Biennial Review of Infertility Vol 2

Biennial Review of Infertility
wwwwwwwwww
Catherine Racowsky • Peter N. Schlegel
Bart C. Fauser • Douglas T. Carrell
Editors
Biennial Review
of Infertility
Volume 2
2011
Editors
Catherine Racowsky, Ph.D. Bart C. Fauser, Prof. Ph.D.
Harvard Medical School University Medical Center Utrecht
Brigham and Women’s Hospital Department of Reproductive Medicine
Department of Obstetrics and Utrecht, The Netherlands
Gynecology Boston, MA, USA [email protected]
[email protected]
Douglas T. Carrell, Ph.D., H.C.L.D
Peter N. Schlegel, M.D. Andrology and IVF Laboratories
New York Presbyterian Hospital Departments of Surgery (Urology)
Weill Cornell Medical Center Obstetrics and Gynecology and
Department of Urology Physiology
New York, NY, USA University of Utah School of Medicine
[email protected] Salt Lake City, UT, USA
[email protected]
ISBN 978-1-4419-8455-5 e-ISBN 978-1-4419-8456-2

DOI 10.1007/978-1-4419-8456-2
Springer New York Dordrecht Heidelberg London
Library of Congress Control Number: 2011928231
© Springer Science+Business Media, LLC 2011

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Springer is part of Springer Science+Business Media (www.springer.com)

We dedicate this book to Robert G. Edwards for his vision,
creativity, and determination in the development of in vitro
fertilization as a successful therapeutic option for infertility
patients and for his enormous influence in the ethics and
politics that challenge our field.
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Preface
The initial volume of Biennial Review of Infertility was published in 2009.

In the preface to that volume we shared our vision that this series would serve
as a forum for evidence-based reviews of cutting-edge topics in the field of
infertility, written by experts in the field and accessible and applicable to
clinicians and researchers alike. We also began a tradition of highlighting and
providing contrasting reviews to emerging and controversial topics. We are
very pleased with the response we have had to Biennial Reviews of Infertility,
Volume 1, and are excited now to present Volume 2.
The exponential growth of technologies and of data generated in research
studies can be both breathtaking and daunting. The expansion in the number
of manuscripts relevant to understanding infertility is growing not only due to
growth in the number of researchers, but also due to emerging technologies,
newly developing fields of study, and broad collaboration between diverse
specialties. This volume includes discussion of cutting-edge topics such as
epigenetics, proteomics, and the role of the environment in infertility, along
with evidence-based discussion of routine clinical procedures. Together,
these diverse topics are likely to benefit a wide spectrum of healthcare profes-
sionals involved in the study and treatment of infertility by providing both
broad perspective and pointed practical advice.
We have all recently felt great excitement with the announcement of the
awarding of the Nobel Prize in Medicine to Dr. Robert G. Edwards for his
contributions to the development of in vitro fertilization as a therapy to millions
of infertile patients. Dr. Edward’s influence has been huge, not only in the
science underpinning infertility, but also in the ethical and political arenas
that impact our field. His contributions and the interface of these disciplines
in our daily practices bring us to reflect on the advances we have made in
understanding and treating infertility, and on the challenges that lay ahead.
Such reflection can aid and inspire us in our quest to discover new insights
through our studies for improved care of our patients. We hope that this volume
of Biennial Review of Infertility can serve as a valuable reference and tool to
implement “best practices” and to aid in the development of more accurate
diagnoses and more effective treatments of infertility.
Boston, MA, USA Catherine Racowsky

New York, NY, USA Peter N. Schlegel
Utrecht, The Netherlands Bart C. Fauser
Salt Lake City, UT, USA Douglas T. Carrell
vii
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Contents
Part I Female Infertility
1 Autoimmunity and Female Infertility: Fact vs. Fiction............ 3

Lawrence N. Odom, Amy M. Cline, and William H. Kutteh
2 Minimal Stimulation IVF............................................................ 11
Ahmad O. Hammoud and Mark Gibson
3 Current Understanding of Anti-Müllerian Hormone.............. 19
Dimitrios G. Goulis, Marina A. Dimitraki,
and Basil C. Tarlatzis
4 The Role of Obesity in Reproduction......................................... 35

Barbara Luke
5 Endometrial Receptivity in Natural and Controlled
Ovarian-Stimulated Cycles......................................................... 43
José A. Horcajadas, José A. Martínez-Conejero,
and Carlos Simón
6 Current Understanding of Mullerian-Inhibiting
Substance...................................................................................... 57
Antonio La Marca, Giovanna Sighinolfi,
and Annibale Volpe
7 Evidence-Based Use of Progesterone During IVF.................... 79
Elena H. Yanushpolsky
8 Monozygotic Twinning and Perinatal Outcomes...................... 91
Kenneth J. Moise Jr. and Ramesh Papanna
9 Multiple Pregnancy Vanishing Twin Syndrome........................ 103
Gabriel de la Fuente, Jose Manuel Puente,
Juan A. García-Velasco, and Antonio Pellicer
Part II Male Infertility
10 The Effect of Cancer Therapies on Sperm:

Current Guidelines...................................................................... 117
Akanksha Mehta and Mark Sigman
ix
x Contents
11 Environmental Insults on Spermatogenesis............................... 133

Stefan S. du Plessis and Ashok Agarwal
12 Sperm DNA Damage: Causes and Guidelines
for Current Clinical Practice...................................................... 155
Aleksander Giwercman, Marcello Spanò, and Mona Bungum
13 The Emerging Role of the Sperm Epigenome
and its Potential Role in Development....................................... 181
Sue Hammoud and Douglas T. Carrell
Part III Assisted Reproduction Techniques
14 ART and Epigenetic Disorders:

Should We Be Concerned?.......................................................... 197
Christopher N. Herndon and Paolo F. Rinaudo
15 Novel Approaches of Sperm Selection for ART:
The Role of Objective Biochemical Markers of Nuclear
and Cytoplasmic Integrity and Sperm Function...................... 211
Gabor Huszar and Denny Sakkas
16 The Role of the Oocyte in Remodeling of Male
Chromatin and DNA Repair: Are Events During
the Zygotic Cell Cycle of Relevance to ART?............................ 227
Liliana Ramos and Peter de Boer
17 Proteomic/Metabolomic Analysis of Embryos:
Current Status for Use in ART................................................... 245
Mandy G. Katz-Jaffe and Susanna McReynolds
18 Ultrasound-Guided Embryo Transfer....................................... 255
Robert L. Gustofson and William B. Schoolcraft
Part IV Evolving Controversies in Contemporary

Reproductive Medicine
19 IMSI as a Valuable Tool for Sperm Selection

During ART.................................................................................. 263
Monica Antinori, Pierre Vanderzwalmen, and Yona Barak
20 Thoughts on IMSI........................................................................ 277
Gianpiero D. Palermo, Jennifer C.Y. Hu, Laura Rienzi,
Roberta Maggiulli, Takumi Takeuchi, Atsumi Yoshida,
Atsushi Tanaka, Hiroshi Kusunoki, Seiji Watanabe,
Queenie V. Neri, and Zev Rosenwaks
Index...................................................................................................... 291
Contributors
Ashok Agarwal, PhD, HCDL Center for Reproductive Medicine,

Cleveland Clinic, Cleveland, OH, USA
Monica Antinori, MD Infertility Unit, RAPRUI Day Hospital, Rome, Italy
Yona Barak, PhD Dr. Yona Barak Laboratories Ltd, Rosh HaAyin, Israel
Mona Bungum, PhD Reproductive Medicine Centre,
Skåne University Hospital, Malmö, Sweden; Department of Clinical
Sciences, Lund University, Malmö, Sweden
Douglas T. Carrell, PhD, HCLD Andrology and IVF Laboratories,
Departments of Surgery (Urology), Obstetrics and Gynecology
and Physiology, University of Utah School of Medicine,
Salt Lake City, UT, USA
Amy M. Cline, MD, PhD Division of Reproductive Endocrinology,
Department of Obstetrics and Gynecology, The University of Tennessee
Health Science Center, Memphis, TN, USA
Peter de Boer, PhD Department of Obstetrics and Gynecology,
Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands
Gabriel de la Fuente, MD IVI Madrid & Rey Juan Carlos University,
Madrid, Spain
Marina A. Dimitraki, MD Section of Reproductive Medicine, First
Department of Obstetrics and Gynecology, Medical School – Aristotle
University of Thessaloniki, Thessaloniki, Greece
Stefan S. du Plessis, PhD Division of Medical Physiology,
Stellenbosch University, Tygerberg, South Africa
Juan A. García-Velasco, MD, PhD IVI Madrid & Rey Juan Carlos Uni-
versity, Madrid, Spain
Aleksander Giwercman, MD, PhD Reproductive Medicine Centre,
Skåne University Hospital, Malmö, Sweden;
Department of Clinical Sciences, Lund University, Malmö, Sweden
Mark Gibson, MD Utah Center for Reproductive Medicine, Department of
Obstetrics and Gynecology, University of Utah School of Medicine,
xi

xii Contributors
Dimitrios G. Goulis, MD, PhD Section of Reproductive Medicine,

First Department of Obstetrics and Gynecology, Medical School – Aristotle
Robert L. Gustofson, MD Colorado Center for Reproductive Medicine,
Lone Tree, CO, USA
Ahmad O. Hammoud, MD, MPH Utah Center for Reproductive
Medicine, Department of Obstetrics and Gynecology, University of Utah,
School of Medicine Salt Lake City, UT, USA
Sue Hammoud, BS Andrology and IVF Laboratories, Department
of Surgery (Urology), University of Utah School of Medicine, Cairns
Laboratory, Huntsman Cancer Institute, Salt Lake City, UT, USA
Christopher N. Herndon, MD Division of Reproductive Endocrinology
and Infertility, Department of Obstetrics, Gynecology and Reproductive
Sciences, University of California San Francisco, San Francisco, CA, USA
José A. Horcajadas, PhD Fundación IVI-Instituto Universitario
IVI-University of Valencia, Valencia, Spain
Gabor Huszar, MD Sperm Physiology Laboratory,
Department of Obstetrics, Gynecology and Reproductive Sciences,
Yale University School of Medicine, New Haven, CT, USA
Jennifer C.Y. Hu, MSc The Ronald O. Perelman and Claudia Cohen
Center for Reproductive Medicine, Weill Cornell Medical Center,
New York, NY, USA
Mandy G. Katz-Jaffe, PhD Colorado Center for Reproductive Medicine,
Lone Tree, CO, USA;
National Foundation for Fertility Research, Lone Tree, CO, USA
Hiroshi Kusunoki, PhD Faunal Diversity Science, Graduate School
of Agriculture, Kobe University, Kobe, Japan
William H. Kutteh, MD, PhD Division of Reproductive Endocrinology,
Antonio La Marca, MD, PhD Mother-Infant Department, Section
of Obstetrics and Gynecology, University of Modena and Reggio Emilia,
Modena, Italy
Barbara Luke, ScD, MPH Department of Obstetrics, Gynecology, and
Reproductive Biology, Michigan State University, East Lansing, MI, USA
Susanna McReynolds, PhD National Foundation for Fertility Research,
Lone Tree, CO, USA
Roberta Maggiulli, MSc Genera Center for Reproductive
Medicine, Valle Giullia Clinic, Rome, Italy
José A. Martínez-Conejero, PhD Fundación IVI-Instituto Universitario
IVI-University of Valencia, Valencia, Spain
Contributors xiii
Akanksha Mehta, MD Division of Urology, Rhode Island Hospital,

