism microarrays

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 vitrification in assisted reproduction
showed no difference in the rate of embryonic aneuploidy Although some drawbacks are present and the number
after CryoTop34 or Cryotip vitrification30 of human MII- of studies is low, data regarding spindle morphology after
stage oocytes. Similar results were obtained after SRF of vitrification of immature human oocytes followed by
mature human oocytes.35,36 Although the published data in vitro maturation are promising. Nevertheless, recent
are reassuring, further work is needed to analyze the chro- studies suggest that the overall efficiency was superior
mosomal status in early embryos and offspring derived after IVM of immature GV-stage oocytes followed by
from vitrified and warmed oocytes in long-term follow-up vitrification when compared to the opposite strategy of
studies. vitrification followed by IVM. It was therefore concluded
that immature oocytes should be vitrified after IVM at
Vitrification of Immature Oocytes the MII stage, although the overall efficiency still remains
Because of the sensitive nature of the MII spindles, vitri- relatively poor.46,50 Protocol optimization and further
fication of immature oocytes at the germinal vesicle (GV) studies are certainly needed to assess safety irrespective
stage followed by IVM represents an opportunity to avoid of the applied strategy.
detrimental cryodamage to the spindle. To date, only a
few births have been reported after cryopreservation of Slush Nitrogen and Beneficial CPAs
immature oocytes followed by IVM,37 or vice versa.38,39 Oocyte vitrification is commonly performed by plung-
Although the efficiency of IVM in animal models and ing the carrier device directly into liquid nitrogen, with
especially in humans is still poor,40 cryopreservation of a cooling rate between 15,000°C and 30,000°C/min.
immature oocytes represents an alternative strategy for The use of slush nitrogen increases the cooling rate up
preserving female fertility in patients for whom hormonal to 135,000°C/min.51 Slush nitrogen avoids vaporization
stimulation is not recommended, or in cases with leftover upon solidification (“Leidenfrost effect”), during which
immature oocytes from stimulated cycles. Studies using small gas bubbles around the specimen result in poor
SRF have shown that the spindle of IVM oocytes was com- temperature transfer rates and cryodamage. It has been
promised after cryopreservation, accompanied by delayed demonstrated that slush nitrogen can increase the clini-
maturation, limited fertilization rates, and altered early cal efficiency,52 and it allows use of lower CPA concen-
embryonic development.41–43 In terms of survival, matura- trations to a level comparable to SRF protocols.53 Using
tion, development, and spontaneous activation rates, the fast cooling by slush nitrogen, healthy meiotic spindles
use of vitrification protocols was reported to be superior were present in nearly 90% of vitrified human denuded
compared to SRF of human oocytes.44–46 Regarding spindle oocytes (VS: 5.5 M EG and 1 M sucrose; electron micro-
and chromosome patterns, Combelles et  al. were able to scope grid).51 In animal models, fewer adverse effects on
show that IVM human MII oocytes vitrified at the GV the spindle morphology were also found in rabbit oocytes
stage (VS: 15% EG, 15% PROH, and 0.5 mol/L sucrose) ­v itrified by slush nitrogen compared to plunging into
exhibit a slightly but not significantly reduced number liquid nitrogen (VS: 20% EG, 20% DMSO, 0.65 mol/L
of bipolar spindles and properly aligned chromosomes trehalose, and 10 mg/mL Ficoll).15 Furthermore, the com-
compared to nonvitrified IVM controls.44 Nevertheless, position of vitrification and devitrification media also
there were more bipolar spindles with some microtubule affects spindle integrity. Supplementation with hydroxy-
irregularities, and a tendency toward completely dispersed apatite nanoparticles, synthetic or organic polymers,
chromosomes, while morphometric parameters regarding and anti-oxidative substances can have beneficial effects
spindle length, width, and microtubule volume were not on intracellular constitution.54,55 Supplementation with
different to controls. However, spindle irregularities and anti-freeze proteins—short polypeptides that permit
displaced chromosomes were less severe after vitrification cellular survival in sub-freezing environments—prior
compared to SRF (1.5 mol/L PROH, 0.3 mol/L sucrose, to vitrification of immature murine oocytes improved
and 137 mmol/L choline chloride), while the general effi- spindle quality after IVM compared to nontreated con-
ciency was low for both. trols.55 This was accompanied by enhanced blastocyst
In animal models, normal spindle and chromosome formation and improved mRNA levels of Mad2 (a spindle
alignment was shown after Cryoleaf (VS: 20% EG and checkpoint protein regulating anaphase onset), Hook1
20% DMSO) and solid surface vitrification (VS: 17.5% EG, (regulating chromosome segregation), and Eg5 (spindle
17.5% propylene glycol, 0.3 M trehalose, and 50 mg/mL maintenance). Additional cryoprotective substances or
polyvinylpyrrolidone) of immature ovine and porcine GV slush nitrogen therefore represent interesting options for
oocytes followed by in vitro maturation,47,48 while OPS vit- next-generation cryopreservation techniques, but further
rification of immature equine (VS: 20% EG, 20% DMSO, comparative clinical studies are needed to evaluate the
and 0.5 mol/L sucrose) and pig (VS: 40% EG) oocytes led protective influence.
to spindle abnormalities and misaligned chromosomes
in IVM MII oocytes.43,49 Irregularities reported in animal Conclusion
models may be caused by differences in CPA permeability, The question of whether and how the meiotic spindle
composition of CPA mixtures, or applied protocols. and  chromosome alignment is affected by cryodamage

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 vitrification of oocytes
has generated much controversy and a general conclusion with the highest experimental experiences. Thus, results
is difficult. Obviously, vitrification of mature human MII may not be generalized to the entire field of reproductive
oocytes appears to alter spindle integrity and chromo- medicine. Further studies are necessary to draw a final
some alignment directly after vitrification and warming, conclusion regarding the safety and efficiency of cryo-
while these changes disappear during recovery of cellu- preservation, either by SRF or vitrification, to elucidate
lar homeostasis. However, the biological significance of unknown downstream alterations or long-lasting effects
intermediate phenotypes with bipolar spindles and only at later stages.
minor dysfunctions is still unknown, and a potential risk
cannot be excluded. Vitrification does not alter chromo- GENOMIC IMPRINTING
some segregation either in meiosis II or in early mitotic DNA methylation and post-translational histone modi-
divisions during embryonic development. No increase fications are two major epigenetic mechanisms that
in birth defects and congenital malformations has been regulate gene activation, or repression, without specifi-
reported in neonatal offspring after vitrification compared cally modifying the DNA sequence. Genomic imprinting
to pregnancies from conventional IVF/ICSI and the gen- represents a highly specialized epigenetic mechanism for
eral population.56,57 There is evidence that fertilization, ensuring mono-allelic gene expression in the offspring.
early embryonic development, implantation, and clinical Accordingly, one parental allele becomes silenced by
pregnancy rates of vitrified and warmed human oocytes repressive modifications established during gameto-
are comparable to fresh oocytes used as part of assisted genesis in one germ cell, while in the second germ cell,
reproductive techniques.58,59 Consequently, the American activating modifications are established to ensure gene
Society for Reproductive Medicine has recently removed expression in the offspring (Figure 12.2a). After fusion
the experimental status of mature human oocyte vitri­ of both gametes, these germ cell-specific genomic modi-
fication.60 Nevertheless, it should be considered that fications remain stable; contrary to this, global demeth-
molecular alterations at the transcript and proteome lev- ylation and reprogramming of both parental genomes
els may have adverse long-lasting effects, and that most occur to ensure totipotency in order to establish cell lin-
of  the  published data were generated in a few clinics eages and cell differentiation. DNA methylation occurs

(a) +

Gene A IC1 Gene B IC2 Gene C

Gene A IC1 Gene B IC2 Gene C

–
Unmethylated CpG Expression
Methylated CpG Repression

(b) Unmethylated loci Hypermethylated loci

me me me
AT CG AT CG TT CG AT AT CG AT CG TT CG AT
Bisulfite
treatment
me me me
AT UG AT UG TT UG AT AT CG AT CG TT CG AT
DNA
amplification
AT TG AT TG TT TG AT AT CG AT CG TT CG AT

Figure 12.2  (See color insert.) Scheme of mono-allelic gene expression in the offspring originated from parental-­
specific hyper- or nonmethylation of maternal and paternal imprinting centers (a). Paternal hypermethyl-
ation of IC1  represses paternal expression of gene B and activates expression of the upstream-located gene A.
Hypermethylation of maternal IC2 results in paternal expression of gene C. Bisulfite treatment induced mutagen-
esis of hyper- and nonmethylated DNA (b). (CpG = cytosine phosphate–guanine dinucleotide.)

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 vitrification in assisted reproduction
Table 12.1  ​Examples of Imprinted Genes in Humans Used in Epigenetic Analysis
Gene Full name Function Human disease

CDKN1C Cyclin-dependent kinase inhibitor 1C Negative regulator of cell proliferation and inhibitor BWS
of several G1 cyclin/Cdk complexes
H19 H19, imprinted maternally expressed Noncoding RNA implicated in growth suppression. BWS, Wilms’
transcript Located in the IGF2 imprinted region tumor
IGF2 Insulin-like growth factor II Member of the insulin family, involved in BWS, RSS,
development and growth Wilms tumor
KCNQ1 Potassium voltage-gated channel, Voltage-gated potassium channel BWS
KQT-like subfamily
KCNQ10T1 KCNQ1 opposite strand/antisense Noncoding antisense transcript to the KCNQ1 BWS
transcript 1 gene. Regulates transcription of multiple target
genes
SNRPN Small nuclear ribonucleoprotein- Involved in mRNA processing and tissue-specific PWS, AS
associated protein N alternative splicing events
UBE3A Ubiquitin-protein ligase E3A Member of the ubiquitin protein degradation PWS, AS
system

Note: AS = Angelman syndrome; BWS = Beckwith–Wiedemann syndrome; PWS = Prader–Willi syndrome; RSS = Russell–Silver

syndrome.

on cytosines at position C5 in cytosine phosphate– Techniques

guanine dinucleotides (CpG). CpGs are located in Detection of epimutations in small amounts of DNA is
GC-rich genomic regions known as imprinting centers not simple, due to the fact that an average cell with one
(ICs) of approximately 1–4 kb in size, and they regulate representative methylation profile does not exist, and
the expression or repression of one or several neighboring imprinting diseases are quite rare.70 Thus, one of the most
genes (Figure 12.2a). challenging tasks is to identify these rare events masked
The establishment and maintenance of genomic within a pool of several oocytes or embryos. Most com-
imprinting is essential for mammalian development, and mon techniques are based either on methylation-sensitive
nearly 150 genes are known to be imprinted (Table 12.1).61 polymerase chain reaction (PCR) or enzymatic DNA
In primordial germ cells, global DNA methylation restriction, or on bisulfite treatment of DNA molecules.
becomes erased, including inherited parental-specific The latter leads to a conversion of cytosine residues to ura-
genomic imprints. De novo methylation of sex-specific cil, while methylcytosine bases remain unaffected (Figure
DNA methylation patterns starts post-natal after the 12.2b). Thus, bisulfite treatment induces distinct muta-
onset of oogenesis, and establishment of  genomic tions depending on the methylation pattern of individual
imprinting is completed in mature oocytes (Figure cytosine residues. Bisulfite treatment followed by DNA
12.3).62,63 DNA methyltransferase 3a (DNMT3A) and its amplification and sequencing (BiSeq) represents the gold
co-factor DNMT3L are crucial for de novo methylation standard for methylation analysis. BiSeq allows analysis
during oogenesis. So far, three maternal factors are of several gene loci of differentially methylated regions
known to prevent demethylation of imprinted genes simultaneously. Recently published studies indicate that
during epigenetic reprogramming at  early embryonic
­ analysis of up to 25 genes in single cells or embryos is pos-
stages—STELLA, ZFP57, and KAP164–66 —while DNMT1 sible at one time, but such multiplex methylome analysis
is essential for restoring DNA methylation patterns of several gene loci is time consuming.71 Current methods
­following DNA replication.67 Loss of the DNA methyla- typically analyze up to five genes, and therefore provide
tion imprinting ­pattern has crucial consequences during information only for a small subset of the entire methy-
embryogenesis and in the offspring due to an altered lome. Whole-genome sequencing could eliminate these
gene–dose effect. STELLA-, DNMT1-, and DNMT3A- restrictions, but this requires microgram quantities of
knockout mutants in mice showed, for instance, an DNA, while there are currently no reliable methods in
altered/arrested embryonic development, or a preco- the picogram range. Nevertheless, recent studies using
cious  death a few weeks after birth.68,69 As the use of amplification-free whole-genome bisulfite sequencing
­cryopreservation either by SRF or by vitrification con­ obtained methylome maps from only nanogram quanti-
tinues to rise worldwide, it is of upmost importance to ties of DNA, which, however, still would correspond to
assess the safety of both techniques with respect to the several hundred to a thousand oocytes.72,73
erasure, establishment, and maintenance of genomic It is also noteworthy to mention that methylome
imprinting. analysis is prone to somatic DNA contamination, and

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 vitrification of oocytes
Imprinting cycle Imprinting Diseases and ART
Human imprinting diseases include Angelman syn-
drome (AS), Beckwith–Wiedemann syndrome (BWS),
and Prader–Willi syndrome (PWS), and they predispose
ce individuals to different tumor types, including ovarian
an
cancer and Wilms’ tumor.61 The BWS critical region at
en
int

chromosome 11p15.5 includes two ICs: IC1 that regulates

Ma

Era
PGC the expression of IGF2 and H19, and IC2 that controls the

sure
Blastocyst
expression of CDKN1C, KCNQ10T1, and KCNQ1. BWS
Zygote is associated with increased methylation at the normally
MII Growing nonmethylated maternal IC1, or a loss of methylation of
oocyte oocyte the normally hypermethylated maternal IC2.61 The criti-
cal regions at chromosome 15q11–q13 for AS and PWS
include an IC that regulates the expression of SNRPN
and UBE3A. AS and PWS children have a higher inci-
dence of altered SNRPN methylation and failures to
E s t a b li s h m e n t express the maternal allele of UBE3A, while loss of the
Genome Imprinted gene IGF2 imprinting profile represents the most common epi-
Methylated Methylated CpG mutation responsible for some cancer types.61,75–77 ARTs,
particularly cryopreservation, interfere with the criti-
Unmethylated Unmethylated CpG cal time windows for the acquisition or maintenance of
epigenetic markers, and doubtlessly pose a risk for alter-
Figure 12.3  (See color insert.) Imprinting cycle in mice. ing DNA methylation patterns either directly or indi-
Erasure, establishment, and maintenance of imprinted rectly by affecting factors involved in epigenetic control.
genes (outer circle) during F0 to F1 transition. Genome- Hormonal stimulation, ex situ fertilization, and in vitro
wide demethylation (including imprinted genes) occurs embryo culture have been reported to increase the inci-
in primordial germ cells (PGCs) until embryonic day dence of epimutations in early pre- and post-implantation
13.5. Post-natal de novo establishment of sex-specific embryos, and in extra-embryonic tissue both in animal
methylation marks occurs during oogenesis (pre-natal models and humans.78–81 The general influence of ART
in males) and maintenance of genomic imprinting with respect to the increased prevalence of imprinting dis-
occurs until the formation of PGCs in the F1 generation. eases in children born after ART-treatment has been hotly
Epigenetic methylation marks of nonimprinted genes debated. Certain recent studies showed no indication of
(inner circle) are also erased in PGCs, followed by meth- an increased risk of imprinting diseases in children con-
ylation events during oogenesis. After fertilization, ceived after ART.82,83 Conversely, some studies described a
active (paternal genome) and passive (maternal genome) four- to nine-fold increase in the risk of BWS for children
demethylation of both parental genomes occurs, fol- conceived in vitro, although the absolute risk remained
lowed by de novo reprogramming during embryonic very low.84,85 Moreover, some studies suggested a link
development. (MII = metaphase II.) between hormonal or ICSI treatment and AS,75,86 while a
recent study did not find a significant association between
the incidences of PWS or AS and IVF or ICSI treatments.87
may exhibit the possibility of stochastic amplification
of just a single or only a few molecules from a start- Cryopreservation and Genomic Imprinting
ing sample of several dozen oocytes or individual Changes in epigenetic patterns were shown following
blastocysts (“amplification bias”). This bias can lead to exposure to extra- and intracellular stresses in vari-
results that are not representative of the studied sample. ous cell types, including oocytes and preimplantation
However, preferential amplification can be excluded embryos.88–92 For instance, oxidative stress leads to
due to stochastic distribution of bisulfite-treated chemical conversion of deoxyguanine to 8-hydroxy-
DNA molecules prior to amplification and sequenc- 2′-deoxyguanosine accompanied by repression of DNA
ing,71,74 while inclusion of the pluripotency-related gene methyltransferase activity at neighboring cytosines,90
OCT4 in the multiplex assay, which is unmethylated an altered IGF2/H19 methylation pattern in the mouse
in early embryos and oocytes and hypermethylated in prostate,91 or a limited expression of DNMTs in human
somatic cells, allows the detection of DNA contamina- embryos that have been cryopreserved.92 Oocyte cryo-
tion. Therefore, although some drawbacks are present, preservation therefore doubtlessly increases the prob-
improved BiSeq protocols represent the best opportu- ability of cellular methylation processes being altered. A
nity to date for studying methylation patterns of several large number of studies investigated the influence of IVF,
gene loci in one assay. ICSI, or in vitro culture on CpG methylation of imprinted

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 vitrification in assisted reproduction
genes, while there is less information regarding the influ- and embryo culture, maintenance of genomic imprint-
ence of cryopreservation either by SRF or vitrification. To ing after cryopreservation seems to be more sensitive to
date, there has been only one study of the CpG methyla- epimutations when compared to the establishment of
tion pattern of two imprinted genes after the vitrification methylation marks after the cryopreservation of imma-
of immature human oocytes followed by in vitro matura- ture germ cells.
tion at the MII stage.93 BiSeq analysis did not reveal sig- Although some epimutations were reported after the
nificant differences for KCNQ1OT1 and H19 methylation, cryopreservation of germ cells and early pre-implantation
although the level of H19 epimutations increased from embryos, data regarding imprinting diseases in children
8% in IVM controls (3/34 analyzed alleles from 48 MII conceived after cryopreservation do not provide evidence
oocytes) to 17% after vitrification and IVM (5/29 ana- for an increased incidence of adverse neonatal outcomes
lyzed alleles from 36 MII oocytes). For KCNQ1OT1, loss or birth defects. Such an analysis was described by Noyes
of methylation was found in 2/37 alleles from 20 IVM et al.57: this study included 392 children conceived after
MII oocytes and in 1/28 alleles from 17 vitrified and IVM oocyte vitrification, 532 children resulting from SRF,
MII oocytes. It was concluded that vitrification does not and 12 children from a combination of both techniques.
cause an increased frequency of epimutations compared Additionally, children born after SRF or vitrification of
to IVM controls,93 although the sample size was relatively cleavage-stage embryos or blastocysts exhibited the same
low and the number of analyzed genes was limited. prevalence of epigenetic diseases as children born after
In animal models, global DNA methylation patterns the transfer of fresh embryos.98–100 However, it should be
were significantly altered either after SRF or vitrification noted that methylation patterns from abortions and still-
of mature bovine oocytes.94 Thus, it seems that cryo- births revealed an increased prevalence for imprinting
preserved oocytes are more vulnerable to epimutations, alterations.101 Thus, inappropriate methylation patterns
but it should be noted that global DNA methylation was of imprinted genes induced by ART treatment may con-
analyzed qualitatively by CLSM, without distinguishing tribute to spontaneous pregnancy loss.
between epigenetic DNA marks and the methylation sta-
tus of imprinted genes. Using a mouse model, the genomic Conclusion
integrity of two imprinted genes was assessed in pups The published data do not provide evidence of an
after the cryopreservation of whole ovaries by SRF fol- increased risk of imprinting mutations after cryopreser-
lowed by orthotopic ovarian re-transplantation and natu- vation of oocytes per se. Epimutations and imprinting
ral mating.95 Methylation-sensitive DNA digestion and diseases were reported in some studies after ART treat-
southern blotting revealed normal H19 and KCNQ1OT1 ment, including hormonal stimulation, ex situ fertiliza-
methylation patterns, suggesting that cryopreservation tion, and embryo culture, but others could not replicate
followed by ovarian transplantation does not disrupt the these findings. ART treatment is mainly a consequence of
proper acquisition of genomic imprinting. underlying subfertility or infertility of the patients. This
Using an alternative methodology to preserve female makes it difficult to distinguish between de novo epimu-
fertility, the genomic integrity of three imprinted genes tations as a consequence of ART or whether potentially
was assessed after CryoTop vitrification of individual present dysfunctions in the parental generation (which
murine pre-antral follicles, followed by in vitro follicu- cause subfertility or infertility) lead to genomic disorders
logenesis and oogenesis.74 Using BiSeq, normal methyla- in the offspring. Thus, the combination of ex situ treat-
tion levels were reported for SNRPN (49/50 alleles), IGF2R ment and the fertility history of patients undergoing ART
(15/15 alleles), and H19 (58/58 alleles) in mature GV is an important factor to keep in mind. Cryopreservation
oocytes compared to nonvitrified (45/47SNRPN ; 16/16IGF2R; of immature oocytes does not seem to have a profound
39/39H19) or in vivo-grown controls (40/40SNRPN ; 16/16IGF2R; impact on the acquisition of genomic imprinting.
38/38H19). Thus, the published data do not provide evi- Methylation patterns after in vitro follicle culture, ortho-
dence of an increased risk of epimutations after the topic re-transplantation followed by endogenous in vivo
cryopreservation of immature oocytes, and acquisition folliculogenesis and oogenesis, as well as after IVM past
of genomic imprinting appears normal compared to con- cryopreservation were comparable to fresh controls.
trols. On the other hand, it was demonstrated using BiSeq However, the influence of human MII oocyte cryopreser-
that vitrification of murine pre-implantation embryos vation has not been investigated so far, while two studies
led to a loss of methylation of the H19/IGF2 IC in early reported an elevated level of epimutations after the vitri-
fetuses.96 Although loss of methylation was also found fication of bovine and murine pre-implantation embryos.
in the IVF group, epimutations were more severe in the It appears that the maintenance of genomic imprinting
vitrification group. In another study using bovine two- in later stages seems to be more sensitive to epimutations
cell embryos and BiSeq, the methylation level of H19 was when compared to ex situ treatment prior to or during
significantly increased in vitrified two-cell embryos and the establishment of DNA methylation. Although it is far
their derived blastocysts.97 In accordance with studies too early to draw conclusions about the risk of imprint-
that reported epigenetic perturbations during IVF, ICSI, ing mutations and birth defects after the vitrification of

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 vitrification of oocytes
oocytes, the current literature is reassuring. An increased 12. Larman MG, Minasi MG, Rienzi L et al. Maintenance
incidence of imprinting diseases in live births after the of the meiotic spindle during vitrification in
cryopreservation of oocytes as well as pre-implantation human and mouse oocytes. Reprod Biomed Online
embryos has not been identified. However, one of the 2007;15:692–700.
most challenging tasks for future work is to verify the 13. Ciotti PM, Porcu E, Notarangelo L et  al. Meiotic
safety of the cryopreservation of immature and mature spindle recovery is faster in vitrification of human
human oocytes or embryos with respect to the acquisi- oocytes compared to slow freezing. Fertil Steril
tion and maintenance of genomic imprinting. 2009;91:2399–407.
14. Keskintepe L, Agca Y, Sher G et  al. High survival
rate of metaphase II human oocytes after first polar
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58. Cobo A, Meseguer M, Remohí J et  al. Use of cryo- 74. Trapphoff T, El Hajj N, Zechner U et al. DNA integ-
banked oocytes in an ovum donation programme: rity, growth pattern, spindle formation, chromo-
A prospective, randomized, controlled, clinical trial. somal constitution and imprinting patterns of mouse
Hum Reprod 2010;25:2239–46. oocytes from vitrified pre-antral follicles. Hum Reprod
59. Potdar N, Gelbaya TA, Nardo LG. Oocyte vitrifica- 2010;25:3025–42.
tion in the 21st century and post-warming fertility 75. Orstavik KH, Eiklid K, van der Hagen CB et  al.
outcomes: A systematic review and meta-analysis. Another case of imprinting defect in a girl with
Reprod Biomed Online 2014;29:159–76. Angelman syndrome who was conceived by intra-
60. Pfeifer S, Goldberg J, McClure R et  al. Mature cytoplasmic semen injection. Am J Hum Genet
oocyte cryopreservation: A guideline. Fertil Steril 2003;72:218–9.
2013;99:37–43. 76. Cox GF, Bürger J, Lip V et al. Intracytoplasmic sperm
61. Kalish JM, Jiang C, Bartolomei MS. Epigenetics injection may increase the risk of imprinting defects.
and imprinting in human disease. Int J Dev Biol Am J Hum Genet 2002;71:162–4.
2014;58:291–8. 77. Mabb AM, Judson MC, Zylka MJ et  al. Angelman
62. Reik W, Dean W, Walter J. Epigenetic repro- syndrome: Insights into genomic imprinting and
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63. Lucifero D, Mann MR, Bartolomei MS et  al. Gene- 78. Market-Velker BA, Fernandes AD, Mann MR. Side-
specific timing and epigenetic memory in oocyte by-side comparison of five commercial media sys-
imprinting. Hum Mol Genet 2004;13:839–49. tems in a mouse model: Suboptimal in vitro culture
64. Nakamura T, Arai Y, Umehara H et  al. PGC7/Stella interferes with imprint maintenance. Biol Reprod
protects against DNA demethylation in early embryo- 2010;83:938–50.
genesis. Nat Cell Biol 2007;9:64–71. 79. Market-Velker BA, Zhang L, Magri LS et  al. Dual
65. Quenneville S, Verde G, Corsinotti A et al. In embry- effects of superovulation: Loss of maternal and pater-
onic stem cells, ZFP57/KAP1 recognize a methyl- nal imprinted methylation in a dose-dependent man-
ated hexanucleotide to affect chromatin and DNA ner. Hum Mol Genet 2010;19:36–51.
methylation of imprinting control regions. Mol Cell 80. Chen SL, Shi XY, Zheng HY et  al. Aberrant DNA
2011;44:361–72. methylation of imprinted H19 gene in human preim-
66. Messerschmidt DM. Should I stay or should I go: plantation embryos. Fertil Steril 2010;94:2356–8.
Protection and maintenance of DNA methylation at 81. Fauque P, Jouannet P, Lesaffre C et al. Assisted repro-
imprinted genes. Epigenetics 2012;7:969–75. ductive technology affects developmental kinetics,
67. Cirio MC, Ratnam S, Ding F et  al. Preimplantation H19 imprinting control region methylation and H19
expression of the somatic form of Dnmt1 suggests a gene expression in individual mouse embryos. BMC
role in the inheritance of genomic imprints. BMC Dev Dev Biol 2007;7:116.
Biol 2008;8:9. 82. Lidegaard O, Pinborg A, Andersen AN. Imprinting
68. Howell CY, Bestor TH, Ding F et  al. Genomic diseases and IVF: Danish National IVF cohort study.
imprinting disrupted by a maternal effect mutation in Hum Reprod 2005;20:950–4.
the Dnmt1 gene. Cell 2001;104:829–38. 83. Zheng HY, Shi XY, Wang LL et  al. Study of DNA
69. Payer B, Saitou M, Barton SC et al. Stella is a maternal methylation patterns of imprinted genes in children
effect gene required for normal early development in born after assisted reproductive technologies reveals
mice. Curr Biol 2003;13:2110–7. no imprinting errors: A pilot study. Exp Ther Med
70. Eroglu A, Layman LC. Role of ART in imprinting dis- 2011;2:751–5.
orders. Semin Reprod Med 2012;30:92–104. 84. DeBaun MR, Niemitz EL, Feinberg AP. Association of
71. El Hajj N, Trapphoff T, Linke M et  al. Limiting in vitro fertilization with Beckwith–Wiedemann syn-
dilution bisulfite (pyro)sequencing reveals par- drome and epigenetic alterations of LIT1 and H19.
ent-specific methylation patterns in single early Am J Hum Genet 2003;72:156–60.

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85. Gicquel C, Gaston V, Mandelbaum J et  al. In vitro methylation profile of H19 and KCNQ1OT1 imprint-
fertilization may increase the risk of Beckwith– ing centers in human oocytes subsequently matured
Wiedemann syndrome related to the abnormal in vitro. Fertil Steril 2011;95:1955–60.
imprinting of the KCN1OT gene. Am J Hum Genet   94. Hu W, Marchesi D, Qiao J et al. Effect of slow freeze
2003;72:1338–41. versus vitrification on the oocyte: An animal model.
86. Ludwig M, Katalinic A, Gross S et al. Increased preva- Fertil Steril 2012;98:752–60.
lence of imprinting defects in patients with Angelman   95. Sauvat F, Capito C, Sarnacki S et al. Immature cryo-
syndrome born to subfertile couples. J Med Genet preserved ovary restores puberty and fertility in mice
2005;42:289–91. without alteration of epigenetic marks. PLoS One
87. Vermeiden JP, Bernardus RE. Are imprinting disor- 2008;3:e1972.
ders more prevalent after human in vitro fertiliza-   96. Wang Z, Xu L, He F. Embryo vitrification affects the
tion or intracytoplasmic sperm injection? Fertil Steril methylation of the H19/Igf2 differentially methylated
2013;99:642–51. domain and the expression of H19 and Igf2. Fertil
88. Yan LY, Yan J, Qiao J et  al. Effects of oocyte vitrifi- Steril 2010;93:2729–33.
cation on histone modifications. Reprod Fertil Dev  97. Zhao XM, Ren JJ, Du WH et  al. Effect of 5-aza-
2010;22:920–5. 2’-deoxycytidine on methylation of the putative
89. Spinaci M, Vallorani C, Bucci D et al. Vitrification of imprinted control region of H19 during the in vitro
pig oocytes induces changes in histone H4 acetylation development of vitrified bovine two-cell embryos.
and histone H3 lysine 9 methylation (H3K9). Vet Res Fertil Steril 2012;98:222–7.
Commun 2012;36:165–71.   98. Kato O, Kawasaki N, Bodri D et al. Neonatal outcome
90. Weitzman SA, Turk PW, Milkowski DH et  al. Free and birth defects in 6623 singletons born following
radical adducts induce alterations in DNA cytosine minimal ovarian stimulation and vitrified versus
methylation. Proc Natl Acad Sci USA 1994;91:1261–4. fresh single embryo transfer. Eur J Obstet Gynecol
91. Bhusari SS, Dobosy JR, Fu V et  al. Superoxide dis- Reprod Biol 2012;161:46–50.
mutase 1 knockdown induces oxidative stress and   99. Pinborg A, Loft A, Aaris Henningsen AK et al. Infant
DNA methylation loss in the prostate. Epigenetics outcome of 957 singletons born after frozen embryo
2010;5:402–9. replacement: The Danish National Cohort Study
92. Petrussa L, Van de Velde H, De Rycke M. Dynamic 1995–2006. Fertil Steril 2010;94:1320–7.
regulation of DNA methyltransferases in human 100. Pinborg A, Henningsen AK, Malchau SS et  al.

oocytes and preimplantation embryos after assisted Congenital anomalies after assisted reproductive
reproductive technologies. Mol Hum Reprod technology. Fertil Steril 2013;99:327–32.
2014;20:861–74. 101. Pliushch G, Schneider E, Weise D et al. Extreme meth-
93. Al-Khtib M, Perret A, Khoueiry R et al. Vitrification ylation values of imprinted genes in human abortions
at the germinal vesicle stage does not affect the and stillbirths. Am J Pathol 2010;176:1084–90.

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13 Metabolic profile of day 3 embryos arising from vitrified oocytes
Damià Castelló and Francisco Domínguez

INTRODUCTION obtained from using vitrified oocytes. Nonetheless, there

The main challenge of assisted reproduction technology is little evidence in the literature regarding the effects
(ART) has always been to achieve pregnancies resulting that these techniques may have on early human embryo
in healthy live births for infertile couples and patients. metabolism.
Embryo selection methods have historically been based
on microscopically evaluating morphology at different VITRIFICATION PROCEDURES
time points that have resulted in a significant improve- One of the most pressing challenges in ART is the
ment in pregnancy rates1,2 and a reduced number of improvement of cryopreservation in order to standard-
multiple pregnancies. Moreover, using new instruments ize its use in clinical practice. There are different kinds
that provide continuous images of embryos has further of human female gamete cryostorage used for both sub-
improved embryo selection and resulting pregnancy fertile and fertile women, including fertility preservation
rates.2,3 patients. The most common method is the use of low tem-
The addition of new genomic, transcriptomic, pro- peratures to completely stop all cellular metabolic and
teomic, and metabolomic technologies that supersede tra- chemical reactions. Work done in the 1930s led to the
ditional morphological methods has led to the proposal of discovery that sperm can survive cryostorage at −160°C,13
new approaches for improving embryo selection that can paving the way for successful embryo cryopreservation
provide more information and/or increase the accuracy and the first pregnancy after cryopreservation in 198314;
of selection. For example, by analyzing embryo culture it has since become a cornerstone of ART.
media, the metabolite content can be determined nonin- Studies have shown that vitrification—the solidifica-
vasively, which might be used to assess embryo viability. tion of an aqueous solution without ice crystal forma-
Nutrients required for embryo development and implan- tion facilitated by high-concentration cryoprotectants
tation may change during in vitro embryo culture, and (CPA)—coupled with very fast cooling rates is a very effi-
these variations might provide us with information about cient method for oocyte cryopreservation.15 However, the
the metabolic activity of the embryo. Indeed, several ret- toxicity of high-concentration CPAs is a major drawback
rospective studies have reported associations between that has led to the development of several devices and
the metabolomic profile and clinical outcomes.4–7 The methodologies to counteract this effect.
metabolism of embryos cultured for in vitro fertiliza- These methods now have pregnancy rates similar to
tion (IVF) often varies because embryos are adapting to those achieved with standard fresh-oocyte procedures,
the stressful conditions that they are subjected to by the sparking the creation of “egg banks” for ovum dona-
variations in culture conditions.8,9 However, other proce- tion.15,16 One controlled clinical trial with a large sam-
dures, such as cryopreservation, are even more demand- ple size failed to prove the superiority of fresh-donated
ing on the embryo, and can adversely affect metabolism oocytes over donated vitrified oocytes in infertility
and development and thus also clinical results. Several treatments.15 Oocyte vitrification also has been very suc-
reports describe the effects of cryopreservation on cessfully used for infertile patients,17 as well as in the
embryos or oocytes; for example, Zhao et  al. report a management of low-response patients.18 Although recent
reduction in the expression of DNA methyltransferase protocols and techniques report high clinical pregnancy
1 (Dnmt1) mRNA expression in mouse oocytes.10 At the and post-birth survival rates from cryopreserved oocytes,
metabolic level, studies also found effects on the metabo- these rates remain low in some cases because of variation
lism of murine embryos frozen in late pre-implantation in the patient’s age, oocyte quality, the type and con-
development.11 In humans, Tachataki et al. reported that centration of CPA used, and the specific protocol used,
freezing embryos altered their gene expression profiles, among other variables.
and that day 2 frozen embryos contained less mRNA
for the tuberous sclerosis gene TSC2 than fresh day 2 -OMICS TECHNOLOGIES
embryos. However, several studies report the births of The introduction of new genomics, transcriptomics, pro-
healthy children after the prior use of different vitrifica- teomics, and metabolomics platforms several years ago
tion approaches,12 which provide information about the has given rise to many new ways to measure and analyze
survival, embryonic development, and clinical outcomes genes, mRNAs, proteins, and metabolites, which may

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 vitrification in assisted reproduction

Genome Transcriptome Proteome Metabolome

DNA mRNA Proteins Metabolites

Figure 13.1 -Omics sciences. New tools in molecular biology for multilayer global analysis, from DNA to
metabolites.

change how embryos are diagnosed and selected (Figure is enabling more complete descriptions of metabolomic
13.1). These -omics technologies study the processes and profiles. These are being used to contrast normal and dis-
interactions between cells, their metabolic byproducts, and ease physiology, including in the early diagnosis of neu-
their phenotypic changes, but necessitate powerful screen- rodegenerative diseases, and for screening fetal disease.
ing and analysis systems. These technologies have changed With regard to ART, evaluating cultured embryos
the classical molecular biology approach, directing it might highlight different metabolic markers and profiles,
toward global analysis. Indeed, recent research using these providing quantitative parameters that we may be able
technologies has aimed to define genetic, proteomic, and to link to their implantation capacity,7 thus significantly
metabolomic embryonic profiles to allow better under- improving the embryo selection process. In addition,
standing of embryo viability.5,19,20 this is a noninvasive technique based on sampling the
metabolites secreted into the culture medium by embryos
METABOLOMICS during in vitro development. This makes the culture
Metabolomics studies the dynamic inventory of metabo- medium a useful source of information about the meta-
lites, using them as small molecular biomarkers to rep- bolic state and health of these embryos that additionally
resent functional phenotypes in biological systems, and can be combined with other evidence (e.g., morphologi-
attempts to determine and quantify the metabolites cal, kinetic, etc.).
associated with physiological and pathological states.21 Different metabolomic parameters have been measured
Low-molecular-weight metabolites are the final prod- in developing embryos using noninvasive techniques.
ucts of cell regulatory processes and can therefore reveal
biological responses to several genetic, nutritional, and
environmental influences.22 The complete array of small-
molecule metabolites contained in a cell or biological Box 13.1  Advantages of Metabolomic Analysis
system constitutes its metabolome.23 Metabolomics is a •• Metabolic control theory25 and experimental stud-
multidisciplinary science requiring the cooperation of ies have shown that the concentration of a particu-
chemists, biologists, and bioinformaticians. lar enzyme within a metabolomic pathway does not
The metabolome greatly varies, has a wide dynamic significantly change the biochemical reactions that
range, and is chemically complex and heterogeneous. In take place but can significantly change metabolite
addition, automatic techniques for its analysis and mea- concentrations.
surement are still limited, and are influenced by the fact •• The metabolome is the final product of genetic
expression and represents the functional level of a
that many metabolites are still unknown, that very few
cell; therefore, changes in the metabolome should
extraction protocols are available, and that extraction be much more biologically relevant than those in the
yields are low.21 Therefore, metabolomics is a science proteome and transcriptome.26
that is complementary to the genomic and proteomic •• Research has shown that metabolic fluids are regu-
disciplines, but with several advantages over them, as lated both by gene expression and environmental
described in Box 13.1.24 There are, however, several spec- factors, and so measuring the final metabolic prod-
troscopy methods available for metabolite detection and ucts (metabolites) gives a more global picture.27
analysis, and the integration of different analysis systems

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 metabolic profile of day 3 embryos arising from vitrified oocytes
For example, an inverse relationship was found between MULTIVARIATE STATISTICAL ANALYSIS
pyruvate absorption and embryo viability.28 Moreover, Multivariate statistical analysis offers a way of obtaining
glucose absorption was higher in human blastocysts with information from the large quantity of data generated
better morphology.29 A recent study also tried to corre- from MS and NMR sample spectra by generating a series
late the presence of the amino acids glycine, leucine, and of multivariable profiles (one for each sample) represented
asparagine in embryo culture media with clinical preg- in a k-dimensional space, where the position (coordi-
nancy rates,8 and another study using embryo culture nates) depend on the value of each descriptor (metabolite)
media from 30 patients with known outcomes related dif- describing the system (sample). Projection methods that
ferent metabolomic profiles to implantation rates.30 convert multidimensional data tables into two-dimen-
However, despite these promising data, use of these sional models are very useful for analyzing profile sets,
techniques in the IVF laboratory has been limited by and are the basis of metabolomic studies. These techniques
their high cost, the need for specialized equipment, and make it possible to detect tendencies in the data because
the long turnaround time required to obtain results, ren- points that cluster represent similar multivariable profiles,
dering it impossible to use this information in a clinical whereas distant points represent different descriptors.
context for fresh oocytes. Although many challenges lie The specific statistical analysis used depends on the
ahead before this technology can be used to predict fresh type of study being developed. Several types of meth-
embryo viability, metabolomics may be beneficial in the ods may be applied to metabolomic data. The most used
cryopreservation field for using vitrified oocytes where multivariable analysis methods are principal component
result turnaround time constraints are less important. analysis (PCA)32 and partial least squares (PLS). PCA is a
Moreover, considering the deep cytoplasmic changes and nonsupervised method used to determine the inner struc-
interactions that occur during the vitrification proce- ture of a dataset without any prior information about it,
dure, it is likely that inferences made from metabolomic whereas PLS is a supervised regression method whose
profiling will be the only way to evaluate these subcel- objective is prediction within a set of data. Figure  13.2
lular disturbances. However, because this is a new tech- shows an example of a PCA.
nique, validation with different approaches using classic
parameters such as morphology and cleavage rate may be THE EFFECT OF VITRIFICATION ON HUMAN
necessary. OOCYTES: METABOLOMIC PROFILE ANALYSIS
OF EMBRYOS
METABOLOMIC ANALYSIS PLATFORMS Oocyte vitrification has been successfully applied in clin-
Platforms for metabolome analysis should be sensitive ical practice, making the establishment of egg-banking
and give a high yield in order to discriminate a high num- programs for ovum donation possible; this technology is
ber of metabolites in a sample. Despite the relative suc- also proving to be an effective approach for fertility pres-
cess of current technologies, because of the wide diversity ervation purposes. An increasing body of literature shows
of complex metabolites contained in any one sample, it the efficiency and consistency of the technology, along
is not possible to analyze the complete metabolome with with the growing number of healthy babies born after
only one platform. oocyte vitrification, which has contributed greatly to the
The most widely used technologies are nuclear mag- definitive validation of this approach. However, despite
netic resonance (NMR) spectroscopy and mass spec- this clinical evidence, it has been argued that there is still
trometry (MS), which is usually performed after liquid a lack of basic research addressing the possible effects of
chromatography (LC) separation; both technologies vitrification on oocytes.
offer broad structural and conformational information As we know, metabolism is intrinsic to embryo health,
on multiple chemical groups from only one procedural and so many studies have focused on identifying non-
step, and each have advantages and disadvantages and invasive metabolic markers associated with successful
offer complementary information about an ample group embryonic development. Special focus has been placed on
of metabolites without the need for preselecting which identifying markers associated with oxidative phosphory-
analytes should be detected.31 The main advantage of lation, Na+/K+-ATPases, redox reactions, and amino acid
MS is its sensitivity and the low minimum sample vol- metabolism.8,33–36 Metabolomic profiling represents an
ume required; however, it is a destructive technique. opportunity to obtain biochemical fingerprints that may
Moreover, not all databases are complete and so, depend- be useful for biological classification or to develop new
ing on the instrument and protocol used, results may diagnostic methods. Moreover, the compounds identified
not be reproducible. In contrast, NMR is a robust, non- may provide insights into the biochemical events leading
destructive technique that is more reproducible than MS to a disease state or classification, and therefore provide
because the data obtained is universal across platforms. opportunities for intervention.
Unfortunately, NMR is less sensitive than MS, needs In 2007, Seli et  al. published the first study using a
larger amounts of input sample, and has a higher metabo- metabolomic approach for assessing the viability of
lite detection threshold. human embryos.5 They used near-infrared and Raman

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 vitrification in assisted reproduction
t2

X1 X2 X3 X2 Comp 1 (t1)

t1

Observation Plane Scores (observations)

X3
Projection
Comp 1 (t2)

p2
x1

p1

x2
x3
X1
Loadings (variables)

Figure 13.2  Principal component analysis. Dots represent relationships between groups of data.

spectroscopy to compare the metabolic profile of embryo- the vitrification group (n = 59), the fresh group (n = 65),
conditioned media from positive and negative implan- and a matched control medium group (n = 66) using cor-
tations after transfer.30 In a follow-up study, viability responding media from the same batch in the same con-
markers were identified using a NMR metabolic platform ditions, but without embryos (Figure 13.3). The samples
and statistically significant differences were found in glu-
tamate concentrations between embryos that resulted in
pregnancies and/or births and those that ended in implan- Spent media samples
tation failure.37 Other recent studies have identified genes, Samples collected
proteins, and metabolomic profiles that can detect which Day 3
oocytes or embryos are viable, and have used these char- Fresh group media (34 patients)
acteristics to measure their implantation capacity.17,20,38 50 μL

Metabolomic profiling
A system has also been developed that aims to obtain n = 65 samples
comprehensive metabolite profiles by quantitatively Vitrification group media (31 patients)
and qualitatively analyzing the metabolites produced/ LC-MS
secreted by an organism, tissue, or even a single cell.39 50 μL
Low-molecular-weight metabolites represent the final n = 59 samples
products of cellular metabolism and therefore reveal the Matched control media
responses of biological systems to a variety of genetic,
50 μL
nutritional, and environmental conditions.22 These non-
invasive, quantitative techniques are the focus of intense n = 66 samples
research to determine their value as predictors of embryo
viability, pregnancy rates,5,38 and even their ability to Figure 13.3  Experimental design of the study.
detect aneuploid embryos,38,40 although a prospective Microdrops were taken from 190 spent culture media
randomized trial found no benefit to using these methods from embryos that had been cultured from day 1 to
for pregnancy prediction.41 day 3. Collection was performed on day 3 by collecting
In 2013, Dominguez et al. evaluated the effects of oocyte 50 μL of culture media. The samples were collected from
vitrification on the global metabolomic profiles of embryos the fresh group (n = 65), the vitrification group (n = 59),
developed from vitrified oocytes compared to the pro- and the matched control media group (n = 66) in the
files observed in embryos from fresh oocytes in an oocyte LC-MS study. Metabolomic analysis was performed in
donor program.42 A total of 190 spent medium samples two rounds due to the instrument capacity limitations.42
were collected from 65 selected patients (34 fresh and 31 (LC-MS =  liquid chromatography mass spectrom-
vitrified oocyte patients, respectively). Each 50 μL sample etry.) (Adapted from Dominguez F et  al., Fertil Steril
was collected from the spent embryo culture medium from 2013;99(2):565–72.)

120
 Second round
(a) First round (c) 2.0
3.0
2.5 1.5
2.0
1.0
1.5
1.0
0.5
0.5
0.0 0.0

t(2)

t(2)
–0.5
–0.5
–1.0
–1.5
–1.0
–2.0
–2.5 –1.5

–3.0
–2.0

0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.0
3.2
3.4
0.0
0.5
1.0
1.5
2.0
2.5
3.0

–3.2
–3.0
–2.8
–2.6
–2.4
–2.2
–2.0
–1.8
–1.6
–1.4
–1.2
–1.0
–0.8
–0.6
–0.4
–0.2
–3.0
–2.5
–2.0
–1.5
–1.0
–0.5
t(1) t(1)
(d)
(b) 2.0
1.4
1.2 1.5
1.0 1.0
0.8 0.5
0.6
0.0
0.4
–0.5
0.2

t(2)
–1.0

t(2)
0.0
–0.2 –1.5
–0.4 –2.0
–0.6 –2.5
–0.8
–3.0
–1.0
–3.5
–1.2

0
1
2
3
4
5
6
7
metabolic profile of day 3 embryos arising from vitrified oocytes

–4
–3
–2
–1
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.0
3.2
3.4

–2.6
–2.4
–2.2
–2.0
–1.8
–1.6
–1.4
–1.2
–1.0
–0.8
–0.6
–0.4
–0.2

Devitrified Fresh t(1) t(1)

Figure 13.4  (See color insert.) Principal component analysis representing all of the metabolites analyzed for each sample in the two rounds. Blue/green triangles
represent the fresh group, and red triangles represent the vitrification group. No separation between groups was observed using positive (a and c) or negative (b and
d) sample ionization in either of the analysis rounds.

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 vitrification in assisted reproduction
were analyzed using ultra-performance LC with MS for oocyte populations, indicating that other metabolic dif-
global metabolomic profiling, and then 10 μL sample ali- ferences between the samples (e.g., patient-to-patient vari-
quots were transferred to microtubes and derivatized for ability or analytical variation) were greater than those
amino acid analysis and analyzed by LC-MS (Figure 13.3). between the sample groups (Figure 13.4a and b). Although
The experimental design of this study involved microdrops univariate statistical analysis revealed a series of metabo-
being taken from 190 spent culture media from embryos lites, including tryptophan and phenylalanine, which were
that had been cultured from day 1 to day 3. Collection was statistically significantly different between these groups,
performed on day 3 by collecting 50 μL of culture medium. none remained significant after Bonferroni’s correction
The samples were collected from the fresh group (n = 59), for multiple testing. Common metabolites involved in
the vitrification group (n = 65), and the matched control embryo development, such as lactate and glucose, were
medium group (n = 66) in the LC-MS study. Metabolomic also checked, and no statistically significant differences
analysis was performed in two rounds due to the instru- were found between the fresh and vitrification groups
ment capacity limitations. In the first round (consisting of (Figure 13.5).
31 fresh and 31 vitrification group embryo media and 33 In the second round of experiments, 29 and 34
matched control spent media), hundreds of metabolites embryo culture media originating from fresh and vit-
(including amino acids) were compared and statistically rified oocytes, respectively, and 33 matched controls
analyzed for differences between the fresh and vitrified were analyzed in the same way. Again, multivariate data
outcome groups. After performing global metabolomic analysis techniques failed to find any differences between
and amino acid profiling, PCA failed to find any statisti- the fresh and vitrified oocyte populations (Figure 13.4b
cally significant differences between the fresh and vitrified and c). However, univariate statistical analysis did reveal

(a) D-Glucose (c) Lactate

0.35
0.055
0.3
0.05
Relative intensity (a.u.)

Relative intensity (a.u.)

0.25 0.045
0.2 0.04
0.15 0.035
0.1 0.03
0.05 0.025

0 0.02
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
Oocyte Oocyte
Fresh Vitrification

(b) L-Tryptophan (d) L-Phenylalanine

0.05 0.034

0.045 0.032
Relative intensity (a.u.)
Relative intensity (a.u.)

0.03
0.04
0.028
0.035
0.026
0.03
0.024
0.025 0.022

0.02 0.02
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
Oocyte Oocyte

Figure 13.5  (See color insert.) Distribution of measurements of (a) glucose, (b) tryptophan, (c) lactate, and (d) phe-
nylalanine in both fresh (green rhombuses) and vitrification groups (red rhombuses).

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 metabolic profile of day 3 embryos arising from vitrified oocytes
some potential markers that were statistically differ- for noninvasive embryo assessment in women
ent between the fresh and vitrified groups, but only one undergoing single embryo transfer. Fertil Steril
of these remained statistically significant after correc- 2010;94(2):535–42.
tion for multiple testing. To identify this metabolite, 8. Houghton FD, Leese HJ. Metabolism and developmen-
mass spectra recorded in the positive and negative ion tal competence of the preimplantation embryo. Eur J
modes were analyzed to determine the most likely par- Obstet Gynecol Reprod Biol 2004;115(Suppl. 1):S92–6.
ent ion m/z value, and the compound was identified as 9. Lane M, Gardner DK. Understanding cellular disrup-
2,5,7,8-tetramethyl-2-(2’carboxyethil)-6-hydroychroman tions during early embryo development that perturb
(alpha-CEHC). The exact mass information obtained viability and fetal development. Reprod Fertil Dev
was checked against the ChemSpider online databases 2005;17(3):371–8.
using the Human Metabolome, LipidMaps, and peptides 10. Zhao XM, Ren JJ, Du WH et al. Effect of vitrification
sub-databases. on promoter CpG island methylation patterns and
This clinical outcome is consistent with other reports: expression levels of DNA methyltransferase 1o, histone
perinatal data also corroborate the safety of the technol- acetyltransferase 1, and deacetylase 1 in metaphase II
ogy, showing no differences for the parameters analyzed mouse oocytes. Fertil Steril 2013;100(1):256–61.
in babies born after oocyte vitrification and those born 11. Emiliani S, Van den Bergh M, Vannin AS, Biramane
after fresh oocyte donation. Although some small dif- J, Englert Y. Comparison of ethylene glycol, 1,2-pro-
ferences in individual metabolites are found, embryos panediol and glycerol for cryopreservation of slow-
developed from vitrified oocytes and those originating cooled mouse zygotes, 4-cell embryos and blastocysts.
from fresh oocytes do not significantly differ in their Hum Reprod 2000;15(4):905–10.
spent medium global metabolomic profiles. This suggests 12. Noyes N, Porcu E, Borini A. Over 900 oocyte cryo-
that the vitrification protocol does not disturb the general preservation babies born with no apparent increase
metabolism of these embryos, a finding that is also sup- in congenital anomalies. Reprod Biomed Online
ported by the clinical results. 2009;18(6):769–76.
13. Bernschtein AD, Petropavlovsky VV. Influence of
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1. Sakkas D, Gardner DK. Noninvasive methods to Biol Med 1937;III:21–2.
assess embryo quality. Curr Opin Obstet Gynecol 14. Trounson A, Mohr L. Human pregnancy following
2005;17(3):283–8. cryopreservation, thawing and transfer of an eight-
2. Wong CC, Loewke KE, Bossert NL et al. Non-invasive cell embryo. Nature 1983;305(5936):707–9.
imaging of human embryos before embryonic 15. Cobo A, Diaz C. Clinical application of oocyte
genome activation predicts development to the blas- vitrification: A systematic review and meta-anal-
tocyst stage. Nat Biotechnol 2010;28(10):1115–21. ysis of randomized controlled trials. Fertil Steril
3. Meseguer M, Rubio I, Cruz M, Basile N, Marcos  J, 2011;96(2):277–85.
Requena A. Embryo incubation and selection in a 16. Stoop D, De Munck N, Jansen E et al. Clinical vali-
time-lapse monitoring system improves pregnancy dation of a closed vitrification system in an oocyte-
outcome compared with a standard incubator: A donation programme. Reprod Biomed Online
retrospective cohort study. Fertil Steril 2012;98(6):​ 2012;24(2):180–5.
1481–9.e10. 17. Rienzi L, Cobo A, Paffoni A et al. Consistent and pre-
4. Scott R, Seli E, Miller K, Sakkas D, Scott K, Burns DH. dictable delivery rates after oocyte vitrification: An
Noninvasive metabolomic profiling of human embryo observational longitudinal cohort multicentric study.
culture media using Raman spectroscopy predicts Hum Reprod 2012;27(6):1606–12.
embryonic reproductive potential: A prospective 18. Cobo A, Garrido N, Crespo J, Jose R, Pellicer A.
blinded pilot study. Fertil Steril 2008;90(1):77–83. Accumulation of oocytes: A new strategy for manag-
5. Seli E, Sakkas D, Scott R, Kwok SC, Rosendahl SM, ing low-responder patients. Reprod Biomed Online
Burns DH. Noninvasive metabolomic profiling 2012;24(4):424–32.
of embryo culture media using Raman and near- 19. Patrizio P, Fragouli E, Bianchi V, Borini A, Wells D.
infrared spectroscopy correlates with reproductive Molecular methods for selection of the ideal oocyte.
potential of embryos in women undergoing in vitro Reprod Biomed Online 2007;15(3):346–53.
fertilization. Fertil Steril 2007;88(5):1350–7. 20. Katz-Jaffe MG, Gardner DK, Schoolcraft WB.
6. Vergouw CG, Botros LL, Roos P et al. Metabolomic pro- Proteomic analysis of individual human embryos to
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embryo viability: A novel, non-invasive method for ity. Fertil Steril 2006;85(1):101–7.
embryo selection. Hum Reprod 2008;23(7):1499–504. 21. Ellis DI, Goodacre R. Metabolic fingerprinting in dis-
7. Seli E, Vergouw CG, Morita H et  al. Noninvasive ease diagnosis: Biomedical applications of infrared and
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22. Singh R, Sinclair KD. Metabolomics: Approaches to 34. Trimarchi JR, Liu L, Porterfield DM, Smith PJ,
assessing oocyte and embryo quality. Theriogenology Keefe DL. Oxidative phosphorylation-dependent
2007;68(Suppl. 1):S56–62. and -independent oxygen consumption by indi-
23. Oliver SG, Winson MK, Kell DB, Baganz F. Systematic vidual preimplantation mouse embryos. Biol Reprod
functional analysis of the yeast genome. Trends 2000;62(6):1866–74.
Biotechnol 1998;16(9):373–8. 35. Manes C, Lai NC. Non-mitochondrial oxygen utiliza-
24. Ellis DI, Dunn WB, Griffin JL, Allwood JW, Goodacre tion by rabbit blastocysts and surface production of
R. Metabolic fingerprinting as a diagnostic tool. superoxide radicals. J Reprod Fertil 1995;104(1):69–75.
Pharmacogenomics 2007;8(9):1243–66. 36. Gardner DK, Lane M. Alleviation of the ‘2-cell block’
25. Kell DB. Revolutionary ideas come round again. and development to the blastocyst of CF1 mouse
Nature 1999;397(6721):644. embryos: Role of amino acids, EDTA and physical
26. Urbanczyk-Wochniak E, Luedemann A, Kopka J parameters. Hum Reprod 1996;11(12):2703–12.
et al. Parallel analysis of transcript and metabolic pro- 37. Seli E, Botros L, Sakkas D, Burns DH. Noninvasive
files: A new approach in systems biology. EMBO Rep metabolomic profiling of embryo culture media
2003;4(10):989–93. using proton nuclear magnetic resonance cor-
27. Johnson HE, Broadhurst D, Goodacre R, Smith AR. relates with reproductive potential of embryos in
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28. Conaghan J, Handyside AH, Winston RM, Leese HJ. 38. Brison DR, Hollywood K, Arnesen R, Goodacre
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Noninvasive assessment of human embryo nutrient 39. Nicholson JK, Lindon JC, Holmes E. Metabolomics:
consumption as a measure of developmental poten- Understanding the metabolic responses of living sys-
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30. Dominguez F, Gadea B, Esteban FJ, Horcajadas JA, statistical analysis of biological NMR spectroscopic
Pellicer A, Simon C. Comparative protein-profile data. Xenobiotica 1999;29(11):1181–9.
analysis of implanted versus non-implanted human 40. Sanchez-Ribas I, Riqueros M, Vime P et  al.
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31. Lenz EM, Wilson ID. Analytical strategies in metabo- 21 human preimplantation embryos. Fertil Steril
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32. Beckwith-Hall BM, Nicholson JK, Nicholls AW et al. 41. Hardarson T, Ahlstrom A, Rogberg L et  al. Non-
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Res Toxicol 1998;11(4):260–72. 42. Dominguez F, Castello D, Remohi J, Simon C, Cobo
33. Thompson JG, Partridge RJ, Houghton FD, Cox CI, A. Effect of vitrification on human oocytes: A meta-
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Fertil 1996;106(2):299–306.

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14 Vitrification of human oocytes for in vitro fertilization patients
Laura Rienzi, Benedetta Iussig, and Filippo Maria Ubaldi

INTRODUCTION option to embryo freezing to avoid potential entanglement

Since the first successes obtained in the second half of the in case of divorce and separation of couples; (iv) IVF pro-
twentieth century,1,2 human sperm freezing and embryo crastination due to no available/inadequate sperm sample;
cryopreservation are today considered indispensable tech- and (v) reported previous implantation failures with good-
niques available to all patients who utilize IVF therapy, quality embryos.4,19,20
such that they are routinely performed, and account for As already mentioned, oocyte cryopreservation proto-
a large percentage of assisted pregnancies. By contrast, cols have evolved considerably over the years in terms of
oocyte cryopreservation has been largely neglected, mostly type and concentration of cryoprotectants used, such as
due to the remarkable technical difficulties related to its cooling rates shifting from “slow freezing” to the more
special cell structure and sensitivity. Nevertheless, since effective “vitrification” methodology. Briefly, whereas
the first human pregnancy reported from frozen eggs,3 slow freezing involves the gradual cell dehydration
considerable efforts have been made to improve on a achieved with the combination of low cryoprotectant
suboptimal protocol. In this regard, the 2004 Italian Law concentrations and slow cooling rates, the principle of
No.  40 that regulates assisted reproductive technology vitrification relies on the initial exposure to high concen-
(ART) is considered a key turning point. In fact, the limit trations of cryoprotectant agents, followed by single-step
of inseminating no more than three oocytes per IVF cycle, ultra-rapid cooling in liquid nitrogen (−196°C) in order
coupled with the legal prohibition against cryopreserving to achieve a solid glass-like state. The introduction of vit-
supernumerary embryos for reasons other than serious, rification has dramatically increased cryopreservation
documented, and not predictable adverse effects affect- efficiency in terms of survival and, more importantly,
ing women’s health, obliged embryologists to invest time pregnancy rates, such that it is today considered to be the
and energy in developing effective cryopreservation meth- method of choice to preserve both gametes and embryos,
ods.4 Fortunately, all of these efforts have ultimately been being finally freed of the restrictive label “experimental.”20
rewarded with excellent results,5–18 such that currently
IVF clinics worldwide are able to enjoy the benefits of this OOCYTE VITRIFICATION IN INFERTILITY
amazingly useful procedure. In fact, apart from restrictive PROGRAMS: RESULTS
legislation and/or ethical concerns, oocyte cryopreserva- As already mentioned, the advent of vitrification
tion is of utmost importance in many aspects of ART, offer- improved many aspects of the entire oocyte cryopreserva-
ing several valid solutions to clinical, logistical, and social tion procedure, while at the same time being reassuringly
problems. First of all, ovum donation programs allow a harmless.21 Notably, recent reports revealed that vitrifi-
more convenient, flexible, and cost-effective approach to cation does not compromise in vitro development and
the coordination between donor and recipient. Moreover, clinical outcomes, being similar to that of its fresh coun-
these programs can be precious tools for fertility preser- terparts.8–14 Unfortunately, many of the studies published
vation both for medical indications (e.g., cancer patients so far refer to ovum donation programs or extremely
undergoing gonadotoxic chemotherapy/radiotherapy and good-prognosis patients, and are therefore strongly
eventual oophorectomy, and for women suffering from biased because of the high-quality oocytes involved.8–11
premature ovarian failure arising from certain genetic dis- The study of the standard infertile population must then
orders) or for social reasons (e.g., for preserving fertility in be considered imperative in order to confirm and validate
post-pubertal females without a partner, and to preserve the clinical value of the technique.
fertility for women approaching menopause). Last but not To our knowledge, the first prospective randomized clin-
least, oocyte cryopreservation may provide useful alter- ical trial conducted in an infertile population establishing
natives and advantages in infertility programs. In fact, it the efficacy of the vitrification procedure for maintaining
allows oocyte cryopreservation in the following cases: (i) oocyte competence to develop in vitro was designed by our
collection of supernumerary eggs to keep for future use, group in 2010.12 This noninferiority trial actually aimed to
thus avoiding the hormonal treatment necessary to sus- compare the in vitro performance of fresh and vitrified–
tain multiple follicular growth; (ii) risk of ovarian hyper- warmed sibling oocytes obtained in consecutive intra-
stimulation syndrome, when ethical concerns and/or legal cytoplasmic sperm injection  (ICSI) cycles in a standard
restrictions limit embryo freezing; (iii) as an alternative infertility program between September 2008 and March

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2009 (thus before the edict of the Italian Constitutional of this particular study, Rienzi and colleagues observed
Court that radically changed the horizon of IVF in Italy a promising trend in the vitrified–warmed group, with a
by definitively allowing embryo freezing). All of the 30% ongoing clinical pregnancy rate and 17.2% ongoing
patients involved were not older than 42 years of age and implantation rate.12
yielding more than six normal-appearing mature oocytes A subsequent prospective longitudinal cohort study
were undergoing ICSI treatment with an ejaculated sperm from our group actually confirmed the in vitro outcomes
count of >500,000 motile spermatozoa/mL (in order to and further focused on clinical results.13 In particular,
exclude negative paternal effect). According to Italian the authors stated a negative trend in the overall clinical
Law No. 40, only three oocytes per patient were injected, outcomes when embryos derived from vitrified oocytes
with the remaining been vitrified according to the proto- were transferred, but there was a very high cumulative
col described by Kuwayama and colleagues.22,23 In order effect on IVF success, comparable to those obtained with
to limit potentially confounding factors, extra care was fresh and cryopreserved embryos25 and more evident in
taken during the oocyte manipulation to minimize extra young women. It is important to note that, in this study,
stress, namely: (i) long exposure to 4-(2-hydroxyethyl)- the “fresh” and “vitrification” groups could not be directly
1-piperazineethanesulfonic acid (HEPES)-buffered media compared: first of all, the second group included all of the
with uncertain temperature control (denudation was car- patients who did not become pregnant in the fresh cycle,
ried out in a closed chamber with temperature plus gas thus potentially revealing a different prognosis. Moreover,
control, with rapid oocyte morphology evaluation by the gonadotropin stimulation and superovulation rather than
stereomicroscope at 40× magnification); (ii) prolonged spontaneous hormonal cycle and surge may account for
denuded oocytes in in vitro culture (denudation applied the different endometrial receptivity. As a consequence,
immediately before treatment); and (iii) oocyte aging the clinical data must be considered in a larger prospec-
(ICSI was performed at 2 hours following warming). As tive trial, and vitrification should be proposed in order to
a consequence, the only difference eventually observed increase the chances of cumulative success.13
between fresh and vitrified oocytes was the vitrification Beyond pregnancy and implantation rates, the true
procedure itself. outcome in IVF is undoubtedly the delivery of a healthy
A total of 124 patients from 124 ICSI cycles met the baby. In this regard, the overall efficiency and consis-
above inclusion criteria (33.8% of treated couples). Fifty tency of oocyte vitrification have been investigated in a
four patients became pregnant in the fresh cycle, and recent observational cohort multicentric study, conducted
therefore were not involved in the warming cycle during between October 2006 and April 2010 in three different
the study period. Of the remaining 70 patients, 40 under- European clinics (GENERA [Gynecology, Endocrinology,
went a first-round warming cycle, for a total of 124 oocytes Embryology and Reproductive Medicine]—Rome, Italy;
warmed. The survival rate was 96.7% (120/124 oocytes Ca’ Granda Ospedale Maggiore Policlinico—Milano,
survived), confirming the superiority of vitrification over Italy; IVI—Valencia, Spain) and involving patients of less
slow freezing as already cited.8,24 The fertilization rate per than 43 years of age enrolled for standard infertility pro-
injected oocyte was not significantly different between grams.15 A total of 486 warming cycles in 450 couples were
fresh (83.3%) and vitrified–warmed sibling oocytes performed, accounting for 2721 vitrified oocytes warmed.
(79.2%); given the high survival rate obtained with the vit- This large study provides three levels of informative data.
rification procedure, the number of degenerated eggs was First of all, the in vitro outcomes (i.e., survival, fertiliza-
negligible and did not affect the overall fertilization rate tion, and embryo development rates) and clinical data
(76.6% per warmed oocytes). Even if a slightly decreased (i.e., pregnancy and implantation rates) were found to be
normal 2 pronuclei (2PN) morphology rate was observed consistent with previously published studies.8–13 Moreover,
in the vitrified–warmed group (which did not reach sta- no significant differences among the three centers were
tistical significance), the cleavage rate and embryo quality observed, except for the direct consequences of different
were similar. In fact, day 2 embryo development and top- legal restrictions (i.e., the number of oocytes injected and
quality embryos were respectively 100% and 52% using the blastocyst stage transfer policy). Finally, the third and
fresh oocytes and 97.9% and 51.6% using their warmed probably most interesting finding consists of the evaluation
oocyte counterparts. Importantly, the evaluation of the of the different factors predictive of a healthy birth. In fact,
vitrification effectiveness should rely on the assessment the logistic regression analysis enabled the identification of
of pregnancy and delivery rates, rather than on merely three elements influencing the final IVF outcome, namely:
in vitro outcomes. In fact, the cryopreservation procedure (i) the maternal age; (ii) the number of vitrified metaphase
may induce much sublethal cellular damage that is not II oocytes; and (iii) the day of transfer after fertilization.
routinely appreciated in an IVF laboratory, but affects Furthermore, the recursive partitioning analysis permitted
embryo development and viability in different and at the design of an intuitive decision-making model able to
later developmental stages, thus leading to implantation predict the probability of delivery according to the above-
failures or miscarriages. Even if the evaluation of preg- mentioned patient characteristics, thus being extremely
nancy and implantation rates was beyond the purpose useful for proper counseling and patient selection. First of

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all, this study established an inverse correlation between In our opinion, the striking results obtained in this study
maternal age and delivery, with a decrease of 7% in delivery are extremely important: first of all, they underline the
rate for each year of increase in maternal age. Moreover, it high effectiveness of vitrification even in poor-prognosis
was calculated that the delivery rate doubles (from 22.6% to infertile patients. Additionally, the clear benefit in terms
46.4%) in cases in which more than eight oocytes were vit- of reduced anxiety levels, coupled with the lower cycle
rified, if the other two variables were fixed. In cases of less cancellation rate, is a major recommendation for con-
than eight oocytes being vitrified, the outcomes are reduced tinuing this type of approach, rather than switching too
for women aged more than 38 years (12.6% versus 27.5%). readily to ovum donation.
Finally, in cases of more than eight available vitrified
oocytes, there was clearly an increased potential for blas- DISCUSSION
tocyst stage transfer to be performed (62.1% versus 40.7%). Oocyte cryopreservation is considered a major tech-
The age-specific probability of live birth with cryo- nique that is offered in IVF clinics worldwide, regardless
preserved oocytes has been more recently estimated by of differing national legislations. In fact, its broad appli-
Pelin and colleagues26 through an individual patient data cability encompasses ovum donation and infertility pro-
meta-analysis. The authors propose 36 years as the upper grams incorporating medical and social egg freezing. The
age limit to accurately counsel patients for a reasonable advent of vitrification for oocyte cryopreservation, finally
chance of success, even if conception and live birth can freed of the label “experimental,”20 represents a significant
occur as late as 44 years of age. In our opinion, these improvement over previous protocols, thus presenting
data confirm the value of egg cryopreservation, despite new opportunities for therapy in assisted reproduction.
the established and distinct age-related decline that will However, in spite of the recent excellent successes, its clini-
occur after any estimated cutoff age. So taken together, cal application is still quite restricted, and is often applied
this information, far from being dogmatic, should be to just a select patient population, namely young women
regarded as a significant instrument for appropriate often enrolled in ovum donation programs. Actually, it has
counseling and informed decision-making. been well established that its high effectiveness can be used
Notably, all of the studies described above assessed the even in the treatment of infertile patients with different
effectiveness of the vitrification procedure in normally prognoses, enabling the achievement of high cumulative
responding women. However, a substantial subpopula- pregnancy and live birth rates.12,13,15,17 Among the various
tion of infertile patients is represented by low responders, factors influencing the outcomes of oocyte vitrification are
namely patients with a low number of oocytes retrieved advanced maternal age, as well as the number and qual-
and suboptimal oocyte maturation and embryo quality, ity of oocytes retrieved, and these remain critical issues to
factors that invariably lead to higher cycle/transfer can- be assessed for these women.15,17,26 Nevertheless, proper
cellation rates. The difficult clinical management of these patient selection coupled with an appropriate counsel-
patients has promoted the evaluation of oocyte accumu- ing and a suitable transfer often enable achievement of
lation through vitrification as an alternative option to remarkable results for these poor-prognosis patient cat-
­disappointing stimulation regimens.17 A recent prospec- egories. It is therefore our firm opinion that the overall
tive study conducted between January 2007 and December efficiency of oocyte vitrification justifies its application in
2009 by the IVI group (Spain) enrolled 724 low-responder routine IVF therapy.
patients (≤5 oocytes retrieved per stimulation cycle; fol-
licular stimulating hormone (FSH) > 11 IU/mL on cycle REFERENCES
day 3; antral follicular count (AFC) < 6 among both ova- 1. Sherman J. Synopsis of the use of frozen human
ries; antimullerian hormone (AMH) < 5 pmol/L) divided semen since 1964: State of the art of human semen
into two groups: 242 patients (594 cycles) who decided to banking. Fertil Steril 1973;24:397–412.
accumulate oocytes through vitrification for later insem- 2. First baby born of frozen embryo. New York Times
ination (LR [low responder]-Accu-Vit) and 482 patients April 1984. http://www.nytimes.com/1984/04/11/us/
(587 cycles) who preferred to undergo standard ovarian first-baby-born-of-frozen-embryo.html.
stimulation and subsequent embryo transfer (LR-fresh). 3. Chen C. Pregnancy after human oocyte cryopreserva-
Notably, there were observed no statistically significant tion. Lancet 1986;1:884–6.
differences between fertilization, cleavage, and top- 4. Benagiano G, Gianaroli L. The new Italian IVF legis-
quality embryo rates between the two groups. On the lation. Reprod Biomed Online 2004;9:117–25.
contrary, there was a fourfold lower cancellation rate in 5. Borini A, Bonu MA, Coticchio G et  al. Pregnancies
the LR-Accu-Vit group (9.1%) compared to the LR-fresh and births after oocyte cryopreservation. Fertil Steril
group (34%), consequently leading to a higher cumula- 2004;82:601–5.
tive live birth rate (36.4% versus 23.7%), in spite of similar 6. Borini A, Lagalla C, Bonu MA et al. Clinical outcome
implantation and live birth rates per transfer. Moreover, of oocyte cryopreservation after slow cooling with a
a positive trend was observed with cryo-embryo trans- protocol utilizing a high sucrose concentration. Hum
fers, which further increased the cumulative outcomes. Reprod 2006;21:512–7.

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7. De Santis L, Cino I, Rabellotti E et al. Oocyte cryo- protocol: Is it really the time to trash the cryopreser-
preservation: Clinical outcome of slow-cooling pro- vation machine? Fertil Steril 2012;97:1101–7.
tocols differing in sucrose concentration. Reprod 17. Cobo A, Garrido N, Crespo J et al. Accumulation of
Biomed Online 2007;14:57–63. oocytes: A new strategy for managing low-responder
8. Cobo A, Kuwayama M, Perez S et al. Comparison of patients. Reprod Biomed Online 2012;24:424–32.
concomitant outcome achieved with fresh and cryo- 18. Cobo A, Garcia-Velasco JA, Domingo J et  al. Is vit-
preserved donor oocytes vitrified by the Cryotop rification of oocytes useful for fertility preservation
method. Fertil Steril 2008;89:1657–64. for age-related fertility decline and in cancer patients?
9. Nagy ZP, Chang CC, Shapiro DB et al. Clinical evalu- Fertil Steril 2013;99:1485–95.
ation of the efficiency of an oocyte donation program 19. Heng BC. Oocyte cryopreservation as an alternative
using egg cryo-banking. Fertil Steril 2009;92:520–6. to embryo cryopreservation—Some pertinent ethical
10. Schoolcraft WB, Keller JL, Schlenker T. Excellent concerns. Reprod Biomed Online 2007;14:64–72.
embryo quality obtained from vitrified oocytes. 20. The Practice Committees of the American Society for
Reprod Biomed Online 2009;19:820–3. Reproductive Medicine and the Society for Assisted
11. Cobo A, Meseguer M, Remohi J et  al. Use of cryo- Reproductive Technology. Mature oocyte cryopreser-
banked oocytes in an ovum donation programme: vation: A guideline. Fertil Steril 2013;99:37–43.
A prospective, randomized, controlled, clinical trial. 21. Chian RC, Huang JY, Tan SL et al. Obstetric and peri-
Hum Reprod 2010;25:2239–46. natal outcome in 200 infants conceived from vitrified
12. Rienzi L, Romano S, Albricci A et al. Embryo devel- oocytes. Reprod Biomed Online 2008;16:608–10.
opment of fresh ‘versus’ vitrified metaphase II oocytes 22. Kuwayama M, Vajta G, Kato O et al. Highly efficient
after ICSI: A prospective randomized sibling-oocyte vitrification method for cryopreservation of human
study. Hum Reprod 2010;25:66–73. oocytes. Reprod Biomed Online 2005;11:300–8.
13. Ubaldi F, Anniballo R, Romano S et  al. Cumulative 23. Kuwayama M. Highly efficient vitrification for cryo-
ongoing pregnancy rate achieved with oocyte vitri- preservation of human oocytes and embryos: The
fication and cleavage state transfer without embryo Cryotop method. Theriogenology 2007;67:73–80.
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Reprod 2010;25:1199–1205. tion. Hum Reprod Update 2007;13:591–605.
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15. Rienzi L, Cobo A, Paffoni A et al. Consistent and pre- 26. Pelin A, Bang H, Oktay K. Age-specific probabil-
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freezing using a 0.2-0.3M sucrose concentration

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15 Oocyte vitrification: Donor “egg banking”
Zsolt Peter Nagy, Ana Cobo, and Ching-Chien Chang

HISTORY AND INDICATIONS FOR OOCYTE is similar to “frozen egg” donation through donor egg
DONATION banks, including: (1) donor recruitment and the type of
In vitro fertilization (IVF) has been an established treat- donors; (2) donor screening and testing; (3)  matching
ment option for couples challenged by infertility since donors to recipients; (4) donor stimulation/egg retrieval
1978.1 However, some couples may not be able to conceive procedures; (5) insemination of donor oocytes/embryo
using their own eggs. Most frequently, the reason is the culture; and (6) preparation of the recipient for embryo
advanced maternal age when no eggs or no viable eggs can transfer. However, there are also a few critically impor-
be obtained. Less frequently, menopause can start early tant differences between fresh and “frozen egg” dona-
due to premature ovarian failure. Additionally, ovaries tions, including: (7) price of treatment; (8) available
can be dysfunctional due to certain medical conditions, or number of donor eggs (and embryos); (9) waiting time;
as a result of treatment against cancer. In all of these situ- and (10) need for “synchronization.”
ations, using donor egg is indicated. In some genetically
inheritable conditions (present in the wife/female part- 1. Donors are typically recruited either by a donor
ner), the couple may also opt for donor oocytes, instead of agency or by the IVF clinic (donor egg banks typi-
using their own.2 cally act as a “combination” of a donor agency and
The first pregnancy using a donor egg was reported by IVF clinic—initially being part of an IVF program,
the Monash group in 1983.3 Within a short period of time, then it may develop into a separate organization to
egg donation was introduced in several countries and recruit donors specifically for a given egg bank).
became an established part of routine IVF treatment.4–9 It Donors can be “known” (where the recipient knows
is of importance to note that there are a number of coun- the identity of the donor) or “unknown” (where the
tries where gamete donation is regulated by law, and can identity of the donor is not known to the recipi-
forbid it completely (like Germany currently), or certain ent). Some countries require by law that even an
restrictions are imposed in many countries, as in the unknown donor should provide their identity to
UK, Italy, Australia, France, and Sweden,10–12 which are the offspring at a certain age when conceived using
just a few examples. On the other hand, there are some their gametes, such as in the UK.14 Depending on
countries where there is no or very minimal regulation a country’s legislation, a donor can be paid, be
on gamete (oocyte) donation. In these countries, the compassionate (not paid), or compensated only
availability of egg donors typically satisfies the demand minimally. “Altruistic” donors are typically a rela-
of the recipients, while in countries with different restric- tive or friend of a patient who requires donor eggs,
tions, typically the demand for egg donors is much higher or may be an IVF patient who is willing to donate
than the availability of donors. As an example, in the some of her own eggs to a recipient (typically for
United States, where there is no specific federal regula- little or no compensation). The amount of compen-
tion on egg donation, about 10,000 treatment cycles were sation for “paid donors” is not restricted in some
performed using egg donations in 2012, as reported by countries and it is mainly dictated by the “market
the Society of Assisted Reproductive Technology, corre- demand,” such as in the United States, Spain, and
sponding to approximately 10% of all IVF cycles (https:// Greece—though typically there is a “historical
w w w.sartcorsonline.com/rptCSR _PublicMultYear. range,” considered to be the highest in the United
aspx?ClinicPKID=0). States, but somewhere between $3,000 and $12,000
per donation.15
SIMILARITIES AND DIFFERENCES BETWEEN 2. Donors are typically “pre-selected” based on dif-
FRESH AND CRYO-BANK DONATIONS ferent physical parameters including: age (in the
Historically, oocyte donations have been performed United States, it is typically younger than 30 years
“fresh,” due to “technical challenges” with egg cryopreser- of age; in Spain, it is younger than 35 years of age);
vation (which will be discussed later on),6 but lately, there body mass index (BMI); personal history, includ-
has been a rapid increase in the use of recently established ing educational level; and medical and family his-
donor egg banks as an alternative option.13 Obviously, tory are also typically recorded, and may play a
there are several aspects in which fresh egg donation role in further selection of the donor. Psychological

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 vitrification in assisted reproduction
evaluation, in addition to medical physical exami- avoided, while maintaining the number and qual-
nation, is typically performed. Genetic testing for ity of oocytes retrieved.18,19 Because egg donors are
the most frequent inheritable genetic diseases (e.g., most at risk for OHSS (due to their young age and
alpha-thalassemia, beta-thalassemia, cystic fibro- tendency to respond overly well to follicle stimulat-
sis, fragile X syndrome, mucolipidosis, sickle cell ing hormone [FSH]), it is now becoming standard
disease, spinal muscular atrophy, and Tay–Sachs to use only the “antagonist/agonist protocol” for all
disease, among others) is also performed. Finally, egg donors.
testing for different infectious diseases is also per- 5. Insemination of donor oocytes and embryo cul-
formed (such as for Treponema pallidum [syphi- ture is performed similarly to other IVF cases.20,21
lis], gonorrhea, West Nile virus, chlamydia, human Depending on the sperm parameters of the partner
immunodeficiency virus [HIV]-1, HIV-2, hepatitis of the recipient, conventional insemination may
C virus [HCV], hepatitis B virus [HBV] [hepatitis be performed, or intracytoplasmic sperm injec-
B core antibody (HBcAb) and Immunoglobulin G tion (ICSI) if sperm parameters are suboptimal.22
(IgG) + Immunoglobulin M (IgM)], HBV [hepatitis If cryopreserved donor oocytes are used, then
B surface antigen (HBsAg)] as required in the United ICSI is the standard procedure for insemination.23
States by the Food and Drug Administration).16 Embryo culture is standard in vitro fertilization
Similar infectious testing is required in the European (IVF) procedure, which is typically performed
Union (EU) based on the EU tissue directives.17 in microdroplets for 3–5 days after insemination,
3. Matching donors to recipients is an important pro- when morphologically adequate embryo(s) are
cess that usually involves both the recipient and the transferred and or cryopreserved.23
donor “agent” (a “donor nurse” from an IVF clinic 6. Preparation of the recipient for embryo transfer is a
or “donor agent” if it is a donor agency or donor egg highly standardized procedure. Uterine preparation
bank). Recipients usually take into consideration is typically carried out using a oral or transdermal
the physical characteristics of the donor, preferen- estrogen or estradiol valerate 6 mg intramuscularly
tially resembling the patient—but educational level, (IM) every 3 days. Vaginal progesterone (Crinone 8%;
medical history, and genetic information are also 90 mg twice a day) or IM progesterone (50 mg once
important factors in the decision-making. On many daily) starts on day 15 of estrogen therapy. Embryo
occasions, there is a website of the clinic or agency, transfer is performed on day 4 (if c­leavage-stage
or the donor egg bank, where available donors are embryo[s] are transferred) or on day 6 of progester-
posted and the recipient can browse the information one therapy (if blastocyst-stage embryo[s] are trans-
to help them select their preferred donor (some exam- ferred). Pregnant recipients continue to receive the
ples: http://www.bhed.com/; http://www.thedonor- same dosages of progesterone and estrogen until an
source.com/; https://myeggbank.com/; https://www. estimated gestational age of 10 weeks.24
donoreggbankusa.com; http://www.theworldegg-   As mentioned earlier, there are some aspects in
bank.com/; http://www.ivi-fertility.com/en/patients/ which there is a major difference between “fresh”
assisted-reproduction-treatments/egg-donation/).  and “frozen” donor egg treatment.
4. Donor ovarian stimulation is performed similarly 7. The price of treatment is typically lower when
to that which an IVF patient undergoes, including oocyte donation is performed through a donor
the use of medications that help to develop several egg bank, using cryopreserved oocytes, compared
follicles at the same time (called follicle-stimulating to “fresh donation.” The reason for this is that egg
hormone [FSH]). When follicles are at the adequate donors are typically producing a larger number of
size (typically, the leading follicles should be over usable oocytes, which are divided among several
17–19 mm in diameter), the final maturation is trig- recipients in the donor egg bank setting, while
gered by an human chorionic gonadotropin (hCG) during fresh donation, typically all eggs from one
injection, and 35–37 hours later, oocytes are aspi- donor are donated to one recipient. Therefore, the
rated from the follicles surgically under sedation. cost of a donor cycle (which includes donor com-
A potential risk of ovarian stimulation is ovarian pensation, testing, screening, medications, IVF
hyperstimulation syndrome (ovarian hyperstimu- costs, etc.) is split between several recipients, result-
lation syndrome [OHSS]; symptoms include diz- ing in significant savings when using a donor egg
ziness/nausea, abdominal bloating, pain in the bank, compared to a fresh donation cycle. There are
abdomen, sudden weight gain, decreased urination, of course some exceptions to this situation: during
and shortness of breath). Since OHSS can be severe, a fresh donation, the eggs from one donor can also
to the point that the patient needs hospitalization, it be split among more recipients, which also makes
is a priority to prevent it. Using the antagonist stim- the donor egg cycle cheaper; however, it becomes
ulation protocol with an agonist trigger (instead of more challenging to synchronize multiple recipi-
hCG), it has been demonstrated that OHSS can be ents with one donor (see below). Alternatively, it is

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 oocyte vitrification
also possible that a donor egg bank is doing “one- only easier to find an acceptable match, but the
to-one” donation (i.e., all eggs cryopreserved from treatment can start within weeks, with no need to
one donor are given to only one recipient), such as wait for the donor to start her stimulation.
is done in some European donor egg banks (e.g., 10. The need for “synchronization” is a clear challenge
Instituto Valenciano de Infertilidad [IVI]). This lat- when using fresh donation, not only for the rea-
ter approach can be explained (in part at least) by son of adjusting the stimulation of the donor with
the fact that donor costs in Spain are significantly the protocol of the recipient, but also in terms of
lower than in the United States; therefore, splitting finding an acceptable time period when both the
donor eggs and donor costs would not result in sig- donor and recipient can undergo the procedures
nificant savings in that specific setting. at the same time. When using donor egg banking,
8. Available numbers of donor eggs (and embryos) both donors and recipients can choose the time
are typically also different in a donor egg bank set- that best fits them, as their treatment procedures
ting compared to fresh (one-to-one) donation. It are independent from each other. Because many
is typical that a recipient receives between five to of the donors are students, they have more specific
eight mature, cryopreserved oocytes from a donor times of the year when they can donate more con-
egg bank, which limits the number of embryos as veniently; and similarly, recipients can also choose
well. During fresh donation, on the other hand, the time period that best fits their schedules. This
commonly all mature eggs are provided to a convenience factor makes donor egg banking so
recipient, usually resulting in a larger number much more successful, in addition to the reduced
of embryos. For example, in a study by Nagy and financial burden with no wait time.
colleagues, when the same donors were used both
for fresh and for “cryo-egg” donation (in different CRYOPRESERVATION AS A TECHNOLOGICAL
donor stimulation cycles), the average number of REQUIREMENT
eggs was 23 and the average number of embryos Cryopreservation has been one of the basic IVF proce-
was 15 per recipient in the fresh donation. In the dures for several decades. Successful cryopreservation of
“cryo-egg” donation cycles, there were seven eggs human semen was reported in 1954.26 Cryopreservation
and six embryos per recipient.25 Importantly, in the of embryos was introduced in humans in 1983 by estab-
same study, there was no difference in the results, lishing a pregnancy using a frozen–thawed cleavage-stage
in that both implantation and clinical pregnancy embryo.27 Since that time, embryo cryopreservation has
rates were similarly high in both groups (“fresh” spread worldwide, and has now become a routine pro-
and “frozen” donor egg).25 The only significant dif- cedure. On the other hand, cryopreservation of human
ference was observed in the number of embryos oocytes has remained ambiguous. Although the first
cryopreserved: in the fresh donation group it was success with a cryopreserved egg was reported in 1986,28
17.3, and in the “cryo” donation group it was 1.6 just 3 years after embryo cryopreservation, the efficiency
per recipient.25 While cryopreserved, supernumer- remained low, making it unsuitable for routine clinical
ary embryos provide a “safety back-up” option if a use. For nearly two decades, there were only a few case
recipient does not get pregnant in the initial cycle reports reflecting the technical challenges with oocyte
(or if the recipient wishes to have more children), cryopreservation.29–31 The first report of pregnancies
most of the time recipients usually wish to have from cryopreserved donor eggs was in 1998 from a study
only one child, as most recipients are typically performed by Tucker et al.32 Nevertheless, there were very
in their forties, when an extended family is not few births reported using cryopreserved oocytes during
planned. This therefore means that the number of these decades, and according to Noyes and colleagues, 33
extra embryos after “cryo-egg” donation is mini- there were fewer than a thousand babies born during the
mized, which ultimately means fewer ethical/moral period of 1986–2008 from cryopreserved eggs.
issues for patients (and clinicians) as to the fate of These low success rates may be attributed to two main
those frozen embryos. causes: (1) the type of cryopreservation technique applied;
9. The waiting time for a treatment cycle is usually and (2) the sensitivity of human oocytes to cryopreserva-
different as well when using a “fresh” donor com- tion. Based on the initial relatively high success rates with
pared to a donor egg bank. Even in a country where embryos using slow freezing, the same technique was
there are no restrictions on egg donation (such as applied for oocyte cryopreservation as well. However, as
the United States), it may take several months to a has been demonstrated later, slow freezing is not the opti-
year to find a fresh donor that a recipient considers mal choice for egg cryopreservation. Secondly, human
to be an acceptable match. However, when using a oocytes are large cells with high cytoplasmic volumes and
donor egg bank, typically there is a wide range of with relatively small surfaces. Additionally, mature, meta-
donors to choose from, whose oocytes are already phase II (MII) oocytes have a strongly temperature-sen-
retrieved and stored cryopreserved. Thus, it is not sitive spindle that can easily be disrupted when exposed

131
 vitrification in assisted reproduction
to lower temperatures, even if just for a few minutes.34 professionals are using a vitrification protocol that is very
Moreover, the membrane properties of a MII oocyte similar to that which Kuwayama developed and referred
are substantially different from a similarly sized zygote, to as the “Kitazato” or “Cryotech” technique.55,56
possibly because of the type and density of aquaporin, a It has also been demonstrated that vitrification has less
protein channel that is responsible in part for water trans- impact on meiotic spindle organization of the oocyte than
port.35 It has also been described in animal models that slow freezing.57,58 Embryos obtained from slow-frozen
exocytosis of cortical granules may occur in response to oocytes, however, did not exhibit higher rates of chromo-
cryopreservation, 36 possibly resulting in the hardening somal abnormalities,38 despite the higher rate of abnor-
of the zona pellucida. This may lead to lower fertilization mal spindle morphology.59 Recently, it was also shown
rates when conventional insemination is performed after that embryos do not have higher aneuploidy rates when
thawing; and since ICSI was not developed until 1992,37 oocytes have been subjected to vitrification.60 Other stud-
this further added to the challenge of egg cryopreserva- ies, looking at the physiology, gene expression, or protein
tion. For these reasons, today, ICSI is regarded as a stan- composition of oocytes (or derived embryos), showed that
dard procedure for inseminating oocytes that have been vitrification preserves better oocyte function than slow
cryopreserved.38,39 Despite much research and protocol freezing.61–65 As a result of much improved outcomes fol-
modification, slow freezing remains largely inefficient for lowing oocyte cryopreservation (in particular using vitri-
human oocyte cryopreservation.40–44 fication), the American Society of Reproductive Medicine
has recently lifted the experimental label from the proce-
VITRIFICATION AS A SUPERIOR TECHNOLOGY dure (as was also recommended by some professionals66),
FOR OOCYTE CRYOPRESERVATION which has made it possible to introduce it in the clinical
Not until the introduction of a technique called vitrifi- routine for different purposes.67
cation in human oocyte cryopreservation45 was it pos-
sible to obtain improved results. During vitrification, the DONOR OOCYTE BANKING: STRUCTURE
same cryoprotectants are used as during slow freezing— AND FUNCTION
propanediol, dimethyl sulfoxide (DMSO), glycerol, and Having the ability to efficiently cryopreserve oocytes has
ethylene glycol (EG) (permeating cryoprotectants); and made it possible to use this technology for various indica-
glucose, sucrose, and Ficoll (nonpermeating cryoprotec- tions. Most professionals were expecting that the major
tants)—but at different concentrations. During vitrifica- use of egg cryopreservation would be for the purpose of
tion, a combination of cryoprotectants (most frequently fertility preservation (both for medical and social rea-
EG/DMSO plus sucrose) has a much higher concentration sons); however, the interest for this currently is still rela-
than that which is applied in slow freezing; however, the tively limited. On the other hand, many IVF patients are
exposure time to those solutions is much shorter.46–48 In now using the option of freezing part of their oocytes
addition to the higher concentration of cryoprotectants, a (instead of freezing embryos), and having only a small
very high speed of cooling has to be achieved in order to number of oocytes inseminated (elective limited insemi-
be able to obtain vitrification. Vitrification occurs when nation), to prevent having larger number of embryos
a solution becomes solid without crystallization; this is created, which later may create ethical/moral problems
also called as a “glass-like” state, which is fundamentally about what to do with them if they no longer wish to use
different than slow freezing, where the extracellular solu- them for reproduction purposes.68 Additionally, egg cryo-
tion crystallizes. To achieve the highest cooling rate, it is preservation may be used to rescue an IVF cycle when the
typical to use a “minimal volume” (such as 0.5 μL) on an husband/partner of the patient is unexpectedly not able
“open” carrier, such as the CryoTop or CryoLock, which to provide a semen sample on the day of egg retrieval.
ensures a cooling rate of approximately 20,000°C/min; However, the major use of oocyte cryopreservation cur-
this is in stark contrast to the cooling rate during slow rently is for the purpose of donor egg banking. This is not
freezing, where, at the most critical stage, a 0.3°C/min really surprising, if we consider that sperm donation has
cooling rate is provided by a programmable freezer.48,49 been done through frozen sperm banking for nearly four
Given the higher cryoprotectant concentrations decades.69,70 Similarly, now with the emergence of donor
applied during vitrification, there is a concern about the egg banks, one can expect that oocyte donation will tran-
potential toxic effects of cryoprotectants on the cell.50 sition from fresh to frozen eggs. This is facilitated by many
However, a recent study by Vanderzwalmen and col- factors. Firstly, many studies that investigated the vitri-
leagues elegantly demonstrated that intracellular cryo- fication technique were using donor oocytes in humans
protectant concentrations are actually higher after slow (it is usually easier to obtain oocytes from donors for
freezing compared to vitrification.51 research purposes), thus there is plenty of evidence avail-
When comparing outcomes of slow freezing with able on the efficient use of donor eggs, where results are
vitrification, it is clear that vitrification is a much more comparable to fresh donation.25,71–77 Additionally, there is
efficient approach for oocyte cryopreservation (and actu- growing evidence that cryopreservation/vitrification of
ally also for embryo cryopreservation).47,52–54 Today, most oocytes does not carry increased risks for birth defects of

132
 oocyte vitrification
babies born from the procedure, which is important from Table 15.1  Outcome Data of Donor Egg Bank USA
the safety point of view for the technique.33,77–80
Donor egg bank
Donor egg banks initially were developed within the
Criteria USA
framework of an already-existing IVF clinic to supply
their own recipients. Some of these clinics later expanded Donation cycles 2,192 egg lots,
their donor pools, creating a larger number of donors and 13,636 frozen eggs
cryopreserved donor eggs being available, which could be Recipient cycles 1,303
offered to other clinics and their patients. Looking at the Mean age of recipients 42.2 years
size, structure, and function of these donor egg banks, Total (mean ± SD) oocytes cryopreserved 13,636
there are potentially three different types: (1) a donor egg per donor (22.17 ± 0.82)
bank that is associated with a single IVF clinic, which only
Total (mean ± SD) oocytes warmed per 8,096 (6.21 ± 0.76)
supplies the recipients of the clinic; (2) a donor egg bank recipient
that is based on collaboration between several IVF clin-
Total (mean ± SD) oocytes survived per 6,966 (5.45 ± 1.61)
ics, and (most or all of) those IVF clinics contribute to the
recipient
“donor pool” by processing donors by performing donor
Total (mean ± SD) oocytes for 6,906 (5.30 + 1.61)
egg retrieval and egg cryopreservation; these cryopreserved
intracytoplasmic sperm insemination
donor eggs are then available for patients/recipients of any
of those clinics that are part of this association; and (3) a Average 2PN intracytoplasmic sperm 76.6%
insemination fertilization rate
donor egg bank with a single or multiple IVF center(s) that
are associated in order to allow donor egg retrieval and % of good-quality embryos on day 3 (per 64.7%
donor egg cryopreservation; these cryopreserved oocytes inseminated oocyte)
are then available to virtually any IVF clinics (and to any % of good-quality embryos on day 5 (per 60.5%
patients) who might wish to use them. This third type embryo subjected to extended culture)
of donor egg bank is probably the most similar to donor Implantation rate 33.6%
sperm banks in structure and function. One would expect Total embryos cryopreserved 1,133
that with further development of donor egg banks, they will Clinical pregnancies (rate/transfer) 49.1%
eventually more likely function like donor sperm banks. Infants born 547
As donor egg banking is a very recent development,
Embryo warming cycles with transfers 269
there are as yet no established procedures on reporting
(originating from cryopreserved donor
their results and obtaining other relevant information. For egg frozen embryos)
this reason, three well-known donor egg banks were con-
Clinical pregnancies from embryo 38.3%
tacted directly, and their outcome data were requested. It
warming cycles
is much appreciated that all three of them were willing to
Infants born from embryo warming 78
provide these data. It is also important to note that, due to
cycles
the differences of patient population, treatment approach,
structure/function, data collection, and reporting, none Source: Courtesy of Donor Egg Bank USA.
of these outcome data should be compared directly. Some
of the basic outcome data from Donor Egg Bank USA
are presented in Table 15.1. Some of the basic outcome
data from My Egg Bank North America are presented in Table 15.2  Outcome Data of My Egg Bank
Table 15.2. Some of the basic outcome data from IVI are North America
presented in Table 15.3.
When looking at some of these outcome parameters, it Egg donor cycles 1,035
is interesting to note that the different donor egg banks do Vitrified oocytes 23,060
have many similarities, even though they were developed Mean oocytes/donor 22.3
independently from each other, as referred to in an earlier Recipient cycles 3,424
publication.75
Oocytes warmed 21,462

SUMMARY Oocytes warmed/recipient 6.3

Donor egg banking is a newly emerged service providing Survival rate 88%
an alternative to fresh oocyte donation. The technological Fertilization rate 78%
base of this service is a recently developed, highly efficient Clinical pregnancies 1,781
oocyte cryopreservation process. Donor egg banking Clinical pregnancy rate 52%
provides several advantages over fresh oocyte donation,
Babies born (to date) 1352
such as no need for synchronization (independent sched-
ules for both the donor and recipient), no waiting (the Source: Courtesy of My Egg Bank North America.

133
 vitrification in assisted reproduction
Table 15.3  Outcome Data of IVI Donor Egg Bank a simplified oocyte donation programme—No more
embryo freezing, synchronisation or disruption of
Oocyte donation cycles 3,467
social life. Singapore Med J 1992;33(4):401–3.
Oocytes cryopreserved (total) 40,741 9. Nakamura MS, Maciel MC, Veiga A, Asch RH.
Oocytes cryopreserved/donor 11.8 Establishing a program of oocyte donation in Brazil.
Number of embryo transfers/donation 3,050/3,382 (90.1%) Fertil Steril 1992;57(2):439–41.
Number of day 3 transfers 1,627 (53.8%) 10. Isaksson S, Skoog Svanberg A, Sydsjo G et  al. Two
Number of blastocyst transfers 1,399 (46.3%) decades after legislation on identifiable donors
in Sweden: Are recipient couples ready to be
Number of embryos replaced 5,695
open about using gamete donation? Hum Reprod
Implantation rate 39.1 2011;26(4):853–60.
Ongoing pregnancy/cycle 1,398 (40.3%) 11. Pennings G. How to kill gamete donation:
Number of babies born “fresh ET” 1,674 Retrospective legislation and donor anonymity. Hum
Number of babies born (deliveries) 632 Reprod 2012;27(10):2881–5.
“cryo-transfers” 12. Salat-Baroux J, Cornet D, Mandelbaum J, Watanabe Y,
Merviel P, Antoine JM. Oocyte donation after the bio-
Source: Courtesy of IVI Donor Egg Bank.
ethics law. Medical, ethical and legal implications drawn
from a series of 300 cases at the Tenon hospital. Bull
recipient can start immediately), large donor selection (an Acad Natl Med 2001;185(2):373–84; discussion 84–5.
easier match procedure), quarantine of eggs is possible, 13. Stoop D. From fresh heterologous oocyte donation
results are similar to fresh egg donation, few or no super- to autologous oocyte banking. Facts Views Vis Obgyn
numerary embryos (fewer moral/ethical dilemmas), and 2012;4(4):271–82.
it is economically less burdensome. It is likely that within 14. Blyth E, Crawshaw M, Daniels K. Policy formation in
a few years we shall experience further development and gamete donation and egg sharing in the UK—A criti-
expansion of donor egg banks, which hopefully will pro- cal appraisal. Soc Sci Med 2004;59(12):2617–26.
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donation in the United States, United Kingdom and
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26. Bunge RG, Keettel WC, Sherman JK. Clinical use M, Flamigni C. Pregnancies and births after oocyte
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1954;5(6):520–9. 42. Quintans CJ, Donaldson MJ, Bertolino MV, Pasqualini
27. Trounson A, Mohr L. Human pregnancy following RS. Birth of two babies using oocytes that were cryo-
cryopreservation, thawing and transfer of an eight- preserved in a choline-based freezing medium. Hum
cell embryo. Nature 1983;305(5936):707–9. Reprod 2002;17(12):3149–52.
28. Chen C. Pregnancy after human oocyte cryopreserva- 43. Boldt J, Cline D, McLaughlin D. Human oocyte cryo-
tion. Lancet 1986;1(8486):884–6. preservation as an adjunct to IVF–embryo transfer
29. van Uem JF, Siebzehnrubl ER, Schuh B, Koch R, cycles. Hum Reprod 2003;18(6):1250–5.
Trotnow S, Lang N. Birth after cryopreservation of 44. Bianchi V, Lappi M, Bonu MA, Borini A. Oocyte
unfertilized oocytes. Lancet 1987;1(8535):752–3. slow freezing using a 0.2–0.3 M sucrose concen-
30. Porcu E, Fabbri R, Seracchioli R, Ciotti PM, Magrini tration protocol: Is it really the time to trash the
O, Flamigni C. Birth of a healthy female after intra- cryopreservation machine? Fertil Steril 2012;97(5):​
cytoplasmic sperm injection of cryopreserved human 1101–7.
oocytes. Fertil Steril 1997;68(4):724–6. 45. Kuleshova L, Gianaroli L, Magli C, Ferraretti A,
31. Tucker MJ, Wright G, Morton P, Shanguo L, Massey J, Trounson A. Birth following vitrification of a small
Kort H. Preliminary experience with human oocyte number of human oocytes: Case report. Hum Reprod
cryopreservation using 1,2-propanediol and sucrose. 1999;14(12):3077–9.
Hum Reprod 1996;11(7):1513–5. 46. Gautam SK, Verma V, Palta P, Chauhan MS, Manik
32. Tucker MJ, Morton PC, Wright G, Sweitzer CL, RS. Effect of type of cryoprotectant on morphol-
Massey JB. Clinical application of human egg cryo- ogy and developmental competence of in vitro-
preservation. Hum Reprod 1998;13(11):3156–9. matured buffalo (Bubalus bubalis) oocytes subjected
33. Noyes N, Porcu E, Borini A. Over 900 oocyte cryo- to slow freezing or vitrification. Reprod Fertil Dev
preservation babies born with no apparent increase 2008;20(4):490–6.
in congenital anomalies. Reprod Biomed Online 47. Smith GD, Serafini PC, Fioravanti J et al. Prospective
2009;18(6):769–76. randomized comparison of human oocyte cryo-
34. Wang WH, Meng L, Hackett RJ, Oldenbourg R, Keefe preservation with slow-rate freezing or vitrification.
DL. Rigorous thermal control during intracytoplas- Fertil Steril 2010;94(6):2088–95.
mic sperm injection stabilizes the meiotic spindle 48. Vajta G, Nagy ZP. Are programmable freezers still
and improves fertilization and pregnancy rates. Fertil needed in the embryo laboratory? Review on vitrifi-
Steril 2002;77(6):1274–7. cation. Reprod Biomed Online 2006;12(6):779–96.
35. Jo JW, Jee BC, Suh CS et al. Effect of maturation on 49. Arav A, Natan Y. Vitrification of oocytes: From basic
the expression of aquaporin 3 in mouse oocyte. Zygote science to clinical application. Adv Exp Med Biol
2011;19(1):9–14. 2013;761:69–83.
36. Chandler DE, Heuser J. Membrane fusion during 50. Arav A, Shehu D, Mattioli M. Osmotic and cytotoxic
secretion: Cortical granule exocytosis in sex urchin study of vitrification of immature bovine oocytes.
eggs as studied by quick-freezing and freeze-fracture. J Reprod Fertil 1993;99(2):353–8.
J Cell Biol 1979;83(1):91–108. 51. Vanderzwalmen P, Connan D, Grobet L et al. Lower
37. Palermo G, Joris H, Devroey P, Van Steirteghem intracellular concentration of cryoprotectants after
AC. Pregnancies after intracytoplasmic injec- vitrification than after slow freezing despite exposure
tion of single spermatozoon into an oocyte. Lancet to higher concentration of cryoprotectant solutions.
1992;340(8810):17–8. Hum Reprod 2013;28(8):2101–10.

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52. Glujovsky D, Riestra B, Sueldo C et  al. Vitrification comparable with freshly isolated gametes. Fertil Steril
versus slow freezing for women undergoing oocyte 2010;94(7):2804–7.
cryopreservation. Cochrane Database Syst Rev 66. Noyes N, Boldt J, Nagy ZP. Oocyte cryopreservation:
2014;9:CD010047. Is it time to remove its experimental label? J Assist
53. Levi Setti PE, Porcu E, Patrizio P et al. Human oocyte Reprod Genet 2010;27(2–3):69–74.
cryopreservation with slow freezing versus vitrifica- 67. The Practice Committee of the American Society for
tion. Results from the National Italian Registry data, Reproductive Medicine. Mature oocyte cryopreserva-
2007–2011. Fertil Steril 2014;102(1):90–5.e2. tion: A guideline. Fertil Steril 2013;99(1):37–43.
54. Cobo A, Diaz C. Clinical application of oocyte 68. Chang CC, Elliott TA, Wright G, Shapiro DB, Toledo
vitrification: A systematic review and meta-anal- AA, Nagy ZP. Prospective controlled study to evaluate
ysis of randomized controlled trials. Fertil Steril laboratory and clinical outcomes of oocyte vitrifica-
2011;96(2):277–85. tion obtained in in vitro fertilization patients aged 30
55. Kuwayama M, Vajta G, Kato O, Leibo SP. Highly to 39 years. Fertil Steril 2013;99(7):1891–7.
efficient vitrification method for cryopreserva- 69. Steinberger E, Perloff WH. Preliminary experience
tion of human oocytes. Reprod Biomed Online with a human sperm bank. Am J Obstet Gynecol
2005;11(3):300–8. 1965;92:577–9.
56. Kuwayama M. Highly efficient vitrification for cryo- 70. Smith KD, Steinberger E. Survival of spermatozoa in
preservation of human oocytes and embryos: The a human sperm bank. Effects of long-term storage in
Cryotop method. Theriogenology 2007;67(1):73–80. liquid nitrogen. JAMA 1973;223(7):774–7.
57. Martinez-Burgos M, Herrero L, Megias D et  al. 71. Sole M, Santalo J, Boada M et al. How does vitrifica-
Vitrification versus slow freezing of oocytes: Effects on tion affect oocyte viability in oocyte donation cycles?
morphologic appearance, meiotic spindle configura- A prospective study to compare outcomes achieved
tion, and DNA damage. Fertil Steril 2011;95(1):374–7. with fresh versus vitrified sibling oocytes. Hum
58. Chang CC, Lin CJ, Sung LY, Kort HI, Tian XC, Nagy Reprod 2013;28(8):2087–92.
ZP. Impact of phase transition on the mouse oocyte 72. Cai LB, Qian XQ, Wang W et al. Oocyte vitrification
spindle during vitrification. Reprod Biomed Online technology has made egg-sharing donation easier in
2011;22(2):184–91. China. Reprod Biomed Online 2012;24(2):186–90.
59. Chen SU, Yang YS. Slow freezing or vitrification 73. Garcia JI, Noriega-Portella L, Noriega-Hoces L.
of oocytes: Their effects on survival and meiotic Efficacy of oocyte vitrification combined with blasto-
spindles, and the time schedule for clinical practice. cyst stage transfer in an egg donation program. Hum
Taiwan J Obstet Gynecol 2009;48(1):15–22. Reprod 2011;26(4):782–90.
60. Forman EJ, Li X, Ferry KM, Scott K, Treff NR, Scott 74. Stoop D, De Munck N, Jansen E et al. Clinical vali-
RT Jr. Oocyte vitrification does not increase the risk dation of a closed vitrification system in an oocyte-
of embryonic aneuploidy or diminish the implanta- donation programme. Reprod Biomed Online
tion potential of blastocysts created after intracyto- 2012;24(2):180–5.
plasmic sperm injection: A novel, paired randomized 75. Cobo A, Remohi J, Chang CC, Nagy ZP. Oocyte cryo-
controlled trial using DNA fingerprinting. Fertil Steril preservation for donor egg banking. Reprod Biomed
2012;98(3):644–9. Online 2011;23(3):341–6.
61. Katz-Jaffe MG, Larman MG, Sheehan CB, Gardner 76. Cobo A, Meseguer M, Remohi J, Pellicer A. Use of
DK. Exposure of mouse oocytes to 1,2-propanediol cryo-banked oocytes in an ovum donation pro-
during slow freezing alters the proteome. Fertil Steril gramme: A prospective, randomized, controlled,
2008;89(5 Suppl):1441–7. clinical trial. Hum Reprod 2010;25(9):2239–46.
62. Gardner DK, Sheehan CB, Rienzi L, Katz-Jaffe 77. Papatheodorou A, Vanderzwalmen P, Panagiotidis
M, Larman MG. Analysis of oocyte physiology to Y et  al. Open versus closed oocyte vitrification sys-
improve cryopreservation procedures. Theriogenology tem: A prospective randomized sibling-oocyte study.
2007;67(1):64–72. Reprod Biomed Online 2013;26(6):595–602.
63. Monzo C, Haouzi D, Roman K, Assou S, Dechaud H, 78. Nagy ZP, Chang CC, Shapiro DB, Bernal DP, Kort HI,
Hamamah S. Slow freezing and vitrification differen- Vajta G. The efficacy and safety of human oocyte vit-
tially modify the gene expression profile of human rification. Semin Reprod Med 2009;27(6):450–5.
metaphase II oocytes. Hum Reprod 2012;27(7):2160–8. 79. Chian RC, Huang JY, Tan SL et al. Obstetric and peri-
64. Dominguez F, Castello D, Remohi J, Simon C, Cobo A. natal outcome in 200 infants conceived from vitrified
Effect of vitrification on human oocytes: A metabolic oocytes. Reprod Biomed Online 2008;16(5):608–10.
profiling study. Fertil Steril 2013;99(2):565–72. 80. Cobo A, Serra V, Garrido N, Olmo I, Pellicer A,
65. Di Pietro C, Vento M, Guglielmino MR et  al. Remohi J. Obstetric and perinatal outcome of
Molecular profiling of human oocytes after vitrifi- babies born from vitrified oocytes. Fertil Steril
cation strongly suggests that they are biologically 2014;102(4):1006–15.e4.