Warren Alpert Medical School at Brown University, Providence, RI, USA
Kenneth J. Moise Jr., MD Department of Obstetrics and Gynecology,
Division of Maternal-Fetal Medicine, Baylor College of Medicine
and the Texas Children’s Fetal Center, Texas Children’s Hospital,
Houston, TX, USA
Queenie V. Neri, BSc, MSc Candidate The Ronald O. Perelman
and Claudia Cohen Center for Reproductive Medicine, Weill Cornell
Medical Center, New York, NY, USA
Lawrence N. Odom, MD Division of Reproductive Endocrinology,
Gianpiero D. Palermo, MD The Ronald O. Perelman and Claudia
Cohen Center for Reproductive Medicine, Weill Cornell Medical College,
New York, NY, USA
Ramesh Papanna, MD, MPH Department of Obstetrics
and Gynecology, Division of Maternal-Fetal Medicine,
Baylor College of Medicine and the Texas Children’s Fetal Center,
Texas Children’s Hospital, Houston, TX, USA
Antonio Pellicer, MD, PhD IVI Valencia & Valencia University, Valencia,
Spain
José Manuel Puente, MD IVI Madrid & Rey Juan Carlos University,
Madrid, Spain
Liliana Ramos, PhD Department of Obstetrics and Gynecology,
Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands
Laura Rienzi, MSc Genera Center for Reproductive Medicine,
Valle Giullia Clinic, Rome, Italy
Paolo F. Rinaudo, MD, PhD Department of Obstetrics, Gynecology
and Reproductive Sciences, Division of Reproductive Endocrinology
and Infertility, University of California, San Francisco, CA, USA
Zev Rosenwaks, MD The Ronald O. Perelman and Claudia Cohen
Center for Reproductive Medicine, Weill Cornell Medical Center,
New York, NY, USA
Denny Sakkas, PhD IVF Laboratories, Department of Obstetrics,
Gynecology and Reproductive Sciences, Yale University School
of Medicine, New Haven, CT, USA
William B. Schoolcraft, MD Colorado Center for Reproductive Medicine,
Lone Tree, CO, USA
Giovanna Sighinolfi, MD Mother-Infant Department, Section
of Obstetrics and Gynecology, University of Modena and Reggio Emilia,
Modena, Italy

xiv Contributors
Mark Sigman, MD Division of Urology, Rhode Island Hospital,

Warren Alpert Medical School, Brown University, Providence, RI, USA
Carlos Simón, MD, PhD Fundación IVI-Instituto
Universitario IVI-University of Valencia, Valencia, Spain;
Valencia Stem Cell Bank, Centro de Investigaciones Principe Felipe,
Valencia, Spain
Marcello Spanò, PhD Laboratory of Toxicology, Unit of Radiation
Biology and Human Health, UTBIORAD-TOSS, ENEA Casaccia
Research Center, Rome, Italy
Takumi Takeuchi, MD, PhD The Reproduction Center, Kiba Park Clinic,
Koto-ku, Tokyo, Japan
Atsushi Tanaka, PhD Saint Mother Hospital, Kitakyushu-City,
Fukuoka, Japan
Basil C. Tarlatzis, MD, PhD Section of Reproductive Medicine, First
Department of Obstetrics and Gynecology, Medical School – Aristotle
Pierre Vanderzwalmen, PhD IVF Centers Prof. Zech, Bregenz, Austria;
Centre Hospitalier Inter Régional Cavell (CHIREC),
Braine l’Alleud, Brussels, Belgium
Annibale Volpe, MD Mother-Infant Department, Section of Obstetrics
and Gynecology, University of Modena and Reggio Emilia, Modena, Italy
Seiji Watanabe, PhD Department of Anatomical Science,
Hirosaki University Graduate School of Medicine, Hirosaki, Japan
Elena H. Yanushpolsky, MD Department of Obstetrics and Gynecology,
Brigham and Women’s Hospital, Boston, MA, USA
Atsumi Yoshida, MD The Reproduction Center, Kiba Park Clinic,
Koto-ku, Tokyo, Japan
Part I
Female Infertility
Autoimmunity and Female
Infertility: Fact vs. Fiction 1
Lawrence N. Odom, Amy M. Cline,
and William H. Kutteh
Abstract
Several autoimmune factors have been investigated as possible influences
on reproductive success of both natural conception and conception by the
use of assisted reproductive technologies. In order for a pregnancy to suc-
ceed, two immunologically and genetically distinct tissues must coexist.
During implantation, local and systemic immune factors, cytokines, and
growth factors may interact with adhesion molecules and other matrix-
associated proteins, glycoproteins, and peptides. Antiphospholipid anti-
bodies are identified more frequently in women undergoing in vitro
fertilization, but their presence does not appear to influence the outcome
of pregnancy, miscarriage, or live birth rates. Antithyroid antibodies are
commonly found in women of reproductive age, but implantation rates
and miscarriage rates are not altered when women have normal thyroid
function. Antinuclear antibodies may be a marker for underlying autoim-
mune disease when coupled with certain signs and symptoms, but low titer
antibodies do not influence in vitro fertilization outcome. Antisperm anti-
bodies are more often associated with fertilization failure when found in
high titers in seminal plasma, on sperm, or in the mucosal immune system
of women. Antiovarian antibodies are uncommon, but most often associ-
ated with ovarian hypofunction. Other autoimmune factors are under
investigation as markers of in vitro fertilization failure.
Keywords
Antiphospholipid antibodies • Antinuclear antibodies • Antithyroid
antibodies • Antiovarian antibodies • Antisperm antibodies • Infertility
• In vitro fertilization
W.H. Kutteh ()
Division of Reproductive Endocrinology,
Department of Obstetrics and Gynecology,
The University of Tennessee Health Science Center,
Memphis, TN, USA
e-mail: [email protected]
C. Racowsky et al. (eds.), Biennial Review of Infertility: Volume 2, 3

DOI 10.1007/978-1-4419-8456-2_1, © Springer Science+Business Media, LLC 2011
4 L.N. Odom et al.
fertilized ova will generate a live birth and 50%

1.1 Introduction appear to fail at time of implantation. Chromo
somal abnormalities alone do not account for
Approximately 10–15% of couples desiring
this number of pregnancy losses, meaning that a
children suffer from infertility. Despite thorough
large number of these losses are otherwise nor-
investigation, the cause of infertility remains
mal human embryos. Human preimplantation
unknown in at least 10% of these couples.
embryos express major histocompatability anti-
“Reproductive autoimmune failure syndrome”
gens theoretically capable of inducing an immune
was originally described by Gleicher et al. [1] in
response [5, 6]. The possibility exists that mater-
women with endometriosis, infertility, and
nal immune responses play a role in the failure of
increased autoantibodies. Autoimmunity refers
implantation [7, 8].
to an immune reaction of the body against sub-
stances normally present in the body. Since the
mention of this reproductive autoimmune failure
syndrome, numerous studies have been per-
1.2 Autoimmune Factors
formed in an attempt to identify specific factors Potentially Related
or antibodies associated with pregnancy loss and to Pregnancy Failure
infertility.
Implantation is one of the most important Recent studies have investigated the role of auto-
aspects of pregnancy that these studies have tar- immune factors in implantation in women under-
geted. Implantation represents a critical develop- going fertility treatment. The most commonly
mental process in that it requires the interaction studied antibodies include antiphospholipid
of immunologically and genetically distinct tis- antibodies, antithyroid antibodies, antinuclear anti
sues. The immune system may influence preg- bodies, antiovarain antibodies, and antisperm
nancy success or failure during any of the critical antibodies.
steps of implantation. First, the blastocyst must
hatch from the zona pellucida to attach to the epi-
thelium of the uterus. Second, apposition occurs 1.2.1 Antiphospholipid Antibodies
when L-selectin on the blastocyst interacts with
the endometrial surface expressing L-selectin Antiphospholipid antibodies are present in an
ligand [2]. Next, hCG secreted from the human estimated 2–5% of the general population [9] and
blastocyst induces troponin expression in human form a heterogenous group of antibodies that tar-
endometrial epithelial cells enriched in pinopo- get negatively charged phospholipids via interac-
des [3]. Finally, the outer trophoblast layer must tion with phospholipid-binding plasma protein
breach the epithelium and invade the underlying [10]. Antiphospholipid antibodies are commonly
stroma and vasculature so as to establish a direct associated with other autoimmune diseases, such
contact with maternal blood flow. as systemic lupus erythematosus, but can also
Attachment occurs only during the “implanta- present in isolation in the form of primary
tion window,” the time period when the epithe- antiphospholipid syndrome [11]. Antiphos
lium is receptive. This period is from day 19–23 pholipid syndrome was first described in 1983 in
of a 28-day menstrual cycle. The preimplantation patients with concurrent lupus, the presence of
embryo must be at a developmental point such anticardiolipin antibodies, and thrombosis [12].
that it is capable of attaching to the endometrium Since this initial presentation, the criteria for
of the uterus. Failure of this synchronization pre- diagnosis have been revised with the most recent
cludes success, as demonstrated in human studies revision in 2006. In order for a patient to be diag-
of implantation [4]. nosed with antiphospholipid syndrome, at least
Implantation is the most important limiting one of the clinical criteria and one of the labora-
factor in human reproduction. Only 25% of all tory criteria must be met as follows [13].
1 Autoimmunity and Female Infertility: Fact vs. Fiction 5
1.2.1.1 Clinical Criteria Antiphospholipid antibodies have been found