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16 Fertility preservation for oncology patients
Kara N. Goldman, Caroline McCaffrey, and Nicole Noyes

INTRODUCTION there is lack of partner or sperm source and, less often,

About 90,000 reproductive-age women are diagnosed ethical and/or religious objections to cryopreserving
with cancer annually in the United States, with the most embryos. The ability to cryopreserve oocytes has revolu-
common malignances being breast, hematologic, gyne- tionized reproductive autonomy for women, and in fact,
cologic, and central nervous system (CNS) cancers.1 when offered the choice at our center between oocyte and
Approximately 25,500 of these cases are breast cancer, embryo cryopreservation, the majority of cancer patients
with the majority of women having local (61%) or regional choose oocyte cryopreservation (OC) as a means to pre-
(32%) disease.2 Almost all of these women require che- serve their fertility.12 After more than 10 years of research
motherapy with or without endocrine therapy for cure.3 on OC, the American Society for Reproductive Medicine
Thankfully, the majority of young women who undergo (ASRM) has lifted its experimental label and this tech-
breast cancer treatments survive their disease (e.g., 99% nology is now considered standard of care as a fertility
of local and 84% of regional breast cancer cases), as do preservation (FP) measure.13,14
those with other cancer diagnoses, albeit curative treat-
ments can lead to a delay in childbearing and/or render APPROACH TO THE FP PATIENT
the patient infertile, if not sterile.2,4 This translates to a Effectively, caring for the FP patient first requires timely
relatively high demand for parenthood following can- referral from the oncologist to the reproductive endo-
cer treatment. Many women express concerns regarding crinologist. The American Society of Clinical Oncology
treatment-related infertility, allowing this risk to influ- and the ASRM recommend that all oncologists discuss
ence whether or not they undergo potentially life-saving FP with patients of reproductive age, as well as with
chemotherapy and/or endocrine therapy.5,6 The concerns the parents or guardians of children and adolescents if
are not unwarranted, as female cancer survivors are therapy has the potential to compromise fertility.15 While
significantly less likely to achieve parenthood, with the FP referrals are increasing as oncologists become bet-
probability of a first child after cancer diminished by 50% ter informed, the proportion of patients referred for FP
compared with the general population.7 counseling remains relatively low; one study estimated
Chemotherapeutic drugs, and particularly alkylating that less than 10% of all eligible patients (<40 years of
agents, cause a significant depletion in the primordial fol- age) were referred, even when FP was completely govern-
licle pool and can result in considerable ovarian reserve ment funded.16–19 Once referred, patients are often faced
compromise. One proposed mechanism for the impair- with a limited time frame during which they can pursue
ment is an upregulation of the PI3K/PTEN/Akt pathway, FP options prior to initiating chemotherapy or radia-
leading to accelerated follicular recruitment and “burn- tion. In one study, women with breast cancer requiring
out.”8 Some cancer-treatment regimens are thought to neo-­adjuvant chemotherapy on average had only 14 days
damage granulosa cells within early growing follicles.9 (range 6–26 days) between FP consultation and initiation
Ovarian damage is evidenced by markedly lower post- of chemotherapy; this abbreviated time frame further
treatment anti-mullerian hormone (AMH) levels, a supports the urgency with which FP must be addressed.20
hormone produced by granulosa cells that inhibits pri- In the United States, the monetary expense of a FP
mordial follicle recruitment.10,11 Serum AMH decline cycle may be cost prohibitive for more than a quarter
associated with chemotherapy is known to be dose and of patients, and cost influences FP decision-making in
regimen dependent, but it is important to appreciate that half of all eligible patients.21,22 Insurance coverage for FP
even low-risk chemotherapeutic regimens can compro- services remains inconsistent, and in most states cover-
mise fertility and result in premature ovarian failure as age is nonexistent, often leaving patients responsible for
measured by AMH levels, antral follicle count, and/or by the full cost of FP services. In states where third-party
clinically evident amenorrhea and subfertility. reimbursement exists for infertility treatment, coverage
Historically, few options existed for the preservation unfortunately does not include preventive FP measures
of fertility, namely undergoing a cycle of in vitro fertil- including oocyte or embryo cryopreservation.23 In coun-
ization (IVF) with embryo cryopreservation. However, tries such as the United States where costs are mostly
the option of creating embryos is not acceptable to all self-incurred, finances more often impact the decision to
women for a variety of reasons, most significantly when pursue FP, and therefore these issues should be addressed

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as early as possible. The immediacy with which treatment refer the patient to the team psychologist. Women begin-
decisions must be made and cycles financed prior to ini- ning FP treatment report higher levels of depression and
tiating treatment is a significant barrier to patients lack- anxiety compared with infertile patients pursuing simi-
ing ample savings. In contrast, in countries such as The lar treatments, and many may benefit from professional
Netherlands, where government medical programs cover help.28 Patients are often surprised by the commitment
FP costs, and therefore financial considerations generally required during an FP cycle, which can result in patient
do not factor into an individual’s decision to pursue FP, attrition following the initial visit.
surveyed patients were noted to have less decisional con- After the initial educational session, the patient will
flict about pursuing FP.19 benefit from consultation with the center’s financial
Given the tremendous investment involved, including department to review her financial resources and options,
time, emotional, physical, and financial costs, it is imper- and to apply for medication grants or other relevant FP
ative that patients are shepherded through the FP process grants. However, it is important that patients understand
by a multidisciplinary team specialized in all facets of that not every patient will qualify for financial assistance,
FP treatment. This multifaceted team should include, at and even if assistance is provided, she may be respon-
a minimum, the patient’s oncologist, reproductive endo- sible for the cost of medication refills and future cryo-
crinologist, nursing staff, embryology laboratory staff, storage expenses. Hopefully, as FP treatment becomes
and patient liaisons. In many institutions, the team also more mainstream, insurance carriers will begin to cover
includes psychologists, social workers, and researchers, as these important services, lessening the patient’s financial
well as stress-relief programs to provide seamless care for burden.
the patient from the earliest moments of cancer diagnosis.
The term “oncofertility” was coined to represent this crit- APPROACH TO THE MINOR PATIENT
ical multidisciplinary relationship, and the most effective Approximately 16,000 children and adolescents under
approach to treating FP patients comes in the form of this the age of 19 are diagnosed with cancer every year, with
patient-centered multidisciplinary care team.24–27 the most common cancers being leukemia, lymphoma,
and CNS tumors.29 Around 1 in 285 children will experi-
Preparing for the FP Process ence cancer before 20 years of age, but as survival rates
Patients should be referred for a FP consultation as soon approach 80%–90% in childhood leukemia and lym-
as possible following cancer diagnosis in order to expe- phoma, there are a growing number of cancer survivors
dite the process prior to chemotherapy or radiation; the for whom survivorship issues like fertility will be an
reproductive endocrinologist’s team should therefore important concern.30 Ovarian stimulation and oocyte
be flexible and able to accommodate last-minute FP retrieval can be performed in young girls with a func-
appointments as the need arises. Immediately following tioning hypothalamic–pituitary–ovarian axis. Ovarian
the initial FP visit, patients benefit from an individual stimulation and OC has even been successfully achieved
educational orientation session with a dedicated FP team in premenarchal girls on the crest of puberty in our cen-
member. Patients are advised to bring a family member, ter, as well as by others, further suggesting that menarche
spouse, or friend with them to this visit, as many patients is not a prerequisite for OC in patients who have not yet
report feeling overwhelmed by the large amount of infor- completed the pubertal transition.31
mation transmitted at their initial consultation, leading The only FP option currently available to very young
to an inability to recall many of the details of this visit. girls (i.e., remote from puberty) is ovarian tissue cryo-
Patients often schedule multiple physician visits in a short preservation (OTC), and only through enrollment in
period of time; however, this often leaves the patient feel- experimental protocols. Transplantation of human cryo-
ing burdened by the quantity and complexity of infor- preserved ovarian tissue into mice suggests that a high
mation. Open communication between patients and number of follicles survive after transplantation, a large
providers is crucial during this often overwhelming time number of primordial follicles remain dormant, and
for the patient and her family. the tissue is responsive to gonadotropins.32 To date, 24
During the initial FP visit, a brief review of the normal live births have been reported worldwide from human
menstrual cycle and ovarian physiology may be provided, ovarian tissue transplantation in postpubertal women,
followed by an explanation of how the menstrual cycle including one who was 17 years of age at the time of tis-
is manipulated to pursue oocyte or embryo cryopreser- sue harvest who now has three children following trans-
vation. A sample calendar is reviewed with the patient plantation.33 The largest case series involving OTC in
to provide a better understanding of her FP schedule. children and adolescents (<16 years) includes 58 patients
Nursing and physician staff can then review medica- with a mean age of 10.4 years, with the youngest child
tion administration, provide hands-on injection training under 1 year of age; unfortunately, no births have yet been
and discuss injection troubleshooting. It is imperative to reported from this cohort.33,34 Due to the young nature
gauge how much information the patient is absorbing, and of this field and the limited number of ovarian tissue
how she is coping psychologically. It is often beneficial to transplantation procedures that have been performed,

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the safety and efficacy of the technology as a standard FP importance is the issue of gamete disposition in the event
measure is yet to be proven. of a minor’s death.37,38 The Tennessee Supreme Court
All of the emotional, physical, medical, and finan- ruled that “the existence of the right (of procreational
cial considerations that apply to FP in adults also apply autonomy) itself dictates that decisional authority rests
to FP in minors, but special considerations must be in the gamete-providers alone,” and this ruling should
addressed. In young and adolescent girls considering FP, extend to all gamete-providers, including children and
it is important to maintain an age-appropriate approach, adolescents.39 Minors undergoing OC who are, by defini-
particularly with regard to the patient’s sexual history. In tion, peri- or post-pubertal, and presumably emotionally
virginal patients, it is best to avoid vaginal ultrasonog- mature enough to pursue FP, should thus be the only ones
raphy during initial evaluation or ovarian stimulation/ with authority to make decisions regarding the disposi-
follicular monitoring in order to lower anxiety and avoid tion of their own gametes. In the case of gametes that are
risk of injury. Transabdominal ultrasonography is more already cryopreserved, no decision should be made as to
appropriate for visualizing the ovaries in these cases. the use or disposition of the gametes until the individ-
Contemporary transabdominal ultrasound machines can ual has reached the age of legal maturity. As a program-
afford adequate visualization, particularly once the ova- matic policy, any minor patient undergoing FP treatment
ries have been stimulated to mature multiple follicles. At should be contacted for re-consent and consideration of
the time of oocyte retrieval, a smaller-shaft vaginal ultra- gamete disposition once adulthood is reached, as minor
sound probe can be utilized, preparing both the patient ownership of and laws surrounding property is limited at
and parents/guardians that minimal bleeding and/or the younger age.
hymenal tearing may occur. The same can be applied to
women of any age who remain virginal or decline the use DISPOSITION OF GAMETES AND/OR EMBRYOS
of vaginal ultrasonography. At our center, we have rou- Consent surrounding the disposition of cryopreserved
tinely used this approach with success. gametes/tissue in a cancer patient, particularly in the
The care team must also be cognizant of the young setting of questionable survival, can be complex. Prior
patient’s developmental age and ability to cope with her to initiating FP treatment, disposition of cryopreserved
recent diagnosis, the demand to be present for frequent oocytes, embryos, or ovarian tissue must be addressed in
follicular monitoring and blood draws, and the possibil- the case of the patient’s death. Options include discarding
ity that she fears needles and injections.35 Every attempt or destroying the gametes/tissue versus donating them/it
should be made to make the young patient feel more for research. Alternatively, the tissue can be donated to
comfortable, including maintaining consistency with her a family member with the intent of having the deceased
nursing and physician care team, and being cognizant of person’s child, albeit posthumous reproduction remains
her emotional maturity level regarding matters as per- controversial. Disposition ambiguity is most commonly
sonal as her fertility. addressed by the following: “When the decedent’s wishes
are unknown, a presumption against using gametes for
Ethical and Legal Issues Surrounding FP in Minors posthumous reproduction should apply.”40 Adding to
FP in children and adolescents under the age of 18 poses the dilemma is the limited property ownership capacity
unique issues due to questions of informed consent, of a minor, again making secondary consent regarding
autonomy, and ownership. While minors are not rec- gamete disposition a practice standard when the patient
ognized as legally competent decision-makers, issues of comes of age.
autonomy become more complicated where reproductive Importantly, if gametes are to be donated for use by
rights are concerned. The constitution protects the repro- another specified individual, an addendum should be
ductive rights of minors just as it does those of adults, included in the consent form naming that individual
and parents cannot deprive minors of future reproduc- and confirming that the individual is aware of this des-
tive capacity. Parental consent is required for medical ignation. In these situations, additional infectious disease
intervention in a minor, but the parent’s right to decide screening and testing should be performed on the patient
on a child’s treatment is not absolute, particularly when it (“oocyte donor”) whenever possible, in order to satisfy
comes to interventions deemed to be “elective”: “First, if requirements of the Food and Drug Administration
the treatment is not medically necessary for the minor, it (FDA) for directed-oocyte donation.
must not be unreasonably harmful. Second, the treatment
must be to the benefit of the minor, and not just to the FP TREATMENT STRATEGY
benefit of the minor’s parents or other family members.”36 Historically, embryo cryopreservation was considered
When caring for minors, providers must balance the standard-of-care FP measure available to reproduc-
responsibilities to the patient as well as to the parents/ tive-age cancer survivors; however, advances in OC in
guardians, and address sensitive issues regarding the experienced hands have now demonstrated success rates
emotional and physical consequences of invasive gyne- comparable to IVF.41–43 The availability of OC has revolu-
cologic procedures in a child. Also of extremely high tionized FP, allowing its pursuit without the requirement

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 vitrification in assisted reproduction
of sperm (directed or donor) at the time of ovarian stimu- (i.e., the early follicular phase), and stimulation typically
lation and cryopreservation. Embryo cryopreservation continues for 10–14 days. For referred FP patients who
may be the treatment of choice for a patient in a stable have only a brief window of opportunity during which
partnership or marriage, although even in this setting, to undergo COS, the menstrual cycle may need to be
it may not be acceptable if ethical objections to embryo “overridden,” with stimulation regimens beginning at a
storage exist.44 In addition, with the high marital divorce time other than the early follicular phase.49 Thus, gonad-
rate occurring in the general population, and in particu- otropin treatment can be randomly initiated within the
lar among survivors of cancer, reproductive autonomy cycle or, alternatively, gonadotropin-releasing hormone
through OC is preferable in many circumstances.45 When (GnRH) antagonist with or without oral contraceptive
appropriate, embryo cryopreservation may still carry a pill co-administration can be given for several days prior
distinct advantage in that blastocyst formation has been to initiating gonadotropin therapy. Studies of women
noted to be consistently higher when using fresh oocytes undergoing “random-start” protocols are encouraging;
rather than following OC with warming; consequently, thus, initiating gonadotropin injections during the mid-
embryo cryopreservation may have the distinct advan- follicular or even mid-luteal phase appears to produce
tage of affording greater numbers of supernumerary outcomes equivalent to a conventional early follicular-
blastocysts to be cryopreserved when compared to cycles phase start.50–52
using frozen–thawed oocytes.43,46
Whether FP should be pursued at all requires clinical Aromatase Inhibitors and Selective Estrogen
judgment along with informed decision-making. Factors Receptor Modulators
include the extent and stage of disease, the patient’s per- Women with estrogen receptor-positive tumors, such as
ceived desire for future parenting, as well as the patient’s those that occur in the breast or endometrium, may benefit
overall physical and mental health, including comorbidi- from the addition of an aromatase inhibitor (e.g., letrozole),
ties. The patient’s oncologist should be directly involved or alternatively a selective estrogen receptor modulator
in the decision-making process; that is, it is not the role of (e.g., tamoxifen), to lessen estrogen stimulation during
the reproductive specialist to judge, but rather to provide COS.53 Despite an absence of randomized controlled trials,
information and counseling regarding the risks and ben- in addition to no proven requirement for this modification
efits of FP options. given the brief treatment interval, this protocol maintains
There is no recommended absolute upper or lower age an excellent safety profile, with serum estradiol maintained
limits within the reproductive span for pursuing OC, but, at relatively lower levels throughout the treatment cycle.54
as with all assisted reproductive technologies, the most Lower estrogen levels may provide an added advantage by
important clinical predictor of outcome is oocyte age at the diminishing the risk of venous thromboembolism (VTE)
time of cryopreservation.47 As discussed above, a mature or in a patient at increased risk due to malignancy. Data
near-mature hypothalamic–pituitary–ovarian axis is the derived from post-menopausal women receiving estrogen-
threshold at which a peri-menarchal or postpubertal ado- containing hormone-­ replacement therapy demonstrate
lescent can pursue ovarian stimulation. Other than meno- that those administered transdermal estradiol (having
pause, no such threshold or optimal upper age limit exists, significantly reduced serum estradiol levels as compared
although at present, success has not yet occurred using to women receiving oral therapy) had a significantly
oocytes cryopreserved past the age of 42 years, and patients lower risk for VTE.55 Theoretically, one could argue that
should be counseled accordingly.48 The appropriate time to all cancer patients should be given a lower-dose estrogen
pursue FP is “as soon as possible” once a cancer diagno- protocol to lessen VTE risk. Future research is needed
sis has been made. In an older woman, only the patient, in this area, and VTE will be discussed again later in the
her oncologist, and her reproductive endocrinologist can chapter. Despite the aforementioned advantages of estro-
decide whether pursuing FP is worth the investment, and gen suppression during COS, these protocols remove the
only after assessing the risks and benefits of FP. ability/advantage of using estrogen levels to gauge ovarian
response to medication, dosing titration, and determina-
CONTROLLED OVARIAN STIMULATION FOR tion of the appropriate timing and administration of the
THE FP PATIENT final maturation trigger. These disadvantages are currently
Ovarian stimulation protocols are tailored to each indi- outweighed by the advantage of diminished circulating
vidual patient. While controlled ovarian stimulation estradiol in a patient with a hormone-sensitive tumor.
(COS) protocols for FP may not differ dramatically from
conventional COS treatment, the following special cir- GnRH Agonist Trigger
cumstances may apply. Traditional ovulation trigger with human chorionic gonad-
otropin (hCG) may be problematic in women who intend
Random-Start Protocols to begin chemotherapy treatment immediately upon
In a typical COS cycle, gonadotropin administration completing FP. hCG retains prolonged activity and has a
begins on day 2 or 3 of the patient’s menstrual bleeding low clearance rate due to a high degree of glycosylation,56

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 fertility preservation for oncology patients
consequently, chemotherapy may be delayed due to symp- both slow cooling and vitrification methods have been
toms of ovarian hyperstimulation, as well as concerns successfully applied to this gamete.64–66 The principle
about low-level hCG representing early pregnancy in a behind both techniques is the avoidance of intracel-
patient receiving chemotherapy. GnRH agonists (GnRHa) lular ice crystal formation, a process known to damage
are used with great success in GnRH antagonist treatment cell membranes and critical intracellular organelles such
cycles as a means of triggering the final stage of oocyte as the meiotic spindle. In recent years, vitrification has
maturation. In a small retrospective study, outcomes in become the predominant methodology employed by
breast cancer patients using a GnRHa trigger were equiva- clinics; importantly, reproducible survival, implanta-
lent to those of women who had been prescribed tradi- tion, and pregnancy rates using this method are now
tional hCG.57 GnRHa may be administered alongside achievable.65–68
low-dose hCG (~1000 units of intramuscular hCG, or the
equivalent dose of recombinant subcutaneous hCG) as a OC LABORATORY PROCESSES
“dual trigger,” if concern exists regarding a patient’s abil- In preparation for OC, ovarian stimulation and oocyte
ity to respond appropriately to a GnRHa trigger with an retrieval are performed in a routine fashion as with tra-
appropriate pituitary luteinizing hormone (LH) release ditional IVF. Immediately following follicular aspiration,
(i.e., peri-pubertal, hypothalamic amenorrhea).58 Women oocytes are transferred to the laboratory and washed
with hypogonadotropic hypogonadism, who have dem- in modified media. They are then placed in bicarbon-
onstrated inappropriately low FSH or LH levels during ate-buffered medium supplemented with protein, and
the early follicular phase or prior to the administration of allowed to incubate at 37°C in 6% carbon dioxide (CO2).
GnRH antagonists, should be flagged as inappropriate can- Approximately 90 minutes later, oocytes are stripped of
didates for a single-agent GnRHa trigger. all cumulus and corona cells using a combination of enzy-
Treatment protocols should be individualized depend- matic (Cumulase, Halozyme Therapeutics, San  Diego,
ing on the patient’s age, diagnosis, hypothalamic status, CA) and mechanical techniques, and oocytes are exam-
and time constraints. In our center, patients with lym- ined for maturity. Metaphase II (mature) oocytes are iden-
phomas have sometimes demonstrated a poorer response tified by the presence of a polar body in the peri-­v itelline
to GnRHa triggers, and in these patients we preferentially space and are preferentially selected for cryopreservation;
utilize a combined GnRHa–hCG trigger for oocyte matu- however, metaphase I oocytes (those lacking both a polar
ration whenever possible. body and a visible germinal vesicle [GV]), as well as GV
oocytes, may also be cryopreserved, but are less likely to
FP AND OC LABORATORY PROTOCOLS result in pregnancy success. Advances in in vitro matu-
The science behind cryobiology and the cryopreservation ration technologies may improve outcomes using imma-
of embryos and oocytes has been well documented and ture oocytes in the future, so for this reason, in cancer
thoroughly reviewed by past authors.59–61 As noted earlier, and young reproductive-age patients having a low oocyte
historically, the only option available to patients seeking yield at retrieval, oocytes of all stages are currently cryo-
FP was embryo cryopreservation using controlled-rate preserved at our center.
slow cooling, originally reported by Testart et al.62 Success
rates using thawed embryos have varied, and depended Slow Cooling and Subsequent Thaw
on an array of factors including the following: embryonic At our center, slow cooling of oocytes has been per-
developmental stage, embryo quality, freezing method, formed using a technique similar to that described by
and post-thaw manipulation (e.g., removal of lysed cells Fabbri et al.69 Briefly, oocytes are first washed in a solu-
and/or disruption of zona pellucida integrity). In recent tion containing phosphate-buffered saline (PBS) and
years, there has been a shift away from slow cooling in favor then transferred to an equilibration solution containing
of vitrification as the choice embryo cryopreservation 1.5 mol/L propanediol (PROH). They are then placed
method, and with this has come significantly improved in a loading solution containing 1.5 mol/L PROH plus
outcomes.63 Despite advances, embryo cryopreservation 0.3 mol/L sucrose. All solutions are supplemented with
continues to fall short in meeting the needs of all patients, protein (Plasmanate, Talecris Biotherapeutics, Inc.,
particularly those lacking a male partner. For this reason, Research Triangle Park, NC; or human serum albumin
it is imperative that fertility clinics become experienced [HSA], Cooper Surgical, Inc., Trumbull, CT). Oocytes
and adept at performing OC and warming. are then loaded into cryopreservation straws (Conception
By virtue of its large size (~120 microns), high water Technologies, San Diego, CA) and transferred into a
content (80%), low surface-to-volume ratio, presence of controlled-rate freezer (Planer Kryo, Planer Products
the meiotic spindle, unique permeability to cryopro- Limited, Sunbury, UK), where the temperature is gradu-
tective agents (CPAs), and spherical shape, the human ally lowered from ±20°C to –7°C at a cooling rate of –2°C/
oocyte presents unique challenges to cryopreservation min. Ice nucleation is induced manually at –7°C. The tem-
and thawing processes. These qualities make the oocyte perature is then decreased to –30°C at a rate of –0.3°C/
exquisitely sensitive to temperature modification, albeit min and then rapidly to –150°C at a rate of –50°C/min.

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 vitrification in assisted reproduction
Straws are then placed into canes and transferred to liq- sequential equilibration solutions containing incremen-
uid nitrogen tanks for storage. tally decreasing concentrations of sucrose (0.5–0 mol/L)
During subsequent thaw cycles, straws containing the for a total of eight dilutions. Oocytes are then washed once
cryopreserved oocytes are removed from liquid nitrogen, more in sucrose-free medium, and then placed in pre-
and briefly air-warmed until all traces of ice have disap- warmed and equilibrated culture medium (Global media,
peared. Straws are then placed into a 30°C water bath for LifeGlobal Group, IVFonline.com) in a 37°C (6% CO2)
40 seconds. The CPAs are removed in a stepwise fash- incubator until ICSI is performed.
ion by passing the oocytes through solutions contain-
ing decreasing concentrations of the agents as follows: UNIQUE CONCERNS IN PATIENTS
1.0 mol/L PROH plus 0.3 mol/L sucrose; 0.5 mol/L PROH WITH CANCER
plus 0.3 mol/L sucrose; 0.3 mol/L sucrose plus PBS; and Venous Thromboembolism
finally into PBS. All solutions are supplemented with Cancer, as well as the chemotherapy and hormonal agents
protein (Plasmanate or HSA). Surviving oocytes are then used in cancer treatment, all have procoagulant effects,
transferred to fresh pre-equilibrated culture media and promoting VTE.70,71 The risk of VTE in cancer patients
incubated in a 37°C and 6% CO2 gas atmosphere until is significantly greater than in the general population,
intracytoplasmic sperm injection (ICSI) is performed. ranging from 0.6% to >18%.72 However, a review of the
available literature on the risk of thrombosis in women
Vitrification Method and Subsequent Warming with cancer and in those undergoing ovarian stimulation
Vitrification differs from slow cooling in that oocytes are draws the following conclusions: (1) the cohort of women
exposed to shorter yet significantly higher concentrations with cancer seeking FP are likely to be at a lower risk
of CPA, and cooling rates are appreciably faster (i.e., ultra- of thrombosis compared to the average cancer patient;
rapid). Quick and high exposure to CPA along with ultra- (2)  avoidance of moderate-to-severe ovarian hyper-
rapid cooling have been described as creating a glass-like stimulation syndrome (OHSS) is likely the most impor-
state in which the oocytes are suspended, hence “vitri- tant way of preventing complications such as VTE; and
fied,” from the Latin vitrum meaning “glass.” Cooling (3) anti-thrombotic prophylaxis should be administered
rates in vitrification often exceed 15,000°C/min and are sparingly, and only to those patients with additional risk
achieved by employing exquisitely small fluid volumes, factors or those who develop OHSS.73
often with direct exposure to liquid nitrogen. Although
several commercially available oocyte vitrification kits Thrombocytopenia
exist, none have yet been approved for usage by the FDA. Women with hematologic malignancies may have an
At our center, vitrification is accomplished as follows: elevated risk of thrombocytopenia, placing these patients
oocytes are first equilibrated in media containing the at an elevated risk of surgical bleeding during oocyte
lowest concentration of CPA (0.30 mol/L ethylene gly- retrieval. As with all patients, a complete blood count
col [EG] plus 0.55 mol/L dimethyl sulfoxide [DMSO]) to should be obtained prior to initiating ovarian stimula-
achieve the first level of dehydration. They are then placed tion, and in patients with thrombocytopenia, the deci-
in sequential equilibration solutions with incrementally sion to pursue FP should be made in consultation with
increasing concentrations of CPA (EG 0.30–1.35 mol/L, the oncologist, the reproductive endocrinologist, and the
and DMSO 0.55–1.10 mol/L) for a total of five dilutions patient. Patients with severe thrombocytopenia may be
over several minutes. Finally, the oocytes are placed in a considered for platelet transfusion immediately preced-
vitrification solution containing 2.7 mol/L EG, 2.1 mol/L ing oocyte retrieval, followed immediately by transfer to
DMSO, and 0.5 mol/L sucrose for 1–1.5 minutes. All an inpatient hospital ward for close monitoring. The risks
solutions are prepared using PBS and supplemented and benefits of treatment should be thoroughly discussed
with protein (Plasmanate). Oocytes are then loaded onto at length before proceeding.
Cryolocks (BioTech Inc., Alpharetta, GA) (usually two
oocytes per carrier) and immediately plunged directly Ovarian and Pelvic Masses
into liquid nitrogen, where they are capped and placed in In women who have an ovarian tumor and are planning
goblets and canes for liquid nitrogen cryostorage. to undergo ovarian stimulation and OC prior to ovarian
When the patient is ready to undergo a warming cycle, surgery, if the contralateral ovary is present and unaffected,
the Cryolocks containing vitrified oocytes are uncapped oocytes can usually be retrieved from that ovary. Oocytes
under liquid nitrogen immediately before warming. The may also be carefully and deliberately retrieved from the
Cryolocks are then quickly moved from the liquid nitro- affected ovary or ovaries; however, if attempting such a
gen, and the tips where the oocytes are located are imme- procedure, careful attention should be paid to avoiding
diately immersed in pre-warmed (37°C) thaw medium. puncture of the tumor during retrieval. If a question exists
The oocytes are then released from the device into 1 mol/L regarding the boundaries of the tumor, it may be prudent
thaw medium. All subsequent handling is performed to avoid retrieval of oocytes in that area. Intraoperative
at room temperature. The oocytes are moved through tumor rupture is thought to result in peritoneal tumor

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 fertility preservation for oncology patients
metastasis through spillage of tumor cells, leading to a cancer or nonmalignant diseases, and is increasingly pro-
higher stage and therefore a poorer prognosis, and for this viding reproductive flexibility to women facing age-related
reason particular caution should be taken when operating fertility decline.85 As the technology spreads worldwide and
in close proximity to a suspicious tumor.74 In addition, in indications for OC expand, cryopreservation will undoubt-
patients that have relatively large tumors in the pelvic area edly continue to open reproductive doors for women.
(e.g., a bladder, cervical, or gastrointestinal malignancy),
increased care must be taken when performing oocyte ACKNOWLEDGMENTS
retrieval as local vasculature may be intensified, increasing We gratefully acknowledge the New York University
the risk of bleeding in these patients. Fertility Center FP team, including physicians, embry-
ologists, nurses, and support staff, for the compassionate
FP OUTCOMES care and service that they provide to patients.
Clinical Stimulation Parameters
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73. Somigliana E, Peccatori FA, Filippi F et  al. Risk of Long-term cryopreservation of human oocytes does
thrombosis in women with malignancies undergoing not increase embryonic aneuploidy. Fertil Steril
ovarian stimulation for fertility preservation. Hum 2015;103(3):662–8.
Reprod Update 2014;20(6):944–51. 83. Noyes N, Porcu E, Borini A. Over 900 oocyte cryo-
74. Sainz de la Cuesta R, Goff BA, Fuller AF Jr et  al. preservation babies born with no apparent increase
Prognostic importance of intraoperative rupture in congenital anomalies. Reprod Biomed Online
of malignant ovarian epithelial neoplasms. Obstet 2009;18(6):769–76.
Gynecol 1994;84(1):1–7. 84. Chian RC, Huang JY, Tan SL et al. Obstetric and peri-
75. Domingo J, Guillen V, Ayllon Y et  al. Ovarian natal outcome in 200 infants conceived from vitrified
response to controlled ovarian hyperstimulation in oocytes. Reprod Biomed Online 2008;16(5):608–10.
cancer patients is diminished even before oncological 85. Stoop D, van der Veen F, Deneyer M et  al. Oocyte
treatment. Fertil Steril 2012;97(4):930–4. banking for anticipated gamete exhaustion (AGE) is a
76. Johnson LNC, Dillon KE, Sammel MD et  al.
preventive intervention, neither social nor nonmedi-
Response to ovarian stimulation in patients facing cal. Reprod Biomed Online 2014;28(5):548–51.

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17 Vitrification of human ovarian tissue
Mona Sheikhi and Outi Hovatta

Cryopreservation of human ovarian tissue has been car- procedure. The samples were vitrified in high-security
ried out for more than 18 years.1,2 Successful studies in straws (Cryobiosystemes–IMV, Aigle, France). The via-
experimental animals with live births following trans- bility of the tissue was confirmed by normal light micro-
plantation of cryostored ovarian tissue in rodents and scopic morphology of the follicles and stromal tissue, the
sheep had been performed earlier.3–6 growth of the follicles in culture, and by the production
In the beginning, slow-programmed freezing was uti- of progesterone, androstenedione, and estradiol. Only the
lized. It was also widely applied clinically. For the time rate of antrum formation was slightly smaller in the tis-
being, we can estimate that more than 30 children have sue following vitrification than it was in the control tissue
been born in this way. They have been summarized in during culture.
several articles. The first live birth after slowly frozen
human ovarian tissue was reported already in 2004,7 fol- VITRIFICATION VERSUS SLOW FREEZING
lowed by many others later on.8–16 Vitrification of human OF HUMAN OVARIAN TISSUE
ovarian tissue was the evident next step for improving In our fertility unit at Karolinska University Hospital,
this methodology. Huddinge, we have offered ovarian tissue cryostor-
Vitrification of rodent ovarian tissue was performed age since 1998, using at first the method that we devel-
at first.17–20 Rodents have a looser structure within their oped at Imperial College at Hammersmith Hospital in
ovarian tissue, but successful vitrification of ovarian tis- London, UK.1 We then improved it by using a serum-free
sue was soon reported in rabbits and nonhuman primates. medium.27 This slow-programmed freezing in our unit
Ovarian tissue survival after transplantation into rat has been proven to be effective, as determined by a live
uteri,21 showing the survival of vitrified–warmed ovarian birth after orthotopic transplantation.28
tissue after uterine transplantation. Sheep was the obvi- We carried out a study in which we compared the slow
ous next species to study,22 metaphase II were obtained controlled-rate freezing using 1,2-propanediol (PrOH)
after in vitro maturation of isolated follicles from vitrified and sucrose as cryoprotectants for the vitrification of pieces
sheep ovarian tissue. Corbiere et al.23 were able to vitrify of ovarian tissue from the same donors who underwent
whole sheep ovaries by perfusing them through their ves- Cesarean section.29 The outcome was measured by light
sels with vitrification solution, and bathing them in the and electron microscopy in fresh, cryostored, and fresh
solution. The survival of the tissue was not optimal in the and cryostored cultured tissue. The cryoprotectants used
sheep ovary, probably due to the large size of the ovary in slow freezing were PrOH–sucrose and EG–sucrose. For
and the time required for the vascularization,24 although vitrification, tissues were incubated for 5 or 10 minutes
the birth of four lambs after auto-transplantation of in three solutions containing a combination of DMSO,
vitrified warmed ovarian cortical tissue into ewes was PrOH, EG, and polyvinylpyrrolidone (PVP). Vitrification
reported. using a combination of PrOH, EG, DMSO, and PVP was
Nonhuman primates have been excellent for model- comparable to slow freezing in terms of preservation of
ling human ovarian vitrification.25 Vitrified ovarian tis- follicles in human ovarian tissue. Ovarian stroma had sig-
sue was obtained from five adult baboons, and the tissue nificantly better morphological integrity after vitrification
was autografted after 5 months. The vitrification medium than after controlled-rate freezing. We did not find any
contained ethylene glycol (EG) and dimethyl sulfoxide light or electron microscopic signs of apoptosis in vitrified
(DMSO) as cryoprotectants. After vitrification, warm- or slow-frozen human ovarian tissue.
ing, and long-term grafting, the follicles were functional Amorim et al.30 compared slow freezing and two vitri-
and morphologically normal as demonstrated by growth fication protocols for human ovarian tissue using EG and
and immunostaining for Ki67, anti-mullerian hormone polymers as cryoprotectants. They demonstrated normal
and growth differentiation factor-9. In addition, the stro- morphology of the follicles after warming and at 1 week
mal tissue appeared normal and vascularized. after xenografting. Vitrification with both protocols
Another group confirmed the feasibility of a closed caused less apoptosis, as shown by terminal deoxynucleo-
vitrification system using macaque ovarian cortical tis- tidyl transferase dUTP nick end labeling (TUNEL) stain-
sue.26 They used a combination of EG and polymers, ing, when compared to slow freezing.
and a combination of EG and DMSO with cooling in Previous studies had used protocols incorporating
liquid nitrogen (LN2) vapor and a two-step warming direct contact with LN2 for the first attempts at ovarian

147
 vitrification in assisted reproduction
tissue vitrification; however, we decided to develop a vit- (a) (b)
rification system in which no direct contact with LN2
was needed.
Vitrification was carried out using 1.8 mL NUNC
cryotubes (Nunclon, Roskilde, Denmark) with an inter-
nal thread cap to make it leak-proof. The tissue pieces
were carefully transferred into the cryotube with a
minimum volume of the vitrification medium. We did
this because it is very difficult to sterilize LN2. There are
reports indicating that microbes are able to survive in
LN2.31 Sterilizing LN2 is difficult, and filters do not neces- Figure 17.1  Transmission electron micrographs of vitri-
sarily remove small viruses. LN2 has a surface tension that fied human ovarian tissue. (a) Human ovarian follicles
prevents filtering without pressure, and applying pressure within cortical tissue that was vitrified using a com-
to this easily evaporating liquid may cause an explosion. bination of dimethyl sulfoxide, 1,2-propanediol, and
Using a vacuum is feasible, but this is not simple, and it ethylene glycol as permeating cryoprotectants. There is
requires special equipment. We studied a closed and sim- a morphologically normal oocyte with well-preserved
ple vitrification method by enclosing the ovarian tissue mitochondria and a granulosa cell with normal mor-
sample within a cryotube that was tightly sealed and then phology. The cellular membranes look unaffected, and
immersed into LN2. According to our experiments, vitri- the extracellular matrix filaments outside the granu-
fication in cryotubes was fast enough to allow full mor- losa cell layer appear intact. Intact protrusions between
phologic integrity of all components of human ovarian the oocyte and the granulosa cell can be clearly seen.
tissue to be maintained, as revealed by light and electron (b) Human ovarian tissue vitrified using only ethylene
microscopy.32 Electron microscopy or caspase-3 immu- glycol as a permeable cryoprotectant. Intact structure of
nostaining did not reveal any signs of apoptosis, and we the membranes, the cytoplasm of the oocyte, the granu-
did not see any necroses in the tissues either. losa cells, and the extracellular matrix can be seen, as
To avoid any performance-related risks during the well as the finger-like protrusions between the oocyte
laboratory procedure that might be overly complicated, and the granulosa cells.
we simplified the methods from the use of three perme-
ating cryoprotectants to one, in which we used only EG
with the addition of nonpermeating Ficoll 70 (w/v). This It is indeed possible to obtain normal electron micro-
approach has resulted in as good an integrity and viability scopic morphology and viability, growth, and hormone
in tissue culture as the combination of the three cryopro- secretion after tissue culture is performed following
tectants used in the previous protocol.33 All of our pro- warming of ovarian tissue. The viability of the ovarian
cedures have been chemically defined and are free of any stroma has been shown to be somewhat better preserved,
xeno-products (Figure 17.1a and b). according to several more recent reports. Vitrification
Since our first article describing human ovarian tissue of human ovarian tissue has not been utilized exten-
vitrification,29 several articles regarding ovarian tissue sively as yet, so at this point we still lack the final proof
vitrification have been published. Many of them confirm of successful vitrification for human ovarian tissue in the
our results,34–36 although some authors have reported form of clinical pregnancies and live births; nevertheless,
necroses after vitrification.37 Importantly, it has been we believe that these will come in due course, probably
noted that estradiol release in culture has been similar in paving the way for vitrification to become the standard
both control and post-vitrification tissue.38 approach to ovarian tissue cryopreservation.
The differences in these results may be explained by
the fact that vitrification is not a single procedure. There REFERENCES
are many contributing factors, such as the time of incuba- 1. Hovatta O, Silye R, Krausz T et al. Cryopreservation
tion and warming, and the concentrations of the various of human ovarian tissue using dimethylsulphoxide
components in the vitrification solutions vary between and propanediol–sucrose as cryoprotectants. Hum
studies. Additionally, the culture conditions also vary. Reprod 1996;11:1268–72.
This procedure is always attempting to strike a balance 2. Newton H, Aubard Y, Rutherford A, Sharma V,
between proper tissue penetration by the cryoprotectants Gosden R. Low temperature storage and grafting of
and their inherent toxicity of the components to the ovar- human ovarian tissue. Hum Reprod 1996;11:1487–91.
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ing and thawing. J Endocrinol 1954;11:197–200.
CONCLUSIONS 4. Green SH, Smith AU, Zuckerman S. The number of
From our experience and a number of other studies, it is oocytes in ovarian autografts after freezing and thaw-
clear that cryostorage of human ovarian tissue is feasible. ing. J Endocrinol 1956;13:330–4.

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5. Carroll J, Gosden RG. Transplantation of frozen– 20. Chen SU, Chien CL, Wu MY et al. Novel direct cover
thawed mouse primordial follicles. Hum Reprod 1993;​ vitrification for cryopreservation of ovarian tissues
8:1163–7. increases follicle viability and pregnancy capability in
6. Gosden RG, Baird DT, Wade JC, Webb R. Restoration mice. Hum Reprod 2006;21:2794–800.
of fertility to oophorectomized sheep by ovarian auto- 21. Kagabu S, Umezu M. Transplantation of cryopre-
grafts stored at −196°C. Hum Reprod 1994;9:597–603. served mouse, Chinese hamster, rabbit, Japanese
7. Donnez J, Dolmans MM, Demylle D et al. Livebirth monkey and rat ovaries into rat recipients. Exp Anim
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ovarian tissue. Lancet 2004;364:1405–10. 22. Al-aghbari AM, Menino AR. Survival of oocytes
8. Meirow D, Levron J, Eldar-Geva T et  al. Pregnancy recovered from vitrified sheep ovarian tissues. Anim
after transplantation of cryopreserved ovarian tissue Reprod Sci 2002;71:101–10.
in a patient with ovarian failure after chemotherapy. 23. Courbiere B, Odagescu V, Baudot A et  al.
N Engl J Med 2005;353:318–21. Cryopreservation of the ovary by vitrification as an
9. Demeestere I, Simon P, Emiliani S, Delbaere A, alternative to slow-cooling protocols. Fertil Steril
Englert Y. Fertility preservation: Successful trans- 2006;86:1243–51.
plantation of cryopreserved ovarian tissue in a young 24. Bordes A, Lornage J, Demirci B et al. Normal gesta-
patient previously treated for Hodgkin’s disease. tions and live births after orthotopic autograft of vit-
Oncologist 2007;12:1437–42. rified–warmed hemi-ovaries into ewes. Hum Reprod
10. Silber SJ, DeRosa M, Pineda J et al. A series of mono- 2005;20:2745–8.
zygotic twins discordant for ovarian failure: Ovary 25. Amorim CA, Jacobs S, Devireddy RV et al. Successful
transplantation (cortical versus microvascular) and vitrification and autografting of baboon (Papio
cryopreservation. Hum Reprod 2008;23:1531–7. anubis) ovarian tissue. Hum Reprod 2013;28:2146–56.
11. Ernst E, Bergholdt S, Jorgensen JS, Andersen CY. The 26. Ting AY, Yeoman RR, Campos JR et al. Morphological
first woman to give birth to two children following and functional preservation of pre-antral follicles after
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Reprod 2010;25:1280–1. tem. Hum Reprod 2013;28:1267–79.
12. Roux C, Amiot C, Agnani G, Aubard Y, Rohrlich 27. Hreinsson J, Zhang P, Swahn ML, Hultenby K,
PS, Piver P. Live birth after ovarian tissue autograft Hovatta O. Cryopreservation of follicles in human
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15. Garcia Rada A. Spanish woman becomes pregnant 29. Keros V, Xella S, Hultenby K et al. Vitrification ver-
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16. Revelli A, Marchino G, Dolfin E et al. Live birth after 30. Amorim CA, Dolmans MM, David A et  al.
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Steril 2013;99:227–30. 31. Tedder RS, Zuckerman MA, Goldstone AH et  al.
17. Sugimoto M, Maeda S, Manabe N, Miyamoto H. Hepatitis B transmission from contaminated cryo-
Development of infantile rat ovaries autotransplanted preservation tank. Lancet 1995;346:137–40.
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2000;53:1093–103. Hovatta O. Clinical grade vitrification of human ovar-
18. Tokieda Y, Ishiwata I, Segino M, Ishikawa H, Sato K. ian tissue: An ultrastructural analysis of follicles and
Establishment of a novel method for cryopreserva- stroma in vitrified tissue. Hum Reprod 2011;26:594–603.
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Successful cryopreservation of mouse ovaries by vit- closed system using only ethylene glycol as a permeat-
rification. Biol Reprod 2003;68:881–7. ing cryoprotectant. Fertil Steril 2013;100:​170–7.

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33. Fabbri R, Vicenti R, Macciocca M et  al. Good pres- Comparison of rapid and conventional freezing.
ervation of stromal cells and no apoptosis in human Cryobiology 2007;55:261–8.
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2014:673537. Griesinger G. Slow-freezing versus vitrification for
34. Herraiz S, Novella-Maestre E, Rodriguez B et  al. human ovarian tissue cryopreservation. Arch Gynecol
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logic patients: Slow freezing versus vitrification, 37. Mathias FJ, D’Souza F, Uppangala S, Salian SR,
effect of different procedures and devices. Fertil Steril Kalthur G, Adiga SK. Ovarian tissue vitrification is
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18 Vitrification of cleavage-stage embryos and blastocysts
and their neonatal outcomes
Tetsunori Mukaida and Chikahiro Oka

INTRODUCTION vitrification solution (EFS40)9 with conventional cryo-

In assisted reproductive technology (ART), cryopreserva- straws was relatively effective for human embryos at the
tion of embryos has become important for the best use of four- to eight-cell stage.10 The effectiveness of vitrification
supernumerary embryos. During the steps of the cryo- was confirmed for human embryos at the 8–16-cell stage11
preservation of embryos, there is a risk of various types and the morula stage,12 also using EG-based solutions.13
of injury.1,2 Among them, the formation of intracellular Over the past decade, there have been several advances
ice appears to be the most damaging. The first strategy for in vitrification technologies such that it can provide high
preventing intracellular ice from forming was to adopt a clinical efficiency along with better clinical outcomes. It is
lower concentration of cryoprotectants and a long slow- proposed that vitrification will become the most suitable
cooling stage. This slow-freezing method has proven method for the cryopreservation of any cells and tissues in
effective for the embryos of a wide range of mamma- the near future. Thus, this chapter will focus on vitrifica-
lian species. Unlike embryos of laboratory animals and tion technologies for cryopreservation in human ART.
domestic animals, in which dimethyl sulfoxide (DMSO),
glycerol, or ethylene glycol (EG) are commonly used PRINCIPLES OF VITRIFICATION
as the cryoprotectants (cryoprotective agents [CPAs]), The basic procedure for vitrification is simple. Embryos
human embryos at early cleavage stages have most often are suspended in a vitrification solution and then plunged
been frozen in a solution of propanediol (PROH) supple- in LN2 or supercooled air. Embryos are warmed rapidly
mented with sucrose, 3 although those at the blastocyst and diluted quickly with a sucrose solution. The most
stage have more frequently been frozen with glycerol and important stage is the exposure of embryos to the vit-
sucrose.4–6 With slow freezing, however, it is difficult to rification solution before rapid cooling. In order to pre-
­eliminate injuries occurring from ice formation com- vent intracellular ice from forming, a longer period of
pletely. Furthermore, the slow-freezing method requires exposure is desirable. However, if the exposure is too
a relatively long period to be undertaken before the long, embryos suffer from the toxicity of the CPA solu-
embryos are finally stored in liquid nitrogen (LN2). tion. Therefore, the optimal exposure time for successful
In 1985, Rall and Fahy7 applied the innovative approach vitrification must be a compromise between preventing
of “vitrification,” in which injuries related to ice crystal the formation of intracellular ice and preventing toxic
formation are minimized by using very high concentra- injury. Ironically, embryos may be injured by the toxicity
tions of CPA together with rapid temperature change. The of the cryoprotectant before enough cryoprotectant can
definition of vitrification is the solidification of a solution permeate inside the embryos. To prevent this, a two-step
at a low temperature without the formation of ice crys- procedure is commonly adopted, in which embryos are
tals, by increasing the viscosity using high cooling rates.1,7 first equilibrated in a more dilute (e.g., 10%) CPA solu-
The rapid cooling process can minimize chilling injury tion, followed by a brief (30–60-second) exposure to a
and osmotic shock to the embryos. With improvements vitrification solution before embryos are cooled with
in recent decades, vitrification has become the most reli- LN2. The optimal exposure time in the vitrification solu-
able strategy, not only because it is technically simple, but tion depends not only on the CPA solution, but also on
also because it can lead to high survival and implantation the temperature, since both the permeability of embryos
rates. To induce vitrification in LN2 or supercooled air,8 the and the toxicity of the CPA are largely influenced by the
solution must contain a high concentration of CPAs. This temperature.6,7
approach simplifies the cooling process, because embryos In vitrification, the selection of the CPA requires
can be rapidly cooled directly in LN2. Although embryos extreme care because its concentration can be as high as
subjected to vitrification are potentially liable to being 6 M, which can make the toxicity of these compounds a
affected by the toxicity of the high concentration of CPAs, key limiting factor in cryobiology. The most appropriate
the method has been refined and proven to be effective characteristics of a penetrating CPA are low toxicity and
for the cryopreservation of embryos at various stages of high permeability. For the cryopreservation of human
development in laboratory and domestic species. In 1998, embryos, PROH and DMSO have been used as the domi-
it was demonstrated that vitrification using an EG-based nant CPAs, although glycerol is used when embryos are

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 vitrification in assisted reproduction
frozen at the blastocyst stage.6 As a less toxic CPA, EG is has proven suitable for human cleavage-stage embryos,10
commonly and widely used.2 However, few comparative including in Mukaida et al. (unpublished data). In 1998,
studies have examined the effect of the CPA on the sur- the effectiveness of this vitrification method for human
vival of vitrified embryos. cleavage-stage embryos was confirmed.10

CLEAVAGE-STAGE EMBRYO VITRIFICATION Vitrification Using the Cryoloop for Human Cleavage-
In 1998, an investigation was conducted to find a suitable Stage Embryos (Tables 18.1 and 18.2)
CPA and suitable conditions for exposing embryos to a An improvement to cleavage-stage ultrarapid vitrification
vitrification solution using eight-cell mouse embryos.10 came with the Cryoloop.14–17 This method is effective for
The survival rates of eight-cell mouse embryos vitrified human cleavage-stage embryos, for which conventional
in various solutions after exposure to the solutions for 0.5 vitrification using a straw was found to be less effective.
and 2 minutes at 20°C and 25°C were measured. The high- The protocol for vitrification using the Cryoloop can
est rates of survival were obtained with EG-based solu- be found in the following section on the vitrification of
tions, regardless of the time and temperature. Although blastocysts.
none of the vitrified embryos were morphologically nor- In 2008, Balaban et  al.18 reported a two-step protocol
mal when the embryos were vitrified after 0.5 minutes of using Cryoloop vitrification for human cleavage-stage
exposure to any mixture of 30% CPA, the survival rate embryos cryopreservation. Their protocol (Table 18.1)
was over 90% when embryos were treated for a longer was originally described by Larman et  al.19 Two steps of
time (2 minutes) at a higher temperature (25°C), or when dehydration and equilibration of CPA were applied prior
embryos were treated with a higher concentration of EG to cooling. The embryos were loaded onto the Cryoloop
(EFS40) at a higher temperature (25°C). (Hampton Research, Aliso Viejo, CA), transferring as little
In addition, a small saccharide (e.g., sucrose) and a medium as possible, typically around 50 nL. For warming,
macromolecule (e.g., Ficoll 70, bovine serum albumin multiple steps of rehydration with several different sucrose
[BSA], or polyvinylpyrrolidone [PVP]) are frequently concentrations were performed. Clinical outcomes were
included in vitrification solutions. These nonpermeating as follows (Table 18.2): a total of 73 women subsequently
agents are much less toxic, and are known to promote underwent vitrified–warmed embryo transfers, where
vitrification of the solution.9 Therefore, their inclusion a mean number of 3.3 embryos were warmed (n = 241).
can reduce the toxicity of the solution by decreasing the The cryosurvival rate was 92.1%, and all blastomeres were
concentration of the permeating agent required for vitri- intact in 72.1% of the embryos after the warming proce-
fication. In addition, inclusion of a saccharide promotes dure. The mean number of embryos transferred was 2.3
shrinkage of embryos, and thus reduces the amount of (n = 168), and clinical pregnancy and ongoing pregnancy
intracellular cryoprotectant, which will also reduce the rates of 49.3% and 45.2% were achieved, respectively. The
toxic effect of the permeating CPA.9 At the same time, implantation rate was 29.7% (n = 50), resulting in a mul-
the osmotic action of saccharides plays an important role tiple pregnancy rate of 36.1% (n = 13: 1 triplet, 12 twins).
in minimizing the swelling of embryos during dilution, At the time of reporting, 8 of the ongoing 33 pregnancies
since a quick dilution is necessary to prevent the toxic had had successful deliveries of healthy children (2 twins,
effects of the CPA solution. 6 singletons). Moreover, in this study, it was shown that vit-
rification was a more effective approach for cryopreserving
PROTOCOLS AND CLINICAL RESULTS OF human embryos than conventional slow freezing.
CLEAVAGE-STAGE EMBRYO VITRIFICATION In 2007, Desai et  al.20 reported the post-vitrification
There are several protocols that have been introduced for development, pregnancy outcomes, and live births for
human cleavage-stage embryo vitrification. However, in Cryoloop vitrification of human cleavage-stage embryos.
these protocols, the basic concept is similar, and the dif- Tables 18.1 and 18.2 include their protocol and results,
ferences between the protocols are related to the type and which presented consecutive vitrification–warming
concentration of CPAs and duration of exposure to CPAs. A cycles performed over a 2.5-year interval.
summary of those protocols and clinical outcome are briefly In 2005, Rama Raju et al.21 reported a modified proto-
described as follows and appear in Tables 18.1 and 18.2. col for the vitrification of human eight-cell embryos using
the Cryoloop technique. The protocol, including the type
Vitrification Using Conventional Cryo-Straws for of CPA and duration of exposure, is different from the one
Cleavage-Stage Embryos by reported by Desai (Table 18.1).20 Table 18.2 includes
This is a two-step protocol for vitrification with straw as results to show the effectiveness of their protocol.
a container using EG-based solutions, EFS20 and EFS40,
and has been described previously.1,10 The two solutions Vitrification Using Cryotops for Human
(EFS20 and EFS40) are used for pretreatment and vitrifi- Cleavage-Stage Embryos
cation, respectively, and contain EG diluted to 20% (v/v) Since the vitrification approaches of the Cryotop and
or 40% (v/v), plus a Ficoll–sucrose solution. This method Cryoloop use similar minimal-volume cooling systems,

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vitrification of cleavage-stage embryos and blastocysts and their neonatal outcomes
Table 18.1  Summary of the Protocols Regarding Concentration, Time, and Properties of the Vitrification
Solution for Day 2–3 Human Embryo Cryopreservation
Mukaida et al.10 Desai et al.20 Rama Raju et al.21 Kuwayama et al.22
Type of container Cryo-straw Cryoloop (1) Cryoloop (2) Cryotop
Temperature Room Warm stage (37°C) Warm stage (37°C) Room (25–27°C)
(25–27°C)
Equilibration EGFS20: 20% 7.5% EG + 7.5% DMSO 10% EG 7.5% EG + 7.5%
EG DMSO
Step 2 min 2 min 5 min 5–10 min*
Vitrification EGFS40: 40% 15% EG + 15% 40% EG + S 15% EG + 15%
EG DMSO + F + S DMSO + S
Step 1 min 35 s 30 s 1 min
Cooling system Vapor-phase Plunged into LN2 directly Plunged into LN2 Plunged into LN2
LN2 (3 min), (ultrarapid cooling) directly (ultra- directly
then plunged rapid cooling) (ultrarapid
into LN2 cooling)
Warming One step Two steps Four steps Two steps
Step 0.5 M S (5 min) 0.25 M S (2 min) 1 M S (2.5 min) 1 M S (1 min)
0.125 M S (3 min) 0.5 M S (2.5 min) 0.5 M S (3 min)
0.25 M S (2.5 min)
0.125 M S (2.5 min)

Note: In the first row, the authors and reference number of each protocol are indicated. In the second row, the types of
containers used for vitrification are indicated. In the third row, the temperature of each protocol performed is
indicated. In the fourth row, the first step of the vitrification protocol that required dehydration and permeation of
the cryoprotective agent (CPA) is described; the type of CPA and duration of exposure to CPA are indicated.
Permeating CPAs are shown in bold. In the fifth row, the second step of the vitrification protocol prior to plunging
LN2 is described, in the same way as the fourth row. In the sixth row, the method of cooling is indicated. In the
seventh row, the warming steps are described as single or multiple steps of different sucrose concentrations.
(DMSO = dimethyl sulfoxide; EG = ethylene glycol; F = Ficoll; LN2 = liquid nitrogen; S = sucrose.)
*The duration of equilibration is adjusted according to the time needed for re-expansion of the vitrified embryos. Cryoloop
(1): reported by Desai et al. in 2007.20 Cryoloop (2): reported by Rama Raju et al. in 2005.21

Table 18.2  Summary of the Clinical Results in Each Vitrification Approach for Day 2–3 Embryos
Mukaida et al.10 Desai et al.20 Rama Raju et al.21 Results of Nagata Clinic
Type of container Cryo-straw Cryoloop (1) Cryoloop (2) Cryotop
Age (years) Not available 34.1 ± 4.5 31.3 ± 4.5 35.0 ± 4.5
No. of cycles 127 77 40 604
346 patients
Survival rate Not available 201/236 121/127 1701/1774
85% 95% 95.9%
Cleavage ratea 486/661 184/236 Not available 1289/1774
76% 78% 72.7%
Pregnancy rate 34/127 34/77 14/40 164/604
26.8% 44.2% 35.0% 27.2%
Implantation Not available 40/201 18/121 192/1442
rate 19.9% 14.9% 13.3%
Delivery rateb 22/127 Not available 13/40 118/604
17% 32.5% 19.5%

Note: Cryoloop (1): reported by Desai et al. in 2007.20 Cryoloop (2): reported by Rama Raju et al. in 2005.21
a Including survival and further cleavage rate.
b Including on-going pregnancy.

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 vitrification in assisted reproduction
the basic concept of these protocols are the same. The fol- Currently, several established blastocyst vitrification
lowing protocol was originally introduced by Kuwayama.22 protocols have been reported. As one of the examples,
For vitrification using a Cryotop, the protocol is simi- this chapter includes our protocol of blastocyst vitrifica-
lar to blastocyst vitrification using a Cryoloop technique tion and a summary of the clinical outcomes for the last
described in the section on blastocyst vitrification. The 10 years, which confirm the safety and the effectiveness
differences with the protocol using the Cryotop are dura- of the Cryoloop technique for the cryopreservation of
tion of equilibration of the CPA for cooling steps and con- human blastocysts.
centration of sucrose for warming steps. Embryos with
70% or more intact blastomeres are considered as survi- PROTOCOL FOR BLASTOCYSTS VITRIFICATION
vors and kept in culture until transfer on the following (FIGURE 18.1)
day (Table 18.1). The protocol for the Cryoloop vitrification of blasto-
Table 18.2 includes the results from the use of the cysts was adopted from the work of Lane and Gardner17
Cryotops at Nagata Clinic to show the effectiveness of this with slight modifications.14–16 Procedures of vitrification
clinical application for human cleavage-stage embryos. involve equilibration and vitrification steps carried out
at 37°C in 7.5% DMSO and 7.5% EG for 2 minutes, and
BLASTOCYST VITRIFICATION 15% DMSO, 15% EG, 1% Ficoll 70, and 0.65 M sucrose
Recent advances in culture systems with sequential media for 30–45 seconds in human tubal fluid (HTF)/human
have made it possible to develop human in vitro fertiliza- serum albumin (HSA). At the end of 30–45 seconds, the
tion (IVF) embryos to the blastocyst stage more routinely. blastocysts are loaded on a small nylon loop (Hamilton
As the blastocyst is better suited to the uterine environ- Research, Laguna Niguel, CA), and are plunged directly
ment and blastocyst formation is one of the more useful into LN2 . They are warmed by placing the tip of the
informational criteria for selecting more viable embryos, Cryoloop into 0.5 M sucrose in HTF/HSA, and are kept
blastocyst transfer has become a promising option for there for 2 minutes, followed by 0.25 M sucrose in HTF/
raising the overall pregnancy rate.23,24 Accordingly, the HSA for 3 minutes. With the use of the Cryoloop as a
need to cryopreserve human blastocysts is increasing. container, the vitrified blastocyst almost floats in the
Menezo et  al.3 cryopreserved human blastocysts that
were developed in a co-culture system using the slow- (a) (b)
freezing method with glycerol and obtained reasonable
clinical results (27% pregnancy rate and 17% implanta-
tion rate). However, results reported by other clinics have
not been consistent.25–27 Menezo et  al.3 speculated that
the cryopreservation outcome might be influenced by the
culture conditions, such as a co-culture system.
Recently, human blastocysts were successfully vitrified
in straws.28 However, our own attempts to vitrify human (c) (d)
blastocysts using straws resulted in only 45% complete
survival (39/86, unpublished data). Vanderzwalmen
et  al.29 also reported a low pregnancy rate with human
blastocysts vitrified in straws. This is probably because
human blastocysts are much less permeable to CPA and
water, since it has been observed that they shrink more
slowly than mouse and bovine blastocysts in the CPA
solution. This suggests that human blastocysts are more
likely to be injured by intracellular ice crystal formation. Figure 18.1  (a) A minute nylon loop (20 μm wide, 0.5–
Increased rates of cooling and warming can help 0.7 mm in diameter) mounted on a stainless steel pipe
circumvent the problem of intracellular ice formation under 100× magnification. (b) A thin layer of the vitrifi-
in less permeable embryos. Faster rates of cooling and cation solution on the nylon loop after dipping the loop
warming can be achieved by minimizing the volume of into the vitrification solution. (c) Under a dissecting
the solution with which embryos are vitrified (i.e., by microscope, the capping portion of the cryovial with the
using minute tools such as electron microscopic grids, 30 Cryoloop attached by a stainless steel handling rod for
open pulled straws, 31 Cryoloops, 2,32 or Cryotop22). We manipulation (held by left hand), and a pulled Pasteur
showed that the transfer of human blastocysts vitrified pipette with blastocysts (held by the right hand). Prior
with Cryoloops can lead to successful births.14 Since this to loading, blastocysts are rinsed several times in small
original report, we have continued to use this vitrifica- drops of final vitrification solution (solution II) on the
tion approach for the cryopreservation of blastocysts on lid of a culture dish. (d) Blastocysts on the loop with a
day 5 and day 6. thin layer of vitrification solution.

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 vitrification of cleavage-stage embryos and blastocysts and their neonatal outcomes
thin filmy layer of the droplet on the nylon loop, and supplemented with exogenous female hormones to over-
heat conduction to the blastocyst becomes homogenous come multiple implantation failures, because uterine
and extremely high. With the extremely high cooling receptivity under CEC has been reported to be better
rate, full equilibration of CPA is not necessary to avoid than during hyperstimulated cycles in donor egg-sharing
ice crystal formation, and inside the cell is a so-called program studies. After two or three failures of implan-
“meta-stable situation.” This is why around 3 minutes tation following fresh transfer attempts, fresh blastocyst
of CPA exposure is enough to reach the vitrified status transfer is intentionally avoided with our clinical concept
inside the cells. This duration of exposure is shorter than based on our clinical experience. One of the reasons for
that of other vitrification approaches. Shorter exposure this is that ovarian hyperstimulation does not always
of CPA is more favorable due to avoiding the exposure of create a suitable uterine receptivity nor environment for
the potentially toxic agent to the cells. implantation, due to super-physiological levels of female
hormones, when compared to controlled endometrial
WARMING OF BLASTOCYSTS, ASSISTED preparation using exogenous hormones.
HATCHING, AND ASSESSMENT OF SURVIVAL On day 5 or 6 after the oocyte pick up, blastocyst devel-
With the cryovial submerged in LN2, the vial is opened opment was examined. On day 5 or 6, each embryo that
with the aid of the stainless steel rod, and the loop con- developed to the blastocyst stage was scored depending
taining blastocysts is removed from the LN2 and placed on the developmental stage, and graded according to
directly and quickly into the well containing the 0.5 mol/L quality criteria35 with slight modifications.15
sucrose solution. Blastocysts immediately fall from the Briefly, blastocysts were first given a numerical score
loop into the solution. Thus, blastocysts are warmed and from 1 to 6 on the basis of their degree of development.
are diluted instantly at around 37°C, adjusted by the stage Secondly, the blastocysts were graded in three ranks
warmer. After 2 minutes, the blastocysts are transferred based on morphological appearance. For example, the
to the 0.25 mol/L sucrose solution. After an additional inner cell mass was graded as A (many tightly packed
3 minutes, blastocysts are washed and are kept in the base cells), B (several loosely grouped cells), or C (few cells);
medium for 5 minutes. During this 5 minutes, assisted and the trophectoderm was graded as A (many cells
zona hatching (AH) is always performed on warmed blas- forming a cohesive epithelium), B (fewer cells forming a
tocysts with either acidic Tyrode’s solution, as previously loose epithelium), or C (very few large cells).
described,33,34 or by multiple shots of a laser pulse to the When patients had their fresh embryos transferred
zona pellucida (ZP) of the warmed blastocysts. Recently, on day 2–3, all the remaining embryos were cultured to
laser equipment manufactured by Research Instrument allow those that developed into blastocysts to be vitrified.
Limited (Saturn 5 Active™ RI, Cornwall) has been able Patients who received transfers of fresh blastocysts had
to create multiple laser pulses automatically along the ZP all their remaining supernumerary blastocysts vitrified.
in the biopsy mode. This approach has allowed embryolo- On  day 5, if at least one supernumerary blastocyst was
gists to perform AH very easily. graded as A or B, all the blastocysts of the patient were
About 2–3 hours after warming, the appearance of vitrified regardless of the developmental stage and the
the blastocysts is examined on an inverted microscope grading. In a few cases, compacted morulae with early
at 400 × magnification, and survival is assessed based on cavity formation were also vitrified with the blastocysts.
the morphological integrity of the blastomeres, inner cell If all the blastocysts of the patient were graded C, they
mass and trophectoderm, and re-expansion of the blas- were not cryopreserved. On day 6, if at least one blasto-
tocoele. The surviving blastocysts are scored as to devel- cyst had a large blastocoele (i.e., scored as 3–6), and was
opmental stage, and are graded according to quality as graded as A or B, all the developed blastocysts scored as
described in the section on the grading of blastocysts. 3–6 were vitrified.

PATIENTS AND GRADING OF BLASTOCYSTS ARTIFICIAL SHRINKAGE OF EXPANDED

For the following three categories of patients groups, BLASTOCYST
vitrified blastocyst transfer has been performed in our In 2003, it was reported that blastocyst survival rates
clinic: group 1—patients who had their fresh embryos were dependent on the developmental stage, and were
transferred on day 2–3, and all the remaining embryos negatively correlated with the expansion of the blas-
were cultured to allow those that developed into blas- tocoele.15 The survival rates of early blastocysts with a
tocysts to be vitrified; group 2—patients who received smaller blastocoele cavity, scored 1 and 2 according to
transfers of fresh blastocysts and had all their remaining quality criteria, 35 were 87% (48/55) and 97% (62/64),
supernumerary blastocysts vitrified; group 3—patients respectively. Also, full blastocysts lacking an expanded
who had no fresh embryo transfer because of ovarian blastocoele cavity, which were scored 3, had a survival
hyperstimulation syndrome (OHSS) symptoms, or who rate of 89% (99/111). The total survival rate of blastocysts
were attempting vitrified blastocyst transfer intention- scored 1–3 together was 91% (209/230). However, the
ally along with a controlled endometrial cycle (CEC) survival rate of both expanded and hatching blastocysts,

155
 vitrification in assisted reproduction
scored 4 and 5, respectively, was 85.0% (288/339), which at the cellular junction of the trophectoderm cells cre-
was significantly lower than that of the group scored 1–3 ates a hole to induce collapsing of the blastocoelic cavity
(p < 0.05). It was therefore postulated that a large blas- (Figure  18.3). The blastocoele of the expanded blasto-
tocoele might lessen potential to survive cryopreserva- cyst shrinks almost immediately. With the use of this
tion due to ice crystal formation during the rapid cooling laser system, it is not necessary to hold and locate the
phase of vitrification. In order to overcome this problem, expanded blastocyst with a holding pipette connected
shrinkage of the blastocoele was thought to be an appro- to a micro-manipulator. The laser technique makes the
priate approach. Several studies reported an increase in procedures simple and convenient.40 Recently, the laser
the survival rate of blastocysts when the volume of the system Saturn 5 Active™ (Research Instrument Limited,
blastocoele was artificially reduced with a glass micro- RI, Cornwall) has been used either for AH or AS as an
needle36 (a  29-gauge needle)37 or micropipetting with a improved system of laser equipment.
hand-drawn Pasteur pipette.38
Since 2003, we have therefore added artificial shrink- CLINICAL RESULTS OF AS PROCEDURES
age (AS) after puncturing the blastocoele with a micro- In order to show the effectiveness of the AS method,
needle, or laser pulse using the Cryoloop technique prior we summarized the results of 270 cycles in Table 18.3.
to vitrification, to improve the survival rate and clinical Results of vitrified expanded and hatching blastocysts in
outcomes of our vitrified blastocyst transfer programs. In our previous study reported in 2003 served as a control
2006, we reported the effectiveness of AS prior to vitri- group (without AS). The survival rate of both expanded
fication, including the confirmation of the safety of this and hatching blastocysts, scored 4 and 5, respectively,
procedure.39 Initially, AS was carried out using a glass was 85.0% (288/339). A statistical difference was noted
micro-needle to collapse the blastocoele (Figure 18.2). between the study and the control groups (p < 0.05).
After complete shrinkage of the blastocyst, it was vitrified When the pregnancy rate of the study group was com-
and stored in a LN2 tank. pared with the control group, a statistically significant
Since September 2004, a laser pulse generated by the improvement was noticed in the AS group (60.2% versus
laser system ZILOS-tk™ (Hamilton Thorn Bioscience 34.1%; p < 0.01).
Inc., Beverly, MA) has been introduced to perform the We also performed preliminary comparisons between
AS, instead of micro-needle puncture. The inner cell the results achieved by using micro-needle or laser
mass should be located away from the targeted point of pulse for blastocoele shrinkage, to show the difference
the laser pulse. One single laser pulse (200 ms) targeted of methodologies for AS. The survival rates achieved

(a) (b) (c)

(d) (e) (f)

Figure 18.2  Artificial shrinkage of expanded blastocyst with the micro-needle. (a) Holding the expanded blastocyst
with a holding micropipette connected to a micro-manipulator. (b) Insertion of the micro-needle inside the blasto-
coele at a point away from the inner cell mass. (c) Puncture through the blastocoele and removing the micro-needle
gradually. (d) Beginning of shrinkage 10 seconds after puncture. (e) Partial shrinkage 30 seconds after puncture.
(f) Complete shrinkage 1 minute after puncture.

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 vitrification of cleavage-stage embryos and blastocysts and their neonatal outcomes

(a) (b) (c)

(d) (e) (f )

Figure 18.3  Artificial shrinkage of an expanded blastocyst with a single laser pulse. (a) Prior to the artificial shrink-
age. (b) A single laser pulse at the point of the cellular junction of the trophectoderm cell at a point away from the
inner cell mass (circle indicated). (c) Beginning of shrinkage 5 seconds after laser shot. (d) Shrinkage 10 seconds
after laser shot, and arrows indicating formation of the peri-vitelline space because of contraction. (e) Shrinkage
20 seconds after laser shot. (f) Almost complete shrinkage 30 seconds after laser shot.

Table 18.3  Characteristics of Patients and Survival of similar. No  statistical difference was observed in the
Vitrified Human Blastocysts with (Study Group) or results achieved with the two methods.39
without (Control Group) Artificial Shrinkage
CLINICAL RESULTS OF VITRIFIED BLASTOCYST
Study group Control group
TRANSFER
No. of patients 245 76 Table 18.4 summarizes the clinical results of our vitrified
Average age (years) 35.6 34.0 blastocyst transfer program using the Cryoloop between
Mean no. of previous in vitro 2.1 2 the years 2000 and 2013.
fertilization/intracytoplasmic Clinical results and perinatal outcomes of vitrified
sperm injection attempts blastocyst transfer using a Cryoloop performed since the
No. of initiated vitrified blastocyst 270 — beginning of 2000 until the end of 2013 (14 years) are
cycles summarized. A total of 12,941 blastocysts originating from
No. of cycles with vitrified 266 85 8,440 cycles were vitrified and warmed. The mean age was
blastocyst transfer 36.6 years.
No. of cancelled cycles due to no 4 (1.5%) — After warming for transfer, 12,339 (95.3%) vitrified
survival of vitrified blastocyst (%) blastocysts survived. In 93 cycles (1.1%), no blastocysts
No. of blastocysts vitrified 502 339
No. of vitrified blastocysts survived 488 288 Table 18.4  Clinical Outcomes of Vitrified Blastocyst
Survival rate 97.2% 85.0%* Transfer at the Tokyo and Hiroshima Human Assisted
No. of vitrified blastocysts 448 — Reproductive Technology (HART) Clinics (2000–2013)
transferred
Criteria Data
Mean no. of blastocysts transferred 1.7 —
Clinical pregnancies 160 29 Total no. of attempted cycles 8440
Clinical pregnancies (%) 60.2% 34.1%** Total no. of warmed–vitrified BL 12,941
Total no. of survived BL 12,339
Note: Control group refers to our previous study (Mukaida Survival rate 95.3%
et al.15). No. of transferred cycles 8347
*p < 0.05, **p < 0.01. Mean no. of BL transferred 1.35
No. of clinical pregnancies (%/BT) 3948 (47.3%)
with the two methods were similar (micro-needle: 97.2% No. of implantations (%) 4362 (38.6%)
­versus laser pulse: 97.5%). The mean number of survived No. of births (babies; boy:girl) 2483 (2757; 1413:1344)
blastocysts transferred was also similar. Clinical preg- No. of miscarriages (%) 967 (24.5%)
nancy, implantation, and miscarriage rates were also = blastocyst; BT = blastocyst transfer.
Note: BL 

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 vitrification in assisted reproduction
survived or surviving blastocysts were obtained, but clinically more effective than slow-cooling methods or
embryo transfer was cancelled because the number not, it is necessary to perform well-established random-
of cells that survived and the quality of the blastocysts ized control studies. Since our cryopreservation program
were not considered to be suitable for transfer. A total of clearly revealed better clinical outcomes obtained with
11,295 blastocysts were transferred in 8,347 cycles. The vitrification when compared with that of slow cooling at
mean number of blastocysts transferred per cycle was the blastocyst stage, to have performed a randomized clini-
1.35. From 2009 to 2013, the mean number was 1.13 per cal trial would have been extremely difficult at our private
cycle. Of 8,347 transfers, 3,948 cycles resulted in clinical ART center. In numerous studies investigating the differ-
pregnancy (confirmed by gestational sac in the uterus); ent embryo cryopreservation techniques, few of them were
the pregnancy rates were 46.8% per warming cycle and properly constructed to truly evaluate whether one tech-
47.3% per transfer. The implantation rate was 38.6% nique was superior to the other or not. In order to compare
(4,362/11,295). embryo cryopreservation methods, a systematic review
A total of 2757 babies were born from 2483 deliveries. and meta-analysis of the literature has been performed by
Since 1413 babies were boys and 1344 were girls, no bias Abdel-Hafez in 2010.41 From the 2700 publications, they
in the sex ratio was observed. Cesarean sections were found 11 prospective and randomized studies. Five were
performed in 1227 deliveries, and the mean gestational excluded because they did not show data as required, and
age was 38.1 weeks. The mean birth weight of overall six were included. In four of the six studies, there was a
births from vitrified blastocysts was 2823 g. However, direct comparison between vitrification and slow freezing
in the singleton delivery outcome (2229 births), the that suggested better results were obtained with vitrifica-
mean gestational age was 39.4 weeks, and mean birth tion. The embryo survival rate was significantly higher
weight was 3071 g. This is not statistically different from with vitrification than with slow freezing, and implanta-
the national statistics on ART conception reported in tion and pregnancy rates were also higher in two of the
2009 in Japan. A total of 240 births were twins (9.7%) studies.22,42 Finally, the group of Desai made the following
and 14 births were triplets (0.6%). Forty three cases had conclusion: results of the current meta-analysis showed
either congenital birth defects or peri-natal complica- that embryo vitrification is superior to slow freezing based
tions (1.6%), including six chromosomal abnormali- on direct comparison of embryo survival and clinical
ties (two trisomy 18 and six trisomy 21), nine multiple pregnancy rates.43 Ongoing pregnancy and implantation
anomalies, one stillbirth due to hydrocephalus, six still- rates were also higher with vitrification as compared with
births of unknown causes during delivery (at 25, 29, 30, slow freezing. In oocyte cryopreservation, several studies
32, 37, and 39 weeks of gestation), two anencephaly, one have also indicated that vitrification appears to be supe-
spina bifida, eleven congenital heart or major vessel mal- rior to slow-freezing methods, leading to improved rates of
formations, three minor anomalies in the hands and/or oocyte survival, fertilization, and embryonic development
feet, one congenital esophageal obstruction, one biliary in vitro.44,45 They showed less disruption of spindle integrity
duct obstruction, one Cornelia de Lange syndrome, and and chromosome alignment in the vitrified human oocytes
one Treacher Collins syndrome. when compared with the slow-frozen human oocytes.
A total of 967 pregnant cycles ended in miscarriage Therefore, they concluded that the lower rate of embryonic
(24.5%). A comparison of 1187 pregnancies established development from the slow-frozen human oocytes might
from fresh blastocyst transfers in our group of clinics be related to the greater level of damage to spindle integrity
during the same period shows that 249 (21.1%) resulted in and chromosome alignment.
miscarriages, and no statistical difference was observed
between them. This was also similar to that which we SUMMARY
reported previously in 2005.40 For embryo cryopreservation, the vitrification method
has many advantages over the classically established con-
RESULTS WITH COMPARISON TO SLOW ventional slow-freezing method: (1) injuries related to
FREEZING ice are less likely to occur; (2) survival of embryos can
There are two main categories of embryo cryopreserva- be maintained at a higher level if conditions for embryo
tion techniques: slow-cooling methods and vitrification. treatment are optimized; and (3) embryos can be cryo-
Slow-cooling methods involve a step-wise programmed preserved by a simple method in a short period with-
decrease in temperature along with manual seeding at out an expensive, sophisticated programmable freezer.
approximately a temperature of −7°C. The duration of Therefore, vitrification is suitable for human embryos in
the procedure requires about 2 hours, as well as the use of which a small number of embryos are cryopreserved fre-
expensive programmed instruments. As described above, quently. Human embryos at early cleavage stages can be
vitrification solidifies the oocytes/embryos into a glass- cryopreserved by conventional vitrification using cryo-
like state, thus avoiding the formation of both intra- and straws or by ultrarapid vitrification using Cryoloops.
extra-cellular ice. Regardless of whether vitrification is Human blastocysts are more efficiently cryopreserved by

158
 vitrification of cleavage-stage embryos and blastocysts and their neonatal outcomes
the ultrarapid approach. Clinical outcomes show that the IMPORTANT POINTS OF THIS CHAPTER
vitrification of blastocysts using the Cryoloop technique 1. In human ART treatment, cryopreservation of
results in high survival and high pregnancy rates, which embryos is one of the most useful approaches for
confirm the safety of this procedure, as seen in our peri- utilizing supernumerary embryos after controlled
natal evaluation. ovarian hyperstimulation to increase the chance of
At first, vitrification was introduced as an alternative pregnancy per IVF attempt (cumulative pregnancy
approach for the cryopreservation of human gametes rate).
and embryos; however, vitrification, with recent techni- 2. To store human eggs/embryos under LN2 while
cal improvements, has become a more reliable strategy, maintaining their viability, intracellular ice crys-
not only because it is very simple, but also because it can tal formation should be avoided completely with
lead to high clinical efficiency, along with better clini- proper equilibration of the CPA for dehydration
cal outcome. In particular, ultrarapid vitrification opens inside the egg/embryo. Also, proper rehydration
a new era for oocyte and blastocyst cryopreservation, as during the warming steps is necessary by reducing
described in this chapter. Classically, adequate equilibra- the osmotic stress with a sucrose solution.
tion of CPA and dehydration is necessary to cryopre- 3. In order to cryopreserve human embryos in LN2,
serve gametes and embryos. However, the extremely high either a conventional slow-cooling method or vit-
cooling rate achieved by direct plunging into LN2 with a rification method may be performed, but there
minimal ­volume (≤0.5 µL) of final vitrification solution, are major differences between them in terms of
including vitrified cells, can obtain high cryosurvival rates cooling rate (slow or ultrarapid) and the nature of
and better viability, and allow us to avoid ice crystal for- equilibrating the embryos with CPA (either high
mation even with the lower concentrations of CPA, which or low concentrations, and different durations of
could cause devitrification (i.e., ice crystal formation) if exposure).
conventional cooling was to be applied. The 2012 annual 4. The vitrification method is becoming increas-
report of the ART registry of Japan, organized by The ingly used due to clinically favorable outcomes
Japanese Society of Obstetrics and Gynecology, showed when compared to the conventional slow-cooling
a remarkable increase in the proportion of cryopreserva- approach, because of the simplicity of the tech-
tion ­activity in clinical ART programs. Briefly, in 2012, a nique itself, and also its higher cryosurvival rates
total of 326,426 ART cycles were performed (IVF: 82,108; and clinical results.
intracytoplasmic sperm injection [ICSI]: 125,229; frozen 5. The blastocyst stage seems to be the most reliable
embryo transfer [FET]: 119,089), and a total of 37,953 and appropriate stage for the cryopreservation of
babies were born (IVF: 4740; ICSI: 5498; FET: 27,715) human embryos with the vitrification approach,
from these ART procedures. When the proportions of live because confirmation of embryo development to
birth outcomes are compared between ART procedures, the blastocyst stage is thought to be one of the best
the summary indicates that almost three-quarters of ART quality-assurance criteria for potential embryo via-
babies born (73.0%) were generated via cryopreservation.46 bility, and additionally, the intracellular permeabil-
The clinical benefits of cryopreservation, as we mentioned ity of the CPA and size/characteristics of the cells
before, are major reasons why there is an increasing pro- of the blastocyst are more suitable for cryopreser-
portion of ART babies born following cryostorage, rather vation. Therefore, high cryosurvival and implan-
than following fresh embryo transfer. The reasons include: tation rates are now being obtained with vitrified
apparently higher pregnancy and implantation rates when human blastocyst programs.
compared with fresh transfers; the avoidance of higher
risks for OHSS; and also the provision of significant con-
venience for the scheduling of transfer timings. This trend DISCLOSURES
will inevitably be continued, and a lot of clinical programs Conflicts of interest: Tetsunori Mukaida and Chikahiro
might be ready to eliminate the transfer of fresh embryos Oka declare that they have no conflicts of interest.
altogether. Human rights statements and informed consent: all
More recently, this ultrarapid vitrification approach procedures followed were in accordance with the ethi-
has been considered as applicable for ovarian tissue and cal standards of the responsible committee on human
stem cell cryopreservation, and with proper preparation experimentation (institutional and national) and with
of ovarian tissue, such as 1 cm × 1 mm segmentation, in the Helsinki Declaration of 1964 and its later amend-
addition to a properly designed container, high survival ments. Informed consent was obtained from all patients
and better post-warming viability may well be expected for being included in the study.
from this vitrification approach.47 Ultimately, vitrifica- Animal studies: all institutional and national guide-
tion will become the most suitable and routinely applied lines for the care and use of laboratory animals were
method for cryopreservation of any cells and tissues. followed.

159
 vitrification in assisted reproduction
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apid vitrification. Reprod Med Biol 2002;1:1–9. metabolism, and blastocyst formation. Hum Reprod
3. Lassalle B, Testart J, Renard JP. Human embryo fea- 2008;23:1976–82.
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tion with the use of 1,2 propanediol. Fertil Steril Maintenance of the meiotic spindle during vitrifica-
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19 Vitrification of human blastocysts: Clinical realities
and neonatal outcomes
Juergen Liebermann

INTRODUCTION cells for cryostorage, high cooling rates in the range of

The announcement in 1972 of the survival of mouse 2500–30,000°C/min or greater, in combination with high
embryos after cryopreservation at −196°C and thawing concentrations of cryoprotectants, are used. During vit-
was groundbreaking.1 Since then, the impact of cryo- rification, water is transformed directly from the liquid
preservation on the growth and improved efficiency of phase into a glassy vitrified state. The physical definition
assisted reproduction in humans has become increasingly of vitrification is the solidification of a solution at low
appreciated. The percentage of frozen human embryos temperature, not by ice crystallization, but by extreme
has risen steadily over the years, with approximately elevation in viscosity during cooling.2,3 A primary strat-
one and a quarter million babies born following cryo- egy for vitrifying cells and tissue is to increase the speed
preservation. Moreover, cryopreservation has also been of thermal conductivity, while decreasing the concentra-
shown to increase overall pregnancy rates, while allow- tion of the vitrificants to reduce their potential toxicity. In
ing for further selection of embryos. Indeed, it is pos- general, the rate of cooling/warming and the concentra-
sible to achieve implantation and pregnancy rates with tion of the cryoprotectant required to achieve vitrifica-
frozen–thawed embryos as high as those achieved with tion are inversely related. In addition, recent publications
fresh embryos. Routine in vitro blastocyst culture and have shown the relatively greater importance of warm-
cryopreservation have been shown to increase pregnancy ing rates over cooling rates with regard to the survival of
rates, while allowing for better selection of embryos. In oocytes subjected to a vitrification procedure.4,5
the early days of human in vitro fertilization (IVF) his- The earliest attempts at using vitrification as an ice-
tory, before reliable cryopreservation protocols, there free cryopreservation method for embryos were first
was an emphasis on transferring as many embryos as reported in 1985.6 In 1993, successful vitrification of
possible into patients. With more reliable cryopreserva- mouse embryos was demonstrated.7 Furthermore, bovine
tion techniques such as “vitrification,” lower numbers oocytes and cleavage-stage embryos were vitrified and
of embryos are now being transferred, resulting in less warmed successfully a few years later.8 In 1999 and 2000,
high-order multiple pregnancies, as well as increased successful pregnancies and deliveries after vitrification
healthy implantations. In addition, decreased numbers and warming of human oocytes were reported.9,10 Since
of embryos are transferred, so increasing the potential that time, and because it seems to be that both entities
for more embryos to be placed into frozen storage, thus appear to be especially chill-sensitive cells in assisted
reducing the number of fresh cycles. The fundamental reproductive technology (ART), oocytes and blasto-
objectives for successful cryostorage of cells in liquid cysts appear to have received a significant boost in sur-
nitrogen (LN2) at −196°C can be summarized as follows: vival rates by avoiding ice crystallization through use
(1) arresting the metabolism reversibly; (2) maintaining of vitrification.11 In general, vitrification solutions (VS)
structural and genetic integrity; (3) achieving acceptable are aqueous cryoprotectant solutions that do not freeze
survival rates after thawing; (4) maintenance of develop- when cooled at high rates to very low temperatures.
mental competency post-thaw; and (5) the technique has Vitrification is very simple, requires no expensive pro-
to be reliable and repeatable. grammable freezing equipment, and relies principally on
Cryopreservation slows or totally prevents unwanted the placement of the embryo in a very small volume of
physical and chemical changes. The major disadvantage of vitrification medium that must be cooled at extreme rates
using cryostorage is that it can lead to the crystallization not obtainable in traditional enclosed cryostorage devices
of water, and thereby can create new and unwanted physi- such as straws and vials.
cal and chemical events that may injure the cells that are Although initially reported in 1985 as a successful
being preserved. Vitrification, however, avoids ice forma- cryopreservation approach for mouse embryos,6 vitrifi-
tion altogether during the cooling process by establishing cation took a backseat for a number of years in human
a glassy or vitreous state, wherein molecular translational assisted reproduction. One perceived “drawback” held by
motions are arrested without structural reorganization embryologists who are not familiar with the vitrification
of the liquid in which the reproductive cells are sus- technique is the use of high concentrations of cryoprotec-
pended. To achieve this glass-like solidification of living tants, which does mean that VS are potentially more toxic

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 vitrification in assisted reproduction
than their counterpart solutions used for conventional between the intra- and extra-cellular compartments. The
slow freezing. This is necessitated by the practical limit high sucrose concentration cannot totally prevent the cell
of the rate of cooling, and the biological limit of tolerance from swelling, but it can reduce the speed and magnitude
of the cells for the concentration of toxic cryoprotectants of swelling.16–18
being used to achieve the cryopreserved state. It is impor- However, the blastocyst as a chill-sensitive entity has
tant to note that recently published papers12–15 have shown a characteristic that is unique to its stage: a fluid-filled
that the use of relatively high concentrations of cryopro- cavity called the blastocoele. A decrease in survival rate
tectants, such as 15% (v/v) ethylene glycol (EG) used in an after vitrification has been noted when the volume of the
equimolar mixture with dimethyl sulfoxide (DMSO), had blastocoele cavity is increased. This is likely due to insuf-
no negative effect on the perinatal outcomes from blasto- ficient permeation of CPAs into the blastocoele, with
cyst transfers following vitrification when compared with residual water in the cavity increasing the potential for ice
those from fresh blastocyst transfers. crystal formation during cooling, and diluting the CPAs
Cryoprotective agents (CPAs) are essential for the reaching the inner cells. Residual fluid in the blastocoele
cryopreservation of cells. Basically, two groups of cryo- can therefore reduce post-warming survival of embryos.
protectants exist: (1) permeating (e.g., glycerol, EG, and Vanderzwalmen et  al.19 showed that survival rates in
DMSO) and (2) nonpermeating (e.g., disaccharides, pro- cryopreserved, expanded blastocysts could be improved
teins, and polymers) agents. The key component of a VS by artificial reduction of the blastocoele cavity, and others
is the permeating agent. These compounds are hydro- consider that blastocoele collapse is necessary pre-vitrifi-
philic nonelectrolytes with a strong dehydrating effect. cation on whichever day the blastocyst forms.19,20
Furthermore, these CPAs are able to depress the “freez- Although one study has suggested that expanded
ing point” of the solution. As the permeating CPA is blastocysts show no significant differences in viability,
responsible for potential toxicity (a key limiting factor in implantation potential, or pregnancy outcome when fro-
cryobiology), different cryoprotectants have been tested zen on day 5 versus day 6,21 our “body of data”13–15 refutes
for their relative toxicity, and the results indicate that EG the comparable implantation rates (IRs) for blastocysts
(MW 62.02) is the least toxic, followed by glycerol. In gen- cryopreserved on days 5 or 6. While there are several pos-
eral, the permeating CPA should be chosen firstly by their sibilities as to why day 6 blastocysts perform less well than
permeating property, and secondly on the basis of their day 5 blastocysts, one explanation could be that the older
potential toxicity. Additionally, these highly permeating blastocysts are often more expanded, with a significantly
cryoprotectants are also more likely to diffuse rapidly out larger blastocoele impacting water loss and CPA absorp-
of the cells, so that the cells quickly regain their original tion. Being mindful of previously published research on
volume upon warming, thus preventing osmotic injury. the artificial collapse (AC) of blastocysts prior to cryo-
The second component of a VS consists of the nonperme- preservation, we looked for an opportunity that could
ating CPAs, such as disaccharides (e.g., sucrose), which potentially help us improve the outcomes for day 6 blas-
do not penetrate the cell membrane, but help to draw out tocysts using AC of the blastocoele prior to vitrification.
more water from cells by osmosis, and therefore lessen the Further, if the concept showed any improvement for the
exposure time of the cells to the toxic effects of the per- day 6 blastocysts, it might also be applied to day 5 blasto-
meating CPAs. The nonpermeating sucrose also acts as cysts that had well-expanded blastocoeles.
an osmotic buffer to reduce the osmotic shock that might AC of the blastocoele can be performed using different
otherwise result from the dilution of the cryoprotectant techniques, such as micro-needles, sucrose solutions, or
after cryostorage. In addition, permeating agents are lasers.20,22–26 In 2003 and 2004, two groups independently
able to bind with intracellular water, and therefore water reported a beneficial effect by applying AC to blastocysts
is very slowly removed from the cell. Hence, the criti- prior to vitrification. Son et  al.22 observed an improved
cal intracellular salt concentration is reached at a lower clinical pregnancy rate (cPR) of 48% and an IR of 29%
temperature. Removal of the CPA during warming can with the use of AC. Hiraoka et al.23 collapsed day 5 and
present a very real problem in terms of trying to reduce day 6 blastocysts by manually pipetting embryos until
toxicity to the cells. Firstly, because of the toxicity of the they collapsed, and achieved a cPR of 50%, with an IR of
VS, quick dilution of them after warming is necessary; 33% after warming. Moreover, Mukaida et al.20 found that
and secondly, during dilution, water permeates more rap- the survival rate of vitrified blastocysts was negatively
idly into the cell than the cryoprotective additive diffuses correlated with the size of the blastocoele. They specu-
out. As a consequence of the excess water inflow, the cells lated that a large blastocoele may disturb the efficacy of
are threatened by injury from osmotic swelling. In this vitrification. They collapsed the blastocoele by punctur-
situation, the nonpermeating sucrose acts as an osmotic ing it with a micro-needle, or by making a hole between
buffer to reduce the osmotic shock. During warming, two trophectoderm cells with a laser pulse. After applying
using a high extracellular concentration of sucrose (e.g., AC, the survival improved from 86% to 97.2%. Moreover,
1.0 M) counterbalances the high concentration of the their pregnancy rate went up from 34.1% to 60.2%, with
CPAs in the cell, as it reduces the difference in osmolarity an IR of 46.7%. Iwayama et al.24 used a laser pulse, or

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 vitrification of human blastocysts
osmotic shock resulting from exposure of the whole pulse length) is applied to the cell junction. The blastocyst
embryo to sucrose, and the IR was significantly higher in is then moved back into the incubator for 5–10 minutes.19
both groups compared to the control group without AC Briefly, blastocysts have to be placed in equilibration solu-
(59.7% and 49.3% versus 34.2%). Furthermore, Hur et al.25 tion, which is the base medium (M199 with 20% serum
looked at the effect of AC achieved using a 29-gauge nee- supplement substitution [SSS] containing 7.5% [v/v] EG
dle or laser pulse on clinical outcomes in fresh transfers, and 7.5% [v/v] DMSO) (see Table 19.1). After 5–7 minutes,
and they observed a significant increase in the cPR in the the blastocysts need to be washed quickly in VS, which is
study group compared to the control group (58.8% versus the base medium containing 15% (v/v) DMSO, 15% (v/v)
39.0%). EG, and 0.5 M sucrose, for 45–60 seconds and transferred
All publications mentioned, including Liebermann onto the HSV using a micropipette. Immediately after the
and Conaghan, 26 conclude that AC has a beneficial effect loading of not more than two blastocysts in less than a
both in frozen blastocyst transfers and for overall cumu- 1-μL drop on the HSV, the straws are heat sealed, then
lative pregnancy and IRs. plunged in LN2, and finally stored inside 5-mL LN2 pre-
filled canes (Visotube Rond, IMV, France). Each single
MATERIALS AND METHODS step is described in detail below.
Materials
1. HSV (High Security Vitrification Kit [catalog # 1. Aseptic techniques are required at all stages. For
022137]; CryoBioSystem) equilibration and vitrification procedures, ensure
2. Heat Sealer (CryoBioSystem) the benchwarmer is at room temperature (~24°C)
3. Polycarbonate micropipettes, 300 μm end hole (see “Addendum,” Notes 3 and 4).
(MidAtlantic Diagnostics# KFPIP-1170-10BS) 2. Take reagents from the refrigerator and allow them
4. Brady TLS 2000 Thermal Label Printer to warm to room temperature.
5. Brady Labels (PTL-19-427) 3. Move blastocysts to freeze into a separate well.
6. 90 × 15 mm Petri dish (Nuncleon # 150362) Bring this dish to the inverted microscope and with
7. Center-well organ culture dish (Falcon 3037) the embryo positioned with the laser objective, use
8. Styrofoam container a single pulse to hit the blastocysts between two
9. Visotubes 10 mm (IMV 5561) trophectoderm cells to collapse the embryo. Place
10. Cryo Canes Aluminum (ThermoScientific 5015-0001) the dish back into the incubator for 5–10 minutes.
4. Label a Petri dish with the patient’s name under the
Reagents lid as follows: HTF-HEPES, equilibration solution
1. Serum substitute supplement (SSS) (Irvine) (ES), and VS.  Prepare 2 × 50 μL of HTF-HEPES
2. Modified human tubal fluid (mHTF) (Irvine) (+20% SSS–mHTF), 2 × 50 μL of ES, and 4 × 50 μL
3. Vit-Kit—Freeze (Irvine Scientific # 90133DSOC) of VS (see Figure 19.1).
4. Vit-Kit—Thaw (Irvine Scientific # 90137DSOC) 5. Brady labels should include the patients’ last name,
first name, accession number, Master Patient Index
Equipment number, and date plus the number and type of
1. Dissecting stereo microscope (Olympus SZX-12, embryos.
Bausch Lomb or Leica) with warming stage 6. Before vitrification, use a stripper tip with a 200-
2. Laminar flow hood (Origio) μm end hole for loading the blastocysts on the top.
3. Inverted microscope (Olympus IX-71) 7. Fill a Styrofoam container with LN2.
4. Infrared 1.48-μm diode laser (Hamilton Thorne—
Zilos laser [Hamilton Thorne Research, Beverly,
MA])
Table 19.1  Summary of Vitrification and Warming
Methods Solutions (Irvine Vit-Kit “Freeze” and “Thaw”)
Stepwise blastocyst vitrification procedure
Composition
Vitrification of blastocysts is undertaken utilizing a
“closed system” (HSV: CryoBio System, L’Aigle, France; Ethylene Dimethyl Sucrose
FDA 510(k) clearance for cleavage-stage embryos in glycol (%) sulfoxide (%) (M)
blastocysts) (see “Addendum,” Notes 1 and 2) after a Equilibration solution (ES) 7.5 7.5 0
two-step loading with cryoprotectant agents at 24°C (see Vitrification solution (VS) 15 15 0.5
“Addendum,” Notes 3 and 4). If AC is done prior to vitri- Thawing solution (TS) 0 0 1.0
fication, then the blastocyst is put on an inverted micro- Diluent solution (DS) 0 0 0.5
scope equipped with a laser system (Zilos-tk, Hamilton
Wash solution (WS) 0 0 0
Thorne), the junction of two trophectoderm cells in each
blastocyst is located, and one shot (100% power, 500 μs Note: M-19 9 + 20% SSS (Wash solution).

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 vitrification in assisted reproduction

ES 16. Store at the cane in a nitrogen tank.

17. Make sure to record the cane location on the freez-
mHTF (Merge drops) ing worksheet and cryo-inventory log.
18. Complete all paperwork and recheck that all vial
2 3 5 locations are logged in the embryo inventory.
1 (Transfer 6 VS Stepwise blastocyst warming procedure
to drop)
7 It is important to mention that, regardless of the day of
ES cryopreservation of the embryo (whether day 5, 6, or 7), at
4 8 thawing, blastocysts should be treated as if they had been
cryopreserved on the fifth day of development. To remove
the cryoprotectants, blastocysts need to be warmed and
diluted in a three-step process. With the HSV submerged
in LN2, the inner straw should be removed, and then
Figure 19.1  Setup for the vitrification procedure on a the ­carrier with the blastocysts can be removed from
plain 90-mm dish lid surface. (VS = vitrification solu- the LN2 and placed directly into a prewarmed (37°C)
tion; ES = equilibration solution; mHTF = modified organ culture dish containing 1 mL of 1.0 M sucrose
human tubal fluid.) (see “Addendum,” Note 9). Blastocysts can be picked up
directly from the HSV and placed in a fresh drop of 1.0 M
sucrose at 24°C, and immediately connected with a drop
8. Each sample that is vitrified will be done in a sepa- of 0.5 M sucrose. After 5 minutes, blastocysts are trans-
rate hood and verified by a second embryologist ferred to a 0.5 M sucrose solution, and connected with
before proceeding. Vitrify good expanded/hatch- drops of base medium for an additional 5 minutes. When
ing blastocysts on day 5/6/7. switching the embryos between different concentrations
9. Remove embryos from culture dishes using a of warming solutions, be sure to fill up the pipette with
stripper tip into the modified human tubal fluid the next-lowest concentration of warming solution before
(mHTF) (drop 1), gently aspirating to remove any picking up the cells for moving into the next dilution
residual culture medium. (see “Addendum,” Note 10). Finally, the blastocysts are
10. Pipette from mHTF (drop 1) to the other drop of washed in the base medium for 3 minutes, then returned
mHTF (drop 2) and immediately merge it with the to culture medium (SAGE+20%SSS) (SAGE Blastocyst
first drop of ES (drop 3). Set the timer for 5 minutes. Medium, Trumbull, CT, USA) in the incubator until
11. After 5 minutes, transfer the embryos to the transfer. Each single step is described in detail below.
remaining drop of ES (drop 4). Set the timer for
3 minutes. Place the embryos on the top of the drop 1. Take reagents from the refrigerator and allow them
and let them settle to the bottom. to warm to room temperature. All cryoprotectants
12. Load the blastocysts in a VS back-loaded strip- are removed at 24°C.
per tip, and rinse through the four droplets of VS 2. Place a 200-μL drop of thawing solution (TS)
(drops 5–8); clean the tip between each droplet. on a Petri dish and place on a warming plate (see
13. Placement into the VS and loading of the Cryotop “Addendum,” Note 9).
should take less than 1 minute, so that the total 3. Label a Petri dish with the patient’s name under the
incubation time in VS is 30 seconds. After 30 sec- lid as follows: TS, diluent solution (DS), and washing
onds, gently transfer them to the tip of the HSV by solution (WS). Prepare 1 × 50 μL of TS, 4 × 50 μL
using a stripper tip to load the blastocyst(s) in as of DS, and 6 × 50 μL of WS (see Figure 19.2).
small a volume (less than 0.5 μL) as possible onto 4. Before warming, use a stripper tip with a 200-µm
the edge of the stick (see “Addendum,” Note 5). end hole for removing the blastocysts from the
14. Visually confirm the placement (see “Addendum,” HSV tip.
Note 6). 5. Fill a Styrofoam container with LN2.
15. Before loading, apply the label to the open end of 6. Confirm the location and identification with a sec-
the empty straw. Load the HSV stick into the empty ond embryologist before warming any HSV kit.
straw, placing the side with the embryos first. Use Warm one kit at a time.
the blue handle to make sure the stick is in as far as 7. Each sample that is warmed is done in a separate
it can go. Then, using the heat sealer, seal the open hood and verified by a second embryologist before
end of the stick and plunge the whole straw into proceeding.
the LN2. Place the straw in a precooled aluminum 8. With the HSV kit under LN2, open the kit by cut-
cane for further storage (see “Addendum,” Notes 7 ting the outer straw. Use the blue handle to remove
and 8). the inner stick.

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 vitrification of human blastocysts
60 mm organ culture Table 19.2  Retrospective Data from 5968 Patients
dish with 1.0 mL TS DS (Average Age 34.3 ± 4.9 Years) with Blastocyst
TS Cryopreservation by Vitrification between 2004
(Merge drops) and September 2014
A B Day of development
D C I Day 5 Day 6 Day 7 Total
(Transfer HS
to drop) J Number of 12,206 10,854 457 23,517
(Merge drops) blastocysts vitrified
DS K HS
E F (52.0%) (46.0%) (2.0%) (100%)

H G
(2.0%). After nearly 11 years of vitrifying blastocysts
using an open (Cryotop) as well as closed (HSV) system,
Figure 19.2  Setup for the warming procedure on a plain and more than 4500 VET with an average number of 1.7
90-mm dish lid surface. embryos transferred, the perinatal outcomes are as fol-
lows: the number of babies delivered until February 2013
9. Submerge the HSV kit directly in the prewarmed is 1495 (766 girls and 729 boys) (Table 19.3). No abnor-
37°C drop containing TS, which should be as close malities were recorded.
as possible to the LN2 Styrofoam container (see Table 19.4 summarizes the data on gestational age
“Addendum,” Note 11). As soon as the HSV kit (GA)  and live birth weight (LBW) from 1363 children
contents liquefy (within 1 second), try to locate the born. A total of 926 babies were from vitrified day 5 and day
blastocyst(s) before removing it (them) with a strip- 6 transfers, whereas 437 babies were born following fresh
per tip. After locating all of the blastocysts, remove elective single-embryo transfers (eSET). Furthermore,
them from the HSV tip and place them in the drop- comparing the 437 newborns from the eSET group to
let of TS (drop A) at room temperature, then con- the 926 newborns from frozen-vitrified group (vitrified
nect this immediately with the first droplet of DS blastocyst transfers [VBTs]), there was a significant dif-
(drop B). Wait for shrinkage and re-expansion. ference in mean age of the patients (p < 0.001). In addi-
10. When they start to wrinkle, connect them with the tion, looking at GA and LBW of newborns derived from
second droplet (drop C) and finally with the third vitrified day 5 blastocysts (n = 561) and day 6 blastocysts
droplet of DS (drop D). (n = 365), the following data were observed: t-test results
11. When they stop reacting and start to re-shrink, indicate no significant difference in GA (p = 0.71) nor
transfer the blastocysts to 0.5 M sucrose (drop E) birth weight (p = 0.124) for the frozen transfers based on
by placing them at the top of this drop so that they day of development. Obviously, GA is highly correlated
float to the bottom. When the reaction is complete, with birth weight, but there is no significant difference in
connect this with first of WS (drop F; wait for about GA between eSET and VBT. However, Table 19.4 shows
90% re-expansion). that one-way analysis of variance (ANOVA) of LBW by
12. After 100% expansion, connect with the second both groups indicates a statistically significant difference
droplet (drop G), then with the third droplet (drop
H) of WS. Turn on the benchwarmer, and finally
Table 19.3  Perinatal Outcome of Vitrified Blastocysts
dilute through a series of three wash drops of hold-
After More than 4100 Transfers between 2004 and 2013
ing solution (HS) (drops I–K).
(Babies Delivered Until February 2013)
13. Place the blastocysts into a culture dish, and put the
dish in the incubator for subsequent culture. Day of development
14. Record the survival and appearance of all blas-
tocysts. Update the log with warming data, and Day 5+ Day 6 Day 5 Day 6
notify the physician of the result (see “Addendum,” Deliveries (total) 1209 748 461
Note 12). Babies born (total) 1495 939 556
Female 766 488 278
RESULTS ON BLASTOCYST VITRIFICATION Male 729 451 278
Between 2004 and September 2014, the Fertility Centers Singletons 931 (77.0) 561 (75.0) 370 (80.0)
of Illinois “IVF Laboratory River North” (Chicago) Twins 270 (22.0) 183 (24.5) 87 (19.0)
has vitrified 23,517 blastocysts from 5968 patients
Triplets 8 (1.0) 4 (0.5) 4 (1.0)
(Table 19.2). The majority of blastocysts were vitrified on
day 5 (52.0%), 46.0% on day 6, with a minority on day 7 Note: Percentages are indicated between brackets.