1. Vascular thrombosis: One or more episodes of in 15% of patient with recurrent first trimester
arterial, venous, or small vessel thrombosis in loss [17] and up to 9% in patients with unex-
any tissue or organ. plained infertility and recurrent implantation
2. Morbidity in pregnancy failure [18]. Treatment of patients with antiphos-
(a) One or more unexplained deaths of a mor- pholipid antibody-associated recurrent pregnancy
phologically normal fetus at or beyond loss with heparin and low-dose aspirin has been
the tenth week of gestation. shown to improve live birth rates [14]. Treatment
(b) One or more premature births of a morpho- of patients with recurrent pregnancy loss and
logically normal neonate prior to the 34th antiphospholipid antibodies with low molecular
week of gestation secondary to eclampsia weight heparin and aspirin has also been shown to
or severe preeclampsia or recognized fea- have similar obstetrical outcomes as well as safety
tures of placental insufficiency. parameters compared to treatment with unfrac-
(c) Three or more unexplained consecutive tionated heparin and aspirin [19]. However, recent
spontaneous miscarriages before the tenth studies have not shown a benefit of treatment with
week of gestation. unexplained recurrent pregnancy loss [20].
Several published reports indicate that posi-
tive APAs are found more frequently in patients
1.2.1.2 Laboratory Criteria undergoing IVF or who have failed IVF [21].
(Must be present on two or more occasions at However, positive antiphospholipid antibodies
least 12 weeks apart): have not been associated with decreased preg-
1. Lupus anticoagulant present. nancy rates in women undergoing IVF [22] and
2. Anticardiolipin antibody; medium or high titer treatment with heparin and aspirin of women
(>40 mg of IgG or IgM phospholipid or >99th undergoing IVF who concurrently test positive
percentile) of IgG or IgM isotype. for antiphospholipid antibodies does not improve
3. Anti-b2-glycoprotein; medium or high titer pregnancy or implantation rates [23].
(>40 mg of IgG or IgM phospholipid or >99th
percentile) of IgG or IgM isotype.
The presence of antiphospholipid antibodies 1.2.2 Antithyroid Antibodies
can have a tremendous impact on reproductive
success. Antiphospholipids interact with the Antithyroid antibodies, specifically thyroglobu-
maternal–fetal interface in multiple aspects and lin and thyroid peroxidase antibodies, are com-
are associated with recurrent spontaneous mis- monly found in patients with Graves’ disease,
carriage [14] as well as preeclampsia, intrauterine postpartum thyroiditis, and Hashimoto’s thy-
growth restriction, and fetal demise. The involve- roiditis. However, antithyroid antibodies have
ment of antiphospholipid antibodies with preg- been reported in healthy individuals and are
nancy is thought to be more of an autoimmune observed more frequently in women during their
factor than a thrombophilic factor as exhibited by reproductive years [24]. The prevalence of anti-
histological studies showing a lack of intravas- thyroid antibodies has been reported in 15–20%
cular or intervillous blood clots in placentas of normal pregnant women and women under-
obtained following spontaneous miscarriage [15]. going assisted reproductive techniques compared
Studies have linked antiphospholipid antibodies to 20–25% in women with recurrent miscarriage
with decreased release of hCG from human [25], and on average, 46% of pregnant women
placental explants, prevention of in vitro tropho- with a diagnosis of hypothyroidism [26].
blast migration and invasion, inhibition of Multiple studies have investigated the role of
trophoblast cell adhesion molecules, and activa- thyroid autoimmunity in infertility, but the inter-
tion of complement on the trophoblast surface pretation of the evidence as a whole is difficult
inducing an inflammatory response [16]. secondary to numerous variations in study design.
6 L.N. Odom et al.
Some studies have shown a significantly increased antinuclear antibody titers were discovered in
relative risk for thyroid autoimmunity among 27% of patients with endometriosis compared to
endometriosis [27, 28], which can also be linked 18% of patients without endometriosis [36].
to infertility. However, others have failed to show Implantation and pregnancy rates have been
this association [29]. Other studies have shown shown to improve with short-term medication-
an association with ovarian causes of infertility, induced immunosuppression. However, this
such as polycystic ovarian syndrome. Janssen et al. treatment has not been shown to improve live
[30] reported a relative risk of 3.2 (CI 1.9–5.6) of birth rates [37] and treatment of patients with
both thyroglobulin and thyroid peroxidase in positive antinuclear antibodies with heparin and
females with infertility and polycystic ovarian aspirin failed to show an improvement in implan-
syndrome compared to age-matched controls, tation and pregnancy rates [38]. A study per-
suggesting that multifactorial causes as opposed formed recently [34] found elevated antinuclear
to isolated thyroid autoimmunity may be respon- antibody titers in 97 (50%) women with recurrent
sible for infertility. pregnancy loss and in only 16 (16%) of age-
Another theory is that hypothyroidism result- matched controls, but also state that the signifi-
ing from thyroid autoantibodies may be respon- cance of this finding is yet to be determined.
sible for the increased incidence of infertility in
this population. Thyroid hormone receptors have
been described in human oocytes where they 1.2.4 Antiovarian Antibodies
assist in the stimulation of granulosa cell func-
tion [31] and trophoblastic differentiation [32]. Antiovarian antibodies include antibodies against
Cramer et al. [33] showed an increased risk of a heterogeneous group of antigens, including
in vitro fertilization in women with infertility and molecular targets in the zona pellucida, theca
elevated levels of thyroid-stimulating hormone, interna, granulosa cells, ooplasm [39], and heat
suggesting an association of hypothyroidism with shock protein 90-b [40]. Studies have suggested
adverse reproductive potential. Studies on the numerous associations of antiovarian antibodies
treatment of subclinical hypothyroidism are with infertility, such as reduced fertilization rates
widely variable [26] and current guidelines on and pregnancy rates, inhibited response to gonad-
screening patients with infertility for thyroid dys- otropin stimulation, altered egg and embryo
function and autoimmunity are conflicted. There development, and possibly implantation failures
are insufficient data to recommend screening [39]. One pilot study showed an improvement in
asymptomatic infertile women for autoimmune pregnancy rate, implantation rate, and live birth
thyroid dysfunction. rate with prednisolone administration to patients
with antiovarian antibodies and at least two pre-
viously failed IVF attempts [41]. The authors
1.2.3 Antinuclear Antibodies [41] concluded that corticosteroids are useful in
a subset of patients with IVF failure and auto-
Antinuclear antibodies are a group of antibodies immunity. Data are still inadequate to provide a
that target nuclear and cytoplasmic antigens. solid link between antiovarian antibodies and
These antigens are essential to cell function infertility, but their presence may be linked to
through playing a role in transcription, transla- ovarian hypofunction.
tion, and cell cycle regulation [34]. A positive
antinuclear antibodies titer is associated with
multiple autoimmune disorders such as systemic 1.2.5 Antisperm Antibodies
lupus erythematosus [34]. The role of antinuclear
antibodies in infertility is largely undetermined; Sperm contain antigens that are foreign to both
however, they have been associated with implan- male and female immune systems. Antisperm
tation failure secondary to an endometriosis- antibody production may be induced in the semi-
induced autoimmune reaction [35]. Elevated nal plasma, in male or female serum, or in the
Table 1.1 Associations of autoantibodies with female infertility

Autoantibody Frequency in infertile women Infertility association Known associations
Antiphospholipid Increased Unproven Recurrent pregnancy loss
Antithyroid No difference Unproven Thyroiditis
Antisperm No difference Unproven Fertilization failure
Antinuclear Slightly increased Unproven Autoimmune disease
Antiovarian Slightly increased Unproven Ovarian failure
cervical mucus when sperm are exposed to the Unfortunately, the majority of available data
immune system [42]. Antisperm antibodies have on the role of immunity in infertility is hindered
been identified in 10–15% of men with infertility by small or poorly conducted studies. This limits
and in 15–20% of women with unexplained infer- the ability to form definitive recommendations
tility. The prevalence and presumed significance for the screening and treatment of autoimmunity
reported depends on the population, source of the in the infertile population. While the existence of
specimen (serum, cervical mucus, semen), and two immunologically distinct organisms during
method of testing. These antibodies are postu- pregnancy suggests an essential role of the immune
lated to interfere with the fecundity process system in fertility, additional studies are needed
through various mechanisms, such as interfer- to suggest treatments and recommendations for
ence with sperm transport within the female gen- immune modulation and screening in patients
ital tract, alteration of sperm capacitation or with infertility (Table 1.1).
acrosomal reaction, interference with fertiliza-
tion, or inhibition of implantation of the early Acknowledgments Funding: Frank Ling Research Grant
in Obstetrics and Gynecology.
embryo. Possible sites where sperm-bound anti-
sperm antibodies might interfere with fertiliza-
tion include sperm binding to zona pellucida,
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Minimal Stimulation IVF
Ahmad O. Hammoud and Mark Gibson
2
Abstract
Minimal stimulation IVF was utilized in the early IVF experiences. It is pro-
posed now as a solution for the unwanted consequences and costs of current
conventional IVF protocols. Minimal stimulation IVF is thought to be a
means to achieve some of the fertility-enhancing effects of IVF while mini-
mizing discomforts, risks (especially of ovarian hyperstimulation syndrome),
and costs. An additional benefit is a marked reduction in the likelihood of
unused embryos. In aggregate, these advantages should increase the access to
and acceptability of IVF for many potential patients. While the per cycle
pregnancy rate in minimal stimulation IVF is lower than that of conventional
protocols, proponents of this method cite increased patient tolerance and
access that allow multiple efforts, with a cumulative success rate that can
approach that of a single cycle of conventional IVF (Curr Opin Obstet Gynecol
22:189–192, 2010). Minimal stimulation IVF is now being offered in many
fertility clinics both to young patients with good prognosis and to poor
responders and women of advanced age as an alternative to conventional pro-
tocols. The renewed interest in minimal stimulation IVF is largely a result of
improved outcomes in the IVF laboratory that have led to higher likelihoods
of viable embryos and better success rates with single embryo transfer.
Keywords
IVF • Hyperstimulation • Gonadotropins • Minimal stimulation • Embryo
transfer
A.O. Hammoud (*)
Utah Center for Reproductive Medicine,
University of Utah School of Medicine,

12 A.O. Hammoud and M. Gibson
with or without concomitant GnRH antagonist

2.1 Classification and Terminology for suppression of the endogenous LH surge.
The field of minimal stimulation IVF is becom-
ing popular and several recent publications have
2.1.3 Mild IVF
described the success with various stimulation
protocols [1–4]. It is not uncommon to refer to
A mild IVF cycle is defined by use of oral agents
minimal stimulation IVF as mild stimulation IVF
(antiestrogens or aromatase inhibitors) and/or low-
or low-dose IVF. The various protocols and
dose gonadotropins to modestly increase oocyte
terminology used in this field make the com-
yields (2–7 oocytes). LH surge suppression with
parison between studies challenging (Table 2.1).
GnRH antagonist and triggering with hCG or
An interested group of experts from the Inter
GnRH agonist is followed by luteal support.
national Society for Mild Approaches in Assisted
Reproduction (ISMAAR) met and proposed the
following classifications [5].
2.1.4 Conventional IVF
This term is used to define scenarios in which

2.1.1 Natural Cycle IVF
conventional gonadotropin dosing is employed to
achieve maximum controlled ovarian hyperstim-
The term Natural cycle IVF should be used when
ulation below the threshold for significant risk of
IVF is carried out with oocytes collected from a
OHSS. In all such scenarios, endogenous LH is
woman’s ovary or ovaries in a spontaneous men-
suppressed with GnRH agonist in long or flare
strual cycle without administration of any medi-
protocols, or with GnRH antogonist, triggering
cation at any time during the cycle. The aim of
employs hCG or GnRH agonist, and luteal sup-
this cycle is to collect a naturally selected single
port is given.
oocyte at the lowest possible cost.
2.1.2 Modified Natural Cycle IVF 2.2 Adoption of Minimal

Stimulation IVF
The term Modified natural cycle should be applied
when exogenous hormones or any drugs are used There are currently several proposed indications
when IVF is being performed during a spontane- for minimal stimulation IVF, including young
ous cycle with the aim of collecting a naturally patients with male factor or tubal factor infertility,
selected single oocyte, but with a reduction in poor responders, and patients with prior implan-
chance of cycle cancelation. Modified natural tation failures [6, 7]. The rationale for its use in
cycle IVF employs hCG triggering of ovulation, poor responders is that comparable oocyte yields
Table 2.1 Different protocols of minimal stimulation IVF

Prevention of
Ovarian stimulation premature LH surge Ovulation trigger Luteal phase support
Natural cycle IVF None None None None
Modified natural None None hCG Yes
cycle IVF Gonadotropins add back GnRH antagonist hCG Yes
Mild IVF Clomiphene, letrozole, GnRH antagonist hCG, or GnRH Yes
early or late low-dose agonist
gonadotropins
Conventional IVF High-dose gonadotropins GnRH agonist or hCG, or GnRH Yes
antagonist agonist
2 Minimal Stimulation IVF 13
are obtained without the costs and intrusiveness 202 natural cycle IVFs were performed [4]. All
of high-dose conventional dosing, but success of women who participated in this study had normal
this approach for these patients has been mixed menstrual function and normal semen parameters
[7]. Adoption of minimal stimulation IVF has in the male partner. The median age was 34 years
been slow, particularly in the United States where with a range of 24–40 years. The protocol for
it is not offered in most centers. The slow accep- natural cycle IVF in this study included initiating
tance of minimal stimulation IVF is thought in follicular scan on day 8 or 9 of a natural cycle,
part to be due to reluctance of clinics to use pro- ultrasound monitoring was repeated as appropri-
tocols that might adversely affect their published ate, and hCG 5,000 IU was administered when
success rates in a competitive marketplace. Other the follicular diameter reached 16–18 mm. There
factors include the often smaller margin of profit- was no follicular growth documented in 21
ability, reduced number of embryo available for cycles. In the 181 cycles where oocyte retrieval
cryopreservation, and health plans that limit the was attempted, pregnancy rate per cycle was
benefits to a certain number of IVF cycles [6]. 12.7% and live birth rate was 8.8%. After four
cycles, the cumulative pregnancy rate was 46%
and cumulative birth rate was 32% [4]. A sub-
group of this cohort received Indomethacin 50 mg
2.3 Comparison of Different three times daily which was administered from
Protocols for Minimal Friday until Monday morning to allow delaying
Stimulation IVF hCG administration so that all retrievals could
occur on week days. Of these subjects, the rate of
2.3.1 Modified Natural Cycle IVF oocyte retrieval was 90.4%, oocyte fertilization
71%, and pregnancy rates per cycle 9.6% [4].
Natural Cycle IVF was the protocol used in the McDougall et al. compared modified natural
early publications describing IVF [8]. Since then, IVF to IVF after stimulation with clomiphene
several changes were introduced to the normal 100 mg daily from day 3–7. The cancelation rate
cycle IVF, mainly the control of the LH surge and in the modified natural IVF cycle (4/14) was
modified oocyte retrieval methods. Natural IVF higher than that in the group stimulated with clo-
cycles have an inherently high cancelation miphene (0/16). The clinical pregnancy rate was
rate because of premature LH surge, premature lower in modified natural IVF group (0%) when
ovulation, and increased risk of failed oocyte compared to that after clomiphene stimulation
retrieval [4]. Because of the unpredictable nature (18% per transfer) [11]. In a later study, Ingerlev
of the natural LH surge, early natural IVF cycles et al. compared modified natural cycle IVF to
required intense and frequent monitoring and clomiphene stimulation. This study included
around the clock availability of the IVF team and good prognosis young patient (<35 years), with
laboratory to achieve a successful retrieval [2]. unexplained, tubal, or severe male factor infertil-
Controlling the timing of ovulation was one of the ity and regular cycles. The proportion of cycle
main achievements that improved the feasibility that resulted in embryo transfer (53.2%) was
of natural cycle IVF. This was achieved with the higher in the clomiphene group when compared
administration of hCG to trigger the ovulation and to that in the modified natural cycle IVF group
later the introduction of GnRH antagonist to sup- (25.4%). The clinical pregnancy rates in the clo-
press the endogenous LH surge. Other less known miphene group (18% per cycle and 33.9% per
methods to prevent a premature LH surge include transfer) were higher than those in the modified
endomethacin and clomiphene use [9, 10]. natural IVF group (3.5 and 13.8%, respectively)
The introduction of hCG injection to trigger [12]. In women with previous poor response (less
ovulation helped reduce the cancelation rates than four follicles), modified natural cycle IVF
with natural IVF. In a study that included had higher implantation rates (14.9%) when com-
35 women with infertility and tubal damage and 17 pared to the GnRH agonist microflare protocol
women with reduced ovarian reserve, a total of (5.5%); however, the ongoing pregnancy rates
were similar (6.1% in the natural cycle group and an average of nine cycles of modified natural
6.9% in microflare protocol) [13]. cycle (using GnRH antagonist) and reported a
While natural IVF cycle with utilization of modest 8% ongoing pregnancy rate per cycle in a
hCG to trigger ovulation is classified as modified cohort of 268 patients aged 18–36 with regular
natural IVF, true modified natural IVF cycle refers ovulatory function [14]. In patient with previ-
mainly to the utilization of GnRH antagonists to ously poor response with conventional IVF, the
prevent premature LH surge. The administration success of modified natural IVF cycle was
of gonadotropins helps supplement the natural reported to be between 0 and 14% [2].
gonadotropins expected to fall after the adminis-
tration of GnRH antagonist, which helps maintain
the follicular growth and the estradiol levels [14]. 2.3.2 Mild IVF
Pelinck et al. described a protocol for modi-
fied natural IVF cycle as follows: During sponta- Mild stimulation IVF can be done using
neous unstimulated cycles, follicular scans were antiestrogens for ovulation induction such as
initiated on cycles days 3 or 8, then repeated daily clomiphene or letrozole or using low-dose
or every other day. A mean follicular diameter of gonadotropins. Typically, endogenous LH is sup-
14 mm was used to determine the need to start the pressed by addition of GnRH antagonist once
GnRH antagonist to prevent premature LH surge. folliculogenesis is underway. Proponents of
At the same day, 150 IU of recombinant FSH milder stimulation propose several advantages to
(rFSH) was also started. The GnRH antagonist this protocol including: the possibilities of a
and rFSH were continued daily until the day of more receptive endometrium and better quality
triggering of ovulation. The follicular growth was embryos as well as less stress for patient and
monitored with daily or every other day morning lower overall costs [16]. This approach may be
follicular scans, LH, and estradiol levels. hCG most effective in young patients with normal
10,000 IU was given when a mean follicular ovarian function and good prognosis. Advantages
diameter of 18 mm and or an estrogen levels of of oral antiestrogen when compared to gonado-
0.8 nmol/L (218 pg/dL) were reached. Cycle can- tropins includes oral administration, lower costs,
celation occurred if there was premature LH and wider availability [2].
surge documented by an elevated LH levels A mild stimulation cycle using clomiphene
³20 IU/L (if the mean follicular diameter was can start with 100 mg of clomiphene from day 3
less than 15 mm), or regardless of the follicular to day 7 of the cycle. Gonadotropins (HMG or
diameter, if the LH levels were ³30 IU/L. Oocyte rFSH) are given at a dose of 150 IU at day 9 of
retrieval occurred 34 h after hCG injection. the cycle. Ultrasound monitoring starts at day 9
Analgesia was only given at patient’s request. In of the cycle and then frequently after that. GnRH
this protocol, embryo transfer occurred on day 3 antagonist is started when the lead follicle reaches
and luteal support was provided through hCG 14 mm in diameter and is continued until the day
injections of 1,500 IU days 5, 8, and 11 after of ovulation induction. hCG is used to trigger
oocytes retrieval [15]. The rate of oocyte retrieval ovulation and retrieval occurs 35 h after the hCG
was 76.9% and the rate of 2PN fertilization was injection. Luteal support is given as IM proges-
68.2% per oocytes. The rate of transfer was terone injection or vaginal suppositories [17].
43.7% per initiate cycle. The success rates of this When compared to conventional IVF, mild
protocol were initially reported as a birth rate of IVF using clomiphene (with or without the GnRH
13.4% per initiated cycles and 30.8% per embryo antagonist) showed similar clinical pregnancy
transfer [15]. In a later study, the cumulative live rate in a retrospective controlled study (37% for
birth rate with this protocol after three cycles in a minimal stimulation and 41% for conventional
cohort of 350 patients was reported as 20.8% per IVF) [17]. Weigert et al. compared the success of
patient [3]. The same group published a study mild IVF using clomiphene followed by gonado-
that looked at the cumulative pregnancy rate after tropins to conventional IVF in a randomized
controlled study. The pregnancy rate per initiated Aromatase inhibitors have been suggested as
cycle in the mild stimulation cycle (35.1%) was another way of reducing the total requirement of
not statistically different from that in the conven- gonadotropins in ovarian stimulation protocols
tional IVF group (29.3%) [18]. Another study for mild IVF [20]. The role of aromatase inhibi-
compared the clomiphene /gonadotropins proto- tors in IVF remains poorly studied. Grabia et al.
col with the GnRH antagonist to conventional studied letrozole (2.5 mg) as an alternative to clo-
long stimulation IVF, in patients undergoing their miphene in minimal stimulation IVF in good
first IVF ICSI for male factor infertility. They prognosis patients. A clinical pregnancy rate of
found similar pregnancy rates in both treatment 27% was reported [21].
groups (41.7 and 40%) [19]. Gonadotropins may be used without prior oral
An alternative protocol for mild stimulation agents in low doses with the intent to reduce the
IVF was developed to alleviate some of the con- costs of medications and avoid complications
cerns associated with the utilization of GnRH associated with standard controlled ovarian hyper-
antagonist including a low LH environment and stimulation. They may be initiated on day 2–3 of
the requirement of a relatively high dose of cycle or in the second half of the follicular phase,
gonadotropins [9]. This protocol relies on con- although early starts more consistently achieve
tinuation of clomiphene to inhibit the LH surge. multifollicular responses [22]. Fernandez-Shaw
A Japanese group reported their experience with et al. reported the efficacy of the low gonadotro-
this protocol in 44,345 cycles. Clomiphene was pins IVF protocol in 79 young women with a
administered at a dose of 50 mg daily until the good prognosis. Patients with polycystic ovarian
day before triggering ovulation using a GnRH syndrome, severe endometriosis, ovarian failure,
agonist. If the patient was found to have multi- or elevated early follicular FSH or estradiol were
follicular growth, gonadotropins were added at a excluded. The stimulation starts similar to the
dose of 150 IU every other day (urinary HMG or traditional long stimulation protocol utilizing
rFSH) until the day before the GnRH agonist GnRH agonist in the luteal phase of the previ-
trigger. GnRH agonist was administered when ous cycle. Pituitary suppression is evaluated on
the follicular diameter reached 18 mm or the day 3 by vaginal ultrasound and estradiol levels
estradiol level was ³300 pg/mL. Follicular scans (<50 pg/mL). Gonadotropins were started on day
were started at day 8. Oocyte retrieval occurred 3 at a low dose of 100 IU rFSH. After 5–6 days of
32–35 h after the ovulation trigger. The rate of stimulation, the dose is increased if needed. hCG
premature LH surge with this protocol was 5.1%. 10,000 IU was given when 1–3 follicles reached
Among this small subset of women with detected 18 mm. Oocyte retrieval was performed 35 h after
LH surges, Oocyte retrieval was scheduled hCG injection. Luteal support was given in the
immediately when LH levels indicated an immi- form of intravaginal micronized progesterone at a
nent ovulation by reaching its peak value and dose of 200 mg TID starting the day of oocytes
documentation of a drop in 4 h. Oocyte retrieval retrieval. The protocol resulted in lower number
was delayed 24 h if LH levels suggested the of embryo when compared to conventional dose
onset of LH surge by documenting increased gonadotropins (150 IU); however, the pregnancy
levels in 4 h. Luteal phase support was provided rates were not different (51.8 vs. 50.7%) [16].
using dydrogesterone at a dose of 30 mg daily.
The embryo transfer occurred at either the four
cells or the blastocyst stages. The use of birth 2.4 Minimal Stimulation IVF
control in the preceding month increased the and Single Embryo Transfer
number of oocytes and embryos available. The
live birth rate for the fresh embryo transfer of One goal of minimal stimulation IVF is to reduce
four cell stage embryos was 5.2%, the frozen the number of multiple pregnancies as well as the
four cell embryo transfer 0.2%, and for the fro- cost of care. The overall number of embryos
zen blastocyst transfer 5.6% [9]. transferred should be, in theory, lower than that
during conventional IVF, and if combined with solution (such as sterile phosphate-buffered
single embryo transfer, the cost saving could be saline) and the fluid is reaspirated. The procedure
considerable [23]. Heijnen et al. compared, in a can be repeated. In one study, the optimal number
randomized controlled study, the success of four of flushing was found to be four, and beyond this,
cycles of mild IVF (gonadotropins started mid the yield of oocyte retrieval is low [25]. Another
follicular phase) with single embryo transfer to study showed that only 4.3% of oocytes can be
three cycles of conventional IVF with transfer of retrieved with the second flushing [27].
two embryos [24]. Participants were good prog- A recent randomized controlled trial com-
nosis women who either had one birth through pared follicular flushing to direct follicular aspi-
IVF or did not have IVF before. The patients were ration in poor responders to conventional IVF
younger than 38 and had regular cycles and BMIs. stimulation. Poor responders were defined as
The duration of stimulation, total dose of medi- patients who on the day of oocytes retrieval had a
cation, number of oocyte retrieved, and number cumulative follicle count of 4–8 follicle ³12 mm
of embryo transferred were lower in the mild and at least two follicles ³16 mm. There was no
IVF group when compared to the conventional difference in the total number of oocytes or
IVF group. The rate of live birth per started fresh number of mature oocytes between the study
mild IVF cycle with single embryo transfer groups. However, retrieval time was two times
(15.8%) was lower when compared with that of longer in patients undergoing follicular flushing
conventional IVF with dual embryo transfer when compared to direct aspiration technique.
(24%). Cumulative pregnancy rate and term Follicular flushing did not improve the fertiliza-
delivery were calculated by adding the rates of tion, implantation, or pregnancy rates [28]. Other
pregnancy of the fresh and frozen cycle that orig- observational studies did not show any benefit
inated from the same treatments. The 1-year from follicular aspiration in the context of con-
cumulative term live birth rate per couple (43.4%) ventional IVF [26, 29].
in the mild IVF group was not inferior to that of In the context of minimal stimulation IVF, fol-
the conventional IVF group (44.7%). The pro- licle flushing may be more important because of
portion of multiple pregnancy rates per couple in the expected low number of oocytes retrieved
1 year in the mild IVF group (0.5%) was signifi- [30]. The competence of oocyte retrieved through
cantly lower than that in the conventional IVF flushing in the context of minimal stimulation
group (13.1%) [24]. IVF was studied by Lozano et al. In this study,
271 minimal stimulation IVF cycles were
included. The oocytes retrieved were divided into
2.5 Follicular Flushing two groups: retrieved with the first aspiration or
retrieved through flushing. Embryo morphology
In protocols where few mature follicles are pres- and implantation rates were higher in the oocytes
ent, optimal yield of oocytes per follicle is critical retrieved through flushing; however, the fertiliza-
[25]. Follicular flushing has been advocated as a tion rate and clinical pregnancy rates were com-
way to improve the yield of oocytes during oocyte parable in both groups [31, 32].
retrieval in the hope of increasing the number of
good quality embryos available for transfer.
Follicular flushing is thought to have a role in 2.6 Cost Considerations
patients undergoing conventional IVF with poor
response or in patients undergoing minimal stim- Cost of fertility treatment and IVF are under strict
ulation IVF [26]. The technique of follicle flush- scrutiny and often cited as the reason for the absence
ing can vary between centers. The most common of insurance coverage of fertility care in the
technique involves the utilization of double lumen United States [33, 34]. Economic considerations
needles that can be used to aspirate the follicular remain the main barrier to increased IVF availabil-
fluid. The follicle is then injected with a sterile ity and utilization in the United States [35].
Minimal stimulation IVF is thought to reduce the good prognosis or patients who failed conven-
overall cost of fertility treatment. The cost reduc- tional IVF. Minimal stimulation IVF has the
tion results from the reduced dose of gonadotropins potential of reducing the side effects of IVF as
and the presence of fewer embryos which can well as the overall cost of care.
reduce the variable costs of IVF laboratories
and may permit lower fees for IVF procedures
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Current Understanding
of Anti-Müllerian Hormone 3
Dimitrios G. Goulis, Marina A. Dimitraki,
and Basil C. Tarlatzis
Abstract
During the last years, anti-Müllerian hormone (AMH) has been trans-
formed into a “hot issue” of Reproductive Medicine, as it has attracted the
attention of many research groups and has found considerable clinical
applications. In this review, we have summarized available evidence on
possible roles of AMH in women with polycystic ovary syndrome (PCOS).
We have arranged the material into three sections. In the first section, we
briefly present the AMH as a molecule, in the second we pay special atten-
tion on AMH involvement in PCOS pathophysiology and, in the third
section, we discuss possible roles of AMH as a predictive factor in women
with PCOS undergoing assisted reproduction technologies (ART).
Serum AMH concentrations, being stable and consistent throughout the
menstrual cycle, constitute a reliable marker of ovarian reserve; thus, AMH
has already found a role in the clinical practice, particularly when combined
with classic markers of ovarian reserve such as age, follicle-stimulating
hormone (FSH), and antral follicle count (AFC). The significance of AMH
in women with PCOS undergoing ART is increasing as well. On top of
being a marker of ovarian reserve, AMH has been used for predicting suc-
cess of ovulation induction and controlled ovarian hyperstimulation proto-
cols, as well as avoidance of ovarian hyperstimulation syndrome (OHSS).
Despite this evidence, many issues remain to be elucidated. AMH
physiology is still obscure, especially its exact role in ovarian folliculo-
genesis, significance of serum and follicular fluid concentrations, and
possible extraovarian actions. As far as PCOS is concerned, there is agree-
ment that AMH concentrations are elevated in women with the syndrome
as compared to normo-ovulatory women. Nevertheless, it is still not known
if this difference is the result of disrupted folliculogenesis, due to increased
D.G. Goulis (*)
Section of Reproductive Medicine,
First Department of Obstetrics and Gynecology,
Medical School – Aristotle University of Thessaloniki,
Thessaloniki, Greece