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 vitrification in assisted reproduction
Table 19.4  Live Birth Weight (LBW) and Gestational Age (GA) of Babies (n = 1363) Delivered
after Fresh Elective Single-Blastocyst Transfers (eSBT; n = 437) Compared to Those Delivered from
Vitrified Blastocyst Transfers (VBT; n = 926) and Related to Day of Development after VBT (Day 5
vs. Day 6)
Day of development
VBT—Day
eSBT—Day 5 5 + Day 6 VBT—Day 5 VBT—Day 6
Average age 31.1 ± 3.1*** 34.7 ± 4.9*** 34.7 ± 5.0 34.8 ± 4.8
(years)
Deliveries 437 926 561 365
Female 238* 479* 288 191
Male 199* 447* 273 174
GA (weeks) 38.1 ± 2.5* 37.9 ± 2.7* 38.0 ± 2.6* 37.9 ± 2.9*
LBW (g) 3294.9 ± 609.4** 3390.1 ± 710.6** 3419.1 ± 712.6* 3345.5 ± 705.8*

Note: T-test (Satterthwaite unequal variance): *p > 0.05: **p = 0.013; ***p < 0.001.

between the means of eSET and VBT (p = 0.013) with and 4500 g (considered as large birth weight), and 33
heavier babies in the VBT group. This observation is con- babies weighed more than 4500 g (considered as macro-
firmed and published by Pinborg et al. [27]. As shown in somic birth weight).
Table 19.4, the χ2 statistic indicates no difference in the The outcomes with regard to day of development and
gender distribution between fresh and vitrified transfers age of the patient between 2004 and July 2014 are summa-
(p = 0.3454). Running a t-test on gender weight combin- rized in Tables 19.6 and 19.7. In good-prognosis patients
ing the gender from fresh with vitrified transfers indi- under 35 years old when transferring day 5 blastocysts,
cates a significant difference in average weight by gender ongoing pregnancy and IRs of 48.0% and 42.3%, respec-
(p < 0.001; Table  19.5). Males were on average 157.2 g tively, were noted (Table 19.6). In contrast, when transfer-
heavier than females. Table 19.5 shows the weight distri- ring day 6 blastocysts in patients younger than 35 years
bution for babies born in both groups. Overall, 110 babies of age, ongoing pregnancy and IRs of 34.0% and 30.0%,
weighed less than 2500 g (considered as low birth weight), respectively, were recorded (Table 19.7).
159 babies born from both groups weighed between 4000 In October 2007, the Fertility Centers of Illinois “IVF
Laboratory River North” moved forward from the use of
Table 19.5  Distribution (%) of Live Birth Weight (LBW) an open carrier system (Cryotop—embryos are in direct
of Babies (n = 1363) Delivered after Fresh Elective Sngle contact with LN2) to a closed system (embryos are sealed
Blastocyst Transfers (eSBT; n = 437) Compared to Those before contact with LN2). Using a closed carrier (HSV) for
Delivered from Vitrified Blastocyst Transfers (VBT; aseptic vitrification, the following data from day 5, day 6,
n = 926) and Gender Specific Live Birth Weight (LBW) and day 7 blastocysts were observed and are summarized
and Gestational Age (GA) between Fresh and Frozen in Table 19.8: (a) cPR: 53.92% versus 41.6% versus 18.5%;
Embryo Transfers Combined (b) ongoing pregnancy rate (oPR): 45.6% versus 32.2%
versus 14.8%; and (c) IR: 42.2% versus 30.2% versus 1.3%
eSBT—Day 5 (n) VBT—Day Total (Table 19.8). As shown in Table 19.8, oPRs, cPRs, and
[%] 5 + Day 6 (n) [%] (n) IRs occurring in the day 5 blastocyst group were signifi-
LBW (g) <2500 34 [7.8] 76 [8.2] 110 cantly higher than when transferring day 6 or even day 7
LBW (g) ≥4000 38 [8.7] 154 [16.6] 192 blastocysts.
LBW (g) ≥400 32 [7.3] 127 [13.7] 159 Between 2007 and July 2014, the Fertility Centers of
and ≤4500 Illinois “IVF Laboratory River North” performed 1675
LBW (g) >4500 6 [1.4] 27 [2.9] 33 VBTs without collapsing prior to vitrification, with a
mean patient age of 35.4 ± 5.0 years (group A), and in
Girls from eSBT Boys from eSBT and 972 VBTs (group B) with a mean patient age of 35.4 ± 4.9
Gender and VBT VBT years, where AC was performed prior to vitrification
N 717 646
(Table 19.9). On average, 1.7 embryos were transferred in
groups A and B, which means 30% of all VBTs were sin-
GA (weeks) 37.9 ± 2.6* 38.0 ± 2.6*
gle-embryo transfers. Survival in group A versus group
LBW (g) 3284.1 ± 663.8** 3441.3 ± 691.0**
B was not significantly different (98.7% versus 99.6%).
Note: Percentages are indicated between brackets; T-test and χ 2 However, there was a significant improvement in group B
test: *p > 0.05; **p < 0.001. compared with group A for the following: (a) cPR: 58.2%

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 vitrification of human blastocysts
Table 19.6  Retrospective Outcome Data (2004–September 2014) at the Fertility Centers of Illinois,
Chicago from Vitrified Day 5 Blastocysts with Regard to Patient Age

Patient age (years)

<35 35–37 38–40 >40 Donor

Average age (years) 31.2 ± 2.4 35.9 ± 0.8 38.9 ± 0.8 42.6 ± 2.1 43.6 ± 4.7
Cycles 1285 539 351 171 252
Transfers 1284 538 349 171 252
Blastocysts survived 98.4 98.2 98.8 98.1 98.9
Blastocysts transferred (mean) 1.8 1.7 1.8 1.8 1.7
Positive pregnancy/VET (%) 62.3 59.9 56.4 52.6 59.1
Clinical pregnancy/VET (%) 55.0 51.0 46.0 41.0 52.0
Ongoing/delivered pregnancies (%) 48.0 41.0 36.0 29.0 42.0
Implantations 954 350 203 87 170
Implantation rate (%) 42.3 38.9 32.5 28.5 38.6

Note: VET = vitrified embryo transfer.

Table 19.7  Retrospective Outcome Data (2004–July 2014) at the Fertility Centers of Illinois, Chicago
from Vitrified Day 6 Blastocysts with Regard to Patient Age
Patient age (years)

<35 35–37 38–40 >40 Donor

Average age (years) 31.2 ± 2.3 36.0 ± 0.8 38.9 ± 0.8 42.6 ± 1.8 43.3 ± 4.8
Cycles 834 439 320 197 147
Transfers 827 434 319 195 146
Blastocysts survived 97.4 98.5 98.5 96.9 99.6
Blastocysts 1.8 1.7 1.8 1.7 1.7
transferred (mean)
Positive pregnancy/ 49.1 44.7 44.5 42.1 45.9
VET (%)
Clinical pregnancy/ 42.0 37.0 39.0 33.0 37.0
VET (%)
Ongoing/delivered 34.0 29.0 30.0 22.0 27.0
pregnancies (%)
Implantations 449 204 159 76 63
Implantation rate (%) 30.0 26.9 27.8 22.4 24.7

Note: VET = vitrified embryo transfer.

versus 43.8%; (b) oPR: 50.9% versus 36.0%; and (c) IR: If we compare day 6 in group A (n = 692; mean age of
44.0% versus 32.7% (Table 19.9). 35.6 ± 4.9 years) with day 6 outcomes in group B (n = 335;
When the vitrified–warmed blastocysts were divided mean age of 36.3 ± 4.9 years), the following data in terms
into day 5 and day 6 groups, the following data were of IR, cPR, and oPR were observed: 26.2%, 36.7%, and
observed (Table 19.10): in 983 VBT transferring day 5 29.0% versus 36.7%, 51.3%, and 43.0%, respectively (Table
blastocysts from group A (n = 983; mean age of 35.3 ± 5.1 19.9). As shown in Table 19.9, IRs, cPRs, and oPRs occur-
years), the IRs, cPRs, and oPRs were 37.4%, 48.7%, and ring in the day 6 blastocysts of group B were significantly
40.9%, respectively, compared to 48.0%, 61.9%, and 55.1%, higher than when transferring day 6 blastocysts from
respectively, of day 5 blastocysts from group B (n = 637; group A (χ2; p < 0.001 for any comparison).
mean age of 35.0 ± 4.9 years). As shown in Table 19.10, In Table 19.11, the results for patients under 35 years
IRs, cPRs, and oPRs occurring from the day 5 blastocysts of age in group A (no assisted collapsing) and B (assisted
in group B were significantly higher than from the day 5 collapsing) are summarized. Comparing day 5 from
blastocyst in group A (χ2; p < 0.001 for any comparison). group A (n = 480; mean age of 31.2 ± 2.3 years) with day

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 vitrification in assisted reproduction
Table 19.8  A Comparison of Retrospective Data from the Cryopreservation Program (Fertility
Centers of Illinois, Chicago) of Vitrified Day 5, Day 6, and Day 7 Blastocysts Using Aseptic
Vitrification Technology between October 2007 and July 2014
Day 5 + Day 6 + Day 7 Day 5 Day 6 Day 7
Patient’s age (years) 35.5 ± 5.0 35.3 ± 2.3 36.0 ± 4.8 35.7 ± 3.7
Transfers 2959 1803 1129 27
Blastocysts warmed 5186 3099 2041 45
Blastocysts survived 5138 (99.1) 3079 (99.4) 2014 (98.7) 45 (100.0)
Blastocysts transferred 5034 3017 1972 45
Blastocysts transferred (mean) 1.7 1.7 1.7 1.7
Implantations 1873 (37.2) 1272 (42.2)a 595 (30.2)a 6 (1.3)a
Positive pregnancy/VET 1709 (57.7) 1132 (62.8)a 567 (50.2)a 10 (37.0)a
Clinical pregnancy/VET 1447 (48.9) 972 (53.9)a 470 (41.6)a 5 (18.5)a
Ongoing/delivered pregnancies 1189 (40.2) 821 (45.6)a 364 (32.2)a 4 (14.8)a
Live births 905 598 303 4

Note: Percentages are indicated between parentheses. VET = vitrified embryo transfer; ap < 0.001.

Table 19.9  A Comparison of Retrospective Data from the Cryopreservation Program (Fertility
Centers of Illinois, Chicago) of Vitrified Blastocysts without Artificial Collapse (AC; Group A) and
with AC (Group B) Using Aseptic Vitrification Technology between 2007 and July 2014
Technique

Group A (without AC) Group B (with AC)

Patient age (years) 35.4 ± 5.0 35.4 ± 4.9
Transfers 1675 972
Blastocysts warmed 3004 1709
Blastocysts survived 2966 (98.7) 1703 (99.6)
Blastocysts transferred 2911 1680
Blastocysts transferred (mean) 1.7 1.7
Implantations 952 (32.7)a 739 (44.0)a
Positive pregnancy/VET 861 (51.4)b 666 (68.5)b
Clinical pregnancy/VET 733 (43.8)b 566 (58.2)b
Ongoing/delivered pregnancies 603 (36.0)b 495 (50.9)b

Note: Percentages are indicated between parentheses. VET = vitrified embryo transfer; ap < 0.01; bp < 0.001.

5 from group B (n = 334; mean age of 31.2 ± 2.2 years), different surface-to-volume ratios; (b) differing cooling
we found the following for IR, cPR, and oPR: 41.1% ver- rate requirements between oocytes, zygotes, cleavage-
sus 52.7%, 51.9% versus 66.5%, and 45.6% versus 59.9%, stage embryos, and blastocysts; and (c) variable chill
respectively. Looking at day 6 outcomes for group A ver- sensitivity between these different developmental stages.
sus group B, we observed the following for IRs, cPRs, and Currently, however, the most widely used protocol
oPRs: 31.4% versus 41.7%, 43.0% versus 58.3%, and 36.1% applied to any embryo stage is the two-step equilibration
versus 50.0%, respectively (see Table 19.11). in an equimolar combination of the cryoprotectants EG
and DMSO, at a concentration of 15% each (v/v), sup-
CONCLUSIONS AND FUTURE DIRECTIONS plemented with 0.5 mol/L sucrose. For the adoption of
Vitrification is a very promising cryopreservation vitrification in ART, as with all new technologies, there
method with many advantages and an increasingly has been initial resistance, but as clinical data have been
consistent clinical track record. A standardized vit- accrued, this technology is becoming more commonly
rification protocol applicable to all stages of the pre- adopted as a standard procedure in many IVF programs
implantation embryo may not be realistic because of (a) worldwide. With this increased use in human assisted

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 vitrification of human blastocysts
Table 19.10  A Comparison of Retrospective Data from the Cryopreservation Program (Fertility Centers of Illinois,
Chicago) of Vitrified Day 5 and Day 6 Blastocysts without Artificial Collapse (AC; Group A) and with AC (Group B)
Using Aseptic Vitrification Technology between 2007 and July 2014
Technique

Group A (without AC) Group B (with AC)

Day 5 Day 6 Day 5 Day 6

Patient age (years) 35.3 ± 5.1 35.6 ± 4.9 35.0 ± 4.9 36.3 ± 4.9
Transfers 983 692 637 335
Blastocysts warmed 1736 1268 1096 613
Blastocysts survived 1721 (99.1) 1245 (98.2) 1092 (99.6) 611 (99.7)
Blastocysts transferred 1687 1224 1083 597
Blastocysts transferred (mean) 1.7 1.8 1.7 1.8
Implantations 631 (37.4)a 321 (26.2)c 520 (48.0)a 219 (36.7)c
Positive pregnancy/VET 558 (56.8)b 303 (43.8)d 455 (71.4)b 211 (63.0)b
Clinical pregnancy/VET 479 (48.7)b 254 (36.7)d 394 (61.9)b 172 (51.3)d
Ongoing/delivered pregnancies 402 (40.9)b 201 (29.0)d 351 (55.1)b 144 (43.0)d

Note: Parentheses are indicated between brackets. VET = vitrified embryo transfer. Day 5: ap < 0.01; bp < 0.001; day 6: cp < 0.01;
dp < 0.001.

Table 19.11  A Comparison of Retrospective Data from the Cryopreservation Program (Fertility Centers of Illinois,
Chicago) of Vitrified Day 5 and Day 6 Blastocysts without Artificial Collapse (AC; Group A) and with AC (Group B)
Using Aseptic Vitrification Technology in Patients Younger than 35 Years of Age between 2007 and July 2014
Technique

Group A (without AC) Group B (with AC)

Day 5 Day 6 Day 5 Day 6

Patient age (years) 31.2 ± 2.3 31.5 ± 0.6 31.2 ± 2.2 31.3 ± 2.6
Transfers 480 316 334 120
Blastocysts warmed 862 591 566 219
Blastocysts survived 848 (98.4) 578 (97.8) 563 (99.5) 218 (99.5)
Blastocysts transferred 829 564 560 218
Blastocysts transferred (mean) 1.7 1.8 1.7 1.8
Implantations 341 (41.1)a 177 (31.4)b 295 (52.7)a 91 (41.7)b
Positive pregnancy/VET 279 (58.1)a 158 (50.0)b 253 (75.7)a 83 (69.2)b
Clinical pregnancy/VET 249 (51.9)a 136 (43.0)b 222 (66.5)a 70 (58.3)b
Ongoing/delivered pregnancies 219 (45.6)a 100 (36.1)b 200 (59.9)a 46 (50.0)b

Note: Values are numbers unless otherwise described; percentages are indicated between parentheses. VET = vitrified embryo transfer.
Day 5: ap < 0.001; day 6: bp < 0.001.

reproduction will come evolution of the vitrification pro- achieve these outcomes, consider these points: (a) with-
cess as it is fine-tuned to clinical needs, so pushing for- out a successful blastocyst vitrification storage program,
ward its development to higher levels of clinical efficiency, extended culture should never be attempted; (b) the blas-
utilization, and universal acceptance. tocyst is composed of more cells and is therefore better
able to compensate for cryoinjury; and (c) the cells are
PRACTICAL IMPLICATIONS FOR VITRIFYING smaller, which makes cryoprotectant penetration faster.
AT THE BLASTOCYST STAGE On average, fewer embryos per patient are cryostored, but
Our data have shown that freezing at the blastocyst stage each one has a greater potential for implantation when
provides excellent survival, implantation, and cPRs. To thawed.

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 vitrification in assisted reproduction
ADDENDUM: SPECIAL NOTES FOR THE surface as soon as possible to avoid any toxic effect
CLINICAL EMBRYOLOGIST of the VS. A stirring motion is recommended when
1. Special care must be given to the selection of the plunging into the warm TS to agitate the cell off
vitrification carrier type. It is necessary to use types the carrier surface without the need to remove it
of carrier or vessel material with rapid heat trans- actively.
fer that also support the process of uniform heat 10. When switching the cells between different con-
exchange to achieve higher cooling rates. centrations of warming solutions, fill up the pipette
2. In addition, although no reports of contamination with the next-lowest concentration of warming
in human IVF following cryopreservation exist, solution, before picking up the blastocysts to move
the user should be encouraged to choose a closed into the next concentration.
carrier system, which, in our experience, works for 11. In general, during vitrification and warming, the
blastocysts without any problems. LN2 Styrofoam box needs to be as close as possible
3. To minimize the toxicity of the cryoprotectant, a to the working area to minimize any lag in cooling
stepwise exposure of cells to precooled concen- and warming rates.
trated solutions (approximate room temperature of 12. Be aware of the expiration dates of the vitrifica-
24°C) is recommended. tion and warming media; once opened, the shelf
4. Utilizing higher concentrations of cryoprotectant life is 6 weeks (Irvine Scientific; according to the
allows shorter exposure times to the cryoprotec- manufacture).
tant, but be careful—the potential toxicity of the
cryoprotectant increases at higher concentrations. ACKNOWLEDGMENTS
As almost all cryoprotectants are toxic to some Juergen Liebermann wants to thank the Fertility Centers
extent, it is important to carefully monitor the of Illinois (FCI) and the embryologists at the FCI IVF
duration of exposure to the final cryoprotectant Laboratory River North (Elissa Pelts, BS; Jill Matthews,
before plunging into LN2. BS; Sara Sanchez, BS; Rebecca Brohammer, BS; Yuri
5. To facilitate vitrification by higher cooling rates, it is Wagner, BS; and Ewelina Pawlowska, MS) for their
also necessary to minimize the volume of the VS as invaluable contributions and support in pushing vitrifi-
much as is practical (preferably less than 1 μL). From cation to become our standard protocol for cryopreser-
this point of view, it is very important to use a small vation of human oocytes and blastocysts within our
pulled pipette. Furthermore, by collecting the blasto- program since 2004.
cysts in one place and loading no more than two blas-
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17. Liebermann J, Nawroth F, Isachenko V et al. Potential 26. Liebermann J, Conaghan J. Artificial collapse prior
importance of vitrification in reproductive medicine. blastocyst vitrification: Improvement of clinical out-
Biol Reprod 2002;67:1671–80. come. J Clin Embryol 2013;16(1):107–118.
18. Liebermann J, Dietl J, Vanderzwalmen P et al. Recent 27. Pinborg A, Henningsen AA, Loft A et al. Large baby
developments in human oocyte, embryo and blasto- syndrome in singleton born after frozen embryo
cyst vitrification: Where are we now? Reprod Biomed transfer (FET): Is it due to maternal factors or the
Online 2003;7:623–33. cryotechnique? Hum Reprod 2014;29:618–27.

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20 Development and hatching of human blastocysts after
vitrification and warming
Joe Conaghan and Sergio Vaccari

INTRODUCTION AH: Is It Necessary after Vitrification?

Blastocyst vitrification is now a routine procedure The utilization of AH for fresh9,10 and frozen11 embryos
that allows patients to store high-quality embryos remains controversial, and the benefits, if any, have been
indefinitely with a high expectation of pregnancy difficult to establish.12,13 The technique was developed
post-warming.1 Patients with multiple blastocysts will and became popular in the early 1990s using enzymatic
routinely transfer just a single fresh embryo (elective digestion or mechanical zona pellucida (ZP) thinning
single-embryo transfer) to avoid multiple pregnancy, 2 or breaching.9,10 Several Cochrane reviews have assessed
with the knowledge that their remaining embryos can the literature over the years, beginning in 200314 with the
be successfully frozen and stored until needed. At the conclusion that AH does increase the odds of a clinical
time of warming, it will again be ideal to use just a single pregnancy in in vitro fertilization (IVF) patients, but cau-
embryo from the freezer to maintain the efficiency and tioning that live birth data were lacking. A subsequent
safety of the process, particularly in younger patients, review showed an additional increase in the multiple preg-
those who experienced a loss or miscarriage after a pre- nancy rate as result of AH.15 However, the pregnancy ben-
vious transfer, or if the embryo is known to be euploid efit trended downward as more studies accumulated,16,17
after chromosome analysis. 3 In attempting to use a sin- and as a result, this procedure has not been universally
gle embryo and to give the patient the greatest possible applied or accepted.18,19 Nevertheless, guidelines for its
chance of pregnancy, the embryologist is charged with use exist from the American Society for Reproductive
recovering, warming, and culturing the embryo, and Medicine,13 although these do not address using the
with accurately assessing the viability of the embryo procedure on frozen embryos except in cases of repeat
before transfer. In addition, the embryologist needs to implantation failure. Discussion has occurred on the use-
be aware of procedures that may limit or enhance the fulness of AH specifically for frozen embryos,10,11,20–27 but
embryo’s chance of implantation,4 such as timing of without much focus on freezing-related changes to the ZP
warming and transfer relative to uterine receptivity, 5 that might specifically necessitate AH after warming.
culturing the embryo for an appropriate  time before
transfer, and assisted hatching (AH).6 MATERIALS AND METHODS
The embryos used in the work described here were all
Assessing Blastocyst Viability Post-Warming surplus to patient needs and were scheduled to be dis-
Immediately after warming and rehydration of the blas- carded. Patients consented to have the embryos thawed
tocyst, some method for determining viability must be and observed prior to discard.
applied to determine whether the embryo has survived Blastocyst vitrification was achieved using a Vit Kit
and is suitable for transfer.7 Although vitrification is (Irvine Scientific, Santa Ana, CA), which contains an
technically difficult, when it is applied correctly, the vast equilibration solution (ES) and a vitrification solution
majority of blastocysts are expected to survive and be (VS). Blastocysts were laser collapsed as described previ-
transferred.8 ously,28 before being incubated in ES for 8 minutes and
VS for 60 seconds at room temperature. Embryos were
Embryo Culture after Warming and before Transfer loaded into Cryotips (Irvine Scientific), which were sealed
It is likely that all blastocysts will be cultured for short before being plunged into liquid nitrogen. Embryos that
periods of time after warming, for anything from just had been biopsied for genetic testing had a 30-µm hole
a few minutes to overnight in the incubator. While it is made in the ZP on day 3 of development. For warming,
the authors’ contention that no embryo can benefit from Cryotips were plunged into a 37°C water bath for 3 sec-
unnecessary time in culture, it is essential to have a dish onds before being washed through solutions of 1, 0.5,
of culture medium equilibrated and ready for the warmed and 0 M sucrose (thaw kit; Irvine Scientific) at room
embryo for holding until transfer. In addition, some short temperature.
period of observation may aid in embryo assessment, and Immediately after warming, embryos were cultured
some re-expansion of the blastocyst in culture may be in well-of-the-well (WOW) dishes29 loaded with G2
evidence of viability after warming.7,8 medium (both from Vitrolife Inc, Gothenburg, Sweden)

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 vitrification in assisted reproduction
supplemented with synthetic serum substitute (SSS: technology system and software, we were able to observe
Irvine Scientific), and placed in gas-controlled incuba- embryo expansion in real time and record events such
tors on the Primo Vision time-lapse imaging system as the cycles of collapse and re-expansion that occur in
(Vitrolife).30 Embryos were imaged every 5 minutes for many embryos.33
up to 72 hours after warming. To assess re-expansion, the volume of each embryo
was measured (Figure 20.3). The average diameter of the
RESULTS embryo was taken and the volume calculated using the
formula πr2. If the embryo was hatching, the volume of
Warming of Blastocysts each half was calculated separately, and then the total
Embryologists assessed embryos for survival during warm- volume calculated by adding the halves. The process was
ing and at the time of placing the blastocysts in culture. In repeated after 1 hour in culture to determine whether any
rare cases, embryos were clearly observed to have died dur- re-expansion had occurred.
ing vitrification and warming, and displayed characteristics Arbitrarily, and in the absence of published guidelines,
such as lysed cells and blackened areas encompassing some we looked for a 20% increase in blastocyst size during the
or all of the cell population (Figure 20.1).31,32 Occasionally, first hour in culture. In a pilot experiment, 47 aneuploid
an embryo looked perfectly normal after warming and embryos that were being discarded by patients were avail-
even began to re-expand during the first few hours in cul- able for study. Only 27 (57%) of the blastocysts showed
ture, only to collapse and die within 6–12 hours of warm- any increase in volume in the first hour after warming
ing (Figure 20.2). These latter embryos were more difficult and just 19 (40%) met the target of re-expanding by 20%.
to identify as dying immediately at warming or during the We then repeated the process by taking measure-
first 4 hours in culture. ments from photographs of embryos being transferred
to patients. Embryos were quickly photographed with a
Re-Expansion of Blastocysts in Culture digital camera, and using software attached to the Zylos
There is no consensus on a method for measuring embryo laser system (Origio Inc, Måløv, Denmark), their diam-
viability after vitrification, although appearance and eters were recorded after warming and after 1 hour in cul-
expansion in culture are used subjectively.7 Many embry- ture. Fifty patients participated in the study, all of whom
ologists like to culture embryos for 1 or more hours after were having elective single-embryo transfers. Thirty-five
warming,23,24 and use criteria such as the appearance of out of 50 embryos expanded by 20% or more after 1 hour,
the embryo and re-expansion of the blastocoele7 as evi- and 22 of these led to clinical pregnancies. Significantly
dence of survival. Using the Primo Vision time-lapse fewer pregnancies were seen among embryos that failed
to re-expand by 20% during the first hour after warming
(Table 20.1).

Time Taken to Escape from the ZP

While the re-expansion profile appeared to predict the
likelihood of implantation, it was also observed that
many embryos that appeared healthy and expanded nor-
mally in culture failed to escape from the confines of the
ZP. In general, for embryos that were considered high
quality before vitrification, such as expanded or hatch-
ing blastocysts with high cell numbers, complete hatch-
ing was achieved in the first 24 hours after warming.
However, the properties of the ZP had clearly changed
as a result of the vitrification procedure, and the elastic-
ity or stretchability appeared to be severely reduced. In
embryos with a small opening in the zona as a result of
laser AH, each embryo had to escape through the exist-
ing hole without any stretching, rupture, or ripping of the
ZP occurring (Figure 20.4). The zona appeared to have
Figure 20.1  Occasionally an embryo is clearly dead after completely hardened and did not give way as the embryo
warming. This zona-free embryo is dark and contracted tried to escape. This delayed the hatching process and
and the outer cells are seen to be lysing, as evidenced by caused embryos to collapse several times during hatch-
the halos around the cells on the outside of the embryo. ing, requiring the embryo to begin re-expanding over
This pattern of cell lysis is similar to that obtained when and over again. Typically, embryos underwent four to
trophectoderm cells are deliberately lysed to isolate or six rounds of collapse and re-expansion and took 24 or
stain only inner cell mass cells. 31,32 more hours to hatch (Figure 20.5). Embryos that had not

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 development and hatching of human blastocysts after vitrification and warming

(a) (b) (c) (d)

Figure 20.2  This blastocyst was assessed as having survived warming and was in a normal collapsed state when
placed in culture (a). The embryo began to expand, with fluid accumulating in the blastocoele during the first hour
in culture (b). At 4 hours, the embryo had collapsed again (c) and was clearly dead and fragmenting after 6 hours (d).

impediment to the hatching process. As embryos re-

expanded and pushed up against the ZP, there was no
expansion or thinning, as is routinely observed with
fresh embryos. All the embryos studied had a small hole
in the ZP as is typical for D3 AH. This hole, by virtue
of its small size and in association with the hardened
85.9 μm zona, made it difficult for embryos to completely hatch.
As mentioned above, high-quality embryos mostly over-
107.9 μm
78.5 μm 88.9 μm came this challenge, but the characteristics of embryos
after warming affected their ability to hatch. In particu-
118.0 μm lar, embryos that had already partially hatched (such as
the one shown in Figure 20.4) struggled to escape the
78.9 μm entrapment of the zona, which had a firm grip around
82.2 μm
the middle of the embryo. The zona behaved like a cor-
set, pinching the embryo and causing the two halves of
the embryo to expand and collapse independently. As a
result, not all embryos in this configuration were able
Figure 20.3  (See color insert.) The volume of each blasto-
to escape and died trying (Figure 20.6). Embryos that
cyst was calculated based on the diameter of the embryo,
appeared normal and underwent good re-expansion
or the parts of the embryo if it was in a figure 8 configu-
were often so trapped that they were unable to free them-
ration, as shown here. The volume was recorded imme-
selves from the constricting ZP, which also affected their
diately after warming and again after 1 hour in culture.
ability to expand uniformly.

Table 20.1  Clinical Outcomes Based on Blastocyst

Expansion in the First Hour after Warming Making Bigger Holes in the ZP
Since embryos appear to struggle to escape from the
Positive serum Clinical zona after vitrification, we looked at the effect of mak-
Patients hCG (%) pregnancy (%) ing bigger openings through which they could hatch, as
All transfers 50 36 (72) 26 (52) shown by other researchers. 26,34 Figure 20.7 is a sche-
≥20% embryo 35 28 (80) 22 (63)† matic of a freshly warmed and collapsed blastocyst
expansion where a significant cut through the zona is being made
<20% embryo 15 8 (53) 4 (27)† with a laser. This procedure aims to remove approxi-
expansion mately one-third of the zona and it is performed imme-
Note: hCG: human chorionic gonadotrophin. Values with the
diately after warming when the embryo is in its most
same superscript † symbol are significantly different from collapsed state. The laser is set on a 450-µs pulse and
each other using Fisher’s exact test. fired 10–15 times. The result of applying the laser like
this is that the embryo “falls” right out of the ZP as soon
escaped from the ZP after 48 hours in culture never man- as it begins to expand (Figure 20.8). The time taken to
aged to hatch fully. hatch is reduced from an average of 24 hours with con-
ventional AH to 4 hours with more aggressive hatching.
Properties of the ZP after Vitrification In addition, the cycles of expansion and contraction are
As seen in Figure 20.4, the ZP appears inflexible after mostly eliminated, since the embryo is not restricted in
vitrification and, at least in vitro, can be a serious any way.

177
 vitrification in assisted reproduction

(a) (b) (c)

(d) (e) (f )

Figure 20.4  Immediately after warming, the collapsed blastocyst (a) is placed in culture. The embryo struggles to
escape through a hole in the zona pellucida that resulted from assisted hatching on day 3 of development. Several
rounds of expansion (b), collapse (c), expansion (d), and collapse (e) occur, before finally the embryo hatches com-
pletely (f). This embryo collapsed four times and took 24 hours to hatch. Note that the size, thickness, and proper-
ties of the zona pellucida do not change in relation to the expansion of the embryo.

in this way stopped expanding in the first 12 hours in cul-

Hours taken for complete hatching (n = 18)
ture and 90% were dead after 48 hours.
60
% fully hatched Does Slow Freezing Affect Zona Properties?
50 Embryos that had been slow frozen over 10 years ago
were thawed and their expansion profile in culture stud-
40 ied. These embryos were created at a time when AH and
% 30 genetic screening of embryos was not so prevalent, and
when zona hardening due to cryopreservation was not
20 considered a risk. Upon thawing, the ZP did show what
appeared to be normal expansion and thinning, but
10
embryos had great difficulty actually breaking the zona
0 and escaping (Figure 20.10).
12 24 48 72
Hours Characteristics of the ZP after Oocyte Vitrification
After observing the behavior of the ZP in response to
Figure 20.5  Embryos that had been hatched on day 3 expansion in vitrified embryos, we were curious to see
(D3) of development and subsequently were vitrified whether vitrified oocytes would have similar trouble with
and warmed were slow to hatch completely from the hatching when they reached the blastocyst stage. Time-
zona pellucida. There was no increase in the number lapse imaging of these blastocysts revealed a pattern
that hatched after 48 hours in culture. that was similar to that seen with slow-frozen embryos
(Figure 20.11). Embryos appeared to expand normally but
were nonetheless unable to hatch.
Hatching and Continued Expansion
Embryos that remained trapped inside the ZP died rela- DISCUSSION
tively quickly (Figure 20.9). Their inability to escape This study has for the first time used time-lapse imag-
through a normal day 3 (D3) AH hole likely had a high ing to characterize the expansion and hatching pro-
energy cost since it required repeated cycles of collapse files for oocytes and blastocysts that were vitrified and
and re-expansion. Twenty percent of blastocysts trapped warmed, and for slow-frozen and thawed embryos.

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 development and hatching of human blastocysts after vitrification and warming

Figure 20.6  This average-quality embryo quickly re-expanded after warming, but was unable to escape from the
zona pellucida. The embryo collapsed and died after three rounds of expansion and collapse, and never had more
than 50% of the cellular mass outside the zona.

the vitrification process will be harder than that around a

fresh embryo, and this should be taken into consideration
at the time of warming and transfer. AH is a procedure
that has not been widely accepted by the medical and sci-
entific community,13 and in detailed studies of its use with
frozen embryo transfers, some authors found a benefit23,24
and some did not.25,37 The work presented here supports
previous studies that show the positive impact of large
holes in the ZP on implantation and pregnancy rates in
frozen embryo transfer cycles.22,26 It is also worth noting
that since hatching occurs almost a day earlier when the
ZP has a large hole, it may be necessary for physicians to
examine the timing of blastocyst warming and transfer
in relation to the patient’s cycle and progesterone therapy.
Vitrification of blastocysts is a wonderful technology
that has allowed embryologists to successfully preserve
embryos with the expectation of high implantation rates
Figure 20.7  Schematic showing the proposed hatching after warming. However, determining exactly which
method for vitrified blastocysts that have not yet re- embryos are viable after vitrification is subjective, and no
expanded. Using a 450-µs laser pulse fired 10–15 times clear method has been established that has been widely
at an area of the zona well removed from the embryo, a accepted and used. In some cases, embryos are trans-
hole is made that is large enough to allow the embryo to ferred immediately after warming based on morphologi-
fall out of the zona as it re-expands. The laser shots are cal assessment alone, but most embryos are likely to be
depicted as a line of dark circles. kept in culture for some defined time period23,24 to deter-
mine whether they are viable and continue to develop
after returning to their pre-vitrification morphology.
The observations suggest that the ZP is modified or hard- But there is no consensus on how long embryos should
ened in a way that could impair blastocyst hatching. This be kept in culture and how much expansion is consid-
observation is consistent with that of Larman et al.,35 who ered normal, nor is there even a method for measuring
showed that the mouse ZP took five-times longer to dis- expansion over time that is related to viability. Since we
solve in chymotrypsin when compared to unvitrified con- now perform IVF treatments in an era in which embryo
trols. Larman et al.35 also showed that the effect was not freezing is routine,38,39 and is used in some clinics for all
due to cryoprotectant exposure alone. It should be noted, embryos to avoid ovarian hyperstimulation,40 to facili-
however, that our observations were made on embryos tate genetic testing,41,42 to return embryos to the uterus
that were subjected to prolonged culture after warm- in a natural cycle,43 or it is simply preferred for conve-
ing, and that they may not in fact reflect the behavior nience,44 it is more important than ever to be able to
of embryos in the uterus. Whether or not embryos have assess embryo viability. Historically, viability is assessed
trouble hatching in the uterus, or even undergo repeated subjectively as discussed above, leading embryologists
collapse and re-expansion cycles, remains unknown. It is to believe that vitrification gives extremely high or even
also possible that factors produced in the uterus,36 or by 100% embryo survival. We have presented evidence that
the embryo itself in its more natural environment, may survival may be less than is first assumed based on the
aid in dissolving or weakening the ZP, such that hatching traditional methods of assessment. Time-lapse imaging
is not difficult. Nevertheless, any ZP that went through clearly shows that embryos that appear perfectly normal,

179
 vitrification in assisted reproduction

Figure 20.8  This embryo had a large hole cut in the zona pellucida before being placed in culture. The embryo
expanded without restriction, never cycled through collapse and re-expansion, and fell out of the zona 3 hours after
warming. The panel on the right shows the large cut in the zona that remained after the embryo had been removed.

100 Often this technology utilizes vitrification of all blasto-

90 cysts to allow embryos to be biopsied on day 5 and 6, and
80 even day 7,47 and thereby having all samples batched and
70
analyzed together. Preserving the embryos also gives the
genetics laboratory more time in which to perform the
60
% of total

analysis, and allows the IVF patient to return for a fro-

50
zen embryo transfer at a convenient time, and when the
40 reproductive organs have returned to normal after hyper-
30 stimulation of the ovaries.
20 To perform a blastocyst biopsy, a hole must be made in
10 the ZP so that a few cells can be recovered for testing. If
0 this hole is too small, the embryo could be damaged dur-
12 24 48 72 ing biopsy and the embryologist will struggle to collect
Hours post-warming cells for analysis. If the hole is too big, the entire embryo
Alive if escaped Alive if trapped in zona may be pulled out of the ZP during the biopsy attempt,
making it technically more difficult to perform the biopsy
and increasing the risk to the embryo. The perfect hole is
Figure 20.9  Embryos that were able to escape from the large enough to allow some cells to herniate through the
zona pellucida continued to re-expand in culture for opening, but small enough to hold the embryo inside the
up to 48 hours. However, embryos that failed to escape ZP during biopsy. If the embryo is then frozen or vitrified,
showed a remarkably reduced ability to keep expand- it is likely that this hole is insufficient for easy hatching,
ing, with only 80% still growing at 12 hours and 10% at and may in fact be detrimental to the embryo. In particu-
48 hours. lar, blastocysts that are not of the absolute ­highest qual-
ity, and those that are in a figure 8 configuration, with
and even begin to re-expand after warming, sometimes some of the embryo inside the ZP and some outside (see
collapse and die in the first 12 hours. Since no embryo Figure 20.4 as an example), are at the highest risk for get-
ever benefited from extended time in culture, embryolo- ting stuck or trapped in the ZP. It is even conceivable that
gists will not always want to keep embryos in the labora- part of the embryo could bud off or get pinched by the
tory for extended periods of time, so some test of viability zona while inside the uterus, leading to a higher-multiple
would be useful. The test that we applied here—looking pregnancy risk.48,49
for a 20% increase in blastocyst size in the first hour after Further studies are needed to validate and confirm the
warming—is not perfect, but some noninvasive test like results presented here. These data were collected over a
this could have value. We plan further refinements of this relatively short period of time, and with only moderate
test, but for now, an opportunity exists for embryologists numbers of embryos studied. Even fewer vitrified oocytes
to develop new assessment methods for warmed embryos and slow-frozen blastocysts were observed. However, as
that are more reliable and less subjective than current with most studies, when we started looking more closely
methods. In addition, we may find that we currently over- at these embryos, we saw things that we did not expect.
estimate embryo survival, and this could lead to further The post-vitrification ZP acted like an iron fist around
refinements in the vitrification methodology. the blastocyst, and was unyielding to repeated hatch-
Pre-implantation genetic screening is rapidly becom- ing attempts by most embryos. Viability, as measured
ing popular as a method for avoiding the transfer of aneu- by continued expansion, was less than we expected, and
ploid embryos and for the high implantation rates that while the small day 3 breach of the ZP facilitated embryo
result from the transfer of a single euploid embryo.45,46 biopsy, it prevented many embryos from hatching after

180
 development and hatching of human blastocysts after vitrification and warming

(a) SlothawnoAH61314 - E9 (b) SlothawnoAH61314 - E9 (c) SlothawnoAH61314 - E9 (d) SlothawnoAH61314 - E9

(e) SlothawnoAH61314 - E9 (f ) SlothawnoAH61314 - E9 (g) SlothawnoAH61314 - E9 (h) SlothawnoAH61314 - E9

Figure 20.10  A slow-frozen and thawed blastocyst allowed to re-expand in culture appears initially to be capable of
expanding without restriction (a–d) and grows significantly in size. However, the embryo is unable to break through the
zona despite 48 hours in culture, and suffers two collapsing (e and g) and re-expansion (f and h) events after which it con-
tinues to expand. Although the zona thinned considerably (visible in g) and the embryo almost outgrew the well in which
it resided, it never hatched.

Figure 20.11  Four vitrified oocytes warmed, fertilized, and cultured to day 6 of development. Pronuclei are visible
in the upper left panel on day 1. The upper right panel shows the embryos on day 3. The lower panels show blastocyst
development on day 5 (left side) and day 6 (right side). Note the largest blastocyst collapsing after thinning and
stretching of the zona pellucida (arrow). None of the embryos hatched spontaneously, although the arrowed embryo
did squeeze a few cells through the zona at the 6 o’clock position.

181
 vitrification in assisted reproduction
vitrification. Not all embryos implant after vitrification, 4. Veleva Z, Orava M, Nuojua-Huttunen S, Tapanainen
even when they are known to be euploid. Some of these JS, Martikainen H. Factors affecting the outcome
failures may be iatrogenic and may be avoided. It appears of frozen-thawed embryo transfer. Hum Reprod
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warming, there will be other embryos that appear 6. Vanderzwalmen P, Bertin G, Debauche C et  al.
to be alive and re-expanding that will die within Vitrification of human blastocysts with the Hemi-
12 hours of warming. Straw carrier: Application of assisted hatching after
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viability after vitrification and warming. A model 7. Lopes AS, Frederickx V, Van Kerkhoven G, Campo R,
based on percentage increase in volume over time Puttemans P, Gordts S. Survival, re-expansion and cell
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vitrification of blastocysts causes ZP hardening. Reprod Genet 2015;32(1):83–90.
4. The small hole made in the ZP of a day 3 embryo 8. Mukaida T, Oka C. Vitrification of oocytes, embryos
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ACKNOWLEDGMENTS trials. Hum Reprod Update 2011;17(4):438–53.
The authors would like to thank the embryologists 13. Practice Committee of the American Society for
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Fertil Steril 2004;82(Suppl. 1):S164–5. alising the inner cell mass and trophectoderm nuclei
19. Practice Committee of the Society for Assisted of mouse blastocysts in situ using polynucleotide-spe-
Reproductive Technology; Practice Committee of the cific fluorochromes. J Exp Zool 1984;231(3):429–34.
American Society for Reproductive Medicine. The 32. Hardy K, Handyside AH, Winston RM. The human
role of assisted hatching in in vitro fertilization: A blastocyst: Cell number, death and allocation dur-
review of the literature. A Committee opinion. Fertil ing late preimplantation development in vitro.
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20. Check JH, Hoover L, Nazari A, O’Shaughnessy A, 33. Mio Y, Maeda K. Time-lapse cinematography of
Summers D. The effect of assisted hatching on preg- dynamic changes occurring during in vitro devel-
nancy rates after frozen embryo transfer. Fertil Steril opment of human embryos. Am J Obstet Gynecol
1996;65(2):254–7. 2008;199(6):660.e1–5.
21. Oehninger S, Mayer J, Muasher S. Impact of differ- 34. Zhang XJ, Yang YZ, Lv Q, Min LH, Li XL, Bai P. Effect
ent clinical variables on pregnancy outcome follow- of the size of zona pellucida thinning by laser assisted
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22. Mantoudis E, Podsiadly BT, Gorgy A, Venkat G, Craft 35. Larman MG, Sheehan CB, Gardner DK. Calcium-free
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predicting the implantation rate of thawed IVF/ DE. Embryonic hatching enzyme strypsin/ISP1 is
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randomized study to assess the benefit of partial zona Hudson C. Clinical rationale for cryopreservation of
pellucida digestion before frozen-thawed embryo entire embryo cohorts in lieu of fresh transfer. Fertil
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26. Hiraoka K, Fuchiwaki M, Hiraoka K et al. Effect of the 39. Shapiro BS, Daneshmand ST, Garner FC, Aguirre M,
size of zona pellucida opening by laser assisted hatch- Hudson C. Freeze-all can be a superior therapy to
ing on clinical outcome of frozen cleaved embryos another fresh cycle in patients with prior fresh blas-
that were cultured to blastocyst after thawing in tocyst implantation failure. Reprod Biomed Online
women with multiple implantation failures of embryo 2014;29(3):286–90.
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2008;25(4):129–35. ger and a freeze-all strategy to prevent ovarian hyper-
27. Valojerdi MR, Eftekhari-Yazdi P, Karimian L, Ashtiani stimulation syndrome: A retrospective study of OHSS
SK. Effect of laser zona pellucida opening on clini- risk and pregnancy rates. Aust N Z J Obstet Gynaecol
cal outcome of assisted reproduction technology in 2014;54(6):581–5.
patients with advanced female age, recurrent implan- 41. Escribá MJ, Zulategui JF, Galán A, Mercader A,
tation failure, or frozen-thawed embryos. Fertil Steril Remohí J, de los Santos MJ. Vitrification of preim-
2008;90(1):84–91. plantation genetically diagnosed human blastocysts
28. Lieberman J, Conaghan J. Artificial collapse prior to and its contribution to the cumulative ongoing preg-
blastocyst vitrification: Improvement of clinical out- nancy rate per cycle by using a closed device. Fertil
comes. J Clin Embryol 2013;16(1):107–19. Steril 2008;89(4):840–6.
29. Vajta G, Korösi T, Du Y et al. The well-of-the-well sys- 42. Zhang X, Trokoudes KM, Pavlides C. Vitrification of
tem: An efficient approach to improve embryo devel- biopsied embryos at cleavage, morula and blastocyst
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30. Pribenszky C, Mátyás S, Kovács P, Losonczi E, Zádori 43. Shapiro BS, Daneshmand ST, Garner FC, Aguirre
J, Vajta G, Pregnancy achieved by transfer of a single M, Hudson C, Thomas S. Evidence of impaired

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endometrial receptivity after ovarian stimulation infertile women with advanced maternal age. Fertil
for in vitro fertilization: A prospective random- Steril 2013;100(3):615–9.
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embryo transfers in high responders. Fertil Steril Neithardt AB, Feinberg RF. Should embryos devel-
2011;96(2):516–8. oping to blastocysts on day 7 be cryopreserved and
44. Roque M. Freeze-all policy: Is it time for that? J Assist transferred: An analysis of pregnancy and implanta-
Reprod Genet 2015;32(2):171–6. tion rates. Fertil Steril 2013;100(4):1008–12.
45. Scott RT Jr, Upham KM, Forman EJ et  al. Blastocyst 48. Kanter JR, Boulet SL, Kawwass JF, Jamieson DJ,
biopsy with comprehensive chromosome screening and Kissin DM. Trends and correlates of monozygotic
fresh embryo transfer significantly increases in vitro twinning after single embryo transfer. Obstet Gynecol
fertilization implantation and delivery rates: A random- 2015;125(1):111–7.
ized controlled trial. Fertil Steril 2013;100(3):697–703. 49. Knopman JM, Krey LC, Oh C, Lee J, McCaffrey C,
46. Schoolcraft WB, Katz-Jaffe MG. Comprehensive Noyes N. What makes them split? Identifying risk
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21 Does storage of vitrified blastocysts have an impact on implantation
potential and birth rate?
Barbara Wirleitner, Nicolas H. Zech, and Pierre Vanderzwalmen

CRYOPRESERVATION OF HUMAN GAMETES In contrast to SF, VIT works (theoretically) by com-

AND EMBRYOS, ITS APPLICATION, AND THE plete avoidance of intra- and extra-cellular ice formation.
IMPORTANCE OF STABILITY DURING STORAGE VIT is the solidification of a supercooled fluid without the
Around 8%–12% of couples of reproductive age experi- formation of crystalline structures. The molecules in the
ence difficulties conceiving, and may seek treatment fluid solidify in a disorganized pattern, and no molecular
using assisted reproductive technology (ART). Since re-arrangements occur. Only when structural changes
the birth of the first in vitro fertilization (IVF) child occur, as happens more commonly with SF, is there a sig-
Louise Brown in 1978, a number of new techniques have nificant risk of poor survival for cryopreserved gametes
been developed. Hormone-stimulation protocols were or embryos.
­established to obtain a number of oocytes per cycle and In order to obtain the vitrified state, viscosity and thus
ovarian ­pick-up. Intracellular sperm injection was imple- the concentrations of CPs in the incubation solutions
mented to treatment couples diagnosed with severe male are notably higher in VIT as compared to SF. Concerns
­infertility. All these techniques have contributed to the arise as the majority of the CPs used may show possible
success of ART, and have led toward a time in which it is harmful effects on cells and embryos, which will be dis-
now standard to obtain more than one (or two) embryos cussed in more detail below. Despite the fact that sev-
per stimulation cycle. Therefore, cryopreservation of eral thousand IVF children worldwide have been born
embryos has become an essential part of IVF. Nowadays, already after  VIT, basic aspects and concerns regarding
many aspects even favor the “freeze-all” strategy, in which this technique remain to be elucidated. New applications
embryos are cryopreserved after ovarian stimulation and such as fertility preservation have led to long storage
IVF, and then transferred in a subsequent cryo-cycle, times of vitrified gametes or embryos, but the question as
avoiding artificial hormonal levels.1 Embryo and oocyte to whether the state of the vitrified embryo is stable over
freezing is applied for fertility preservation in cancer time remains to be elucidated.
patients, and also in women planning delayed child bear-
ing. Due to this technological progression, the storage CONCERNS ABOUT SAFETY OF
time of cryopreserved embryos has arisen as an impor- CRYOPRESERVED CELLS AND TISSUE
tant issue. According to differing legislation in different Stability of Cryopreserved Cells and Tissue during
countries, storage of cryopreserved gametes and embryos Storage in SF
may be utilized for several years, or even decades. The main danger for embryos cryopreserved by SF where
crystallization occurs is the accumulation of radiation
TECHNIQUES FOR CRYOPRESERVATION over time. The influence of a cumulative dose of radia-
OF EMBRYOS AND OOCYTES tion has been studied. The authors found that a radiation
In human IVF, cryopreservation started with use of slow dosage equivalent to 2000 years of normal background
freezing (SF) technology in the 1980s. With SF, embryos radiation levels do not impair embryo survival or
are exposed to cryoprotectants (CPs) such as 1,2-propane- increase the incidence of mutations in offspring.2–4 A
diol (PrOH) or dimethyl sulfoxide (DMSO), followed by few studies on SF embryos and the impact of time have
a gradual lowering of the temperature down to the “seed- been published; however, the results remain controver-
ing” temperature just below the freezing point (at around sial. In SF mouse embryos, live births were reported after
−6°C to −7°C), at which point extracellular ice crystal for- 20 years of storage,5,6 and good embryo survival was also
mation is artificially induced. During this process, cells observed in other species, including sheep7 and rabbits8
are slowly dehydrated, and intracellular concentrations after cryopreservation for 13 and 14 years, respectively.
of CPs increase. SF has proven to be an excellent tech- In humans, successful pregnancies have been reported
nique for zygotes and cleavage-stage embryos; however, after the transfer of SF cleavage-stage embryos stored for
for oocytes and blastocysts, survival rates after SF have 7.5 and 8.9 years.9,10 Similarly, a live birth was obtained
mostly been unsatisfyingly low. Another technique had after the transfer of a SF zygote stored for 8 years.11 In a
to be found for these stages, and for this, the vitrification larger study, the authors found no reduction in pregnancy
(VIT) procedure has been established as more optimal. rates with prolonged storage time in SF.12 In contrast,

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other authors observed a reduction of 20% in the survival is accompanied by overgrowth, enlarged organs, and
rate of SF human embryos when stored for 6–15 months, overweight, has been described.19 Recent studies report
compared to 1 month of storage.13 In a multiple stepwise increased birth weights of children born after SF as well as
logistic regression analysis, the clinical outcomes for SF VIT embryo transfer.20,21 In VIT, predominantly ethylene
cleavage-stage embryos stored for 1–6 months or 24–108 glycol (EG) and DMSO are used, whereas the majority of
months were evaluated. No adverse effects on embryo SF protocols use PrOH, which was found to display higher
survival or implantation rates were observed, but a ten- genotoxicity. PrOH produces DNA damage in vitro, lead-
dency toward a lower pregnancy rate after prolonged ing to chromosomal mutations.22,23 Further, PrOH shows
cryopreservation was reported.14 cytotoxic effects, as it lowers the survival rate of embryos
and induces parthenogenetic activation.24
Special Aspects of the Vitrified State As stated at the beginning of this chapter, in VIT,
In addition to the aforementioned risk of accumulated concerns arise from the use of solutions with high con-
radiation over time, further concerns arise with VIT. The centrations of CPs. However, the incubation time in CPs
stability of VIT cells during storage is questionable, as it before cooling in liquid nitrogen is far shorter in VIT as
represents the solidification of a supercooled viscous fluid compared to SF. Recently, a study was conducted analyz-
without the formation of crystalline structures. It was ing the intracellular concentrations of CPs in SF and VIT
shown that considerable molecular mobility persists near in order to evaluate their possible harmful effects. It was
and just under the glass transition temperature (TG), reduc- shown that only a small percentage of CPs transverse the
ing the presumed stability of the vitrified state. VIT on the cell membrane during VIT. Further, it was observed that
molecular level is the loss of translational and rotational the intracellular concentration of CPs in VIT zygotes is
degrees of freedom over a particular measurement times- even lower than in SF.25 Chapter 3 focuses on this topic
cale, leaving only bond vibrations within a fixed molecular in more detail.
structure. Residual molecular mobility below the TG allows Although it is known that temperature is important
VIT objects to very slowly contract, release heat, and for evaluating the toxicity of CPs, and the duration and
decrease entropy during relaxation toward equilibrium. protocol for the incubation of cells with CPs are especially
Although diffusion is practically nonexistent below the TG, critical, the duration and temperature during storage
small local movements of molecules related to relaxation could also play a role. Little is known yet regarding the
are important and have consequences for cryobiology.15 possible impact of CPs during storage time.
This is due to that fact that amorphous solids are formed
when liquids are cooled very fast and molecules move too Direct Contact with Liquid Nitrogen in Open-Device
slowly to orient themselves. It is commonly admitted in the VIT and Long-Term Storage
field of thermodynamic cryobiology that once the vitrified Additional potential damage for VIT cells arise from
state is formed, it ages. Aged amorphous materials show the use of “open” devices. In order to increase cooling
decreased physical and chemical reactivity when com- rates, gametes and embryos vitrified on open VIT car-
pared to un-aged materials. rier devices are plunged directly in liquid nitrogen. Apart
In seeds stored in liquid nitrogen, apparent time- from the potential risk of contamination during VIT,
dependent deteriorations were proven over a period storage in open devices risks cross-contamination and
of years. One possible reason for this is a degradation physicochemical problems during storage. As a techno-
mechanism driven purely by vibrations of adjacent logically young innovation in VIT, closed carrier devices
molecules.16,17 were implemented in order to circumvent the direct con-
tact of embryos with liquid nitrogen, with the goal of pre-
Concentrations of CPs venting infection with pathogens during VIT, as well as
CPs are inevitable in SF as well as VIT. In contrast to SF, cross-contamination with reactive chemical compounds
far higher concentrations of CPs are applied in VIT in the during storage.26,27 Impairment of cells and embryos due
incubation solution, and their potentially toxic effects on to chemical reactions in liquid nitrogen during the stor-
cells and embryos have to be noted. However, the toxic- age time is avoided. This aspect of aseptic storage is of
ity of CPs decreases with temperature and biological and particular interest for long-term storage.
biochemical activity. Therefore, duration and tempera-
ture during incubation with CPs are especially crucial. RESULTS FROM VITRIFIED BLASTOCYSTS
Frequently applied in SF as well as VIT is DMSO, AFTER LONG-TERM STORAGE
which is known to alter DNA methylation and poten- Study Design to Evaluate the Effect of Storage Time
tially change the epigenetic profile of cells.18 Congenital on Vitrified Blastocysts
abnormalities might be induced by these epigenetic As VIT is a young technique in ART, little is known
alterations. A consistently discussed phenomenon in this about the stability of VIT cells over time and the health
context is the “large offspring syndrome” in animals. In of the babies born after cryostoring embryos and oocytes
humans, the “Beckwith–Wiedemann syndrome,” which for several years. In a retrospective study, the impact of