20 D.G. Goulis et al.
number of small antral follicles, or the cause of it, due to AMH inhibition
on folliculogenesis. Data on AMH pathophysiology in adolescent girls
with PCOS are particularly scarce.
The answers to these questions will broaden the spectrum of AMH
clinical applications. Adjustment of overall ART strategy and individual-
ization of protocols according to AMH concentrations seems to constitute
possibilities for the near future. More distant applications could include
use of AMH in hormonal contraception, given its inhibitory action on
follicular development, or even development of AMH-antagonists in the
therapeutic approach of women with PCOS.
Keywords
Anti-Müllerian hormone • Müllerian inhibiting substance • Polycystic
ovary syndrome • Transforming growth factor-b • Estradiol • AMH
receptor, type I • AMH receptor, type II • Gonadotropins • Follicle-
stimulating hormone • Human chorionic gonadotropin • Luteinizing
hormone • Androgens • Testosterone • Hyperandrogenism • Insulin •
Insulin resistance • Hyperinsulinemia • Bone morphogenetic proteins •
Inhinins • Inhibin B • Activins • Smads • Ovary • Oocyte • Aromatase •
Follicle, primordial • Follicle dominant • Antral follicle count •
Granulosa cells • Theca cell • Metformin • Clomiphene citrate • In vitro
fertilization • Controlled ovarian hyperstimulation • Ovarian hyperstimu-
lation syndrome
3.1 Introduction r eference to current clinical applications of serum

AMH measurement.
During the last years, anti-Müllerian hormone In the second section, we pay special
(AMH) has been transformed into a “hot issue” attention on AMH involvement in PCOS. Aspects
of Reproductive Medicine, as it has attracted the of ovarian physiology and PCOS pathophysiol-
attention of many research groups and has found ogy are discussed as well as serum AMH concen-
considerable clinical applications. A MEDLINE trations in women with PCOS. We also present
search performed in July 2010 revealed almost data on correlation of AMH with other important
1,200 papers under the medical subheading parameters of PCOS, such as gonadotropins,
(MeSH) term “anti-Müllerian hormone.” Of androgens, and indices of insulin resistance.
them, more than 50 were specifically focused on Finally, in the third section, we discuss possi-
women with polycystic ovary syndrome (PCOS). ble roles of AMH as a predictive factor in women
In this review, we have attempted to summa- with PCOS undergoing assisted reproduction
rize available evidence on possible roles of AMH technologies (ART). Thus, we present data on
in women with PCOS. We have arranged the two techniques (ovulation induction and
material into three sections. In the first section, controlled ovarian hyperstimulation) as well as
we briefly present the AMH as a molecule. We one complication (ovarian hyperstimulation
discuss the gene and the protein, its expression syndrome, OHSS), in an attempt to clarify if
and actions, as well as the controversial issue of the outcome/presence of them could be pre-
serum concentrations during a spontaneous dicted by routine application of serum AMH
menstrual cycle. The section closes with a measurement.
3 Current Understanding of Anti-Müllerian Hormone 21
CAGAC, with a relatively low specificity or with

3.2 Anti-Müllerian Hormone cofactors into a super-complex that can modulate
ligand-specific gene expression [7].
3.2.1 The Gene and the Protein
The human AMH gene has a length of 2,750 base-

pairs, is divided into five exons, and maps on 3.2.2 Expression
chromosome 19 p13.3 [1]. Its product, AMH, is a
dimeric glycoprotein [2], a member of the trans- AMH is secreted by the human testis from the
forming growth factor-b (TGF-b) superfamily, sixth week of gestational age (eighth week of
that also includes inhibins, activins, bone mor- amenorrhea) and provokes irreversible Müllerian
phogenetic proteins (BMPs), and a wide range of duct regression, which is completed by the end of
growth and differentiation factors (GDFs) [3]. ninth week [8]. The AMH receptors are expressed
The proteins in this family have a broad range of in the fetuses of both sexes at the time when
functions in mesenchymal – epithelial interaction, sexual differentiation is triggered. Thus, AMH is
cell growth, extracellular matrix production, and one of the earliest Sertoli cell-specific proteins
tissue remodeling [4]. They are produced as expressed by the gonad [9]. Except for a transient
dimeric precursors and undergo posttranslational decline in the perinatal period [5, 10, 11], testicu-
processing for activation; cleavage and dissocia- lar AMH secretion is maintained at high levels
tion is required for biologically active C-terminal until puberty, when Sertoli cell maturation is
fragments to be released [5]. characterized by a decreasing AMH activity in all
As for other members of the TGF family, species studied [12–20] (Fig. 3.1). The decline of
AMH signals through membrane receptors, the AMH production by Sertoli cells during puberty
TGF/activin group of type I receptors (ALK-1, -4 in the boy is related to the stage of pubertal devel-
and -5), and the BMP/GDF group of type II opment. The most significant decrease in serum
receptors (ALK-2, -3 and -6). They all have AMH is observed between stages II and III of
intrinsic protein kinase activity that catalyzes pubertal development [21], in coincidence with
phosphorylation of proteins on serine and threo- the increase of intratesticular testosterone con-
nine residues. The AMH receptors consist of two centration [22].
nonidentical subunits, each of which has a single Ovarian granulosa cells, the homologous to
membrane spanning region and an intracellular testicular Sertoli cells, also produce AMH [23],
kinase domain [6]. Binding of AMH to its recep- but with several differences: AMH expression
tor causes it to complex with and phosphorylate a only begins at the perinatal period [19, 24, 25],
signal-transducing subunit. The activated recep- remains low throughout reproductive life, and
tor complex associates with and phosphorylates becomes undetectable after menopause. Thus,
cytosolic proteins called Smads, which enter the gonadal AMH secretion shows a clear-cut sexual
nucleus and activate transcription of specific dimorphism in prepubertal ages, when serum
genes. Smads fall into three different classes, AMH concentrations are significantly lower in
receptor-regulated R-Smads, inhibitory Smads, females; in adults, serum AMH concentrations
and the common Smad-4 [6]. The R-Smads-2 are similarly low in both sexes [10, 26, 27],
and -3 are phosphorylated by the TGF/activin whereas show a progressive decline along repro-
type I receptors ALK-4 and -5, whereas the ductive life in women [28].
R-Smads-1, -5, and -8 are phosphorylated by the Ovarian AMH expression, observed as late
BMP/GDF type II receptors ALK-2, -3, and -6. as gestational week 32 [19], seems to be absent
The phosphorylated Smads dimerize with the in primordial follicles, theca cells, or oocytes
common Smad-4 to form heteromeric complexes [25, 29–31], but is highest in granulosa cells of
that translocate to the nucleus. They act by preantral and small antral follicles (Fig. 3.2).
binding to the Smad-responsive DNA element Interestingly, AMH is expressed in follicles that
Fig. 3.1 Profiles of serum anti-Müllerian hormone (AMH) tion in males after the neonatal period (reproduced from
and testosterone (T) in the male. The serum AMH concen- Rey [133])
tration is inversely proportional to the serum T concentra-
Fig. 3.2 The role of anti-Müllerian hormone (AMH) in Initial recruitment takes place as a continuous process,
the two main compartments of normal ovarian follicle whereas cyclic recruitment is driven by a rise in FSH
development (the red center represents the oocyte, the serum levels at the end of a previous menstrual cycle.
grey area represents the granulosa cell layer, and the The inhibitory effects of AMH are shown (a) on the ini-
white area represents follicle fluid in the antrum). AMH tial recruitment of primary follicles from the resting pri-
is expressed in small and large preantral follicles (bro- mordial follicle pool and (b) on the sensitivity of antral
ken arrows) and in small antral follicles (unbroken follicles for FSH (reproduced with permission from
arrow), and the latter mainly contributes to serum levels. Broekmans et al. [134])
have undergone recruitment from the primordial Within these follicles, AMH expression is not
follicle pool, but have not been selected for always evenly distributed, as expression may be
dominance. highest in the granulosa cells immediately
The intrafollicular concentrations of AMH surrounding the antrum and around the oocyte
become progressively lower with increasing fol- [25, 29–31, 33]. This gradient of AMH expression
licle diameters [32]. At the larger antral follicle within a follicle may reflect functional differences
stage (>8 mm), AMH expression diminishes. between the granulosa cells surrounding the
oocyte and the more peripheral ones, such as for AMH even as a coregulator of steroidogenesis
differences in proliferation capacity and steroido- in granulosa cells, as AMH concentrations appear
genic activity [30]. These functional differences to be related to estradiol concentrations in folli-
may arise under the influence of factors produced cular fluid from small antral follicles [43].
by the oocyte, as it has been shown for GDF-9 Finally, AMH may play a significant physiolog-
[34]. Follicles showing signs of atresia also have ical role in controlling involution of the normal
decreased or no AMH expression; expression is breast [44].
completely lost in corpus luteum.
3.2.4 Serum Concentrations During

3.2.3 Actions a Spontaneous Menstrual Cycle
In human ovary, possible actions of AMH include Numerous studies have shown that the AMH
inhibition of follicular activation and growth, inhi assay has high reproducibility in repeated mea-
bition of follicle-stimulating hormone (FSH)- surements [45] and AMH concentrations, unlike
dependent growth, inhibition of granulose cells steroids, gonadotropins, and peptides, such as
growth, and inhibition of aromatase (Fig. 3.2). inhibin B, have been reported to be constant
There is evidence that AMH undertakes throughout the menstrual cycle [46–49]. Indeed,
important intrafollicular functions around the serum AMH concentrations have been measured
time of normal follicular selection, in the midfol- at different times during the menstrual cycle, sug-
licular phase of the menstrual cycle [32]. Thus, gesting extremely subtle or nonexistent fluctua-
AMH may serve to inhibit recruitment of primor- tion [27, 46]. It may be hypothesized that minimal
dial follicles into the pool of growing follicles to fluctuations in serum AMH concentrations may
prevent early depletion [31, 34, 35] and decrease be consistent with continuous noncyclic growth
follicle sensitivity to gonadotropin stimulation, of small follicles. Thus, AMH may constitute a
to control the number of large preantral and small unique endocrine parameter for the investigation
antral follicles that reach the preovulatory stage of ovarian function.
[35, 36]. In contrast to the above results, some investi-
As it is well established that follicles are more gators have demonstrated significant cyclical
sensitive to FSH in the absence of AMH, primor- fluctuations in AMH concentrations, with a rapid
dial follicles are recruited at a faster rate under decrease in the early luteal phase [50, 51]. They
these conditions, resulting in premature exhaus- report that the changes in AMH concentrations
tion of the primordial follicle pool [37, 38]. after ovulation are slight, yet statistically signifi-
In addition, diminished expression of AMH cant, and suggest that these changes may influ-
within the follicles reduces the threshold level for ence the circulating gonadotropin and steroid
FSH, allowing follicles to continue growing and hormone concentrations. Excursions from mean
to ovulate in the next cycle [36, 39]. levels of 3–219% have been reported [50, 51].
Oocytes upregulate AMH expression in gran- In the clinical setting, the inter- and intracycle
ulosa cells in a manner that is dependent upon variability in serum AMH concentrations may be
the developmental stage of the oocyte [40]. This considered to be low enough to permit the use of
observation leads to the hypothesis that oocytes serum AMH as a cycle-independent marker for
in the pool of growing follicles could control the ovarian reserve and ovarian activity [52]. In addi-
pool of primordial follicles by modulating the tion, AMH concentrations appear to be unmodi-
expression of the inhibiting factor AMH [41]. In fied in conditions under which endogenous
addition, considerable attention has focused on gonadotropin release is substantially diminished,
the possible role of AMH in a new signaling strengthening the concept that AMH levels reflect
pathway between granulosa and theca cells in the the continuous, FSH-independent, noncyclic
developing follicle [42]. Studies suggest a role growth of small follicles in the ovary.
3.2.5 Clinical Applications women with PCOS [66]. In mice, exogenous