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 does storage of vitrified blastocysts have an impact on implantation potential and birth rate?
storage time on aseptically cryostored VIT blastocysts Table 21.2  Clinical Outcomes after the Transfer of
was studied by evaluating the outcomes of 603 embryo Vitrified–Warmed Blastocysts in Relation to Storage
transfers of vitrified–warmed blastocysts during a period Time
of 3 years.28 Survival rates, implantation potentials, and
Ongoing
birth rates, as well as the health of the children born, were
Pregnancy pregnancy Abortion Birth
analyzed.
Storage time rate (%) rate (%) rate (%) rate (%)
All blastocysts were vitrified in closed devices (Vitrisafe)
in EG/DMSO solutions as described previously.26 The 0–3 months 48.0 40.0 7.0 33.0
straws were kept in conventional liquid nitrogen storage 3–6 months 38.5 30.2 1.8 27.8
tanks until embryo transfer, for a period of up to 6 years. 6–12 months 39.4 33.3 5.1 28.3
At 3–6 hours prior to embryo transfer, blastocysts were 12–24 months 50.0 33.7 2.2 32.6
warmed in sucrose solutions. Endometrial development 24–36 months 53.3 47.8 8.5 38.9
was supported by estrogen supplementation and proges- 36–48 months 48.2 40.7 — 40.7
terone administration. Fourteen days after embryo trans- 48–60 months 46.2 38.5 7.4 26.9
fer (ET), patient urine was tested for β-hCG; for positive
patients, an ultrasound was performed at 6–8 weeks of
gestation to confirm the presence of a fetal heartbeat. In a recurrent implantation failure (data not shown). In addi-
later follow-up, the birth rate and the health of the children tion, when considering abortion or birth rates, no sig-
born were evaluated. nificant differences between the different storage groups
The results were grouped according to storage time were detected (Table 21.3).
of blastocysts; no statistically significant difference in
female age at the time of VIT was observed. Birth Weight and Gestational Age of Babies Born
After 38 multiple gestations and 147 singleton gestations,
Survival of Vitrified Blastocysts over Time 226 babies were born. Only three babies were born with
A similar number of VIT blastocysts were warmed per very low birth weights of <1500 g (1.3%), and three babies
ET (mean 3.2–3.6 embryos) in all groups. Survival rates of  >4500 g were reported (1.3%). Mean gestational age
as evaluated 3 hours post-warming ranged from 81.8% to at birth was 39.1 weeks for singletons and 36.0 weeks for
89.9% (Table 21.1). No statistically significant differences twins.
were observed between the groups, and no significant To study the birth weight of newborns in more detail,
decreases in blastocyst survival over time were observed. sibling children were compared. Patients who had previ-
ously given birth to a singleton in their fresh cycle, and
Clinical Outcomes after Transfer of Vitrified then delivered another singleton after subsequent cryo-
Blastocysts over Time ET of VIT blastocysts were selected. In these 42 patients,
Similar to survival rates, no statistically significant differ- the mean birth weights of children born after fresh ET
ences in the pregnancy rates between the storage groups was 3198 g, while the siblings born after cryo-ET weighed
were observed. No declines between blastocysts stored for an average of 3406 g, and so no statistically significant
0–3 months (48.0%) and those stored for 48–60 months difference was observed.
(46.2%) were found (Table 21.2). A trend toward a decline
in pregnancy rate in blastocysts stored for 6–12 months Table 21.3  Gestational Age and Birth Weight of
and for 12–24 months was seen (38.5% and 39.4%, respec- Children Born after the Vitrification of Blastocysts
tively), which could be explained by patient histories.
Parameter Singleton gestation Multiple gestation
Patients in these groups were more often diagnosed with
No. of children 147 79
Mean birth weight (g) 3400 ± 562 2508 ± 406
Table 21.1  Survival Rates of Vitrified–Warmed
Blastocysts in Relation to Storage Time <1500 g 2 1
<2500 g 2 38
Mean female age at Survival 2500–4000 g 124 40
Storage time No. of cycles vitrification (years) rate (%)
4001–4500 g 16 —
0–3 months 100 34.5 83.0 >4500 g 3 —
3–6 months 169 35.0 89.9 Mean gestation 39.1 36.0
6–12 months 99 35.2 84.3 age (weeks)
12–24 months 92 34.4 82.0 <28 weeks 1 —
24–36 months 90 34.4 81.8 <32 weeks 1 1
36–48 months 27 33.6 87.8 <37 weeks 13 19
48–60 months 26 32.4 83.1 Term birth 132 18

187
 vitrification in assisted reproduction
Congenital Malformation in Babies Born after freeze–thaw procedure. In the presented patient cohort
Transfer of Vitrified Blastocysts with VIT blastocyst transfer, 2% of singletons were born
In the follow-up, multiple births and the health of babies >4500 g, which is a far lower rate than the reported 4.5%
were evaluated according to storage time. Two babies after SF, or 2.9% after fresh ET.34
were  born with multiple malformations: one girl after There was an abortion rate in patients after the trans-
ET of a blastocyst stored for 12–24 months was diag- fer of VIT blastocysts of 4.3% after a positive heartbeat
nosed with stenosis, polysyndactyly, a palatine cleft, and was observed. The incidence of stillbirth was 2 in 191
a ventricular septum defect due to a deletion on chromo- births (1.0%). Severe malformations were reported in 4
some  7. Another child derived after ET of a blastocyst out of 224 children (1.8%). These findings support other
stored for 48–60 months was diagnosed with hydrone- papers showing that VIT and the fresh transfer of blasto-
phrosis, clubfeet, and a single umbilical artery. In this cysts have similar neonatal outcomes.35
case, no genetic background for the malformations was In summary, the data reassure use that VIT is a safe
found. One minor malformation was reported for a technique, and that cryostorage time does not have
twin girl born with a cleft palate after blastocyst storage a negative impact on outcomes and children born.
for 36–48 months. Two children with trisomy 21 were However, it has to be kept in mind that the data were
reported, both in the 6–12-month storage group. Further, observed using a “closed” aseptic device, thereby avoid-
two stillbirths were reported (both singletons), both after ing direct contact between the embryo and the liquid
storage for 0–6 months. In both cases, no medical reason nitrogen during the VIT procedure, and importantly
for the stillbirth was detected. No trend toward a higher during the storage period. This hermetically sealed pro-
incidence of congenital malformation after a longer stor- tection can also be obtained in SF. The ensuing results
age time of VIT blastocysts was found, although a larger might therefore not be directly translated to the use of
number of children will need to be analyzed to entirely VIT with open devices, where this hermetically sealed
exclude any negative impact. protection of the embryos is not provided. In open
devices, the constant contact with liquid nitrogen con-
IS THE STORAGE OF VITRIFIED EMBRYOS taining reactive chemical compounds might potentially
(GAMETES) SAFE OVER TIME? promote embryo damage.
The aim of cryopreservation in ART is to preserve gametes
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water and triacylglycerols in fern spores relating to stor- Reprod 2009;24:2158–72.
age at cryogenic temperatures. Cryobiology 2007;55:1–9. 31. Wada I, Macnamee MC, Wick K, Bradfield JM,
18. Iwatani M, Ikegami K, Kremenska Y et al. Dimethyl Brinsden PR. Birth characteristics and perinatal
sulfoxide has an impact on epigenetic profile in outcome of babies conceived from cryopreserved
mouse embryoid body. Stem Cells 2006;24:2549–56. embryos. Hum Reprod 1994;9:543–6.
19. Dupont C, Armant DR, Brenner CA. Epigenetics: 32. Belva F, Henriet S, Van den Abbeel E et al. Neonatal
Definition, mechanisms and clinical perspective. outcome of 937 children born after transfer of cryo-
Semin Reprod Med 2009;27:351–7. preserved embryos obtained by ICSI and IVF and
20. Pinborg A, Henningsen AA, Loft A, Malchau SS, comparison with outcome data of fresh ICSI and IVF
Forman J, Andersen AN. Large baby syndrome in cycles. Hum Reprod 2008;23:2227–38.
singletons born after frozen embryo transfer (FET): Is 33. Revel A, Safran A, Laufer N, Lewin A, Reubinov BE,
it due to maternal factors or the cryotechnique? Hum Simon A. Twin delivery following 12 years of human
Reprod 2014;29(3):618–27. embryo cryopreservation: Case report. Hum Reprod
21. Liu SY1, Teng B, Fu J, Li X, Zheng Y, Sun XX. Obstetric 2004;19:328–9.
and neonatal outcomes after transfer of vitrified early 34. Sazonova A, Källen K, Thurin-Kjellberg A,
cleavage embryos. Hum Reprod 2013;28(8):2093–100. Wennerholm UB, Bergh C. Neonatal and maternal
22. Aye M, Di Giorio C, De Mo M, Botta A, Perrin J, outcomes comparing women undergoing two in vitro
Courbiere B. Assessment of the genotoxicity of three fertilization (IVF) singleton pregnancies and women
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Dimethylsulfoxide, ethylene glycol and propylene 2013;99:575–9.
glycol. Food Chem Toxicol 2010;48:1905–12. 35. Wikland M, Hardarson T, Hillensjo T et al. Obstetric
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Botta A, Courbiere B. Comet assay on mouse oocytes: Reprod 2010;25:1699–707. 

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22 Ovarian tissue vitrification—Clinical realities and outcomes
Sherman Silber

PREFACE OVARIAN TISSUE VITRIFICATION: CLINICAL

The developed world is in the midst of a widespread infer- REALITIES AND OUTCOMES
tility epidemic. Economies in Japan, the United States, Fresh Series of Identical Twins with Premature
southern Europe, and even China are threatened by a Ovarian Failure
decreasing population of young people having to support Let us take the clinical evolution of this technology in
an increasing population of elderly people and retirees.1 logical order. The first successful fresh human ovary
Infertility clinics are emerging throughout the world in transplantation was reported between a pair of remark-
huge numbers because of a worldwide decline in fertil- able monozygotic twins discordant for premature ovar-
ity as women age and become less fertile.2 In her teenage ian failure (POF) using a cortical grafting technique.7
years, a woman has a 0.2% chance of being infertile, and This key event allowed us to assess the results of fresh
by her early twenties, it is up to 2%. By her early thirties, it transplantation unclouded by the confusion that might
is up to 20%.2,3 Most modern women today do not think have been caused by freezing. The transplantation tech-
of having a baby until their mid-thirties, and by then, nique has subsequently been refined over a larger series
over 25% are infertile, simply because of aging and the of nine consecutive successful fresh ovary transplants
decline in the number and quality of their oocytes. This is in identical twins (plus two fresh allotransplants to be
clearly demonstrated by the high pregnancy rate via using treated separately), with resumption of normal hormonal
donor oocytes from young women placed into the uter- cycling and menstruation in all cases, eventually leading
uses of older women.2–4 to 14 pregnancies and 11 healthy babies born from the
Until recently, oocyte freezing had very poor to no 9 fresh identical twin recipients.8–11 This unusual con-
success, and so ovary tissue slow freezing was the only secutive series of fresh ovary cortical transplants helped
preservation method we could rely upon. Of course, now us also to refine the techniques necessary for successful
we also have a favorable option of retrieving oocytes after preservation of fertility for cancer patients using ovarian
ovarian stimulation and egg retrieval, using vitrification tissue freezing, with six additional successful pregnancies
instead of slow freezing for cryopreservation.5,6 from nine frozen transplants. This unusual series also
However, as we will discuss later in this chapter, many helped to establish a method for distinguishing between
programs are not even aware of their terrible results the egg loss from transplant ischemia versus the egg loss
with oocyte freezing because they are either using a from cryopreservation. We now can report long-term
brainless commercial product, or they simply are not follow-up (up to 8 years) of this original series of fresh
using the best protocol perfectly. Success with oocyte transplants, and add to it our more recent experience
freezing should be 95%–99%, but most clinics come with cryopreserved ovarian tissue.
nowhere near this. We will explain this in this chap- Micro-hematoma formation under the graft was
ter, but meanwhile, we expect that there will be many avoided by micro-bipolar cautery and micro-pressure
unhappy “fooled” women complaining about this in stitches of 9-0 nylon. Constant pulsatile irrigation with
the next 5–10 years. Nonetheless, ovarian tissue freez- heparinized saline prevented adhesions (Figure 22.1a–d).
ing and transplantation still have great advantages over
egg freezing. There does not need to be a prior delaying Ovarian Cryopreservation
stimulation cycle, as ovarian tissue freezing would delay All of the frozen cases in the past that transplanted back
cancer treatment by only a few days. Furthermore, one into the patient utilized the slow-freeze approach.12–14
cycle of ovarian stimulation and egg freezing does not However, we now use vitrification exclusively for cryo-
ensure successful pregnancy as much as an entire ovary preservation in humans because of the results of in vitro
would, and finally, transplanting ovarian tissue back not viability analysis in humans, as well as in vivo transplant
only restores fertility, but also restores endocrine func- studies in the bovine and human.11,15
tion. We hope that in this book and in this chapter, we The goal of the in vitro study was to determine which
can clarify this so that all clinics can avoid the confu- method produced a higher cell survival rate: slow freeze
sion of the literatus and get the 95% results that patients or vitrification. The high viability (92%) of oocytes in con-
expect. trol (fresh) specimens indicated only minimal damage to

191
 vitrification in assisted reproduction

(a) (b)

(c) (d)

Figure 22.1  Steps in the procedure of ovarian transplantation between monozygotic (MZ) twin sisters: (a) prepa-
ration of donor ovarian cortex by dissection in a Petri dish on ice; (b) preparation of recipient ovarian medulla;
(c) attaching donor cortical tissue to recipient ovarian medulla; (d) attaching thawed donor cortical tissue for re-
transplant to the recipient medulla.

the eggs.11 Overall, 2301 oocytes were examined from (HM; HEPES-buffered TCM-199 solution supplemented
16 specimens. Results within each of the three groups with 20% serum substitution DMSO; cat. no. D2650;
revealed no significant differences between fresh and vit- Sigma Aldrich, St. Louis, MO) for 25 minutes, followed
rified tissue, but the viability of slow freeze-cryopreserved by a second equilibration in 20% EG and 20% DMSO
tissue was less than half that of vitrified tissue or controls with 0.5 mol/L sucrose for 15 minutes. Ovarian tissues
(42%) (p < 0.01). Transmission electron microscopy has are then placed in a minimum volume of solution (virtu-
also been used to analyze ovarian tissue that had been ally “dry”) onto a thin metal strip (Cryotissue: Kitazato
either cryopreserved by slow freezing or vitrified by ultra- BioPharma, Fujinomiya, Japan), and submerged directly
rapid freezing, showing vitrification to be superior.16 into sterile liquid nitrogen,17 following which the strip is
Standard H&E histology showed no difference between inserted into a protective container and placed into a liq-
pre-freeze ovarian tissue and post-vitrification ovarian uid nitrogen storage tank (Figure 22.3).
tissue (Figure 22.2a and b). For thawing, the protective cover is removed and the
Finally, quantitative histologic study of primordial fol- Cryotissue metal strip is immersed directly into 40 mL
licles in the bovine after vitrification and transplantation HM solution at 37°C supplemented with 1.0 mol/L
back to the cow 2 months later remarkably showed no fol- sucrose for 1 minute. Then, ovary tissues are transferred
licle loss. into 15 mL of 0.5 mol/L sucrose HM solution for 5 min-
The basic science concept of vitrification, whether for utes at room temperature, and washed twice in HM
eggs, embryos, or tissue, is to completely avoid any ice solution for 10 minutes before viability analysis or trans-
crystal formation by using a very high concentration of plantation. No ice crystal formation occurs during any of
cryoprotectant and a very rapid rate (virtually “instant”) these vitrification procedures.15
of cooling. This is quite different from classic slow-freeze One of our twin recipients became pregnant at 39 years
cooling, which relies on a partial and very gradual removal of age without medical assistance after her fifth menses,
of water from the cell by encouraging ice crystal formation 8 months after transplantation. She delivered a healthy
preferentially on the outside of the cell, drawing water out. baby girl at full-term, then conceived again at 42 years of
Using the vitrification technique, cortex tissue of each age, and delivered a healthy baby boy, again at full-term,
ovary is cut into slices of 1 × 10 × 10 mm. Ovarian tissues 4 years after her transplant. Her ovary is still functioning
are initially equilibrated in 7.5% ethylene glycol (EG) and to date after 7 years, and she conceived again at 45 years
7.5% dimethyl sulfoxide (DMSO) in handling medium of age with another healthy boy.

192
 ovarian tissue vitrification

(a) (b)

Figure 22.2  ​Histology (a) pre- and (b) post-vitrification of ovarian tissue.

Table 22.1  Worldwide Frozen Ovarian Cortex Tissue

Transplant Pregnancies
Case # Diagnosis Babies Which Center
1 Hodgkin’s 1 Donnez
2 Neuro tumor 1 Donnez
3 Non-Hodgkin’s 1 Meirow
4 Hodgkin’s 1 Demeestere
5 Ewing’s 3 Andersen
6 Hodgkin’s 1 Andersen
7 Premature 1 Silber
ovarian failure
8 Hodgkin’s 2 Silber
9 Polyangiitis 1 Piver
10 Breast cancer 2 Pellicer
11 Sickle cell 1 Piver
12 Hodgkin’s 2 Revel
Totals: 12 patients 17 babies 8 centers
Fresh + frozen 28 babies Silber – 14 babies
Figure 22.3  Ovarian tissue slice.
Note: The estimated outcomes presented in this table are based
on a survey performed in October 2013 by Dr. Silber.
This newly favorable experience with ovarian cortex
grafting is not limited just to our center.18 Equally robust
results are being experienced in Belgium, France, Spain, risk for POF, of whom 16 had spare frozen tissue sub-
Denmark, and Israel. Frozen ovarian grafts (even with jected to detailed viability testing before cryopreserva-
the slow-freeze technique) in Denmark are lasting over tion and after thaw. Only one had ovarian metastasis, a
5 years, and many spontaneous pregnancies have been young woman with widespread breast cancer metasta-
reported with no need for in vitro fertilization (IVF) or sis throughout her entire body. Otherwise, none of our
other ancillary treatment. At the time of this writing, other 61 cases had any tumor cells in their ovaries. The
over 28 healthy babies have been born from ovarian tissue reason for the remarkable absence of ovarian metasta-
grafting fresh and frozen, and most involved no IVF and sis might possibly be due to the fibrous avascular nature
resulted from regular intercourse with no special treat- of the ovarian cortex (Anderson C, personal commu-
ment (Table 22.1). nication). In fact, the reason why fetal ovarian tubules
(which in the fetal male become seminiferous tubules)
Frozen Cortical Ovarian Transplantation invade the fibrous cortex and become follicles is that the
The most common benefit of ovarian transplantation dense fibrous tissue of the cortex (which in the fetal and
is not the unusual cases of fresh grafting in identi- adult testis is just tunica albuginia) is needed to suppress
cal twins, but rather the protection of the fertility and the resting follicles from developing all at once prema-
future endocrine function of young women undergo- turely. In addition to these 68 pathological cases, seven
ing cancer treatment.11,15,19–26 Since 1996, we have fro- women have had ovarian tissue frozen simply to allow
zen ovary tissue for 68 young women with cancer or at them to have the possibility of bearing children at an

193
 vitrification in assisted reproduction
older age, because they had to delay childbearing for EGG AND EMBRYO VITRIFICATION
strong p
­ ersonal or economic reasons. As mentioned in the preface, many centers that perform
oocyte vitrification are not doing it well, and their pro-
Future Prospects for Ovarian Tissue Transplantation tocols lead to terrible egg survival rates. Among their
After ovarian transplantation, all patients were able to errors are too rapid and changing osmolality with dan-
attempt natural conception every month without medi- gerously aggressive osmotic shifts. Also, there is a com-
cal assistance. In fact, the commonly held view that egg mon failure in failing to create a rapid enough “freeze,”
freezing is a proven technique and ovary tissue trans- and worse yet, not a rapid enough “thaw.” The commer-
plantation is “experimental” is belied by the fact that cial kits that are designed to make this “easy” often fail in
most of the successful pregnancies resulting from fer- this regard, and closed freezing is worse than open freez-
tility preservation in cancer patients thus far have been ing for rapid “freeze and thaw.” Vitrification for freezing
from frozen ovary tissue, and few at the date of this writ- eggs or embryos was first suggested in the mid-1980s.32,33
ing have come from frozen oocytes.18 Of course eventu- However, it was not until 2005 that a highly efficient
ally they will have long term results to report, but not yet. method was published, which stimulated a huge wave of
However, for cancer patients, ovarian tissue does have a justified enthusiasm for this approach to egg and embryo
better record currently than egg freezing. Most of our freezing.4,6,34,35 But the details of this successful “bridge
cured cancer patients who have “young” ovarian tissue technique” have been lost, or have given way to poor-
frozen feel almost grateful that they had cancer, because quality commercialization (Figure 22.4a–f).
otherwise they would share the same fear that all mod- The concept behind vitrification is not just its potential
ern, liberated women have about their “biological clock.” simplicity (given that no freezing machine is required),
At the time of this writing, we are aware of numer- but that it must completely eliminate ice crystal forma-
ous other births after implanting ovarian tissue, to a tion. Instead of clinical IVF programs having to weigh
total of over 37 live births thus far.18,26–31 Thus, despite carefully the risks to pregnancy rate posed by embryo or
initial skepticism, this technique is now gaining world- egg freezing, both can now be cryopreserved without con-
wide acceptance and is being enthusiastically received by cern in virtually any case in which there would be a clini-
young women of reproductive age with cancer. cal advantage. With the new vitrification methodology,

(a) (b) (c)

HEPES ES 3 min VS 60–90 s

ES 3 min VS 60–90 s
HEPES
ES 9 min
ES 9 min
ES 3 min ES 3 min
All drops 20–30 µL

(d) (e) (f )

Figure 22.4  (a) The “bridge” technique for oocyte freezing. (b) Setup for “bridge” equilibration. (c) First “bridge”
between ES and isotonic HEPES media; 3 minutes. (d) Second “bridge” equilibration; 3 minutes. (e) Transfer to full
concentration ES; 9 minutes. (f) Transfer from ES to VS; 60–90 seconds. High cryoprotectant concentrations are
not toxic. It is only the rapid osmotic shifts that kill the egg or embryo and give the incorrect impression of toxicity.
To avoid over-rapid osmotic shifts (that are more poorly tolerated by the egg than the embryo), the original “bridge”
technique is best. ES solution droplets are first “bridged” over to the iso-osmotic solution the egg is in, and 3 min-
utes later, another droplet of ES solution is “bridged” over to the original solution very gradually and continuously
raising the osmolality of the solution the oocyte is resting in.

194
 ovarian tissue vitrification
there seems to be no difference between fresh and cryo- an expansion of the reproductive lifespan in any young
preserved eggs or embryos, so long as the principles per- woman who wishes to delay childbearing, or delay her age
fected in 2005 are followed. of menopause.
For vitrification, the cryoprotectant solution is a
combination of EG and DMSO (cat. no. D2650; Sigma REFERENCES
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solution (15 mol/L EG and 15 mol/L DMSO in 20% SSS Overcoming Infertility, 2nd ed. Little, Brown: Boston,
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uid nitrogen. The Cryotop containing the embryo is then tion of human oocytes. Reprod Biomed Online
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concentrations of sucrose solutions to remove the cryopro- transplantation between monozygotic twins dis-
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0.5 mol/L, and 0.25 mol/L sucrose). A wash solution failure. N Engl J Med 2007;356:1382–4.
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100% wash solution.6 This protocol was designed to avoid transplantation (cortical versus microvascular) and
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The high concentrations of cryoprotectant are actu- pregnancy after microsurgical transplantation of an
ally not toxic. The appearance of toxicity comes only from intact ovary. N Engl J Med 2008;359(24):2617–8.
too rapid an osmotic shift. The ultrarapid rate of cooling 11. Silber SJ, Kagawa N, Kuwayama M, Gosden R.
(−23,000°C/min) and the high-end concentration of cryo- Duration of fertility after fresh and frozen ovary
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With vitrification, mature retrieved oocytes can of fertility to oophorectomized sheep by ovarian
be successfully cryopreserved with 95% success. For autografts stored at −196 degrees C. Hum Reprod
embryos, there really is no difference at all between fresh 1994;9(4):597–603.
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New technology in cryopreservation via vitrification rate and dehydration regimen on the histologi-
allows us to remove ovary tissue and freeze it to protect cal appearance of human ovarian cortex following
it from sterilizing cancer treatment in young women, as cryopreservation in 1,2-propanediol. Hum Reprod
well as to freeze individual mature eggs. Our latest pub- 1999;14(8):2061–8.
lished data reveals a very high pregnancy rate with either 14. Newton H, Aubard Y, Rutherford A, Sharma V, Gosden
fresh or frozen ovary tissue transplants of over 76%.37 It R. Low temperature storage and grafting of human
also allows us to stop the aging of the ovary and eggs, ovarian tissue. Hum Reprod 1996;11(7):1487–91.
which is the major cause of the current worldwide infer- 15. Kagawa N, Silber S, Kuwayama M. Successful vitri-
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tial of young women with cancer, and will also allow Biomed Online 2009;18(4):568–77.

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16. Keros V, Xella S, Hultenby K et al. Vitrification versus pregnancy after transplantation of cryopreserved
controlled-rate freezing in cryopreservation of human ovarian tissue: a review of 60 cases of reimplantation.
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17. Fuentes F, Dubettier R. Air separation method and 27. Donnez J, Dolmans MM, Demylle D et al. Livebirth
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2011;43(6):437–50. N Engl J Med 2005;353(3):318–21.
19. Bleyer WA. The impact of childhood cancer on 29. Andersen CY, Rosendahl M, Byskov AG et  al. Two
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Cancer Incidence and Survival among Children and 30. Demeestere I, Simon P, Emiliani S, Delbaere A,
Adolescents: United States SEER Program, 1976–1995. Englert Y. Fertility preservation: Successful trans-
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21. Jeruss JS, Woodruff TK. Preservation of fertility patient previously treated for Hodgkin’s disease.
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23 Vitrification of human testicular tissue, spermatogonia,
and spermatozoa
Christine Wyns, Gael Abou-Ghannam, and Jonathan Poels

INTRODUCTION Since extrapolation of observations in animals to

Improvement of cryopreservation techniques for human humans is limited by the differing sizes of sperm heads,
male germ cells is highly warranted for optimal clinical which in turn appear to be correlated to cryostability,11–13
management of male infertility in the context of assisted this chapter will focus on human spermatozoa. Only a
reproduction programs, as well as fertility preservation few teams have studied the vitrification of human sper-
for cancer patients. matozoa.14–17 Isachenko and colleagues were the first to
Vitrification has been developed as an innovative report the successful vitrification of human spermato-
strategy for gamete and gonad preservation, with a view zoa in the absence of toxic permeable CPAs by directly
to minimizing cell or tissue damage due to ice crystal plunging a sperm suspension into LN2.14,15 Changes in the
formation and solution effects, as observed during slow mitochondrial membrane potential, as a marker of mito-
freezing. Vitrification involves a transition from a liquid chondrial activity, were found to be dependent on the vit-
phase to a vitreous phase, which occurs at a low tempera- rification medium. The best results were achieved with a
ture following very fast cooling by directly immersing combination of 0.25 M sucrose and albumin, providing
of samples in liquid nitrogen (LN2).1 Use of high cool- protection for about 65% of spermatozoa.18 Furthermore,
ing rates means water molecules have less time to escape CPA-free cryopreservation by vitrification was achieved
from cells, preventing cell shrinkage and osmotic cell using very fast cooling rates (ranging from 150–250°C/
damage. Addition of high cryoprotectant (CPA) concen- min to 7.2  ×  105°C/min) and instant warming in a
trations increases viscosity, thereby preventing move- warm medium, with an approximately 40% reduction in
ment of water molecules with solute concentrations and motility of spermatozoa, but unaffected DNA integrity.19
ice crystallization. Ease of use, speed, and absence of need Overall, depending on the vitrification method used and
for freezing equipment have contributed to the rapidly the quality and preparation of the original sperm sample,
growing interest in this technique. motility levels of up to 60% could be reached for normo-
Application of vitrification to mature or immature zoospermic ejaculates, 20 and 20% for oligozoospermic
male germ cell suspensions and immature gonadal tissue, samples after warming.21,22 Open systems with direct
as well as lessons learned from reported empirical proto- contact with LN2 are preferentially used to achieve the
cols, will be reviewed in this chapter. highest cooling rates, but because these systems are at
risk of microbiological contamination, methods allowing
VITRIFICATION OF SPERMATOZOA aseptic vitrification have been developed.23
It is widely known that cryopreservation has detrimental Vitrification of spermatozoa has been compared to
effects on spermatozoa.2,3 Spermatozoa are cryo-sensitive standard LN2 vapor freezing. A significant improvement
cells, showing a decrease in sperm motility of 50%–90% of 11.6% in post-thaw motility was achieved when vitrify-
after cryopreservation by conventional slow freezing ing swim-up prepared spermatozoa with no CPAs,14 and
using permeable CPAs, whether programmed or not as almost two-fold higher residual motility after vitrification
with LN2 vapor freezing.4–6 Classical vitrification using using 0.25 M sucrose and 1% LGPS (LifeGlobal Protein
high concentrations of permeable CPAs is also unsuitable supplement, IVF online, Guelph, Ontario) and fast warm-
for spermatozoon preservation due to lethal osmotic and ing at 37°C for 10 seconds was observed.24 In addition,
cytotoxic effects.7,8 Insights into the cryobiology of sper- no statistical differences were noted for parameters such
matozoa related to their physical features have been sum- as viability, recovery rate, or percentage of morphologi-
marized.9 Briefly, their intracellular milieu, containing cally normal spermatozoa with nondamaged DNA, and
large amounts of proteins, nucleotides, and sugars and levels of membrane changes related to “cryocapacitation”
a small quantity of water,10 is responsible for their high were also similar.21 Higher rates of plasma and acroso-
intracellular viscosity that determines the possibility of mal membrane integrity were attained.21,22 An increased
vitrifying these cells without permeable CPA and at rela- mitochondrial membrane potential was also reported
tively low cooling rates.11 The small size of the spermato- after vitrification,17 although the results were not always
zoon head may be an additional advantage for ensuring consistent, probably due to use of different vitrification
intracellular vitrification. protocols.18 While lower DNA fragmentation levels were

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 vitrification in assisted reproduction
observed after vitrification by some authors,17 others did VITRIFICATION OF SSC SUSPENSIONS
not find any clear benefit in terms of DNA integrity.24,25 Only one study has reported the vitrification of human
When compared to controlled programmed slow freez- diploid germ cell suspensions. In this study, cell viabil-
ing, similar motility and DNA integrity were achieved ity was higher with vitrification using open pulled straws
with both techniques.20 Solid-surface vitrification (SSV), compared to three different slow-freezing protocols,35
as a variant form of vitrification, has also been compared but similar recovery and viability rates were previously
to the slow-freezing technique.16 Using sperm-freeze CPA achieved by others with slow freezing.36
(Fertipro NV, Beernem, Belgium) for both protocols,
sperm motility, morphology, and vitality were not found LESSONS LEARNED FROM VITRIFICATION
to be different. However, a slightly significant difference OF ITT IN ANIMALS
was detected in favor of SSV for DNA integrity and mor- ITT vitrification has been investigated in different spe-
phological parameters, such as tail defects. cies,37–41 including nonhuman primates.42 Protocols were
Since there is evidence that vitrification of the intra- empirically developed or adapted from those established
cellular compartment of spermatozoa occurs even with for other reproductive cells or tissues. Table 23.1 summa-
slow cooling rates, thereby avoiding intracellular ice for- rizes studies on vitrification protocols for ITT, showing
mation,9 this questions the benefits of using very high the CPAs and systems used.
cooling rates to cryopreserve spermatozoa. Indeed, ultra- Since open systems, with their advantage of higher
rapid freezing in vapor before immersion in LN2, yielding cooling rates, run the risk of microbiological contami-
five- to ten-times higher cooling rates than those used in nation by direct contact with LN2, therefore closed sys-
conventional slow freezing, has also been investigated, tems have been used by most teams. For closed-system
both with the addition of CPAs26–29 and without.19,26,28 vitrification, different tissue carriers are available: closed
Compared to CPA-free vitrification, which allowed 60% plastic cryovials,38,43 the straw-in-straw method,44 and
residual motility, this method was not found to differ aluminum boats partially immersed in LN2, known as
in terms of sperm motility, fertilization ability, or DNA SSV.39,45,46 Higher cooling rates were obtained with alu-
integrity outcomes.19 minum, being a better temperature conductor, which
Placing vitrified samples directly in a warm solution may explain the discrepant results in studies on the vitri-
involves the use of very high warming rates, which is of fication of pig ITT, with the best outcomes achieved with
paramount importance for the successful vitrification SSV by Abrishami et al.39
of spermatozoa. Indeed, if rewarming takes place too Most vitrification protocols use a combination of two
slowly, recrystallization of the vitrified intracellular solu- permeable CPAs, the most common being dimethyl sulfox-
tion occurs, with the development of large intracellular ide (DMSO) and ethylene glycol (EG). Observations point
crystals.30 Furthermore, due to its physical features, the to the importance of decreasing exposure times to high
intracellular compartment of the sperm cell will vitrify concentrations of CPAs in order to minimize their toxic-
during rapid cooling, but will not devitrify until it reaches ity. ITT vitrification was initially studied in pigs.38,39 After
approximately −30°C during warming. This causes dam- vitrification and xenotransplantation, Zeng et al.38 observed
age to spermatozoa as a result of an osmotic imbalance, decreased germ cell viability and similar differentiation
with exposure of the outer surface of the plasmalemma to potential to conventional slow freezing, albeit delayed
the stresses of osmotic shock due to highly concentrated compared to fresh tissue grafts.38 When comparing differ-
extracellular fluid.9 ent exposure times to CPAs, Abrishami et al. found better
The reproductive potential of vitrified sperm was cell viability after 5 minutes of equilibration in DMSO, and
finally proved by obtaining healthy live births, reported complete and similar differentiation to fresh tissue when
after intracytoplasmic sperm injection with vitrified sper- exposed for 5 or 15 minutes to glycerol, or for 5 minutes to
matozoa,31 following intrauterine insemination of vitri- DMSO. Furthermore, the proportion of elongated sperma-
fied semen,32 and after rapid freezing of testicular sperm.33 tids was higher when exposure times were the shortest.39
Promising results were also obtained in mice using
VITRIFICATION OF IMMATURE MALE GERM an open vitrification system and comparing it with slow
CELLS OR TISSUE freezing with 0.7 M DMSO.40 Although markers of cyto-
Cryopreservation of spermatogonial stem cells (SSCs) or toxicity and cellular necrosis were lower after vitrifica-
immature testicular tissue (ITT) containing SSCs is so tion and warming, higher apoptosis levels and decreased
far the only approach that can be proposed to preserve intratubular cell density were observed after 1 day of
fertility in young boys whose fertility is threatened by organotypic culture of the tissue. However, this differ-
gonadotoxic treatments, since spermatozoa are not pro- ence in cell density was not detected on day 3 of culture
duced before puberty. To restore fertility from cryostored because of increased cell proliferation. This observation,
tissue, auto-transplantation of testicular cells, cellular plus the similar diameters of seminiferous tubules found
aggregates, or tissue, as well as in vitro maturation of in both fresh and vitrified–warmed tissue, suggest that the
SSCs, need to be considered.34 tissue is not—or is only slightly—affected by vitrification.

198
 vitrification of human testicular tissue, spermatogonia, and spermatozoa
Table 23.1  Vitrification Protocols for Immature seminiferous tubule integrity was achieved in fragments
Testicular Tissue in Animals of up to 16 mm3 when 1 mm-thick slices were vitrified.42
Appropriate fragment size is a major concern for vitri-
Vitrification
fication, since tissue damage increases with size due to
Vitrification systems:
longer penetration times. This leads to the overexposure
References Species cryoprotectants open/closed
of surface cells to CPA concentrations and slower cooling
Bono-Mestre Zebrafish DMSO 20%, EG Closed rates, which are related to greater amounts of tissue and
et al., 200937 20%, FBS 20% enlarged vapor coats around fragments when immersed
Zeng et al., Pigs EG, NaCl 0.9%, Closed in LN2.46 Since vitrification is time consuming, establish-
200938 raffinose 0.5 M ing the maximum size of fragments yielding optimal out-
Abrishami Pigs VS1: DMSO 15%, Closed comes after vitrification may be crucial for laboratories.
et al., 201039 EG 15%, The importance of appropriate warming rates has also
FBS 20%,
been the focus of vitrification studies. The superiority of a
sucrose 0.5 M
rapid warming protocol over a slower one has been dem-
VS2: glycerol 7%,
onstrated. Indeed, while no difference in graft vascular-
EG 15%,
FBS 20%,
ization was observed between fresh and vitrified–warmed
sucrose 0.5 M tissue at 40°C, vascularization was less developed when
Curaba et al., Mice DMSO 20%, Open warming at room temperature.41 Besides achieving com-
201140 EG 20%, plete SSC differentiation after vitrification, efforts in the
HSA 25 mg/mL field were finally rewarded by the demonstration of true
Gouk et al., Mice EG 40%, Closed reproductive potential in pigs47 and Japanese quails,48
201144 sucrose 0.6 M with normal reproductive development reported in pigs
Poels et al., Macacca DMSO 15%, Open produced with sperm from vitrified xenografted ITT.49
201242 mulatta EG 15%, Such encouraging results in animals confirm the poten-
sucrose 0.5 M, tial of vitrification in the field of fertility preservation.
HSA 25 mg/mL
Baert et al., Mice DMSO 15%, Closed VITRIFICATION OF ITT IN HUMANS
201245 EG 20%, Vitrification of human ITT has been evaluated in vitro
sucrose 0.5 M, in an organotypic culture system50 and in vivo in a nude
FBS 20% mouse transplantation model.51 Prior to vitrification,
Liu et al., Japanese DMSO 15%, Open the tissue was pretreated with an equilibration solution
201241 quail EG 15%, consisting of 7.5% EG, 7.5% DMSO, and 0.25 M sucrose,
sucrose 0.5 M, supplemented with 25 mg/mL human serum albumin
FBS 20%
(HSA) for 10 minutes at 4°C. It was then transferred to
Gholami et al., Mice DMSO 15%, Closed
the vitrification solution containing 15% EG, 15% DMSO,
201343 EG 15%,
0.5 M sucrose, and 25 mg/mL HSA. After removal of the
FBS 20%
surrounding vitrification medium, the tissue was placed
Note: DMSO  = dimethyl sulfoxide; EG  = ethylene glycol; in open cryostraws and plunged into ultraviolet-sterilized
FBS = fetal bovine serum; HSA = human serum albu- LN2, according to the protocol of Parmegiani et  al.52 It
min; VS = vitrification solution.
was stored in sealed, precooled cryotubes. For warming,
the cryotubes were removed from the LN2 and the straws
Furthermore, vitrification did not appear to increase the were quickly immersed in a 35°C warming solution con-
expression of apoptosis-related genes in SSCs.43 Higher taining sucrose (1 mol/L) supplemented with 25 mg/mL
cell viability was also reported after vitrification in a HSA, before being transferred to three consecutive baths
closed system compared to both slow and rapid freezing of warming solutions with decreasing sucrose concentra-
(71.5% of cells were viable after spermatogonial enrich- tions. Ten-day organotypic culture of vitrified–warmed
ment versus 82.9% in control tissue).44 Recovery of com- tissue showed in vitro spermatogonial survival, but did
plete spermatogenesis similar to fresh tissue samples was not allow further functional evaluation of human SSCs
eventually achieved after vitrification in a closed system after vitrification.50
and allografting.45 As shown in Figure 23.1, good preservation of semi-
The potential of vitrification to preserve ITT of non- niferous tubule integrity was obtained after transplanta-
human primates has been studied by means of xenograft- tion of the vitrified ITT, with strong cell cohesion and
ing in a nude mouse model. Besides the preservation of adhesion to the basement membrane and no sclerosis.51
seminiferous tubule integrity and the survival and prolif- A comparative study investigating the functionality
eration of spermatogonia, the functionality of Leydig cells of tissue after transplantation in a nude mouse model
was demonstrated.42 The homogeneous distribution of showed that its developmental potential was similar with

199
 vitrification in assisted reproduction

(a) (b) (c)

(d) (e) (f )

Figure 23.1  (See color insert.) Fresh and vitrified human immature testicular tissue grafted for 6 months to castrated
nude mice. Hematoxylin–eosin staining of fresh grafted (a and d) and vitrified grafted (b and e) tissue of 2- and 9-year-
old donors, respectively. Spermatogonial differentiation to the pachytene stage was achieved in vitrified grafted tissue
of the 9-year-old donor (e, asterisk). Immunostaining of spermatogonia with MAGE-A4 antibody can be seen in the
vitrified grafted tissue of the 2-year-old (c) and 9-year-old (f) donors. Magnification 400×. Scale bar: 10 µm.

both vitrification and conventional slow freezing51 cur- carrier systems [open versus closed]). So far, there is no
rently used in clinical practice.53 Survival, proliferation evidence that vitrification of ITT is superior to the slow
capacity, and early differentiation up to the pachytene freezing already used in clinical practice in the context of
stage were demonstrated in vitrified SSCs, and functional clinical trials. Until the reproductive potential of frozen
Leydig cells were observed in vitrified tissue. Moreover, ITT protocols has been proved for SSC cryopreservation,
the functional potential of the tissue appeared to be methods still need to be further investigated.
similar to fresh tissue grafts, indicating that not only the One key motivation for pursuing research on gamete
cryopreservation method, but possibly also the xeno- and gonad vitrification is to widen the diffusion of the
transplantation model may be implicated.51 technique to centers where there is no availability of cryo-
Although vitrification offers a number of theoretical preservation equipment.
advantages over slow freezing (e.g., avoidance of ice crys-
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24 Vitrification in pluripotent stem cell banking:
Requirements and technical solutions for large-scale biobanks
Julia C. Neubauer, Axel F. Beier, Frank Stracke, and Heiko Zimmermann

INTRODUCTION normal cell lines. They usually grow as three-dimensional

Pluripotent stem cells (PSCs) have been of central interest cell clusters (“colonies”), and are often cultivated on a
in biomedical research since the first isolation of human feeder layer (a nonproliferating murine cell line) to main-
embryonic stem cells (hESCs) in 1998.1 PSCs have unique tain their pluripotency.1 Additionally, PSCs need cellular
potential for novel therapies of degenerative diseases,2,3 contacts to each other in order to survive, which leads to
due to their ability to differentiate into cells of all three serious problems during cryopreservation. Because of the
germ layers4,5 and their theoretical property of unlimited colony’s three-dimensional structure and size (<100 µm
proliferation.6 Studying these cells advances the under- to >1 mm), fast and homogenous removal of the ther-
standing of human developmental processes as well as mal energy while cooling and permeation of cryoprotec-
cellular differentiation processes. Furthermore, with the tants into the inner part of cell clusters before freezing is
discovery of “induced” PSCs (iPSCs), new possibilities extremely limited.12–14 This increases the risk of large ice
in the area of drug discovery and compound develop- crystal formation within the colony, disruption of cell–
ment have appeared: whereas hESCs can only be isolated cell contacts, and serious cell damage. Although the use
from the inner cell mass of blastocysts during the early of a selective Rho-associated kinase inhibitor (ROCKi,
development of embryos usually created by in vitro fer- Y2763215) significantly improves the survival of cells dur-
tilization, iPSCs are artificially generated by epigen- ing passaging and dissociation,16,17 mechanisms of cell
etic reprogramming techniques from somatic cells.7,8 death during cryopreservation are still not completely
This offers for the first time the possibility of producing understood, and recovery rates remain insufficient.
patient- or disease-specific PSCs and integrating specific Slow-rate cooling and rapid thawing protocols are
mutations (e.g., using Clustered Regularly Interspaced the current state of the art in the banking of various cell
Short Palindromic Repeats/CRISPR associated protein types. These allow sterile storage of samples in the vapor
[CRISPR/Cas]-technology 9) to generate model cells for phase of liquid nitrogen, the cryopreservation of large
compound screenings. New cell handling tools (e.g., sur- quantities of PSCs, and they are comparatively easy to
face-based hanging drop cultivation10) will enable single- perform.18 However, such protocols yield very low recov-
cell diagnostic and new high-throughput/high-­content ery rates, and commercial providers of hESCs guaran-
screening for drug discovery. In the future, PSCs may tee a recovery of just 0.1%–1%.19 To increase efficiency,
even be used to replace damaged or nonfunctional cells some cryobanks, especially in the field of reproductive
and tissue, enabling personalized regenerative medicine. medicine, use rapid-freezing protocols in small-volume
However, prospective applications will need high- straws.20,21 In this “vitrification” process, the liquid
quality stem cells or stem cell-derived progenitors that solidifies without crystallization and the growth of ice
are permanently available, reliable, and well character- crystals. The procedure of vitrification was discovered
ized.11 Until now, thousands of different stem cell lines and extensively investigated by Luyet and colleagues in
have been isolated and cultivated by researchers in vari- 1967,22,23 and only some years later was it proven to be
ous scientific fields all over the world, using different a very efficient tool for the preservation of sensitive and
protocols for the isolation, identification, and cultivation complex cell systems (e.g., oocytes in reproduction medi-
of the cells. Hence, the standardization of processes and cine).24 The idea behind vitrification-based storage is to
reproducibility of protocols are limited. Additionally, prevent the specimen from biochemical degeneration by
long-term culture may change cell characteristics and low temperatures, without the corruptive effects of phase
increase the risk of contamination with viruses or myco- separation and biomolecular reorganization that occurs
plasma. Therefore, cryobanking of defined and fully char- during slow equilibrated cooling. Hence, unlike slowly
acterized human PSCs is in the focus of several European cooling the sample to a stable equilibrium state, fast vit-
Union-funded projects, even coordinated by the pharma- rification results in a metastable off-equilibrium state of
ceutical industry (www.ebisc.org), illustrating the high the sample—virtually a “snap-shot” of its room tempera-
relevance of this cell system. ture molecular composition. Physically, all liquids of the
One major problem of PSCs is the biological com- sample solidify to a glass-like state by an increase of the
plexity of these cells, which is significantly higher than viscosity without any ice crystallization. The cell volume

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 vitrification in assisted reproduction

Vitrified water (glass) Crystalline water (ice)

Devitrification

2750 3000 3250 3500 3750 2750 3000 3250 3500 3750
Wavenumber/cm–1 Wavenumber/cm–1

EA
Energy

Molecular order

Figure 24.1  (See color insert.) Schematic of the devitrification process in an energy diagram, the corresponding
molecular arrangements, and the Raman spectra of liquid water (15°C) and ice (−115°C). Liquid water resembles the
spectral shape of vitrified water quite well. It was used here as the vitrification of macroscopic amounts of pure water
is practically impossible. EA is the activation energy for devitrification.

remains unchanged, and the biomolecules persist in their usage in Good Manufacturing Practice (GMP)-compliant
room temperature conformations. But the vitrified state workflows.
is fragile, tending to change to the stable, crystallized The aim of this chapter is therefore the derivation of
state under the release of the latent heat. This process is requirements for large-scale biobanks concerning effi-
called devitrification, and can be observed by Raman cient cell handling protocols and storage technologies for
spectroscopy (Figure 24.1). Once the devitrification pro- PSCs on the basis of demanding vitrification procedures.
cess is triggered, the entire sample will irreversibly crys- Furthermore, solutions using state-of-the-art technolo-
tallize, resulting in complete sample loss. gies are shown and a reference workflow is described.
In order to avoid devitrification, the sample must
strictly be kept below its glass transition temperature PROCEDURES AND WORKFLOWS FOR THE
(TG). Put simply, at temperatures above the TG, the ther- VITRIFICATION OF HUMAN PSCs
mal energy of the glassy sample exceeds the activation Human PSCs (hPSCs) are very sophisticated cell systems,
energy (EA) of molecular reorientation and crystalliza- as these cells grow in multicellular colonies. hPSCs can be
tion may start. Whereas the TG of pure water is at about maintained in their undifferentiated state by co-culturing
−137°C,25,26 high concentrations of vitrification additives with mouse or human embryonic fibroblasts, or using
like dimethyl sulfoxide (DMSO), ethylene glycol (EG) growth media containing special growth factors and cyto-
and various carbon hydrates can be used to raise the TG to kines. The applications of hPSCs range from regenera-
a certain extent,27 but commonly used vitrification media tive medicine to developmental biology, as these cells are
still possess TG values significantly below −100°C.28 capable of differentiating into cells of all three germ layers.
Although vitrification procedures in straws are very There is an increasing demand for permanently avail-
efficient, they can only be applied for the storage of able hPSCs and hPSC-derived cells, but freezing and stor-
small sample volumes with only up to ten-cell clumps. age at cryogenic temperatures is still the only strategy for
Additionally, the workflow is difficult to perform even for storing viable biological material over long periods with
trained staff, and minor changes of the incubation times minimal alterations, sufficient viability, and conservation
can lead to a complete loss of samples. Hence, automation of functionality.
and upscaling of this complex procedure is not possible, Most cryopreservation protocols of hPSCs are cur-
so vitrification is not yet suitable for the operation of large rently based on freezing cells in suspension using slow-
biobanks. Finally, vitrified samples have to be stored in rate protocols. For adherent cell systems, like PSCs,
liquid nitrogen, risking contamination with viruses or these procedures include cell detachment from the cul-
cross-contamination with other cell types,29,30 preventing tivation surface using cell-damaging bio-active factors

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 vitrification in pluripotent stem cell banking
(i.e., enzymes and divalent cation chelators) and re-adhe- embryos.21 This protocol was then optimized and slightly
sion after thawing. Detachment of adherent cells and cell modified by Richards et al.36 in 2004 (Figure 24.2).
colonies affects cell–cell and cell–matrix junctions, mem- The corresponding procedure includes dissociation of
brane integrity, and cell morphology. In this impaired undifferentiated hESC clumps by mechanical cutting into
state, cells are additionally exposed to the highly stressful fragments consisting of 300–400 cells. For the vitrification
mechanisms of freezing (i.e., solution effects or intracel- procedure, a sterile four-well cell culture plate containing
lular ice) during slow-rate protocols. Furthermore, re- the following solutions is prepared at room temperature:
establishment of full adhesion after thawing leads to a (i) well 1 contains the holding medium ES–HEPES–HSA
prolonged recovery time and a delayed proliferation. (embryonic stem–2-(4-(2-hydroxyethyl)-1-piperazinyl)-
These effects have been intensively investigated for ethane sulfonic acid (a buffer)–human serum albumin)
hESCs. One of the first studies in this field in 2001 revealed consisting of 80% DMEM (Dulbeccos Modified Eagle
that only 16% of the hESC clumps slowly frozen in 10% Medium) (vol/vol) (high-glucose DMEM) and 20% HSA
DMSO could be recovered.31 Additionally, the hESC colo- (vol/vol) buffered to 20 mM HEPES; (ii) well 2 contains
nies frozen in suspension possessed a smaller colony size the vitrification solution 1 (VS1) consisting of 80% ES–
in comparison to the nonfrozen control after cultivation HEPES–HSA (vol/vol), 10% DMSO (vol/vol), and 10% EG
for 2 weeks, and showed significantly higher grades of dif- (vol/vol); (iii) well 3 contains the vitrification solution 2
ferentiation. Until now, combination of different cryopro- (VS2) consisting of 30% ES–HEPES–HSA (vol/vol), 30%
tective agents (CPAs)32 and dissociation of cell aggregates ES–HEPES–HSA with 1 M sucrose (vol/vol), 20% DMSO
into single cells using the addition of the ROCKi Y2763216 (vol/vol), and 20% EG (vol/vol); and (iv) well 4 contains
improved the cryopreservation efficiency of hPSCs, the warming solution 1 (WS1) consisting of 80% ES–
achieving viability of over 85% and 80% confluency after HEPES–HSA (vol/vol) and 20% ES–HEPES–HSA with
3–4 days with maintained pluripotency.33 1 M sucrose (vol/vol).
Nevertheless, detachment of adherent cells and cell An embryo straw is prepared by loading it through
colonies, and in particular the disruption of cell–cell con- the open end with VS2 until about 20 mm of the straw
tacts during dissociation into single cells, causes massive is filled using a syringe with a connector, provided with
cell stress and significantly affects the cell physiology. the straws (Figure 24.3). Then, about 5 mm of the straw is
Therefore, cryopreservation of adherent cells in their filled with air by aspiration.
native states reduces, on the one hand, cellular stress The preparation of the cell clumps for vitrification is
induced by cell detachment and dissociation, and on the a multi-step procedure with a gradual increase of the
other hand it limits the necessary recovery time after- medium osmolarity before cryopreservation and a grad-
wards, as re-attachment of cells is not necessary at all. ual adjustment to physiological conditions after thawing.
But cryopreservation of adherent hESCs with slow-rate For vitrification, the dissected colony fragments are ini-
protocols also resulted in a low efficiency, as cell viability tially washed once in modified phosphate-buffered saline
decreased to approximately 30% within 90 minutes after (PBS +/+). Then, five to eight colony fragments are trans-
thawing.34,35 These data indicate that beside detachment ferred to well 1 of the four-well dish containing the hold-
and dissociation of cells, ice crystal formation inside and ing medium ES–HEPES–HSA using a sterile glass Pasteur
outside of cells during slow-rate freezing is the limiting pipette to remove the PBS. The hESC fragments are then
factor for successful cryopreservation. transferred to well 2 containing VS1 and are incubated for
To avoid ice nucleation inside the cells, vitrification is 1 min. For the final loading of the straw, a 20-μL drop of
the method of choice and has already been successfully VS2 is placed into the middle of a 35-mm Petri dish.
applied to hPSCs.31 But vitrification in straws still means When the minute-long incubation in VS1 is over, the
detachment of cells, is only applicable for low numbers of hESC fragments are transferred to well 3 containing
cell clumps, and is very labor intensive and time sensitive. VS2 for 5 seconds. Then, the clumps are transferred to
Therefore, combination of surface-based freezing with the 20-μL drop of VS2 placed in the middle of a 35-mm
the vitrification technology would enable efficient cryo- Petri dish using a sterile glass Pasteur pipette, with a
preservation of high cell numbers with low cell stress, minimal volume of VS2. From there, the hESC fragments
even in ready-to-use multiwell formats suitable for direct are immediately loaded into the prepared embryo straw,
integration in high-throughput screenings. followed by a 5-mm air gap and about 20 mm of WS1
(Figure 24.3). Both ends of the straw are directly sealed
Surface-Based Vitrification using a heat sealer, and the straw is plunged into liquid
Development of an efficient surface-based vitrification nitrogen as quickly as possible.
approach requires a closer look at the established and For thawing, again a sterile four-well cell culture plate
well-functioning vitrification procedures of hPSCs in containing the following solutions is prepared at room
straws. Reubinoff et al. established the open pulled straw temperature: (i) well 1 contains WS1; (ii) well 2 con-
vitrification method for hESCs in 2001,31 which had tains warming solution 2 (WS2) consisting of 90% ES–
already been highly effective for the cryopreservation of HEPES–HSA (vol/vol) and 10% ES–HEPES–HSA with

205
 vitrification in assisted reproduction

Standard cell culture protocol

Cultivation Cell culture medium
Incubator at 37°C

Vitrification protocol
Preparation Manual picking of fragments
Preparation of straw
Cleanbench
Vitrification protocol
Incubation Incubation of fragments in VS1/VS2;
manual transfer of fragments
Cleanbench
Straw
Vitrification protocol
Loading Loading of fragments into straw
Sealing of straw
Cleanbench

Vitrification protocol
Vitrification Submerge into LN2
Outside cleanbench

Vitrificiation protocol
Storage Transport submerged into LN2
Storage in LN2 or gas
phase of nitrogen tank

Thawing protocol
Thawing Transport submerged into LN2
Thawing in water at RT
Straw
Thawing protocol
Opening of straw on one end
Washing Washing steps in WS1 and WS2
Manual transfer of fragments
Cleanbench
Standard cell culture protocol
Plating and Replating in cell culture medium
cultivation Incubator at 37°C

Figure 24.2  (See color insert.) Workflow of the open pulled straw vitrification method for human embryonic stem
cells by Richards et al. (LN2 = liquid nitrogen; VS = vitrification solution; WS = warming solution.) (From Richards
M et al. Stem Cells 2004;22:779–89. With permission.)

hESC Cotton
WS1 VS2 Connector
clumps plug
Air Air

Embryo Pipette tip 3-mL

straw syringe

Figure 24.3  Schematic drawing of the filled embryo straw connected to the syringe, according to Richards et al.
(hESC = human embryonic stem cell; WS = warming solution.) (From Richards M et al. Stem Cells 2004;22:779–89.
With permission.)

206
 vitrification in pluripotent stem cell banking
1 M sucrose (vol/vol); and (iii) wells 3 and 4 contain the conditions (coating with 0.1% gelatin and co-cultivation
holding medium. with mitotically inactivated primary mouse embryo
The straw is removed from the nitrogen storage and fibroblast [PMEF] cells) onto the coverslips.
is plunged immediately into a vessel containing water at After cultivation for 5–7 days, hESCs are vitrified by
room temperature for 5 second. Then, the straw is dis- taking the Thermanox© coverslips at the handlebar and
infected by wiping it with 70% ethanol or isopropanol, incubating them for 1 minute in cell culture medium
and opened using sterile scissors cutting both ends of the containing 10% DMSO and 10% EG. Then, coverslips are
straw. The entire content of the straw is placed into well incubated for 5 seconds in culture medium containing
1 containing WS1, and is incubated for 1 minute. Then, 20% DMSO, 20% EG, and 300 mM sucrose. Finally, the
the hESC clumps are transferred into well 2 containing coverslips are directly plunged into liquid nitrogen and
WS2 and are incubated for 5 minutes. In the last step, the stored in the vapor phase of a nitrogen storage tank.
clumps are transferred into well 3 containing the holding For thawing, the Thermanox© coverslips are removed
medium for 5 minutes, then finally transferred into well from the nitrogen storage tank and are directly incubated
4 for washing, also containing the holding medium. Now for 1 minute in culture medium containing 200 mM
the clumps are ready for re-plating or usage. sucrose. Afterwards, cells are incubated for 5 minutes
The vitrification protocol of Richards et  al. possesses in culture medium containing 100 mM sucrose, and are
high complexity, is very labor intensive, and can be some- then transferred to normal cell culture medium for fur-
what error prone. Even small deviations from the protocol ther cultivation or application.
regarding incubation times can result in significant cell For comparison with slow-rate freezing of adherent
loss due to the severe cellular stress caused by the high hESC colonies, hESCs are seeded using the standard
osmolarity of the vitrification solutions. Even the origi- cultivation conditions (coating with 0.1% gelatin and
nal manuscript of Reubinoff et al.31 reported only survival co-cultivation with mitotically inactivated PMEFs) into
of about 30% of the undifferentiated hESC fragments, a 35-mm cell culture dish with identification marks on
whereas Richards et al.36 reached over 75%. Additionally, the underside. After cultivation for 4–6 days, the culture
only a maximum of ten hESC fragments can be vitrified at medium is removed and 1 mL of the standard cryopreser-
one time, requiring mechanical dissection of the colonies. vation medium recommended by WiCell containing 60%
Therefore, transfer of the protocol from Richards culture medium, 30% FCS (fetal calf serum), and 10%
et al.36 to adherent cell systems would result in an easy- DMSO is added and incubated for 30 minutes at 4°C.
to-handle vitrification procedure for higher cell numbers. Then, cells are frozen at −1°C/min from 4°C to −80°C
Incubation times can be observed more strictly, as cells using a computer-controlled freezer, and are stored in the
are immobilized on the cultivation surface, so transfer vapor phase of a nitrogen storage tank.
and handling is simplified. Additionally, heat transfer For thawing, the cell culture dish is removed from
of adherent, flat cells and cell clumps is higher than of the nitrogen storage tank and incubated in a water bath
detached cells and colonies with a round morphology. without submerging the lid. Afterwards, 2 mL cell culture
Hence, the vitrification solutions and incubation times medium is added drop-wise for dilution of the cryopreser-
proposed by Richards et al.36 have been applied to adherent vation medium. Finally, the medium is changed with cell
hESCs cultivated on special cell culture-treated coverslips, culture medium for further cultivation or application.
so-called Thermanox©, with a diameter of 13 mm37 (Figure Viability of hESC colonies after adherent vitrification
24.4). In addition, a standard slow-rate protocol has been and slow-rate freezing was determined using live/dead
used with adherent hESCs to investigate whether avoid- staining directly after thawing, after 24 hours, and after
ance of cell stress during cell detachment and dissociation 48 hours. Microscopic images showed that, with slow-
may significantly improve cryopreservation efficiency. rate protocols, most hESC colonies were still adherent
For this, Thermanox© coverslips are prepared by mak- directly after thawing, but with large dead areas, mainly
ing two parallel, about 3-mm long cuts into the plastic in the center of the colonies (Figure 24.6a). Twenty-four
coverslip using sterile scissors. Afterwards, the resulting hours after thawing, the colonies possessed a disinte-
part is bent up, building a handlebar for better handling grated morphology, as most of the dead cells detached
of the coverslip (Figure 24.5a and b). from the surface (Figure 24.6c). Proliferation of the sur-
Additionally, some identification marks (e.g., lines) viving cells for 48 hours induced recovery of colony mor-
are made onto the untreated side of the coverslip using a phology (Figure 24.6e).
sterile scalpel to enable recognition and distinction of the In comparison, hardly any cell or colony loss was
hESC colonies. Afterwards, the coverslips must be ster- detected using adherent vitrification directly after thaw-
ilized by incubation in 70% ethanol for 20 minutes, and ing (Figure 24.6b), after 24 hours (Figure 24.6d), or after
subsequent ultraviolet radiation for 15 minutes. 48 hours (Figure 24.6f). Most cells were viable and colony
Then, up to five prepared Thermanox© coverslips morphology was compact at every time point.
are placed in a 60-mm cell culture dish (Figure 24.5c), Based on the microscopic fluorescence images, the
and hESCs are seeded using the standard cultivation viable area of the same colony was determined before and

207
 vitrification in assisted reproduction

Modification step
Modification Attach handlebar to plastic coverslips
UV sterilize coverslips
Modification
Standard cell culture protocol
Cultivation Cell culture medium
Incubator at 37°C

Vitrification protocol
Incubation CPA incubation in VS1/VS2
Transfer with sterile tweezers
Cleanbench
Vitrification protocol
Vitrification Submerging of coverslips into LN2
using sterile tweezers
Cleanbench/laminar flow
Vitrification protocol
Transportation Collection and transfer of samples in
submerged dish inside LN2 dewar
Outside cleanbench

Vitrification protocol
Storage Storage in LN2 or gas
phase of nitrogen tank
Thawing protocol
Transport submerged in LN2
Thawing Rapid thawing in WS1 (37°C) using
sterile tweezers
Cleanbench/laminar flow
Thawing protocol
Washing Washing steps in WS1 and WS2
Cleanbench

Standard cell culture protocol

Cultivation Cell culture medium
Incubator at 37°C

Figure 24.4  (See color insert.) Workflow of the surface-based vitrification method for human embryonic stem cells
by Beier et  al. (CPA = cryoprotective agent; LN2 = liquid nitrogen; UV = ultraviolet; VS = vitrification solution;
WS = warming solution.) (From Beier AF et al. Cryobiology 2011;63:175–85. With permission.)

after cryopreservation. For this, pictures of identical colo- cell proliferation was determined, with colony areas of
nies have been taken before and after cryopreservation on 167% ± 38% and 254% ± 81%, respectively.
the basis of the identification marks on the underside of the In addition to colony recovery and survival, main-
35-mm cell culture dish or Thermanox© coverslips. Based tained expression of stemness markers is absolutely cru-
on these images, direct comparison of the colony survival cial for efficient cryopreservation of hESCs. Therefore, the
is possible.37 After slow-rate freezing of adherent hESC expression of the transcription factor Oct-4 and cell sur-
colonies, 51% ± 22% of the colony area remained adherent face marker Tra-1-81 was measured using flow cytomet-
and viable directly after thawing (Figure 24.7). Twenty- ric analysis (Figure 24.8). Results showed that stemness
four hours later, the viable colony area slightly decreased to markers were maintained after surface-based vitrifica-
44% ± 25%. At only 48 hours after thawing, slowly frozen tion (Figure 24.8a and c) and were higher than in the non-
hESC colonies recovered, started to proliferate again, and frozen control colonies (Figure 24.8b and d).
reached an adherent and viable colony area of 73% ± 36%. The data revealed that vitrification can be applied to
In contrast, hardly any cell loss was detected using sur- adherent cells and results in highly efficient recovery
face-based vitrification, as 89% ± 10% of the hESC colo- rates of large cell numbers with maintained stemness.
nies remained viable and adherent directly after thawing Additionally, comparison with slow-rate freezing of
(Figure 24.7). At 24 and 48 hours later, considerable adherent hESC colonies showed that high cooling rates

208
 vitrification in pluripotent stem cell banking

(a) (b)

(c) (d)

Figure 24.5  Preparation and handling of the Thermanox© coverslips for vitrification. Addition of a handlebar for
better treatment (a and b), arrangement of coverslips in a 60-mL cell culture dish for cell seeding (c) and handling
of coverslips during cell incubation in different cryo-media (d). (From Beier AF et al. Cryobiology 2011;63:175–85.
With permission.)

(a) (b) 400%

Slow-rate freezing
Vitrification on thermanox *
300% Nonfrozen control
Vital residual area

*
200%

(c) (d) *
100%

0%
0h 24 h 48 h
Time after thawing
* Significantly different from slow-rate samples, p < 0.0001
(e) (f )
Figure 24.7  Viable and adherent colony areas at different
time points after adherent slow-rate freezing and vitrifi-
cation. Colony areas of the same colony were c­ ompared
before and after adherent slow-rate freezing and vitri-
fication. Additionally, the colony areas of nonfrozen
human embryonic stem cell colonies were determined.
Figure 24.6  (See color insert.) Microscopic fluorescence (From Beier AF et al. Cryobiology 2011;63:175–85. With
images of human embryonic stem cells at different time permission.)
points after slow-rate freezing (a, c, and e) and adherent
vitrification (b, d, and f). Cells have been stained with complete colony loss.38 Decrease of viable and adherent
live/dead staining (fluorescein diacetate/ethidium bro- hESC colonies 24 hours after thawing is a sign of apopto-
mide [FDA/EB]) directly after thawing (a and b), after sis, probably induced by alterations in cytoplasm caused
24 hours (c and d) and after 48 hours (e and f). Scale bars by intracellular ice formation during slow-rate freezing.39
indicate 200 µm (a–f) and 2 mm (inserts). (From Beier Hence, intracellular ice crystal formation seems to be the
AF et al. Cryobiology 2011;63:175–85. With permission.) critical factor during the cryopreservation of hESCs, and
surface-based vitrification is the optimal method for the
are necessary to enable cell survival, as slow-rate freezing efficient cryopreservation of ready-to-use cells.
led to significant loss of cell numbers and viability. Due to The method of surface-based vitrification has already
their three-dimensional colony structure and high num- been successfully applied to several other sensitive cell
bers of tight and gap junctions, optimal cell dehydration types that are difficult to cryopreserve using standard
is impeded and intracellular ice crystals have the possibil- slow-rate protocols; for example, human iPSCs and iPSC-
ity to propagate throughout the whole colony, resulting in derived hepatocyte-like cells.

209
 vitrification in assisted reproduction

Vitrification on thermanox Control colonies

(a) (b)

175

375
(95.7%) M1
(93.8%) M1

Events

Events
Oct-4

0 0
100 101 102 103 104 100 101 102 103 104
FL2-H FL2-H
(c) (d)
166

175
(95.6%) M1
Events

Events
(94.3%) M1
Tra-1-81

0 0
100 101 102 103 104 100 101 102 103 104
FL2-H FL2-H

Figure 24.8  Flow cytometric analysis of stemness markers after surface-based vitrification. Expression levels of
transcription factor Oct-4 (a and b) and cell surface marker Tra-1-81 (c and d) of human embryonic stem cells pas-
saged once after surface-based vitrification (a and c) and of nonfrozen control colonies (b and d) were determined.
(From Beier AF et al. Cryobiology 2011;63:175–85. With permission.)

But surface-based vitrification of cells on Thermanox© disposable, the so called TWIST substrate, which strictly
coverslips also possesses several limitations. The polymer separates cells and liquid nitrogen using a two chamber
of the coverslips is not fully resistant to the high thermal system was developed (Figure 24.9). This TWIST substrate
requirements of cell cultivation and vitrification (tem- is based on a cell culture-treated imaging dish with a thin
perature range from +37°C to −196°C). Hence, thermal foil as the cultivation surface.40 The foil possesses good
cracks occur in about 5%–10% of the coverslips, resulting optical quality and high thermal conductivity.
in disruption and damage of the affected hESC colonies.37 The TWIST substrate consists of a sterile, closable
Additionally, the cells on the coverslips have direct contact ­cultivation chamber and an open liquid nitrogen cham-
with liquid nitrogen in order to reach the necessary high ber. The concept of the TWIST substrate is based on using
cooling rates for vitrification. Usually, however, liquid it in two different positions: for cultivation, the substrate
nitrogen is not sterile and cross-contamination between is used in an “upright position” (Figure 24.9a), with the
samples is possible, as various bacteria and viruses are able cultivation chamber on top. In this position, cells can
to survive at these temperatures.29 Although the produc- be seeded, expanded, monitored, and prepared for cryo-
tion of sterile liquid nitrogen is possible, costs would sig- preservation sealed with a lid guaranteeing a closed and
nificantly increase for sterile banking of cells under GxP sterile environment. Additionally, the “upright position”
regulations using the surface-based vitrification tech- is used for washing steps, cell recovery, and cultivation
nique on Thermanox© coverslips. Therefore, adaptation of after thawing. Then, there is an “upside-down position”
this highly efficient method has been done to make this a with the liquid nitrogen chamber on top (Figure 24.9b). In
GMP-compliant vitrification device.40 this position, the vitrification itself is executed by filling
liquid nitrogen into this chamber, onto the cultivation foil.
GMP-Compliant Surface-Based Vitrification On the other side of the foil, the cells are now covered with
To guarantee GMP compliance of the surface-based vitri- only a very thin liquid layer. In this position, the cells are
fication method, direct contact of cells with liquid nitrogen also stored in liquid nitrogen storage tanks. For thawing,
has to be avoided without significantly decelerating the 37°C warm water is filled in the liquid nitrogen chamber,
cooling rate of the procedure. Therefore, a self-assembled enabling a very fast thawing of the adherent cells.

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 vitrification in pluripotent stem cell banking

(a) Lid “cultivation (b)

compartment”
Nitrogen or pre-
Media (culture/ heated water
CPA/washing)

Adherent LN2
cell clusters

Cultivation CPA film Adherent

LN2 surface cell clusters

Figure 24.9  (See color insert.) Outline and application of the TWIST substrate. The TWIST substrate is a two-
chamber system with a closable, sterile cultivation chamber and a LN2 chamber. In the cultivation chamber, cells
are seeded, expanded, monitored, and prepared for cryopreservation by incubation in CPAs (a); for surface-based
vitrification, the cryo-medium is removed, the substrate is turned over (“twisted”), and LN2 is filled into the LN2
chamber (b). (CPA = cryoprotective agent; LN2 = liquid nitrogen.) (From Liu BL, McGrath J. Acta Biochim Biophys
Sin (Shanghai) 2005;37:814–8. With permission.)

Figure 24.10 illustrates every step of the GMP-compliant TWIST substrate was as high as for the nonfrozen control
surface-based vitrification workflow and shows the corre- colonies (Figure 24.12).
sponding position of the TWIST substrate. Cells remain These data revealed that efficient surface-based vit-
adherent for the complete procedure, and no direct con- rification is adaptable to GxP regulations by ­avoiding
tact with potentially contaminated liquid nitrogen is direct contact of cells with liquid nitrogen and by using a
necessary. closed cultivation chamber to guarantee sterility. Based
For evaluation of the GMP-compliant surface-based on the TWIST substrate, vitrification of ready-to-use
vitrification procedure, hESC colonies were cultivated cells in different formats is possible, including patient-
and vitrified in the TWIST substrate using the workflow derived primary cell material or disease-relevant,
and the solutions described above. Preparation of media induced pluripotent stem (iPS) cell-derived model
used and analysis of the viable and adherent hESC colony systems in 96- or 384-multiwell formats for high-
area was performed as described in Beier et al.37 throughput screenings in the field of drug discovery or
Pictures of the hESC colonies have been taken before target finding. Furthermore, large-scale cryopreserva-
and after vitrification, with cells stained using a live/dead tion and storage of highly sensitive cells for therapeutic
dye  at different time points after thawing (Figure  24.11). approaches, in which large cell numbers are necessary,
Hardly any cell loss was detected directly after ­thawing is possible by development of an adapted TWIST sub-
(Figure 24.11e) or 24 hours later (Figure 24.11f). Addi­ strate with a large cultivation surface (e.g., T175 cm 2
tionally, vitrified colonies showed a compact and tight mor- vitrification flask).
phology without any signs of differentiation after passing Nevertheless, the GMP-compliant surface-based vitri-
and further cultivation (Figure 24.11i and j). Nevertheless, fication also has some drawbacks: although the number
slight localized cell loss was determined in some areas at of thermal cracks was reduced using a cell culture-treated
the border of the cultivation surface (Figure 24.11f, asterisk). foil for cell cultivation and vitrification to <5%, further
Quantitative analysis of the microscopic images optimization of the polymer used is necessary for com-
showed that 99% ± 1% viable and adherent hESC colonies plete avoidance of cracks. Additionally, some cell loss was
were detected directly after thawing (data not shown). detected at the border of the cultivation area, as here the
After 24 hours, the colony area increased to 155% ± 24% cryo-medium could not be removed completely, resulting
in comparison to 161% ± 32% of the nonfrozen control. in a thicker liquid layer at the edge. Therefore, the thermal
In addition, maintenance of stemness markers after capacity is increased in these areas, and the applied cool-
GMP-compliant surface-based vitrification was deter- ing rate is lower than in the middle part of the TWIST
mined using flow cytometric analysis. The results showed substrate, preventing successful vitrification of the hESC
that expression of the transcription factor Oct-4 and the colonies. In conclusion, formation of a liquid meniscus
cell surface marker Tra-1-81 after vitrification using the has to be avoided in order to guarantee a homogenous

211
 vitrification in assisted reproduction

Standard cell culture protocol

Cultivation Cell culture medium
Incubator at 37°C

Vitrification protocol
Incubation CPA incubation in VS1/VS2
Cleanbench
TWIST
Vitrification protocol
Vitrification Liquid nitrogen addition
Outside cleanbench

Vitrification protocol
Transportation Liquid nitrogen present in nitrogen
compartment

Vitrification protocol
Storage Liquid nitrogen evaporates from nitrogen
compartment while in storage
Gas-phase of nitrogen storage tank

Thawing protocol
Transportation Refill liquid nitrogen in nitrogen
compartment before transportation

Thawing protocol
Thawing Remove liquid nitrogen and add water
(37°C) to nitrogen compartment
TWIST
Thawing protocol
Washing Washing steps in WS1/WS2
Cleanbench

Standard cell culture protocol

Cultivation Cell culture medium
Incubator at 37°C

Figure 24.10  (See color insert.) Workflow of the GMP-compliant surface-based vitrification procedure. Every step
of the workflow and the corresponding positions of the TWIST substrate are summarized here. (CPA = cryoprotec-
tive agent; VS = vitrification solution; WS = warming solution.) (From Beier AF et al. Cryobiology 2013;66:8–16.
With permission.)

low thermal capacity and ultra-fast cooling rates using In addition to DMSO and EG, there are a variety of
the surface-based vitrification method. potential CPAs reported to be feasible for vitrification.44–47
Another drawback of vitrification protocols in general In particular, lower-strength glass formers are reported to
is the use of very high concentrations of toxic CPAs.41,42 show reduced toxicity when compared to strong glass form-
These high concentrations, usually in the range of 40%, are ers like DMSO.43 Strong glass formers, on the other hand,
required to achieve successful vitrification of the samples need lower cooling rates to vitrify. Figure 24.13 shows a
without ice crystallization.43 Due to the toxicity and high comparison of several potential CPAs according to their
osmolarity of the applied media, incubation steps within effectiveness in surface-based vitrification of hESC colo-
the vitrification protocols are generally very short in order nies. The efficiency has been determined by viability assess-
to keep the detrimental effects on the cells at a minimum. ment directly after thawing and after a 24-hour recovery
However, due to the nature of some vitrification workflows, period. In addition to DMSO and EG, the substances for-
the handling of the samples can be awkward and cumber- mamide, 1,2-propanediol (PD), and 2,3-butanediol were
some, leading to a high dependency of protocol effective- tested. Also included in the study were various mixtures
ness on the manual skills of the operator. Therefore, the of the tested components, since it is common to mix good
development of less toxic CPA media compositions and the glass-forming CPAs with poorer glass formers in order to
precise definition of CPA incubation times could lead to an eliminate ice formation, while at the same time incurring
improved applicability of vitrification techniques. a reduced toxicity penalty (Figure 24.13, variations 1–4).45

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 vitrification in pluripotent stem cell banking

Before vitrification After thawing Passage after 5 days

(a) (e) (i)

Twisted vitrification
(b)* (f )* (j)

(c) (g) (k)

Nonfrozen control

(d) (h) (l)

Figure 24.11  (See color insert.) Microscopic images of human embryonic stem cell (hESC) colonies before and after
vitrification using the GMP-compliant surface-based vitrification method. Pictures of the same hESC colonies have
been taken before (a–d) and after vitrification (e and f), in comparison to a nonfrozen control (g and h). For deter-
mination of the viable and adherent colony area, hESC colonies have been stained with a live/dead dye (FDA/EB)
directly after thawing (b) or 24 hours after thawing (f). The nonfrozen hESC colonies have been stained on the day
of vitrification (g) or 24 hours later (h). Vitrified (i and j) and nonfrozen hESC colonies (k and l) have been passaged
and cultivated further to determine signs of differentiation. Red arrows indicate thicker, probably differentiated
areas of the hESC colonies that are lost after vitrification (e) in comparison to the nonfrozen control (g). Scale bars
indicate 1 mm (a–h) and 500 µm (i–l). (From Beier AF et al. Cryobiology 2013;66:8–16. With permission.)

DMSO, EG, and PD all showed high post-thawing a minimum. The same goes for the incubation times in
survival rates, while formamide and 2,3-butanediol led the respective media. These should be chosen to be long
to an almost complete cell loss immediately after thaw- enough to enable sufficient penetration of the hESC and
ing. While most of the tested substances and mixtures iPSC colonies to allow for an efficient avoidance of ice
resulted in very high cell survival and good recovery crystallization in the whole sample, while keeping nega-
rates, the least spontaneous post-thawing differentiation tive toxicity effects as low as possible.
rate was achieved with 20% PD and 20% EG, suggesting a To emphasize this, the effects of different concentra-
reduced toxicity of this medium composition when com- tions and incubation times of the commonly used CPA
pared to other mixtures (Figure 24.13, variation 3; data on medium, containing equal volumes of DMSO and EG,
differentiation rate not shown). The concentration of the on adherent hESC colonies have been evaluated (Figure
PD/EG medium used could be further reduced to 20% PD 24.14).
and 15% EG without a reduction in post-thaw viability The concentration range was chosen from 20% up to
(Figure 24.13, variation 4). This indicates that the poten- 70% total CPA concentration, and evaluated according
tially less toxic PD/EG combination might be a valuable to the vital residual areas of the colonies before and after
alternative for the commonly used DMSO/EG medium vitrification. The results showed an optimal concentra-
in the surface-based vitrification of hESC and iPSC colo- tion of CPAs at around 40% (Figure 24.14a and b). Lower
nies. However, extensive long-term analysis on the toxic- concentrations led to an insufficient ability of the cryo-
ity of this medium composition is needed to ensure its protective media to avoid ice crystallization, leading to
effectiveness in hESC and human iPSC banking. the loss of complete colonies (data not shown). Higher
Another working point for the improvement of vitrifi- concentrations (50%–70%) led to cell death, which was
cation techniques for human ESCs and iPSCs is the clear probably caused by the high toxicity of the applied media.
definition of CPA concentrations and incubation times in Incubation time dependency was evaluated by variation
the applied workflows. The optimal CPA concentration of the surface-based vitrification protocol described above
should allow sufficient avoidance of ice crystallization in regard to incubation times (Figure 24.14c–e and  g).
while keeping toxic detrimental effects of the media at The  standard protocol implies a 60-second incubation

213
 vitrification in assisted reproduction

Twisted vitrification Control colonies

(a) (b)

150

100
(89.5%) M1
(94.4%) M1

Events

Events
Oct-4

0 0 0
100 101 102 103 104 10 101 102 103 104
FL2-H FL2-H
(c) (d)
150

128
(98.1%) M1
(86.7%) M1
Events

Events
Tra-1-81

0 0 0 0
10 101 102 103 104 10 101 102 103 104
FL2-H FL2-H

Figure 24.12 Flow cytometric analysis of stemness markers after GMP-compliant surface-based vitrification.
Expression levels of the transcription factor Oct-4 (a and b) and cell surface marker Tra-1-81 (c and d) of human
embryonic stem cells passaged once after surface-based vitrification in the TWIST substrate (a and c), and of nonfro-
zen control colonies (b and d) were determined. (From Beier AF et al. Cryobiology 2013;66:8–16. With permission.)

Screening for CPA alternatives Control Propanediol

200%

150% 300 μm 300 μm

Formamide Butanediol
Vital residual area

100%

50% 300 μm 300 μm

Var. 1 Var. 2
0%
Control
DMSO
Ethylene glycol

Propanediol
Butanediol
Var. 1
Var. 2
Var. 3
Var. 4
Control
DMSO
Ethylene glycol

Propanediol
Butanediol
Var. 1
Var. 2
Var. 3
Var. 4
Formamide

Formamide

300 μm 300 μm

Var. 3 Var. 4

0h 24 h
CPA/Time after thawing
300 μm 300 μm

Figure 24.13  (See color insert.) Comparison of potential vitrification CPAs according to their effectiveness in sur-
face-based vitrification. Concentrations of CPAs have been 40% except for in Var. 4, which shows a less-concen-
trated version of Var. 3. Images have been stained with EB and FDA and show the corresponding colonies directly
after thawing. Var. 1–4 show different mixtures of the CPAs used. Var. 1: 10% propanediol, 10% formamide, 10%
ethylene glycol, and 10% butanediol. Var. 2: 13% propanediol, 13% ethylene glycol, and 13% butanediol. Var. 3:
20% propanediol and 20% ethylene glycol. Var. 4: 20% propanediol and 15% ethylene glycol. (CPA = cryoprotective
agent; DMSO = dimethyl sulfoxide; Var. = variation.)

214
 vitrification in pluripotent stem cell banking
(a) Dependency on CPA concentration (c) Dependency on incubation time
200% 140%
120%
Vital residual area

Vital residual area

150% 100%
100% 80%
60%
50% 40%
0% 20%
Control
20%
30%
40%
50%
60%
70%
Control
20%
30%
40%
50%
60%
70%
0%
1 step 2 step 2 step 2 step 2 step
5s 2s 5s 30 s 60 s
0h 24 h
Workflow
CPA concentration and time after thawing

(d) (e)
(b)
>7 mol
Concentration high;
Osmolarity- and toxicity-
based damages expected
5.57 mol 500 μm 500 μm
Optimal concentration;
Successful vitrification (f ) (g)
most likely

<5.5 mol Concentration low;

Cell damages through ice
crystallization expected 500 μm 500 μm

Figure 24.14  (See color insert.) Evaluation of optimal CPA concentration range (a and b) and incubation time (c) in
surface-based vitrification. CPA concentration range experiments were done with media containing equal amounts
of dimethyl sulfoxide and ethylene glycol in comparison to a nonfrozen control sample (d). The effects of too short
an incubation time (e), too long an incubation time (g), and damage by inadequate surface material are shown.
Images have been stained with FDA/EB. (CPA = cryoprotective agent.)

step in media containing 10% DMSO and 10% EG, fol- Evaluation, Authorisation and Restriction of Chemicals,
lowed by a 5-second incubation step in media comprising it is expected that 68,000 chemicals have to be tested in
20% DMSO and 20% EG. This second high-concentration about 54 million animal trials.48 Today, there are great
incubation step was varied in incubation time. Analysis of expectations that hPSCs can replace and reduce the nec-
post-thaw viability showed an optimal incubation time of essary amount of animal testing. For the performance
between 5 and 30 seconds. Shorter incubation times led to of large high-throughput screenings of several thousand
incomplete CPA penetration of the thicker inner parts of compounds and chemicals, cells have to be available in
the colonies, resulting in major cell death in these areas miniaturized small-volume compartments, like 96- or
(Figure 24.14e). Longer incubation times (>30 seconds) 384-multiwell plates, to reduce costs of compounds and
led to high survival rates, but resulted in a more “frisky” media, as well as the necessary cell number. Additionally,
colony morphology after thawing (Figure 24.14g), prob- standard cultivation of cells in two dimensions as mono-
ably due to toxicity based damage to cell–cell contacts and layers affects cell characteristics49 and is not comparable
cell membranes. Also, spontaneous differentiation rate to the three-dimensional environment within the human
appears to be higher (data not shown). body. Therefore, results from candidate screens for drug
discovery and development that are performed on cells
Integration in Automated Workflows cultivated as monolayers are often not transferrable,
Due to their capability to differentiate into cells of all and fail in human clinical trials. To maintain cell–cell
three germ layers, PSCs are also an important model sys- interactions and the regulatory networks necessary for
tem for toxicology screens. In particular, human iPSCs expressive cell reactions after exposure to chemicals or
that can be derived from patients with specific genetic drug candidates, cultivation of cells as 3D micro-tissues
disorders offer new possibilities for the development of is necessary. Recent advances in micro-tissue production
effective drug treatments. Until now, approval of novel have highlighted the potential of scaffold-free cell aggre-
drugs has mainly been based on successful animal test- gates in maintaining tissue-specific functionality.50 One
ing. In the context of the Regulation on Registration, common procedure for the formation of micro-tissue is

215
 vitrification in assisted reproduction
the hanging drop method. Here, small microliter drops rate for the vitrified than for the slow-frozen samples,
of cell suspension are placed on the inner side of the lid with values between 16% ± 3% and 24% ± 6%, depend-
of a bacterial cell culture dish, over several microliters ing on the different basal media and additives used.
of water or PBS acting as the evaporation buffer. Within Additionally, expression of a stemness marker was inves-
the drop, all cells are concentrated in the lower part of tigated at 7 days post-thaw (Figure 24.15e–g), showing
the drop due to gravity, forming one homogeneous cell low signs of differentiation in all samples.
aggregate. The hanging drop cultivation induces dif- Furthermore, the pellet freezing of cell aggregates in
ferentiation of PSCs (e.g., into cardiomyocytes or neu- small sample volumes can also be used for cells culti-
rospheres) by preventing cell–matrix contacts, and is vated on micro-carriers (Figure 24.16) (e.g., for prevent-
therefore also an important tool for the generation of ing differentiation of human iPSCs in the hanging drop).
disease-specific cell models. To avoid differentiation For this, human iPSCs adherent on micro-carrier form
and maintain the expression of stemness markers, pro- a three-dimensional cell–micro-carrier agglomerate
vision of adhesion surfaces in the drop by the addition (Figure 24.16d) in 20-µL drops that were dropped into
of micro-carriers is possible.10 For enabling permanent liquid nitrogen, using the same vitrification and thawing
availability of micro-tissues for high-throughput screen- solutions as mentioned above (Figure 24.16a).
ings, cryopreservation strategies have to be developed, The viability of vitrified human iPSCs adherent on
preferably able to be directly integrated into the hanging micro-carriers has been investigated using live/dead
drop workflow. One promising strategy for integrated staining (FDA/EB) directly after thawing and 24 hours
cryopreservation of micro-tissues is the adaptation of later, normalized to the nonfrozen control (Figure 24.16b),
pellet-freezing procedures for the hanging drop tech- and was analyzed as described elsewhere.14 Directly after
nology. Using this method, human iPSC aggregates in thawing, human iPSCs showed high viability values. But
20-µL drops are dropped into liquid nitrogen, using the 24 hours later, a significant decrease was detectable, prob-
same vitrification and warming solutions as mentioned ably due to apoptosis processes within the first 8 hours.
above (Figure 24.15a). Assessment of stemness was also performed on vitrified
After vitrification and thawing, human iPSC aggre- human iPSCs adherent on micro-carriers using anti-
gates have been re-plated and cultivated further (Figure HESCA-2 antibody with Alexa-Fluor-488 directly after
24.15d) in comparison to a nonfrozen control (Figure thawing (Figure 24.16e) and 24 hours later (Figure 24.16f)
24.15b) and human iPSC aggregates frozen in standard in comparison to a nonfrozen control (Figure 24.16c
cryovials using slow-rate protocols (Figure 24.15c). and  d). At all of the time points after thawing, human
Analysis of the adhesion rate after 24 hours normalized iPSCs showed expression of the stemness marker that was
to the nonfrozen control resulted in a higher adhesion comparable to the control.

Control Crystallization Vitrification

(a) (b) (c) (d)

hiPSC 500 μm
aggregates

(e) (f ) (g)

20 μL

LN2 50 μm

Figure 24.15  (See color insert.) Cryopreservation of hiPSC aggregates using slow-rate protocols and pellet vitri-
fication. For pellet vitrification, 20-µL drops of vitrification medium containing one hiPSC aggregate each were
dropped into LN2 (a). Adhesion of aggregates was monitored after slow-rate freezing (c) and pellet vitrification (d),
in comparison to a nonfrozen control (b). Stemness was analyzed with immunofluorescence staining using anti-
HESCA-2 antibody with Alexa-Fluor-488 after cultivation for 7 days (e–g). (hiPSC = human induced pluripotent
stem cell; LN2 = liquid nitrogen.)

216
 vitrification in pluripotent stem cell banking

Control
(a) (b) (c) (d)
100
90
80

Viability (%, standardized)

70
hiPSC on 200 μm
60
microcarrier
50
After thawing
40
(e) (f )
30
20
20 μL
10
0
LN2 0 h after 24 h after
Control
thawing thawing

Figure 24.16  (See color insert.) Cryopreservation of hiPSC on micro-carriers using pellet vitrification. For pellet
vitrification, 20-µL drops of vitrification medium containing hiPSCs adherent on micro-carriers were dropped
into LN2 (a). Viability of vitrified cells was analyzed using live/dead staining (FDA/EB) directly after thawing and
24 hours later (b). Stemness of a nonfrozen control (c and d) directly after thawing (e) and 24 hours later (f) was deter-
mined with immunofluorescence staining using anti-HESCA-2 antibody with Alexa-Fluor-488. (hiPSC = human
induced pluripotent stem cell; LN2 = liquid nitrogen.)

Hence, a first workflow could be established for cre- micro-tissue formation, and by dripping the drops con-
ating the preconditions to integrate the vitrification pro- taining the micro-tissues into liquid nitrogen placed
cedure into automation workflows for high-throughput beyond the perforated hanging-drop plates.
screenings or cell differentiation. Full integration can be Integration of the micro-tissue formation into robotic
realized by using commercially available hanging-drop platforms also offers the possibility of performing min-
plates on pipette or liquid-handling robots (Figure 24.17). iaturized compound screens on these systems. The drop
These hanging-drop plates possess a perforated surface, access from above enables liquid-handling units to per-
so that pipette robots have access from above to generate form medium changes during cultivation and add CPAs
droplets, perform medium exchange, or add compounds. for cryopreservation, and also add compounds for drug
With this setup, complete automation of the pellet vit- discovery and development. One possible application
rification workflow described above is possible by add- of this is the embryonic stem cell test for analyzing the
ing the different vitrification solutions after automated embryotoxicity of compounds on mouse embryonic stem

(a) (b)

Figure 24.17  Robotic platforms for the integration of stem cell workflows. The Freedom EVO 200 platform (TECAN
Group AG, Männedorf, Switzerland) enables very dynamic and flexible adaptation of different stem cell workflows
(a), whereas the CompacT SelecT platform (TAP Biosystems, Royston, UK/Sartorius Stedim Biotech, Aubagne,
France) is an established system used inter alia by pharmaceutical industries (b).

217
 vitrification in assisted reproduction
cells during differentiation into cardiomyocytes in the vitrified samples caused by devitrification. Additionally,
hanging drop. the humidity in the ambient air causes the formation of
Furthermore, cell culture platforms are already used ice and frost on the stored samples during the storage
for the automated production of biologicals (e.g., of HIV process, increasing the risk of contamination and sig-
pseudoviruses for the development of HIV vaccines).51 nificantly reducing the readability of labels and barcodes.
Finally, manual removal and insertion of the storage rack
STORAGE, MANIPULATION, AND CRYO- is accompanied by uncontrolled collisions and vibrations,
PHYSICAL VALIDATION OF VITRIFIED SAMPLES which result in sufficient EA to induce ice crystallization
Another major concern in the use of surface-based vitri- in metastable, vitrified samples.
fication techniques is the dependence of sample viability Hence, standard cryostorage technologies used in
on storage temperature. Maintenance of the correct tem- state-of-the-art biobanks are not applicable for the effi-
perature during banking, and especially during transpor- cient storage of vitrified samples, and can also lead to the
tation to and from the laboratory to the storage tank, has reduced sample quality of slow-frozen samples. In order
great impact on cell survivability, recovery, and preser- to address these drawbacks, the hermetic storage concept
vation of pluripotency. Temperatures of vitrified samples was developed in collaboration with ASKION GmbH
cannot exceed the TG (around −130°C), or devitrification (Gera, Germany), combined with internal and optional
can cause a complete loss of viability by rapid ice crystal- external automation of the storage process (Figure 24.20).
lization throughout the complete sample.43 These short- For this, a vacuum-isolated access tower is mounted on
comings hamper the more widespread use of vitrification conventional cryotanks that can be cooled down to about
techniques, since regular banking systems often show −130°C with dry nitrogen gas, preventing condensation of
fluctuations in temperature through opening and closing ice from ambient air (Figure 24.20a). The frozen samples
of the tanks, and therefore cannot guarantee a constant are transported in liquid nitrogen-cooled transport vessels
sample temperature below −130°C. This makes the devel- that are connected to the cooled access tower. The samples
opment of specialized banking systems for vitrified sam- are then transferred from the transport vessel to the final
ples a major criterion when aiming for widespread use of storage position by an integrated gripper. Hence, a closed
vitrification techniques. cold chain and an ice-free environment are guaranteed
during sample input and output. In addition, each rack is
Prevention of Devitrification during Long-Term Storage mechanically connected to a special lift system, enabling
The importance of correct storage temperature is shown smooth movement of the storage racks, significantly reduc-
in Figure 24.18. Vitrified hESC samples were stored at ing the collisions and vibration of the samples. Especially
−80°C in a commercially available freezer and at −170°C for vitrified samples, this feature prevents devitrification
in the gas phase of a liquid nitrogen storage tank. Samples and increases sample quality. Several cryotanks can be
were thawed after 1 day, 1 week, and 1 month, and their connected by a rail system for automated transport and
viability was evaluated after a 24-hour recovery period. connection of the cooled transport vessel containing the
The results show a clear dependence of cell survivability samples with the access tower (Figure 24.20b). The sample
on storage temperature. After only 24 hours of storage at management software in combination with integrated
−80°C, the viability of the vitrified samples went down barcode readers enable permanent sample tracking and
by more than 90%, as compared to samples stored below storage of sample history and information. Additionally,
the TG. This implies that reliable liquid nitrogen banking temperature sensors at different positions inside the stor-
systems are a basic and definitive prerequisite for the effi- age system enable the preparation of a temperature profile
cient application of vitrification workflows in the field of for each sample during the whole storage duration.
hESC and iPSC research and clinical application. Comparison of storage systems with the protective
Current biobanking technologies are based on liquid hood concept and conventional storage systems showed
nitrogen storage tanks for the storage of samples in the that, for slow-frozen peripheral blood mononuclear cells,
vapor phase of liquid nitrogen. Within these systems, a temperature fluctuations during sample storage using the
temperature of about −160°C is guaranteed, so storage of conventional liquid nitrogen tank resulted in reduced cell
vitrified samples is possible. But during sample input and viability, recovery, and T-cell functionality in contrast to
output, the closed cold chain cannot be maintained, as the storage system with an access tower.52
the sample racks of the storage tanks have to be pulled out In summary, new storage and biobanking systems are
to room temperature. Handling of high sample numbers available that enable efficient and vibration-free storage of
can take several minutes, leading to a significant warm- vitrified samples, preventing warming of samples above
ing of the complete sample rack up to above −80°C during the TG and hence devitrification.
sample input and output (Figure 24.19).
This can lead to an adverse effect on slow-frozen Safe Handling of Vitrified Samples
samples in standard cryovials due to recrystallization Beside the storage process, handling of frozen samples
processes, but may also result in complete cell loss in bears the risk of exceeding the TG, thus causing ice

218
 vitrification in pluripotent stem cell banking

1 Day 1 Week 1 Month

(a) (b) (c)
Storage at –80°C

200 μm 200 μm 200 μm

500 μm 100 μm
100 μm

(d) (e) (f )
Storage at –170°C

200 μm 200 μm 200 μm

500 μm 500 μm 500 μm

(g) Control
250% Dependency on storage temperature
(h)

200%

150%
Vital residual area

100%

(i)
50%

0%
Control 1 Day 1 Week 1 Month 1 Day 1 Week 1 Month
–170°C –80°C
Storage temperature and time

Figure 24.18  (See color insert.) Comparison of different storage temperatures for vitrified human embryonic stem
cell colonies. Cells have been vitrified via surface-based vitrification and stored at −80°C (a–c) and −170°C (d–f).
Samples were thawed after 1 day (a and d), 1 week (b and e), and 1 month (c and f). Evaluation was done after a
24-hour recovery period via staining with FDA/EB and comparison of the vital residual areas (g). A nonfrozen
control sample has been included (h and i).

crystallization in vitrified samples. Especially in large creating a temperature gradient from −170°C to about 8°C
biobanks, re-sorting of frozen samples is often necessary (Figure 24.21b). Frozen samples can be relabeled or re-
(e.g., during preparation of sample shipment or for opti- sorted inside the cryo-workbench without interrupting
mizing the storage capacity). the cold chain. Additionally, up to three independently
Therefore, a working space that can be cooled down operating controlled-rate freezers are integrated in the
to −130°C for the handling of frozen, especially vitri- cryo-workbench (Figure 24.21c). These freezers are based
fied samples, is necessary to guarantee sample quality. on an elevator principle, making use of the temperature
The cryo-workbench developed in collaboration with gradient inside the working space. The freezer consists
ASKION GmbH (Figure 24.21a) is cooled down by pump- of a platform for taking up the samples with two tem-
ing liquid nitrogen into the base of the handling room, perature sensors for measuring the ambient temperature

219
 vitrification in assisted reproduction
(a)
Pre- Transfer to Viability and Post-
Freezing Transfer to
freezing Storage thawing Thawing functionality freezing
procedure storage tank
preparation device analysis preparation

Transfer from other Transfer to other facilities

facilities

(b)
50
Temperature (°C)

Uncontrolled
0
environment
–50 0°C
Idealized temperature graph
–100 Controlled
–150 environment
–200 –130°C

Time (relative units)

Figure 24.19  Overview of the workflow and temperature profile during sample storage. General workflow of sample
processing, cryopreservation, and storage in biobanks today (a) and related temperature profiles for the cryopreser-
vation and storage of samples (b).

(a)
Access
tower

Sample
gripper

Conventional
cryotank

Storage
architecture

(b)
Transport
vessel
Rail
Transport system
station

Figure 24.20  Automated biobank with vacuum-isolated access tower mounted on a conventional liquid nitrogen cryo-
tank (ASKION GmbH, Gera, Germany). Plan and cross-section of the storage tank with access tower (a) and image
of the automated biobank with transport station and rail system (b). The access tower can be cooled down to about
−130°C with dry nitrogen gas, guaranteeing a closed cold chain and preventing condensation of ice from ambient air.

220
 vitrification in pluripotent stem cell banking
(a) (b) gradient between the storage space and the ambient air
can be passed through by fiber optics, which are compa-
rably insensitive to temperature influences, so the optical
8°C probe in the cold can be connected to the light source and
–100°C
the Raman detection instrument at ambient temperature.
Raman spectroscopy detects molecular vibrations like
–170°C
infrared spectroscopy, but is based on inelastic scatter-
ing53,54 and may thus be acquired by emission spectros-
copy setups akin to fluorescence. No exogenous markers
are needed since the molecular vibrations of water can be
(c) (d) used directly. Due to the different intermolecular forces
and arrangements between liquid/vitrified water and
crystalline ice, their molecular vibration spectra can be
clearly discriminated (Figures 24.1 and 24.22).
Utilizing a confocal probe geometry assessment of
a sample becomes possible. By this means, a suspicious
sample may be inspected for ice formation at storage tem-
perature. Because the spectral behaviors of liquid and
Figure 24.21 Cryo-workbench with integrated con- glassy water as well as ice are known, the sample status
trolled-rate freezer. Image of the cryo-workbench (a) can be directly determined without any need for compar-
and plan of the system with the applied temperature ison. It is even possible to observe ice formation from the
gradient (b) with integrated controlled-rate freezer (c). vitrified state in real time. Figure 24.22 shows a sequence
This handling system enables the manipulation of fro- of Raman spectra of an initially vitrified aqueous solu-
zen samples during re-sorting, relabeling, or for tak- tion of DMSO (20% by volume), EG (20%), and 300 mM
ing inventory with an uninterrupted cool chain below sucrose, which was rewarmed at 1 K/min, beginning at
−130°C (d). −135°C. At about −120°C, ice formation starts, giving rise
to the striking ice band at 3100 cm−1. After a few minutes,
and the temperature in a reference sample. The platform the entire sample volume is crystalized (an analogous
moves downwards inside the handling room, resulting study is shown in Doerr et al.28).
in a sensor-controlled temperature decrease according
to the temperature profile that is programmed. Finally,
the frozen samples can be transferred to the cooled
transport vessels and are transferred to the storage tank
(Figure 24.21d). Additionally, sample tracking and taking
inventory of samples is implemented by a barcode-based
sample management system.
In summary, today, innovative biobanking systems are
Raman scatter

available guaranteeing a closed cold chain and sample tem-

perature below −130°C, while also making the safe han-
dling and storage of vitrified samples in biobanks possible.

Noninvasive Validation of Vitrified Samples

The TG can be easily exceeded during retrieval of sam-
ples, transport, or due to technical failures. If a sample
or even an entire sample collection has experienced such 3000
ss n

an interrupted cool chain, risk of crystallization—and 3200

re tio
og za

hence destruction—of the biological content is high. Wa 3400

pr talli

ven
um
ys

Unfortunately, the event of ice crystallization is invis- ber 3600

Cr

/cm –1
ible to visual inspection. An interim reanimation of the
sample for inspection purposes is not possible since it is
costly, time-intensive, and significantly harms the sam- Figure 24.22  (See color insert.) Waterfall plot of a Raman
ple. To overcome this drawback, Raman spectroscopy spectral sequence showing ice formation from a vitrified
can be used to discriminate unscathed, vitrified samples medium. The medium composition was 300 mM sucrose
from crystalized ones without rewarming. Using a smart solution with 20% dimethyl sulfoxide and 20% ethylene
confocal probe design even allows for assessment of the glycol. The medium was vitrified at a temperature of
sample state in a closed container. The drastic thermal −135°C, then heated to −95°C at a rate of 1 K/min.

221
 vitrification in assisted reproduction
The benefit of the technique is, however, limited if fluo- especially the possibility of automating the cryopreserva-
rescent species are present, which cover the much weaker tion of cells in a ready-to-use format upon thawing in
Raman emission. While we found cellular auto-fluores- high-throughput applications, as this will overcome the
cence to be a minor problem, fluorescent media additives drawback of there being a clear lack of surface-based ster-
like phenol red may considerably hamper any Raman ile freezing that could be integrated in industry-standard
measurement. plate- or flask-based workflows ready for automation pro-
Raman spectroscopy reveals information about any cesses in high-throughput applications. Surface-based
(main) constituent of a sample, as long as it is not a noble freezing has the double advantage of: (i) cell dissociation/
gas or a metallic compound, so it has the potential to tell detachment not being necessary, therefore retaining nor-
us about the status of slowly frozen samples, as well.55–57 mal cell–cell and cell–matrix interactions to reduce cell
But here the analysis and interpretation of the data are stress; and (ii) cell re-attachment on thawing not being
much more complicated, since we can no longer make necessary, thereby promoting recovery, thus reducing cell
use of the predominant constituent—water. In future, death and manufacturing timelines.
this technique could offer new possibilities for the non-
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25 Scrying the future: The ongoing transformation of reproductive
medicine through vitrification
Kevin S. Richter, James R. Graham, and Michael J. Tucker

Reproductive medicine is in the midst of an exhilarating while fresh or following cryopreservation. Thus, any out-
revolution. Few innovations have had as transformative come differences are more accurately reflective of the rela-
an impact on the field of assisted reproduction as vitrifi- tive viability of the embryos transferred. National results
cation. This impact will only increase as development and for the United States, reported by the Society for Assisted
refinement of vitrification technology and methodology Reproductive Technology (SART), reveal that, in transfers
continues. The benefits associated with vitrification of of embryos derived from donor oocytes, live birth rates are
cells and tissues have profound consequences for nearly consistently much higher for fresh transfers compared to
every aspect of assisted reproductive practices. Damage- transfers of cryopreserved embryos. From 2003 to 2013, live
free storage of gametes and embryos, made possible for birth rates for cryopreserved embryo transfers were 28% to
the first time through vitrification, dramatically improves 41% lower compared to fresh transfers (Figure 25.2), despite
the efficacy and practicality of a variety of current proce- the fact that similar numbers of embryos were transferred
dures and unlocks an entirely new realm of possibilities per cycle. An essentially equal viability deficit has been
for fertilization and embryo transfer strategies, oocyte reported for embryo transfers to gestational carriers, in
donation, and elective fertility preservation. which the live birth rate for fresh transfers was 51% com-
pared to 34% for cryopreserved embryo transfers to gesta-
EMBRYO CRYOPRESERVATION tional carriers not having a fresh transfer first.3
The earliest reported pregnancies and successful live
births following transfers of previously cryopreserved VITRIFICATION ENHANCES CRYOPRESERVED
embryos were published in 1983 and 1984.1,2 Since then, EMBRYO VIABILITY
embryo cryopreservation has been increasingly incorpo- Until recently, embryo cryopreservation has been per-
rated as a standard adjunct to fresh in vitro fertilization formed almost exclusively via “slow-freezing” protocols.
(IVF) treatment cycles. From 2003 to 2013, the number Mounting evidence increasingly demonstrates that embryo
of cryopreserved embryo transfer procedures performed vitrification, as an alternative to traditional slow freezing,
in the United States nearly tripled, growing from 17,494 can substantially reduce if not virtually eliminate this his-
in 2003 to 40,643 in 2013 (Figure 25.1). During that time torical “cryopreservation deficit.” A systematic review and
span, the proportion of all embryo transfer procedures meta-analysis of post-cryopreservation survival rates has
that involved cryopreserved rather than fresh embryos reported that cryosurvival is significantly higher with vit-
also more than doubled, from 18.7% in 2003 to 38.4% in rification versus slow freezing of both cleavage-stage and
2013 (Figure 25.1). The rates of increase over time in both blastocyst-stage embryos.4 Nearly all (97.5%) of the vitri-
the number and percentage of cryopreserved embryo fied cleavage-stage embryos survived compared to 84.1%
transfers remained relatively constant from 2003 to 2009, of the slow-frozen cleavage-stage embryos, while 89.9%
but since then, both of these rates have been progressively of the vitrified blastocysts survived compared to 75.4% of
accelerating. the slow-frozen blastocysts. A randomized trial published
As a consequence of the widespread and increasing use soon afterwards added to the evidence that cleavage-
of embryo cryopreservation, hundreds of thousands of stage vitrification, compared to slow-freezing, resulted in
children have been born worldwide. However, despite this significantly higher cryosurvival (94.8% versus 88.7%).5
substantial success, it is well established that, historically, In addition, pyruvate uptake (an indicator of metabolic
the implantation potential of cryopreserved embryos has rate), embryos with 100% blastomere survival (77.9% ver-
been significantly lower than that of embryos transferred sus 51.4%), and subsequent blastocyst formation (60.3%
while fresh. The viability deficit of cryopreserved embryos versus 49.5%) were all found to be significantly higher
relative to fresh embryos is clearly evident in a comparison with vitrification. A more recent systematic review and
of outcomes of fresh versus cryopreserved embryo transfers meta-analysis evaluating pregnancy outcomes found that
to donor oocyte recipients. Unlike autologous transfers, both clinical pregnancy rates (38.7% versus 33.1%) and
recipients of donor oocytes are not subject to the potential ongoing pregnancy rates (34.7% versus 27.1%), as well as
disruption of endometrial receptivity caused by stimulation implantation rates (29.3% versus 24.2%), were significantly
medications, and protocols for endometrial preparation are higher with embryo vitrification versus slow freezing.6 In
identical regardless of whether embryos are transferred a population-based cohort study of all (more than 30,000)

225
 vitrification in assisted reproduction
60,000 45%

40%
50,000
35%

40,000 30%

25%
30,000
20%

20,000 15%

Total number of cryopreserved embryo transfers 10%

10,000
Percentage of all embryo transfer procedures 5%

0 0%
2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013

Figure 25.1  Total annual numbers of cryopreserved embryo transfer procedures (nondonor and donor oocytes
combined) and the percentage of cryopreserved embryo transfers among all embryo transfers (fresh and cryopre-
served) performed in the United States from 2003 to 2013. (Adapted from data reported by the Society for Assisted
Reproductive Technology [www.sart.org].)