AMH inhibits FSH-mediated follicle growth [36].
According to the published evidence, determi- In humans, several investigators have demon-
nation of serum AMH concentrations could be strated higher AMH concentrations in amenor-
useful in the following clinical conditions: rhoeic compared to oligomenorrhoeic women with
1. In children, for the presence of testicular tissue PCOS [67]. Alternatively, high AMH values could
[10, 53, 54], when serum testosterone concen- reflect a more evident impairment in follicular
trations are very low. development and granulosa cell function in the
2. In the differential diagnosis of patients with ovaries of amenorrhoeic compared to oligomen-
intersex disorders [53, 54]. AMH concentra- orrhoeic women with PCOS. Increased ovarian
tions are an instructive marker [55] in the eval- AMH production may exert a paracrine negative
uation of androgen insensitivity syndrome or control on follicle growth, sufficient to prevent
steroidogenic disorders, particularly in the selection of a dominant follicle.
infant or young male, in whom the normally The cause of the increased AMH production
low and fluctuating testosterone concentra- in PCOS is unknown. However, the intraovarian
tions cannot be detected, unless stimulated hyperandrogenism may be the main culprit for
with human chorionic gonadotropin (hCG) or the follicular arrest observed in PCOS, promoting
luteinizing hormone (LH). early follicular growth and leading to an excess
3. In patients with bilateral nonpalpable gonads of 2–5 mm follicles [68]. Insulin is the second
[56]. candidate for the cause of the increase of AMH
4. In females with granulosa cell tumors [57–60], concentrations in women with PCOS.
where AMH concentrations can be dramati- According to recent data, genetic variation of
cally elevated. ALK-2 is associated with AMH concentrations
and follicle number in women with PCOS, sug-
gesting that this type I receptor for AMH/BMP
3.3 Anti-Müllerian Hormone in signaling contributes to the disturbed folliculo-
Polycystic Ovary Syndrome genesis in women with PCOS [69].
3.3.1 Pathophysiology
3.3.2 Serum Concentrations
Many studies of ovarian histology have shown
that the number of primordial follicles is the same Based on the observation that AMH is predomi-
in women with PCOS compared to normo-ovula- nantly expressed by small follicles, several
tory controls, but the number of developing and, studies have concluded that AMH serum con-
subsequently, atretic follicles is doubled [61, 62]. centrations are increased in women with PCOS
Thus, in women with PCOS, ovaries with poly- [36, 70]. AMH measurement has been found to
cystic morphology differ from normal ovaries in offer a relatively high specificity and sensitivity
that follicle development is arrested at the stage (92 and 67%, respectively) as a diagnostic
where, under normal conditions, dominant follicle marker for PCOS [71]. Furthermore, it seems
selection would have taken place [63–65]. that AMH concentrations correlate with the
On a histology level, AMH concentrations were extent of ovarian dysfunction in these women,
found to be, on average, 75 times higher in granu- as represented by elevated LH or testosterone
losa cells from polycystic ovaries, compared with levels and an increased follicle number and/or
concentrations in normal ovaries [42]. Although ovarian volume, as established on ultrasound.
there is positive correlation between AMH con- These elevated serum AMH concentrations in
centrations and follicle number in women with women with PCOS might indicate an increased
PCOS, it is unclear whether AMH has a regulatory ovarian reserve [72].
role in follicle development or whether this is a In a longitudinal study [73], serum AMH
consequence of increased antral follicle number in concentrations were found to decline over time
both in PCOS group and in controls. However, action. Though insulin resistance is amplified by
the reduction in women with PCOS was lower. increasing obesity, women with PCOS are more
These results may indicate a sustained reproduc- insulin resistant than can be accounted for by
tive lifespan in these patients. their obesity per se [81–83]. Moreover, in women
It has been shown that metformin administra- with PCOS, ovarian antral follicle counts and
tion in women with PCOS is associated with a ovarian volume correlate positively with endog-
reduction in AMH concentrations in both serum enous and exogenous hyperinsulinemia [84, 85].
and antral follicles, suggesting that the measure- Hyperinsulinemia may stimulate the develop-
ment of AMH could be used to evaluate the treatment of antral follicles, increase the sensitivity of
ment efficacy with insulin sensitizers [66]. granulosa cells to FSH, and thus, increase the
number of follicles and ovarian volume [86].
Finally, insulin has been shown to promote in vitro
3.3.3 AMH and Androgens secretion of androgens by ovarian theca and
stromal tissue [87] and, in women with PCOS, to
Serum AMH has been positively correlated to stimulate testosterone biosynthesis by binding to
androgen concentrations [72, 74, 75]. Women its own receptor in theca cells [88].
with hyperandrogenism and polycystic ovary La Marca et al. [89] found no correlation
morphology (PCO) had higher serum AMH con- between serum AMH and androgen concentra-
centrations compared to women with PCO and tions, but did observe a direct correlation between
normal androgen concentrations [75]. AMH and insulin insensitivity, reporting that the
Androgen production per theca cell is equally raised AMH may be secondary to an effect of
increased in anovulatory and ovulatory women insulin on androgen concentrations. In addition,
with PCOS [76]. However, the total number of women with PCOS have been reported to have a
follicles found in the anovulatory ovary is higher, positive correlation between AMH and the 2-h
resulting in increased total androgen concentra- insulin concentrations [90]. However, other
tions [77]. This fact may not only explain the investigators have failed to find a direct correla-
higher AMH concentrations in women with PCOS tion between insulin and AMH concentrations
as a whole, but also the significantly higher pro- [74, 75] and reported that, even when insulin
duction of AMH by anovulatory ovaries. concentrations have been reduced with treatment,
Nevertheless, the results of a recent study rather a fall in serum AMH has not followed directly
contradict this hypothesis [78]. Although, at the [78, 91].
beginning of the study, there was a direct correla- Quantitative ultrasound evaluation of follicle
tion between AMH and androgen concentrations distribution in PCO suggests a possible role for
in women with PCOS, after 6 months of androgen insulin in the dysregulation of folliculogenesis
suppression with dexamethasone, the AMH con- [68]. Some study groups [92] have shown that
centrations remained unchanged [78]. However, follicular development in response to the induc-
several possibilities exist that the concentration tion of ovulation with gonadotropins is qualita-
of androgen within the ovary is the determining tively different in insulin-resistant women with
factor and that a different control mechanism PCOS. Treatment with the insulin sensitizer met-
would have to be present in the ovary than in the formin reduces the number of antral follicles,
testis for androgens to cause the rise of AMH, ovarian volume, and circulating AMH concentra-
described in women with PCOS. tions [66]. It is most likely that the decrease is
simply related to the decrease of follicle number,
but the contribution of the improvement of hyper-
3.3.4 AMH and Insulin Resistance androgenism, insulin action, or menstrual pattern
cannot be excluded. Short-term metformin treat-
PCOS is considered to be a syndrome of pre- ment was reported to result in improvements in
served [79], if not increased [80], ovarian sensi- hyperandrogenism, menstrual cyclicity, reduc-
tivity to insulin with systemic resistance to insulin tion in the number of antral follicles, and insulin
resistance [91], while long-term metformin

administration is required for a reduction in AMH
3.4 Anti-Müllerian Hormone
concentrations [66, 93]. in Assisted Reproduction
Technologies
3.3.5 AMH and Gonadotropins 3.4.1 Ovulation Induction
FSH directs the cyclic recruitment and forms the There is limited evidence on the clinical relevance
basis of the menstrual cycle by enabling the of serum AMH concentrations in women in
secretion of the steroid estradiol from the domi- whom clomiphene citrate ovulation induction
nant follicle. It has been hypothesized that AMH previously failed [72]. An AMH concentration of
could be one of the factors involved in determin- 1.2 ng/mL could be used to predict response to
ing the responsiveness of ovarian follicles to FSH clomiphene citrate in obese women with PCOS,
during cyclic recruitment, playing an inhibitory with a sensitivity of 71.0% and a specificity of
role in the FSH-dependent follicle growth 65.7% [98]. However, the response to clomiphene
(Fig. 3.2). AMH also plays a role in the fine- citrate in obese patients with PCOS seems to be
tuning of the FSH threshold for dominant follicle dependent on initial AMH concentrations. In
selection. Both in vitro and in vivo studies have addition, in the same group of women, pregnancy
shown that large antral follicles are more sensi- rates and miscarriage rates were similar in patients
tive to FSH in the absence of AMH [38]. with moderately and severely elevated AMH
An inverse correlation between AMH and serum concentrations [98]. Although, among
estradiol concentrations in follicular fluid of women with PCOS, those who have the highest
small antral follicles has been demonstrated, concentrations of AMH seem to respond less well
which indicates a close interdependent regulation to ovulation induction [99], elevated AMH serum
between AMH production and FSH activity [43]. concentration is a marker of limited predictive
In addition, FSH has been reported to decrease power in these patients, as far as adverse treat-
the expression of AMH and its type II receptors ment outcome is concerned.
in granulosa cells [30]. In anovulatory women with PCOS, a gentle
Correlation between FSH and AMH concen- increase in serum FSH concentrations reduces the
trations has been observed in several studies AMH excess, thus relieving the inhibition from
[70, 74, 89, 94–96]. Whereas FSH and AMH were the latter on aromatase expression by selectable
negatively correlated in women without PCOS, a follicles [100]; this mechanism allows the devel-
positive correlation was found in the PCOS opment of a dominant follicle. In addition, it has
group. This observation leads to speculation on been shown [92] that follicular development, as a
the role of AMH as direct modulator of the FSH response to ovulation induction with gonadotro-
sensitivity of individual follicles in PCOS, which pins, is qualitatively different in insulin-resistant
in turn may also explain previous data on follicular women with PCOS. Treatment with the insulin
recruitment and delayed ovarian aging in women sensitizer metformin reduces the number of antral
with PCOS [73]. follicles, ovarian volume, and circulating AMH
It has been demonstrated [97] that exogenous concentrations [66].
AMH administration reduced aromatase expres-
sion and the number of LH receptors in cultured
granulose cells. Positive correlation has been 3.4.2 Controlled Ovarian
reported between AMH and LH in women with Hyperstimulation
PCOS. In these women, increased LH concentra-
tions might be a significant link between disor- Ovarian hyperstimulation by exogenous FSH has
ders of ovulation and the observed increase in allowed for the investigation of the contribution
serum AMH concentration. of different follicular stages on AMH serum
c oncentrations, as it forces many small antral increasing basal AMH concentrations, indicating
follicles to be transformed into large dominant that serum AMH may definitely be considered a
follicles. Indeed, hyperstimulation results in a better marker for quantitative than for qualitative
remarkable decline in serum AMH concentra- aspects of ART [123].
tions [101, 102]. Under such conditions, serum Maximal receiver operating characteristic
AMH concentrations correlate with the number (ROC) curve inflections, which differentiate
of smaller (<12 mm diameter) but not larger between better and poorer delivery chances in
(>12 mm) follicles [70, 89, 103, 104]. In addi- women with diminished ovarian reserve, indepen-
tion, a significant reduction has been observed in dent of age, were at AMH 1.05 ng/mL (improved
concentrations of AMH protein in the conditioned odds for live birth 4.6, 95% confidence interval
medium from granulose cells from women with 2.3–9.1), although live births occurred even with
PCOS, which had been treated with FSH [42]. undetectable AMH [128]. Pregnancy wastage was
AMH seems to be a better predictor for very low at AMH concentrations less than 0.04 ng/
oocyte maturation and successful in vitro fertil- mL, but significantly increased at AMH 0.41–1.05 ng/
ization (IVF) treatment than traditional markers mL, resulting in similarly low live birth rates at all
[105–107]. While FSH mostly reflects the last 2 AMH concentrations less than 1.05 ng/mL and
weeks of follicular maturation, when follicles significantly improved live birth rates at AMH
become gonadotropin sensitive, AMH is mostly more than 1.06 ng/mL [128].
representative of the young, postprimordial to In a recent study [129], AMH concentrations
preantral follicle pool going through earlier were measured in follicular fluid collected at the
stages of folliculogenesis [41, 108, 109]. time of oocyte retrieval for IVF from women with
More specifically, lower serum AMH concen- PCOS. Although AMH was higher compared to
trations preceding or during ART were strongly ovulatory women, the concentrations in both
associated with reduced oocyte yield and low small and large follicles were found to be lower
oocyte quality [106, 107]. In a similar way, higher in those women who began a pregnancy. This
day 3 serum AMH concentrations were associ- finding indicates that even following a stimula-
ated with greater number of retrieved oocytes tion protocol, it is those women with PCOS pro-
[50, 70, 95, 104, 105, 107, 110–122]. As a confir- ducing the relatively lower levels of AMH who
mation, serum AMH concentrations have been have the best outcome. In addition, it has been
shown to be tenfold lower in the canceled cycles suggested that there is an association between
[41]; thus, in prediction of a canceled cycle, follicular fluid AMH concentrations and the qual-
AMH seems to be a better marker compared with ity of embryos in patients with PCOS [130].
FSH or inhibin B [123] and an equal one to AFC
[52]. For the prediction of nonpregnancy, both
serum AMH levels and AFC were shown to be 3.4.3 Ovarian Hyperstimulation
similarly poor performers [124]. Syndrome
A number of investigators have tried to iden-
tify threshold AMH concentrations that are able Considerable attention has been attracted on the
to distinguish between pregnancy and nonpreg- finding that elevated serum AMH concentrations
nancy in an IVF procedure [110, 112, 115, 116]. could be associated with greater probability of
However, the majority of them indicated that developing OHSS. A trend, but not statistically
AMH measurement is not useful for predicting significant difference (p = 0.052), was reported in
this end-point [95, 104, 114, 115, 125–127]. Up day 3–5 AMH concentrations between those
to the present, only one study has been published women who developed OHSS and those who did
relating serum AMH concentrations to the live not [113]. Inconsistent with these findings, however,
birth rate following IVF [122]. In this prospective another study noted sixfold higher serum AMH
study of 340 patients, it was demonstrated that concentrations in women with OHSS [131].
the live birth rate dramatically increases with A recent study [132] has clearly demonstrated
that basal AMH measurement works as well as

the AFC and estradiol levels on the day of hCG in
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The Role of Obesity in Reproduction
Barbara Luke
4
Abstract
Obesity has risen to epidemic proportions worldwide and affects more
than two thirds of US adults. In both genders, obesity is associated with
impaired fertility, primarily due to disorders of the reproductive hormonal
profile. Across the reproductive spectrum, obesity is associated with
greater risks for adverse health outcomes, including higher rates of infer-
tility, subfertility, early pregnancy loss, fetal deaths and stillbirths, con-
genital anomalies, and pregnancy complications. The excess reproductive
morbidity associated with obesity may increase with longer duration,
making the current trends among children and young adults particularly
critical in terms of their future reproductive potential. Clinical and epide-
miologic studies strongly implicate prenatal growth restriction followed
by early childhood catch-up growth with development of symptoms of
polycystic ovary syndrome by early adolescence. Abnormal glycemic
parameters, including high dietary glycemic load, fasting, and 2-h glucose
levels, and fasting insulin have also been linked to adverse reproductive
outcomes, further increased in the presence of obesity. A recent national
study of ART reported reduced clinical pregnancy rate with increasing
BMI with autologous but not donor oocytes and reduced live birth rate
with increasing BMI regardless of oocyte source. These findings point to
the need for dietary periconceptional and prenatal therapies targeted at
improving the metabolic environment in obese women. Weight loss should
be the first-line treatment for overweight men and women considering
pregnancy. In addition to dietary modifications to facilitate weight loss,
lifestyle factors such as regular physical exercise, elimination of tobacco
use and alcohol consumption, behavior modification, and stress manage-
ment may be of benefit.
B. Luke (*)
Department of Obstetrics, Gynecology,
and Reproductive Biology, Michigan State University,
East Lansing, MI, USA