60%
Our Own Experience of Transitioning from Slow
Freezing to the Vitrification of Blastocysts
Live birth per embryo transfer

50%
Shady Grove Fertility Reproductive Science Center,
40% encouraged by the increasing published evidence sup-
porting embryo vitrification, made the transition from
30% slow-freeze protocols8 used through December 2008 to
vitrification9 for all cryopreserved embryos beginning in
20% January 2009. Embryos were cryopreserved at the blasto-
Fresh embryo transfer
Cryo embryo transfer cyst stage on day 5, 6, or 7 after oocyte retrieval and fertil-
10%
ization, according to the day on which they developed to
0%
the expanded blastocyst stage, with a clearly visible inner
cell mass (ICM) and trophectoderm. Only good-quality
03

04

05

06

07

08

09

10

11

12

13

blastocysts were cryopreserved (minimum ICM/trophec-

20

20

20

20

20

20

20

20

20

20

20

toderm score of BB according to Gardner and Schoolcraft’s

Figure 25.2  Annual live birth rates per embryo trans- grading system10). The morphological criteria for supernu-
fer procedure for in vitro fertilization cycles in which merary blastocyst cryopreservation were the same regard-
donor oocytes were used, comparing outcomes of fresh less of the day on which they were cryopreserved, and
embryo transfers to transfers of embryos that had been remained the same before and after the switch to vitrifica-
cryopreserved, among all reported cycles performed tion. Between January 2003 and April 2012, we performed
in the United States from 2003 to 2013. (Adapted from 4597 transfers of 7598 autologous cryopreserved blasto-
data reported by the Society for Assisted Reproductive cysts, consisting of 2842 transfers using slow freezing and
Technology [www.sart.org].) 1755 transfers using vitrification protocols (Table 25.1),
providing what is to date perhaps the largest reported sin-
autologous cryopreserved blastocyst transfer cycles per- gle-center clinical experience comparing slow freezing to
formed in Australia and New Zealand over a 3-year period, vitrification of human embryos.
vitrification compared to slow freezing resulted in more Blastocysts cryopreserved by vitrification exhibited
transfers per warming/thawing attempt (95.6% versus substantially reduced cryodamage compared to those
90.6%), more transferred embryos per warmed/thawed cryopreserved using slow-freeze protocols. As has been
blastocyst (84.5% versus 71.4%), and higher pregnancy reported by others,11–15 vitrified blastocysts were more
and birth rates per transfer (32.7% versus 23.8% and 24.8% likely to survive warming (96% versus 92%). In addition,
versus 17.7%).7 surviving blastocysts had a much higher percentage of

226
 scrying the future
Table 25.1  Treatment Outcomes Compared between significant relative reduction of 34%, and pregnancy loss
Slow Freezing and Vitrification among Autologous following ultrasound confirmation of clinical pregnancy
Cryopreserved Blastocyst Transfer Cycles was significantly lower as well, with a relative reduction
of 17%.
Slow freezing Vitrification p-value
Predictably, clinical pregnancy rates and live-birth
Cycles 2842 1755 — rates resulting from single-blastocyst transfers declined
Age at cryopreservation 33.5 33.7 0.078 significantly with increasing patient age at the time of
(years) the fresh oocyte retrieval and embryo cryopreserva-
Embryo survival per 91.9% 95.6% <0.0001 tion, whether embryos were cryopreserved using slow-
cycle freeze or vitrification protocols (Figure 25.4; p < 0.0001
Intact cells per 88.7% 95.3% <0.0001 for all). Within each age group, clinical pregnancy rates
surviving embryo were higher with vitrification than with slow freezing,
Embryos per transfer 1.71 1.56 <0.0001 although the difference was not statistically significant
Total embryos 4862 2736 — for the 41–42-year age group (<35 years: 54% versus 30%,
transferred p < 0.0001; 35–37 years: 53% versus 21%, p < 0.0001;
Positive hCG per 1304 (45.9%) 1204 (68.6%) <0.0001 38–40 years: 31% versus 19%, p = 0.03; 41–42 years: 32%
transfer versus 17%, p = 0.32). Live-birth rates were also higher for
Biochemical pregnancy 312 (23.9%) 190 (15.8%) <0.0001 vitrification versus slow freezing within each age group,
per positive hCG but statistically significantly so only for patients under 38
Clinical pregnancy per 992 (34.9%) 1014 (57.8%) <0.0001 years (<35 years: 42% versus 22%, p < 0.0001; 35–37 years:
transfer 39% versus 17%, p < 0.0001; 38–40 years: 19% versus 12%,
Implantation per 1212 (24.9%) 1285 (47.0%) <0.0001 p = 0.16; 41–42 years: 24% versus 4%, p = 0.1). Statistical
embryo transferred power was very limited in the 41–42-year age group, with
Live birth per transfer 717 (25.2%) 780 (44.4%) <0.0001 sample sizes of only 23 and 25 transfers for slow freezing
Live-born children per 829 (17.0%) 939 (34.3%) <0.0001 and vitrification, respectively.
transferred embryo Whether using slow freezing or vitrification, success
Clinical pregnancy loss 275 (27.8%) 234 (23.1%) 0.017 rates among single-blastocyst transfer cycles declined as
the time it took for embryos to develop to the expanded
blastocyst stage, indicated by the day of cryopreservation,
intact cells (95% versus 89%). A comparison of the fre- increased (Figure 25.5). Among slow-frozen embryos,
quency distributions of observed post-warming cryo- clinical pregnancy rates differed significantly between
damage between slow freeze and vitrification illustrates day 5 and day 6 cryopreservation (42% versus 27%,
substantial differences (Figure 25.3). Most vitrified p < 0.0001) and between day 6 and day 7 cryopreser-
embryos (73%) had little or no visible cryodamage (≤5% vation (27% versus 10%, p < 0.0001). Live birth rates
cell loss), while only 34% of the slow-frozen embryos also declined significantly with each progressive day in
fared as well. Conversely, only 8% of the vitrified embryos the slow-freeze group (day 5 versus 6: 34% versus 19%,
suffered greater than 10% cell loss, while 40% of the slow- p < 0.0001; day 6 versus 7: 19% versus 6%, p < 0.0001).
frozen embryos lost more than 10% of their cells. Under Among transfers of single vitrified blastocysts, the mod-
2% of vitrified embryos were less than 80% intact, com- est declines in pregnancy and birth rates from day 5 to
pared to 12% of the slow-frozen embryos. day 6 cryopreservation were not statistically significant
Despite the transfer of significantly fewer blastocysts (pregnancy: 54% versus 50%, p = 0.34; birth: 42% versus
per cycle, pregnancy and birth outcomes were much 37%, p = 0.1). However, day 7 vitrification was associated
better following vitrification (Table 25.1). Compared to with significantly lower outcomes for both pregnancy
slow freezing, vitrification resulted in significantly more (15%) and birth (10%) compared to either day 5 or day 6
positive serum human chorionic gonadotropin (hCG) vitrification (p < 0.0001 for all).
tests (relative increase of 49%). Similar to a few previous Within each cryopreservation day, vitrification com-
reports,14–16 we observed significantly improved implan- pared to slow freezing was consistently associated with
tation, pregnancy, and birth rates. Our data indicated higher clinical pregnancy (day 5: 54% versus 42%,
relative increases of 89% for implantation, 66% for clini- p = 0.008; day 6: 50% versus 27%, p < 0.0001; day 7: 15%
cal pregnancy, and 76% for live births with vitrification versus 10%, p = 0.4) and live-birth rates (day 5: 42% ver-
compared to slow freezing. Vitrified blastocysts were sus 34%, p = 0.05; day 6: 37% versus 19%, p < 0.0001;
twice as likely to result in live-born children compared day 7: 10% versus 6%, p = 0.4), although the statistical sig-
to slow-frozen blastocysts. Conversely, early pregnancy nificance of these trends could not be confirmed within
loss occurring after confirmation of positive serum hCG the day 7 cryopreservation groups. The small sample size
but before ultrasound confirmation of clinical preg- of blastocysts vitrified on day 7 (n = 41) limited the power
nancy (i.e. biochemical pregnancies) had a statistically of comparisons within this day.

227
 vitrification in assisted reproduction
50%

45%

40% Slow freezing

35% Vitrification
Proportion of all cycles

30%

25%

20%

15%

10%

5%

0%
100 95–99 90–94 85–89 80–84 75–79 70–74 60–69 <60
Mean percentage of intact cells post‐thaw

Figure 25.3  Frequency distribution of observed post-warming cryodamage, quantified as the percentage of intact
cells among all blastocysts surviving the cryopreservation and warming process, compared between slow freezing
(black) and vitrification (grey).

60% 60%
Slow freezing Slow freezing
50% 50%
Vitrification Vitrification
Pregnancy and birth
Pregnancy and birth

40% 40%

30% 30%

20% 20%

10% 10%

0% 0%
<35 35–37 38–40 41–42 Day 5 Day 6 Day 7
Patient age at embroyo cryopreservation (years) Day of blastocyst expansion/cryopreservation

Figure 25.4 Clinical pregnancy and live-birth rates Figure 25.5 Clinical pregnancy and live-birth rates
among autologous single cryopreserved blastocyst trans- among autologous single cryopreserved blastocyst trans-
fers, according to patient age at the time of the fresh in fers, according to the day after oocyte retrieval on which
vitro fertilization cycle from which embryos were cryo- embryos developed to the expanded blastocyst stage and
preserved. Compared between slow freezing (pregnancy were cryopreserved. Compared between slow freezing
in downward pattern and birth in black) and vitrification (pregnancy in downward pattern and birth in black) and
(pregnancy in upward pattern and birth in grey). vitrification (pregnancy in upward pattern and birth in
grey).
Clinical pregnancy and live-birth rates among sin-
gle-blastocyst transfer cycles both declined steadily and of 99 slow-frozen embryo transfers and none of the six
rapidly as the percentage of intact cells in transferred vitrified embryo transfers with fewer than 75% intact
embryos declined, regardless of the method of cryo- cells resulted in live births. Even among embryos with
preservation (Figure 25.6; p < 0.0001 for all,). Only one relatively little cryodamage (≤10% cell loss), the negative

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 scrying the future
60% cryopreserved embryo transfers consistently remained at
Slow freezing approximately 41% lower relative to fresh transfers from
50% 2003 to 2008 (when nearly all cryopreservation was by
Vitrification
conventional slow-freezing protocols), while thereafter
Pregnancy and birth

40% the relative deficit in birth rates for cryopreserved rela-

tive to fresh transfers has declined annually (39% in 2009;
30%
37% in 2010; 35% in 2011; and 34% in 2012; 28% in 2013)
as embryo vitrification has been increasingly incorpo-
20%
rated into clinical practices.
10%
SHIFTING PARADIGMS IN EMBRYO
0% TRANSFER STRATEGIES
100 95–99 90–94 75–89 <75 The greatly enhanced survivability and viability associ-
Percentage of intact cells post-warming ated with embryo vitrification suggests the need for a par-
adigm shift in the approach to assisted reproduction. The
Figure 25.6 Clinical pregnancy and live-birth rates conventional approach to IVF is to transfer the highest-
among autologous single cryopreserved blastocyst quality embryos while fresh, usually more than one per
transfers, according to the percentage of intact cells transfer, under the assumption that this will maximize
following cryopreservation and warming. Compared their chances for implantation by avoiding the damaging
between slow freezing (pregnancy in downward pat- effects of cryopreservation. As refinements in vitrification
tern and birth in black) and vitrification (pregnancy in protocols lead to continued reductions in cryodamage,
upward pattern and birth in grey). further limiting the adverse effects of cryopreservation on
embryo viability, the rationale for multiple embryo trans-
fer, or even fresh embryo transfer at all, wanes. With little
effect of cryodamage was evident. When the analyses compromise to embryo viability, clinical outcomes with
were limited to blastocysts that were at least 90% intact, cryopreserved embryos can approach those achieved with
there were still statistically significant declines in both fresh transfers. In fact, live-birth rates have been reported
pregnancy and birth rates among both slow-frozen and to be equivalent for vitrified compared to freshly trans-
vitrified blastocysts (p = 0.0009 and p = 0.0017 for slow ferred blastocysts of equal morphological quality.17
freezing; p = 0.0066 and p = 0.0052 for vitrification). Outcomes of vitrified embryo transfers may even sur-
Within each of the top three categories of cell survival, pass those of fresh transfers given that cryopreserved
vitrification was associated with significantly higher embryo transfer circumvents the known adverse effects
pregnancy rates than slow freezing (100%: 58% versus of ovarian hyperstimulation on endometrial receptivity
40%, p = 0.0004; 95%–99%: 52% versus 37%, p = 0.0003; and endometrium–embryo developmental synchrony.18
90%–94%: 47% versus 27%, p = 0.0002). Live-birth rates Dramatic hyperstimulation-induced alterations in endo-
were also significantly higher for vitrification compared metrial histology (regardless of the stimulation protocol
to slow freezing in each of the top three cell survival cat- used or the fertility status of the subjects) and a clear
egories (100%: 46% versus 31%, p = 0.0026; 95%–99%: negative association between the severity of these altera-
40% versus 27%, p = 0.0008; 90%–94%: 33% versus 19%, tions and the likelihood of pregnancy have recently been
p = 0.004). The significantly higher (by 13%–20%) preg- documented,19 providing strong evidence for compro-
nancy and birth rates for vitrification versus slow freezing, mised endometrial quality in stimulated cycles. Indeed,
even among blastocysts in which observable cryodamage a meta-analysis of 633 cycles randomized to either fresh
appeared to be the same, is particularly interesting. This or cryopreserved embryo transfer reported relative differ-
result suggests that while differences in visible cryodam- ences exceeding 30% in favor of cryopreserved embryo
age partially explain differences in pregnancy and birth transfer for both clinical pregnancy (50% versus 38%) and
rates between vitrification and slow freezing, slow freez- ongoing pregnancy (47% versus 36%).20 One of the trials
ing relative to vitrification causes additional damage to included in this meta-analysis, in addition to significant
embryos that is not apparent upon microscopic examina- improvements in clinical and ongoing pregnancy rates,
tion and yet adversely affects embryo viability. also reported a dramatic improvement in implantation
In light of this accumulating evidence of greater viabil- rates with cryopreservation (71% versus 39%).21 A retro-
ity retention associated with embryo vitrification, it may spective study of single fresh or cryopreserved blastocyst
not be a coincidence that among donor oocyte trans- transfers that were well matched for age, day of blasto-
fer cycles the relative difference in birth rates between cyst expansion, blastocyst and ICM diameter, and troph-
cryopreserved and fresh transfers has been attenuating ectoderm cell count demonstrated significantly higher
noticeably in recent years as vitrification has begun to clinical pregnancy with cryopreservation (56% versus
replace slow freezing (Figure 25.2). Live-birth rates from 27%).22 Among our own patients undergoing transfers of

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 vitrification in assisted reproduction
thousands of vitrified blastocysts at Shady Grove Fertility can result from a single oocyte retrieval procedure, as
RSC, we observed an implantation rate of 47% among all well as maximizing the perinatal health of any resulting
autologous transfers regardless of age and an implanta- children. Firstly, a succession of single-embryo trans-
tion rate of 54% among patients under 35 years old at the fers eliminates the drawbacks of “putting one’s eggs all
time of blastocyst vitrification, success rates that are well in one basket.” Assuming that cycle-to-cycle variability
above the most recently reported (www.sart.org, 2013) in endometrial receptivity exists (which is practically a
implantation rates for autologous fresh transfers of those given), a succession of single-embryo transfers would
under 35 years of age for either our center specifically minimize the potential for all viable embryos to be
(42%) or for the nation as a whole (39%). transferred to a nonreceptive uterus. It would have the
In addition to equivalent if not better pregnancy and additional advantage of avoiding any possible competi-
live-birth rates with vitrified rather than freshly trans- tion among viable embryos for implantation sites, grow-
ferred embryos, there is a growing body of evidence dem- ing space, nutrient supply, and other maternal resources,
onstrating that cryopreserved embryo transfers result thus maximizing the potential for the optimal growth of
in substantially better perinatal outcomes than fresh each embryo.
embryo transfers. A systematic review and meta-analysis We have actually documented this effect among cryo-
of singleton IVF pregnancies concluded that, compared to preserved embryo transfers in our own data from Shady
fresh embryo transfers, cryopreserved embryo transfers Grove Fertility RSC. In an analysis of vitrified blastocyst
were significantly less likely to be associated with peri- transfers,32 we found a relative increase of 13.6% in the
natal mortality (RR = 0.68), pre-term birth (RR = 0.84), number of live-born children per transferred embryo
low birth weight (RR = 0.69), small for gestational age among cryopreserved blastocysts transferred singly
(RR = 0.45), and antepartum hemorrhage (RR = 0.67).23 rather than in pairs (adjusted means = 38.08% versus
More recent studies have found that pregnancies resulting 33.52%). This 13.6% relative difference suggests that,
from cryopreserved rather than fresh embryo transfers given the same number of vitrified blastocysts, 13.6%
were less likely to experience third-trimester bleeding or more children could be born if they were transferred one
placenta previa,24 and provided further confirmation that at a time rather than two at a time. Predictably, the chil-
the resulting children were born later and heavier,17,24–26 dren resulting from single vitrified blastocyst transfers
and thus were more comparable to children from natural were also healthier at birth, being born on average 1 week
conceptions. It has also been reported that blastogenesis later (and only one-sixth as likely to be very or extremely
birth defects are significantly more common with fresh pre-term) and 581 g (20%) heavier (and only one-tenth as
embryo transfers, but not with cryopreserved embryo likely to be very low birth weight) compared to children
transfers, compared to natural conceptions.27 born from transfers of two vitrified blastocysts.
Based on such evidence of higher success rates and
improved perinatal outcomes associated with cryopre- EMBRYO VITRIFICATION WHEN FRESH
served embryos, there is increasing advocacy for aban- TRANSFER IS CONTRAINDICATED
doning fresh embryo transfers altogether in favor of In addition to the more general benefits of embryo vitri-
transferring cryopreserved embryos into a more hospita- fication already described, there are a variety of special
ble and receptive endometrial environment undisrupted case situations in which fresh embryo transfer may be
by ovarian hyperstimulation necessary to produce a sup- particularly contraindicated. One common contraindica-
raphysiological cohort of mature oocytes.28–31 With the tion to fresh embryo transfer is either the presence of sig-
minimization of cryodamage possible through vitrifica- nificant symptoms of ovarian hyperstimulation syndrome
tion, it now appears likely that vitrification of all embryos (OHSS) prior to scheduled transfer and/or conditions
for transfer in a later unstimulated cycle may provide the suggesting a high risk for developing moderate-to-severe
highest potential for success, and may soon become the OHSS (e.g. polycystic ovary syndrome [PCOS], previ-
norm for IVF treatments. ous history of OHSS, high or rapidly increasing serum
Under these conditions, the imperative to transfer estradiol before hCG trigger, large number of developing
multiple embryos while still fresh, and in fact the clini- follicles during ovarian stimulation, or large number of
cal benefits of multiple embryo transfer at all (except retrieved oocytes33,34). OHSS is a potentially serious—in
perhaps, but not necessarily, in cases of poor embryo rare cases even life threatening—complication that gener-
quality), virtually disappear. With the minimization of ally only occurs in the context of ovarian stimulation in
cryodamage resulting in equivalent or better chances of conjunction with IVF treatment.35 It is the leading cause
achieving pregnancy following vitrification rather than of IVF-related mortality among nonpregnant women
fresh transfer, transfer of multiple embryos (whether (approximately three deaths per 100,000 stimulated
fresh or cryopreserved) will not increase the chances of cycles).36 It is induced and exacerbated by exogenous hCG
eventual success from an oocyte retrieval cycle. In fact, from the trigger injection and/or by the endogenous hCG
transfer of embryos one at a time will maximize the produced by implanted embryo(s).34 In the presence or
potential for success and the number of children that with high risk of OHSS, postponing transfer will therefore

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 scrying the future
either lessen the severity of OHSS or prevent moderate-to- enhancement of whole-genomic sequencing50 will make
severe OHSS requiring intervention from occurring at all. available a growing abundance of increasingly detailed
Fresh embryo transfer may also be contraindicated information upon which assessments of embryo viability
when there are indications of unusually poor endome- and health can be made. Vitrification of biopsied embryos
trial receptivity during the fresh cycle. In addition to the would provide ample time for conducting and interpret-
already discussed general disruption to normal endo- ing increasingly complex genetic analyses, offer appropri-
metrial development caused by ovarian hyperstimula- ate genetic counseling to patients, and allow patients and
tion for IVF, some specific conditions are known to be clinicians to consider and make informed clinical deci-
predictive of unusually low chances of success with fresh sions based on this information.
embryo transfer. For example, a thin endometrial lin-
ing at the time of trigger administration is known to be OOCYTE VITRIFICATION
associated with significantly lower pregnancy rates.37–40 Compared to the historically widespread practice of
Elevated serum progesterone levels, defined variously embryo cryopreservation, oocyte cryopreservation has
by a threshold between 0.8 and 3.0 ng/mL, at the time of been, until recently, no more than a minor footnote in
trigger administration have also been linked to signifi- the field of assisted reproduction. However, with the
cantly poorer success rates with autologous fresh embryo rise of vitrification, oocyte cryopreservation is rapidly
transfers.41 There are also situations in which unantici- becoming an integral and major component of assisted
pated health issues, personal issues, emergencies and the reproductive technology (ART). Clinical application of
like prevent undergoing an embryo transfer procedure as oocyte cryopreservation had been impeded by disap-
planned. pointingly low success rates with the slow-freezing pro-
In all such occasions and in similar circumstances, tocols used for nearly all attempts until 2003. Through
in the absence of a reliable and effective embryo cryo- 2002, fewer than 80 births from cryopreserved oocytes
preservation program, forgoing fresh embryo transfer had been reported worldwide.51 By the end of 2008, the
could necessitate discarding the entire embryo cohort total number of births from cryopreserved oocytes, while
and wastage of the entire treatment cycle. A suboptimal still very low relative to overall ART births, had grown to
cryopreservation program would reduce embryo viability over 900, with 532 through slow freezing and 392 through
and decrease the chances of success once a suitable time vitrification.51
for embryo transfer arrives. With the high viability main- Oocyte survival is significantly higher with vitrifi-
tained through the use of current vitrification protocols, cation,52,53 and multiple groups have published reports
however, embryos can be stored for later use with high of post-vitrification oocyte survival rates greater than
chances of success in all of these situations. 95%.54–57 Fertilization rates, embryo cleavage rates, and
top-quality embryo rates are also all significantly higher
PRE-IMPLANTATION GENETIC SCREENING with vitrification compared to slow freezing.52 A review
AND EMBRYO VITRIFICATION of all published series reports from 1998 to 2008 dem-
The ability to minimize cryodamage through the use onstrated that the number of children born per thawed/
of vitrification also has important implications for the warmed oocyte was more than twice as high with vitrifi-
practicality of incorporating pre-implantation genetic cation than with slow freezing (2.4% versus 5.2%), and by
screening (PGS) as a clinically advantageous adjunct to 2008, the majority of newborns were the result of oocyte
IVF treatment. The first iteration of PGS, using fluores- vitrification rather than slow freezing.51
cence in situ hybridization analysis of a minority subset Fertilization, embryonic development, embryo ­quality,
of the chromosome complement derived from a cleavage- implantation, pregnancy and birth rates, and obstet-
stage biopsy, has proven to be detrimental.42 This failure ric and perinatal complications following oocyte vitri­
of early PGS attempts appears to be largely due to dra- fication have been shown in numerous studies to be
matic reductions in embryonic developmental potential comparable to those of fresh oocytes.52,54,56–60 Electron
(39% relative reduction in implantation with progression microscopy has revealed that vitrification of mature
to a live-born infant) when a cleavage-stage biopsy is per- human oocytes maintains good ultrastructural preserva-
formed.43 More recently, alternative methods incorporat- tion,61 and metabolic profiling has failed to disclose any
ing comprehensive chromosome screening (CCS)44.45 and significant differences between embryos derived from
trophectoderm biopsy at the blastocyst stage, while as-yet vitrified versus fresh oocytes.62 Such studies demon-
not conclusively proven, appear to hold more promise of strate that oocytes can be cryopreserved with little or no
benefit.45–49 Blastocyst-stage biopsy appears to preserve damage using vitrification. The consistency and repeat-
developmental potential much more so than cleavage- ability63 of such positive outcomes with this method of
stage biopsy,43 but leaves little time for evaluation before oocyte cryopreservation has spurred the increasingly
embryos must be transferred if transferred while fresh. rapid adoption of oocyte vitrification into assisted repro-
CCS enables analysis of all 23 pairs of chromosomes, duction programs. In 2013, the American Society for
and as the technology matures, the emergence and Reproductive Medicine (ASRM) and the SART issued

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 vitrification in assisted reproduction
a joint committee opinion,64 endorsed by the American clinical pregnancies had been established using oocytes
College of Obstetrics and Gynecology,65 concluding that dispensed by these banks. In 2013, the first year for which
oocyte vitrification and warming should no longer be donor oocyte banking data was included in the annual
considered experimental based on good evidence that summary report of SART member clinics, cryopreserved
fertilization and pregnancy rates are similar for vitri- banked donor eggs accounted for more than 20% of all
fied–warmed oocytes as with fresh oocytes when used for donor egg embryo transfers performed in the United
IVF/intracytoplasmic sperm injection treatment, with no States, and these banked donor egg cycles resulted in 960
apparent increase in chromosomal abnormalities, birth live births.
defects, or developmental deficits in resulting offspring, Such egg banks provide a multitude of benefits to
further opening the door to the use of oocyte vitrification recipient patients. No treatment delay or synchronization
in standard clinical practice. is necessary; donor oocytes have already been retrieved
and can be thawed/warmed whenever the recipient
DONOR VITRIFIED OOCYTE BANKING is ready to use them. Cycles are never cancelled due to
Oocyte donation by healthy young women plays a sub- inadequate donor response. Eggs are not thawed/warmed
stantial role in assisted reproduction, providing repro- until the intended recipient demonstrates adequate endo-
ductive options for women whose oocyte quality is poor metrial development, so poor development in a given
or whose ovarian reserves are depleted. In the United cycle is relatively inconsequential. Sharing large cohorts
States, more than 17,000 transfers of embryos derived of oocytes is simple; each recipient from a donor egg bank
from donor oocytes were performed in 2013, accounting typically receives five to seven mature eggs, so a single
for 13.8% of all embryo transfers performed. The ability donor retrieval can usually provide enough eggs to supply
to successfully cryopreserve oocytes enables substantial two or three (and sometimes more) recipients. The sim-
changes in the ways in which donor oocytes can be pro- plicity of cohort sharing allows egg banks to distribute
vided to patients in need. donor oocytes to a much larger number of recipients, and
Traditionally, oocyte donation could only be per- at a significantly reduced cost per patient. Cryopreserved
formed fresh. With fresh transfer, careful synchroniza- oocytes can be safely shipped between distant treatment
tion of donor and recipient cycles is required to ensure facilities, so they can provide recipients with a much
that recipients are maximally receptive when embryos larger and more diverse donor pool from which to select
are ready to implant. Cycle synchronization is even more compared to a fresh donor program that must depend on
complicated and challenging when fresh donor cohorts the potential donors available at the time in the local geo-
are shared among multiple recipients, a practice that graphic area. In addition, oocyte banking, in contrast to
makes more efficient and cost-effective use of the large fresh oocyte donation, allows for quarantining and thor-
oocyte cohorts often produced by donors,66–69 but which ough evaluation for infectious agents prior to clinical use,
is only practical in relatively large programs with numer- as is standard practice for donor sperm. Given these many
ous concurrently cycling recipients. In most cases, donor and substantial advantages, as well as the clinical out-
cohorts used fresh are unshared, often resulting in more comes for vitrified oocytes that are comparable to fresh
viable oocytes and embryos than patients require for oocytes, it seems inevitable that egg donation—likely in
their family-building goals. An ever-present risk in fresh the near future—is destined to follow the model of sperm
oocyte donation cycles is that cycles may be cancelled due donation, with fresh donations being entirely replaced
to an unexpectedly poor response by the donor, resulting with cryopreserved (mainly vitrified) donor egg banking.
in the cancellation of retrieval or, in the case of shared
cohorts, retrieval of too few oocytes to share among all MEDICALLY INDICATED FERTILITY
intended recipients. Cycles may also be cancelled due PRESERVATION THROUGH OOCYTE
to inadequate endometrial development in the intended VITRIFICATION
recipient, in which case, without the option of oocyte Fertility preservation through oocyte cryopreservation
cryopreservation, the donated oocytes would go unused for medical indications has been in use for some time
and wasted. In either case, the intended recipient incurs on a very limited scale, and with only limited success in
considerable time and expense for cycle cancellation. the past due to the substantially reduced viability asso-
Advancements in oocyte cryopreservation, par- ciated with earlier oocyte cryopreservation protocols.
ticularly vitrification, have led to the establishment of Despite limited success, these attempts continued, since
numerous donor egg banks. By early 2012 (while oocyte they often provided the best—and sometimes the only—
cryopreservation was still labeled as “experimental” by chances for these women to bear children in the future.
societal guidelines), there were already at least seven The current potential of oocyte vitrification greatly
commercial egg banks operating in the United States (all enhances the applicability and success of medically indi-
but one of them cryopreserving by vitrification), main- cated fertility preservation.
taining more than 3000 oocytes obtained from nearly One of the primary medical indications for fertility pres-
300 individual donors.70 As of April 2012, more than 600 ervation is anticipated gonadotoxic medical treatments

232
 scrying the future
among women of childbearing years.64 Chemotherapy entirely new industry.80,81 Only a short time ago, elective
or radiation therapy, which often reduce fertility, are oocyte cryopreservation was essentially unheard of, as it
frequently administered as treatments for a variety of was considered too expensive and unreliable to be rec-
oncological conditions, as well as some autoimmune and ommended without a compelling medical reason, given
hematological diseases. Oocyte vitrification has been the low success rates associated with the oocyte cryo-
used with good success among women diagnosed with preservation techniques available at the time. However,
cancer who used their vitrified oocytes following suc- spurred by the emergence of compelling evidence dem-
cessful cancer treatments.71 Additionally, oophorectomy onstrating success rates with vitrified oocytes rivaling
may be required by some patients for either malignant those of fresh eggs, as well as the repeal of the ASRM/
or benign conditions, or undergone prophylactically in SART “experimental” label, the floodgates have opened
women with BRCA mutations or other genetic conditions to an explosive expansion of this fledgling industry.
associated with a high risk for ovarian cancer. It is esti- There are well over 100 independent medical centers in
mated that one in every 46 women in the United States the United States alone that now routinely perform elec-
will develop invasive cancers by 39 years of age,72 demon- tive oocyte cryopreservation as a standard clinical prac-
strating the wide applicability of a truly effective method tice for women seeking to store a number of their eggs
like oocyte vitrification to preserved fertility options for for the future.
these patients. In the past, embryo cryopreservation was The trends of delaying reproduction until a later
the most reliable option available to young women fac- age, particularly in more industrialized societies, and
ing such treatments, but many women in this position are the increasingly rapid decline in female fertility poten-
without a partner with whom they intend to build a fam- tial beginning at approximately 30 years of age are well
ily. Given the proven success of oocyte vitrification, this understood. These trends are largely responsible for the
option should be, and likely will become, a standard com- growing demand for and use of ART in general. However,
ponent of pretreatment counseling for younger women delaying treatment until a time when reproduction is
planning gonadotoxic therapies. desired (e.g., when the woman is in or past her mid-30s)
Additional medical indications for oocyte vitrification is likely to substantially reduce her chances of success.
include a variety of genetic conditions associated with Therefore, with oocyte vitrification becoming a clinically
premature ovarian failure, such as Turner syndrome, viable option, this opportunity is now being offered and
fragile X syndrome, and deletions of the X chromosome. marketed to women who are not yet ready to start a fam-
If these conditions are diagnosed at an early age, oocyte ily, but who anticipate wanting to do so in the future. For
vitrification is likely to provide such women with the best such women, oocyte vitrification enables them to collect
chances of conceiving with their own eggs. and store what are expected to be relatively viable eggs
Other potential but as-yet unproven alternatives while they are still young, for availability when they are
to mature oocyte retrieval and vitrification for medi- ready to start building a family at a later age when their
cally indicated fertility preservation are currently being fertility potential is diminishing. While far from a guar-
investigated. Collection of immature oocytes followed antee, banking of their own eggs while young provides
by in vitro maturation and vitrification holds potential these women with some degree of insurance, if and when
as an option for fertility preservation without the delay they eventually decide they are ready to have children.
required for ovarian stimulation and retrieval of mature A survey of reproductive-aged women (21–40 years) con-
oocytes.73–75 This approach could be particularly advan- ducted in 2010, when there was little awareness of the
tageous when a delay in gonadotoxic therapy is contra- efficacy and possibilities of oocyte vitrification, indicated
indicated. Cryopreservation of ovarian cortical tissue or that nearly a third of the respondents might consider elec-
whole ovaries for later transplantation has also met with tive oocyte cryopreservation for themselves.82 Since then,
some initial success, and may eventually become a viable public awareness and acceptance of fertility preservation
treatment option both for women and prepubertal girls. possibilities available through oocyte vitrification have
Preliminary experience with ovary and ovarian tissue increased considerably. Very recently, major companies
cryopreservation suggests that viability retention may be including Facebook and Apple announced that they will
much better with vitrification rather than slow freezing, cover the costs of elective egg cryopreservation for fer-
with better morphological integrity of stroma, higher tility preservation as a benefit to their employees, signal-
rates of oocyte survival, reduced follicular DNA damage, ing a high acceptance level for this procedure. Given that
and increased post-transplantation activation of the rest- elective oocyte cryopreservation could be much more
ing follicle pool.76–79 broadly applicable to any young women contemplating
delayed but future reproduction, and not just to patients
ELECTIVE OOCYTE VITRIFICATION currently experiencing reproductive difficulties, demand
The success of oocyte vitrification has led to the emer- for fertility preservation procedures could conceivably
gence of elective fertility preservation in anticipation of eventually far surpass demand for traditional assisted
the normal age-related decline in fertility potential as an reproductive practices.

233
 vitrification in assisted reproduction
OTHER APPLICATIONS OF OOCYTE techniques,85–88 an advantage that has significant clinical
VITRIFICATION value for men with severe oligozoospermia and azoosper-
In addition to donor egg banking and fertility preserva- mic men requiring surgical sperm retrieval.
tion, the ability to achieve clinical results with oocyte vit-
rification that are comparable to using fresh oocytes has CONCLUSIONS
several other clinically important applications. The transformative impact of vitrification on the field of
Oocyte vitrification can be an invaluable option in assisted reproduction is only just beginning to be real-
autologous IVF cycles in which there is an unanticipated ized. Embryo cryopreservation will continue to play
unavailability of sperm on the day of oocyte retrieval. an increasingly dominant role in IVF treatment cycles,
While infrequent, there are occasions in which the male eventually supplanting fresh embryo transfers entirely,
partner is unable to be present on the day of retrieval or due to the higher birth rates per transferred embryo that
otherwise unable to produce a usable sample. In such can be achieved through embryo vitrification, coupled
cases, without a viable oocyte cryopreservation program, with the healthier perinatal outcomes of those resulting
the retrieved eggs would simply be discarded and the children. In a continuing effort to maximize the number
entire cycle wasted—a potentially devastating consequence of children who are born per oocyte retrieved and also
for couples investing considerable effort and expense to maximize the health of those children, single-embryo
to overcome their infertility. With the option of oocyte transfer, aided by oocyte and embryo vitrification, will
vitrification, the retrieved eggs can be reliably stored for become the norm rather than the exception. The allur-
later use with little clinical consequence. Similarly, for ing but elusive promise of PGS, coupled with vitrification
couples in which the male suffers from severe male fac- to provide ample time for the analysis and interpretation
tor infertility, this ability to store oocytes can allow for of increasingly vast quantities of accessible genetic infor-
repeated sperm recovery attempts to accumulate enough mation, may finally be realized. Vitrified donor oocyte
usable sperm before the eggs are warmed for use. banking will replace fresh oocyte donation, simplify-
The success of oocyte vitrification also provides an ing scheduling and treatment, increasing availability to
important alternative to the insemination of entire more patients in need, expanding the pool of donors from
cohorts of retrieved oocytes. Many patients have reli- which each recipient may choose, enabling more effec-
gious, moral, and/or ethical reservations (or legal restric- tive screening of infectious agents, and reducing treat-
tions, as has been the case in Italy) against creating more ment costs. Oocyte vitrification is also providing the first
embryos than they will necessarily use in their infertility truly effective treatment for medically indicated fertility
treatments. For these patients, oocyte vitrification may preservation, and has led to the birth of an entirely new
provide a more acceptable alternative while still maxi- industry of elective fertility preservation that may even-
mizing the clinical utility of the eggs that are retrieved. tually eclipse traditional assisted reproduction in patient
Small numbers of eggs can be inseminated at a time, volume.
referred to as elective limited insemination, reducing or
preventing the creation of surplus untransferred embryos REFERENCES
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237
 Prior to vitrification Directly after vitrification 2 hours after vitrification
(a) (b) (c)

Polarization microscopy
20 μm

(d) α-tubulin DNA (e) α-tubulin DNA (f) α-tubulin DNA

Confocal microscopy

10 μm

Figure 12.1  Spindle dynamics after CryoTop vitrification of murine metaphase II (MII) oocytes analyzed by nonin-
vasive polarization light microscopy (a–c) or by confocal laser scanning microscopy (d–f) (Trapphoff T., ­unpublished
data). Spindle characteristics prior to vitrification (a and d), directly after vitrification (b and e), and after recovery
for 2 hours at 37°C (c and f). The kind of information obtained on spindle integrity differs between polarization
light and fluorescence microscopy directly after vitrification and warming (b and e). Arrowhead: slightly aberrant
spindle, arrow: displaced chromosome; both phenotypes occurred in about a third of in vivo-ovulated and vitrified
murine MII oocytes directly after warming (unpublished data).

(a) +

Gene A IC1 Gene B IC2 Gene C

Gene A IC1 Gene B IC2 Gene C

Figure 12.2  Scheme of mono-allelic gene expression in
– the offspring originated from parental-specific hyper-
Unmethylated CpG Expression
Methylated CpG Repression or nonmethylation of maternal and paternal imprinting
(b) Unmethylated loci Hypermethylated loci
centers (a). Paternal hypermethylation of IC1 represses
me me me paternal expression of gene B and activates expression
Bisulfite
AT CG AT CG TT CG AT AT CG AT CG TT CG AT of the upstream-located gene A. Hypermethylation of
treatment
me me me maternal IC2 results in paternal expression of gene C.
AT UG AT UG TT UG AT AT CG AT CG TT CG AT Bisulfite treatment induced mutagenesis of hyper- and
DNA
amplification nonmethylated DNA (b). (CpG = cytosine phosphate–
AT TG AT TG TT TG AT AT CG AT CG TT CG AT
guanine dinucleotide.)

Imprinting cycle Figure 12.3  ​Imprinting cycle in mice. Erasure, establish-

ment, and maintenance of imprinted genes (outer circle)
during F0 to F1 transition. Genome-wide demethylation
ce
an (including imprinted genes) occurs in primordial germ
en

cells (PGCs) until embryonic day 13.5. Post-natal de

int
Ma

novo establishment of sex-specific methylation marks

Era

PGC
sure

Blastocyst
occurs during oogenesis (pre-natal in males) and
Zygote
Growing
maintenance of genomic imprinting occurs until the
MII
oocyte oocyte formation of PGCs in the F1 generation. Epigenetic
methylation marks of nonimprinted genes (inner circle)
are also erased in PGCs, followed by methylation events
during oogenesis. After fertilization, active (paternal
E s t a b li s h m e n t genome) and passive (maternal genome) demethyl-
Genome Imprinted gene
ation of both parental genomes occurs, followed by de
Methylated Methylated CpG
novo reprogramming during embryonic development.
Unmethylated Unmethylated CpG
(MII = metaphase II.)
 (a) First round
(c) Second round
2.0
3.0
2.5 1.5
2.0
1.0
1.5
1.0
0.5
0.5
0.0 0.0

t(2)
t(2)
–0.5
–0.5
–1.0
–1.5
–1.0
–2.0
–2.5 –1.5
–3.0
–2.0

–3.2
–3.0
–2.8
–2.6
–2.4
–2.2
–2.0
–1.8
–1.6
–1.4
–1.2
–1.0
–0.8
–0.6
–0.4
–0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.0
3.2
3.4

–3.0

–2.5

–2.0

–1.5

–1.0

–0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0
t(1) t(1)

(b) (d)
1.4 2.0
1.2 1.5
1.0 1.0
0.8 0.5
0.6
0.0
0.4
–0.5
0.2

t(2)
–1.0
t(2)

0.0
–0.2 –1.5
–0.4 –2.0
–0.6 –2.5
–0.8
–3.0
–1.0
–3.5
–1.2
–4

–3

–2

–1

–2.6
–2.4
–2.2
–2.0
–1.8
–1.6
–1.4
–1.2
–1.0
–0.8
–0.6
–0.4
–0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.0
3.2
3.4
Devitrified Fresh t(1) t(1)

Figure 13.4  Principal component analysis representing all of the metabolites analyzed for each sample in the two
rounds. Blue/green triangles represent the fresh group, and red triangles represent the vitrification group. No sepa-
ration between groups was observed using positive (a and c) or negative (b and d) sample ionization in either of the
analysis rounds.

(a) D-Glucose (c) Lactate

0.35
0.055
0.3
Relative intensity (a.u.)

0.05
Relative intensity (a.u.)

0.25 0.045
0.2 0.04
0.15 0.035
0.1 0.03
0.05 0.025
0 0.02
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
Oocyte Oocyte
Fresh Vitrification

(b) L-Tryptophan (d) L-Phenylalanine

0.05 0.034

0.045 0.032
Relative intensity (a.u.)
Relative intensity (a.u.)

0.03
0.04
0.028
0.035
0.026
0.03
0.024
0.025 0.022
0.02 0.02
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
Oocyte Oocyte

Figure 13.5  Distribution of measurements of (a) glucose, (b) tryptophan, (c) lactate, and (d) phenylalanine in both
fresh (green rhombuses) and vitrification groups (red rhombuses).

85.9 μm

107.9 μm
78.5 μm 88.9 μm

118.0 μm Figure 20.3  The volume of each blastocyst was calcu-

lated based on the diameter of the embryo, or the parts
78.9 μm 82.2 μm of the embryo if it was in a figure 8 configuration, as
shown here. The volume was recorded immediately after
warming and again after 1 hour in culture.
 (a) (b) (c)

(d) (e) (f )

Figure 23.1  Fresh and vitrified human immature testicular tissue grafted for 6 months to castrated nude mice.
Hematoxylin–eosin staining of fresh grafted (a and d) and vitrified grafted (b and e) tissue of 2- and 9-year-old
donors, respectively. Spermatogonial differentiation to the pachytene stage was achieved in vitrified grafted tissue
of the 9-year-old donor (e, asterisk). Immunostaining of spermatogonia with MAGE-A4 antibody can be seen in the
vitrified grafted tissue of the 2-year-old (c) and 9-year-old (f) donors. Magnification 400×. Scale bar: 10 µm.

Vitrified water (glass) Crystalline water (ice)

Devitrification

2750 3000 3250 3500 3750 2750 3000 3250 3500 3750
Wavenumber/cm–1 Wavenumber/cm–1
EA
Energy

Molecular order

Figure 24.1  Schematic of the devitrification process in an energy diagram, the corresponding molecular arrange-
ments, and the Raman spectra of liquid water (15°C) and ice (−115°C). Liquid water resembles the spectral shape of
vitrified water quite well. It was used here as the vitrification of macroscopic amounts of pure water is practically
impossible. EA is the activation energy for devitrification.

Standard cell culture protocol

Cultivation Cell culture medium
Incubator at 37°C
Vitrification protocol
Preparation Manual picking of fragments
Preparation of straw
Cleanbench
Vitrification protocol
Incubation Incubation of fragments in VS1/VS2;
manual transfer of fragments
Cleanbench
Straw
Vitrification protocol
Loading Loading of fragments into straw
Sealing of straw
Cleanbench
Vitrification protocol
Vitrification Submerge into LN2
Outside cleanbench
Vitrificiation protocol
Storage Transport submerged into LN2
Storage in LN2 or gas
phase of nitrogen tank
Thawing protocol
Thawing Transport submerged into LN2
Thawing in water at RT
Straw Thawing protocol
Opening of straw on one end Figure 24.2 Workflow of the open pulled straw vitri-
Washing
Washing steps in WS1 and WS2
Manual transfer of fragments
fication method for human embryonic stem cells by
Cleanbench Richards et al. (LN2 = liquid nitrogen; VS = vitrification
Plating and
cultivation
Standard cell culture protocol
Replating in cell culture medium
solution; WS = warming solution.) (From Richards M
Incubator at 37°C et al. Stem Cells 2004;22:779–89. With permission.)
 Modification step
Modification Attach handlebar to plastic coverslips
UV sterilize coverslips
Modification
Standard cell culture protocol
Cultivation Cell culture medium
Incubator at 37°C

Vitrification protocol
Incubation CPA incubation in VS1/VS2
Transfer with sterile tweezers
Cleanbench
Vitrification protocol
Vitrification Submerging of coverslips into LN2
using sterile tweezers
Cleanbench/laminar flow
Vitrification protocol
Transportation Collection and transfer of samples in
submerged dish inside LN2 dewar
Outside cleanbench

Vitrification protocol
Storage Storage in LN2 or gas
phase of nitrogen tank
Thawing protocol
Transport submerged in LN2
Thawing Rapid thawing in WS1 (37°C) using
sterile tweezers
Cleanbench/laminar flow
Thawing protocol
Washing Washing steps in WS1 and WS2
Cleanbench

Standard cell culture protocol

Cultivation Cell culture medium
Incubator at 37°C

Figure 24.4  Workflow of the surface-based vitrification method for human embryonic stem cells by Beier et  al.
(CPA = cryoprotective agent; LN2 = liquid nitrogen; UV = ultraviolet; VS = vitrification solution; WS = warming
solution.) (From Beier AF et al. Cryobiology 2011;63:175–85. With permission.)

(a) (b)

(a) Lid “cultivation (b)

compartment”
Nitrogen or pre-
Media (culture/ heated water
(c) (d) CPA/washing)
Adherent LN2
cell clusters

Cultivation CPA film Adherent

(e) (f ) LN2 surface cell clusters

Figure 24.9 Outline and application of the TWIST

substrate. The TWIST substrate is a two-chamber sys-
tem with a closable, sterile cultivation chamber and
Figure 24.6  Microscopic fluorescence images of human a LN2 chamber. In the cultivation chamber, cells are
embryonic stem cells at different time points after slow- seeded, expanded, monitored, and prepared for cryo-
rate freezing (a, c, and e) and adherent vitrification (b, preservation by incubation in CPAs (a); for surface-
d, and f). Cells have been stained with live/dead stain- based vitrification, the cryo-medium is removed, the
ing (fluorescein diacetate/ethidium bromide [FDA/ substrate is turned over (“twisted”), and LN2 is filled
EB]) directly after thawing (a and b), after 24 hours (c into the LN2 chamber (b). (CPA = cryoprotective agent;
and d) and after 48 hours (e and f). Scale bars indicate LN2 = liquid nitrogen.) (From Liu BL, McGrath J. Acta
200 µm (a–f) and 2 mm (inserts). (From Beier AF et al. Biochim Biophys Sin (Shanghai) 2005;37:814–8. With
Cryobiology 2011;63:175–85. With permission.) permission.)
 Standard cell culture protocol
Cultivation Cell culture medium
Incubator at 37°C

Vitrification protocol
Incubation CPA incubation in VS1/VS2
Cleanbench
TWIST
Vitrification protocol
Vitrification Liquid nitrogen addition
Outside cleanbench

Vitrification protocol
Transportation Liquid nitrogen present in nitrogen
compartment

Vitrification protocol
Storage Liquid nitrogen evaporates from nitrogen
compartment while in storage
Gas-phase of nitrogen storage tank

Thawing protocol
Transportation Refill liquid nitrogen in nitrogen
compartment before transportation

Thawing protocol
Thawing Remove liquid nitrogen and add water
(37°C) to nitrogen compartment
TWIST
Thawing protocol
Washing Washing steps in WS1/WS2
Cleanbench

Standard cell culture protocol

Cultivation Cell culture medium
Incubator at 37°C

Figure 24.10  Workflow of the GMP-compliant surface-based vitrification procedure. Every step of the workflow and
the corresponding positions of the TWIST substrate are summarized here. (CPA = cryoprotective agent; VS = vitri-
fication solution; WS = warming solution.) (From Beier AF et al. Cryobiology 2013;66:8–16. With permission.)

Before vitrification After thawing Passage after 5 days

Figure 24.11  Microscopic images of human embryonic
(a) (e) (i)
stem cell (hESC) colonies before and after vitrification
using the GMP-compliant surface-based vitrification
Twisted vitrification

method. Pictures of the same hESC colonies have been

taken before (a–d) and after vitrification (e and f), in
(b)* (f )* (j) comparison to a nonfrozen control (g and h). For deter-
mination of the viable and adherent colony area, hESC
colonies have been stained with a live/dead dye (FDA/
EB) directly after thawing (b) or 24 hour after thawing
(f). The nonfrozen hESC colonies have been stained on
(c) (g) (k)
the day of vitrification (g) or 24 hours later (h). Vitrified
(i and j) and nonfrozen hESC colonies (k and l) have
Nonfrozen control

been passaged and cultivated further to determine signs

of differentiation. Red arrows indicate thicker, probably
(d) (h) (l) differentiated areas of the hESC colonies that are lost
after vitrification (e) in comparison to the nonfrozen
control (g). Scale bars indicate 1 mm (a–h) and 500 µm
(i–l). (From Beier AF et al. Cryobiology 2013;66:8–16.
With permission.)
 Screening for CPA alternatives Control Propanediol
200%

150% 300 μm 300 μm

Formamide Butanediol
Vital residual area

100%

50% 300 μm 300 μm

Var. 1 Var. 2
0%
Control
DMSO
Ethylene glycol

Propanediol
Butanediol
Var. 1
Var. 2
Var. 3
Var. 4
Control
DMSO
Ethylene glycol

Propanediol
Butanediol
Var. 1
Var. 2
Var. 3
Var. 4
Formamide

Formamide
300 μm 300 μm

Var. 3 Var. 4

0h 24 h
CPA/Time after thawing
300 μm 300 μm

Figure 24.13  Comparison of potential vitrification CPAs according to their effectiveness in surface-based vitrifica-
tion. Concentrations of CPAs have been 40% except for in Var. 4, which shows a less-concentrated version of Var. 3.
Images have been stained with EB and FDA and show the corresponding colonies directly after thawing. Var. 1–4
show different mixtures of the CPAs used. Var. 1: 10% propanediol, 10% formamide, 10% ethylene glycol, and 10%
butanediol. Var. 2: 13% propanediol, 13% ethylene glycol, and 13% butanediol. Var. 3: 20% propanediol and 20%
ethylene glycol. Var. 4: 20% propanediol and 15% ethylene glycol. (CPA = cryoprotective agent; DMSO = dimethyl
sulfoxide; Var. = variation.)

(a) Dependency on CPA concentration (c) Dependency on incubation time

200% 140%
120%
Vital residual area

Vital residual area

150% 100%
100% 80%
60%
50% 40%
0% 20%
Control
20%
30%
40%
50%
60%
70%
Control
20%
30%
40%
50%
60%
70%

0%
1 step 2 step 2 step 2 step 2 step
5s 2s 5s 30 s 60 s
0h 24 h
Workflow
CPA concentration and time after thawing

(d) (e)
(b)
>7 mol
Concentration high;
Osmolarity- and toxicity-
based damages expected
5.57 mol 500 μm 500 μm
Optimal concentration;
Successful vitrification (f ) (g)
most likely

<5.5 mol Concentration low;

Cell damages through ice
crystallization expected 500 μm 500 μm

Figure 24.14  Evaluation of optimal CPA concentration range (a and b) and incubation time (c) in surface-based
vitrification. CPA concentration range experiments were done with media containing equal amounts of dimethyl
sulfoxide and ethylene glycol in comparison to a nonfrozen control sample (d). The effects of too short an incubation
time (e), too long an incubation time (g), and damage by inadequate surface material are shown. Images have been
stained with FDA/EB. (CPA = cryoprotective agent.)
 Control Crystallization Vitrification
(a) (b) (c) (d)

hiPSC 500 μm
aggregates

(e) (f ) (g)

20 μL

LN2 50 μm

Figure 24.15  Cryopreservation of hiPSC aggregates using slow-rate protocols and pellet vitrification. For pellet
vitrification, 20-µL drops of vitrification medium containing one hiPSC aggregate each were dropped into LN2
(a). Adhesion of aggregates was monitored after slow-rate freezing (c) and pellet vitrification (d), in comparison to
a nonfrozen control (b). Stemness was analyzed with immunofluorescence staining using anti-HESCA-2 antibody
with Alexa-Fluor-488 after cultivation for 7 days (e–g). (hiPSC = human induced pluripotent stem cell; LN2 = liquid
nitrogen.)

Control
(a) (b) (c) (d)
100
90
80
Viability (%, standardized)

70
hiPSC on 200 μm
60
microcarrier
50
After thawing
40
(e) (f )
30
20
20 μL
10
0
LN2 0 h after 24 h after
Control
thawing thawing

Figure 24.16  Cryopreservation of hiPSC on micro-carriers using pellet vitrification. For pellet vitrification, 20-µL
drops of vitrification medium containing hiPSCs adherent on micro-carriers were dropped into LN2 (a). Viability
of vitrified cells was analyzed using live/dead staining (FDA/EB) directly after thawing and 24 hours later (b).
Stemness of a nonfrozen control (c and d) directly after thawing (e) and 24 hours later (f) was determined with
immunofluorescence staining using anti-HESCA-2 antibody with Alexa-Fluor-488. (hiPSC = human induced plu-
ripotent stem cell; LN2 = liquid nitrogen.)
 1 Day 1 Week 1 Month
(a) (b) (c)

Storage at –80°C
200 μm 200 μm 200 μm

500 μm 100 μm
100 μm

(d) (e) (f )
Storage at –170°C

200 μm 200 μm 200 μm

500 μm 500 μm 500 μm

(g) Control
250% Dependency on storage temperature
(h)

200%

150%
Vital residual area

100%

(i)
50%

0%
Control 1 Day 1 Week 1 Month 1 Day 1 Week 1 Month
–170°C –80°C
Storage temperature and time

Figure 24.18  Comparison of different storage temperatures for vitrified human embryonic stem cell colonies. Cells have
been vitrified via surface-based vitrification and stored at −80°C (a–c) and −170°C (d–f). Samples were thawed after 1
day (a and d), 1 week (b and e), and 1 month (c and f). Evaluation was done after a 24-hour recovery period via staining
with FDA/EB and comparison of the vital residual areas (g). A nonfrozen control sample has been included (h and i).
Raman scatter

Figure 24.22 Waterfall plot of a Raman spectral

3000
sequence showing ice formation from a vitrified
ss n

3200
re tio

medium. The medium composition was 300 mM sucrose

og za

Wa 3400
pr talli

ven
um solution with 20% dimethyl sulfoxide and 20% ethylene
ys

ber 3600 glycol. The medium was vitrified at a temperature of

Cr

/cm –1
−135°C, then heated to −95°C at a rate of 1 K/min.
MEDICINE / REPRODUCTIVE MEDICINE

Vitrification
in Assisted
Reproduction
Second Edition
Vitrification in Assisted Reproduction presents standard and new cryopreservation
techniques in detail, outlining those that have resulted in success, and providing
recommended means for overcoming typically encountered problems.

This new edition provides a much broader range of clinical application and data
to demonstrate its contribution to the use of vitrified oocytes and embryos. The
book also discusses new areas in the field of assisted reproductive technology
such as oocyte banking, preimplantion diagnostics at the blastocyst stage, and the
burgeoning adoption of elective single embryo transfer.

Written by expert scientists and clinical embryologists, this book will help you to
consistently and predictably apply vitrification as an important therapeutic strategy
in assisted reproduction.

K23395
ISBN: 978-1-4822-4257-7
90000

9 781482 242577