36 B. Luke
Keywords
Overweight • Obesity • Body mass index • Excess reproductive morbidity
• Prenatal growth restriction • Abnormal glycemic parameters • Insulin
resistance • Metabolic environment
4.1 Introduction 4.2 Definitions
As the most common chronic disease in the According to the World Health Organization
United States, overweight or obesity affects more [25], obesity is a disease defined as the condition
than two thirds of adults [1]. The prevalence of of excess body fat to the extent that health is
obesity has more than doubled since the 1970s impaired. The most widely accepted measure is
and is a leading cause of morbidity and mortality, the body mass index (BMI, weight (kg)/height
second only to tobacco use [2]. In both genders, (m)2), with cutpoints of 25 kg/m2 (overweight)
obesity is associated with impaired fertility, and 30 kg/m2 (obese), respectively, recommended
primarily due to disorders of the reproductive by the National Heart, Lung, and Blood Institute’s
hormonal profile. In obese men, this can result in and North American Association for the Study of
hypotestosteronemia and even hypogonadotropic Obesity expert committee [26] (see Table 4.1). In
hypogonadism; in obese women, it can result in addition, this expert committee recommends
hyperandrogenism and the metabolic abnormali- using waist circumference cutpoints of 40 in.
ties which characterize the polycystic ovary syn- (102 cm) for men and 35 in. (88 cm) for women
drome (PCOS). The abdominal obesity phenotype to define central obesity. This measure may be
amplifies these associations. even more useful than BMI because of its greater
Obesity is associated with greater risks for predictive value for future health risks, as well as
adverse health outcomes across the reproductive ease of measurement [26–28]. BMI is not the
spectrum, including higher rates of infertility best measure to reflect body fat and does not
[3–5], subfertility (increased time-to-pregnancy) account for racial and ethnic differences in body
[6–8], early pregnancy loss [9–15], fetal deaths, build [29]. Specifically, the proportion of Asians
stillbirths and neonatal deaths [16–19], congeni- at high risk for type 2 diabetes and cardiovascular
tal anomalies [20], as well as pregnancy compli- disease is considerable at lower cutoffs for over-
cations [21, 22]. The excess reproductive weight. The World Health Organization Expert
morbidity associated with obesity may increase Consultation recommended retaining the current
with longer duration, making the current trends BMI cutoffs, but adding additional cutoff points
among children and young adults particularly of 23, 27.5, 32.5, and 37.5 kg/m2 for public health
critical in terms of their future reproductive action (see Table 4.1).
potential. In the United States between 1971–
1974 and 2005–2006, the proportion of young
adults (ages 18–29 years) who were obese tripled 4.3 Male Reproductive Health
(8–24%), compared to a doubling among most and Obesity
other adult age groups during the same time
period [23]. Recent findings from the Study of It has been suggested that semen quality has
Women’s Health across the Nation indicate that been declining during the second half of the last
adolescent obesity is associated with a century [30–33], triggering debate regarding
threefold increased risk of lifetime nulliparity possible causes [34–37]. This trend corresponds
and a fourfold increased risk of lifetime with the international rise in obesity [1, 38].
nulligravidity [24]. Excess weight in adult men has been directly
4 The Role of Obesity in Reproduction 37
Table 4.1 The World Health Organization’s international classification of adult underweight, normal weight, overweight,
and obesity according to BMI
BMI (kg/m2)
Classification Subclassification Principal cutoff points Additional cutoff points
Underweight <18.5
Severe thinness <16.0
Moderate thinness 16.00 – 16.99
Mild thinness 17.00 – 18.49
Normal weight 18.50 – 24.99 18.50 – 22.99
23.00 – 24.99
Overweight ³25.00 25.00 – 27.49
Preobese 25.00 – 29.99 27.50 – 29.99
Obese ³30.0
Class I 30.00 – 34.99 30.00 – 32.49
32.50 – 34.99
Class II 35.00 – 39.99 35.00 – 37.49
37.50 – 39.99
Class III ³40.0
and indirectly related to biological changes that Most published studies demonstrate an improve-
could reduce fertility [39]. Most, but not all stud- ment in the reproductive hormonal profile, includ-
ies, have reported an association between male ing an increase in SHBG, inhibin B, and total
infertility and elevated BMI. testosterone levels as well as a reduction in estra-
Many studies have reported that adult BMI in diol levels [60–63].
males is dose-dependently associated with the
levels of reproductive hormones, including: (1)
lower concentrations of testosterone, sex hor- 4.4 Female Reproductive Health
mone-binding globulin (SHBG), and inhibin B; and Obesity
and (2) higher concentrations of estradiol, but
unaffected or only slightly lower concentrations In concert with the rise in obesity, there has been
of lutenizing hormone and follicle-stimulating a long-term trend in delaying childbearing and an
hormone [40–47]. The results regarding semen increased use of infertility treatments to achieve
quality are inconsistent, with some studies reporting conception. Infertility affects an estimated 12%
a significant association between overweight and of women of reproductive age [64]. Research
low semen quality [40, 41, 48–51], and other stud- suggests that perinatal outcome may be worse for
ies reporting no association [42, 43, 47, 52–54]. women with assisted vs. spontaneous concep-
Several researchers have suggested that the tions, including greater risks for preterm birth
adverse effect of male obesity on semen parame- (<32 and <37 weeks), low birth weight and very
ters is mediated by serum leptin, altering intracel- low birth weight, small-for-gestational age, cesar-
lular metabolism and peripheral tissue receptors ean delivery, NICU admission, and perinatal
in Leydig and germ cells [55–57]. Leptin inhibits mortality [65, 66]. An important underlying
testicular steroidogenesis, decreasing testosterone mechanism may be a genetic predisposition to
synthesis in Leydig cells and inhibiting sperm factors associated with infertility, including
maturation, as well as suppressing Sertoli cell allelic variants in cytokine genes known to stimu-
function [58, 59]. late inflammation or those known to downregu-
Although weight loss is the logical first-line late the anti-inflammatory response. Ness [67]
therapy for obesity, there are few studies docu- suggests that although women with a robust
menting its effect on fertility among obese men. inflammatory response may be more likely to
38 B. Luke
survive to reproduce, their reproductive experi- develop high dehydroepiandrosterone sulfate

ences may be less successful than women who (DHEAS) and low SHBG levels [85]. Precocious
are less responsive. Obesity has been shown to be puberty (appearance of pubic hair before age
a chronic inflammatory state with increased eight) has also been demonstrated as part of this
expression of proinflammatory factors and a sequence, as well as anovulatory and hyperinsu-
reduction in anti-inflammatory factors [68, 69]. linemic hyperandrogenism in late adolescence
In women with assisted conceptions, obesity may and adulthood [80–82]. Insulin resistance has
further potentiate this inflammatory response, been cited as a key mechanism linking prenatal
increasing the known risks for adverse reproduc- growth restraint to early menarche [77], with
tive outcomes, including fetal loss and stillbirths insulin-sensitizing therapy improving ovulation
associated with higher body weight [16–18]. rates [78, 79].
Inflammation and dyslipidemia early in pregnancy
have been shown to be independently associated
with preterm birth [70, 71]. In the presence of 4.6 Diet, Obesity, and Adverse
obesity, these factors are even greater and also Reproductive Outcomes
include significant impairment of endothelial
function [72, 73]. Obesity is associated with alterations in carbohy-
Among women, the reproductive axis is drate and fat metabolism central to the develop-
closely linked to nutritional status, with ame ment of insulin resistance. A diet with a high
norrhea, anovulation, subfertility, and infertility glycemic index has been associated with infertil-
occurring with higher body weights. Obese ity, fetal loss, congenital anomalies, prematurity,
women are more likely to experience irregu- as well as macrosomia. Greater carbohydrate
lar menstrual cycles, anovulation, and signs of intake and dietary glycemic load have been asso-
androgen excess, particularly when the excess ciated with an increased risk of infertility due to
weight occurred during adolescence [74, 75]. anovulation [86]. Jovanovic et al. [87] demon-
Obesity is strongly associated with PCOS, an strated a threefold increased risk of pregnancy
abdominal phenotype of fat distribution, hyper- losses at glycemic extremes in both normal and
androgenism, and insulin resistance with com- diabetic pregnancies, as measured by plasma gly-
pensatory hyperinsulinemia [76]. Obese women cated protein and fructosamine levels. A diet with
have a lower chance of pregnancy following a high glycemic load is associated with a twofold
in vitro fertilization, require higher dosage of increased risk of neural tube defects [88, 89];
gonadotropins, and have an increased miscar- among women with BMIs greater than 29, this
riage rate [9–15]. risk increases to more than fourfold [89]. Among
normal-weight women treated with ART, Wei
et al. [90] reported greater risk for preterm birth
4.5 Prenatal Growth and PCOS associated with abnormal preconception glyce-
mic parameters, including higher fasting and 2-h
A growing body of research implicates prenatal glucose levels, fasting insulin, and homeostatic
growth to timing of puberty and subsequent model assessment of insulin resistance.
symptoms of PCOS [77–85]. Even after achiev-
ing a normal body size by age two, children
born small for their gestational age tend to 4.7 Endometrial vs. Oocyte
become relatively adipose, hyperinsulinemic, Factors
and hypoadiponectinemic, with physiologic
evidence of low-grade inflammation [83, 84]. The endocrine and metabolic environment may
By 6 years of age, these children are more likely influence oocyte quality, and therefore, embryo
to develop visceral adiposity, even with normal development and subsequent implantation and
body weight. By 8 years of age, children born pregnancy outcome. One possible mechanism for
small for gestational age with catch-up growth the lower pregnancy rate associated with obesity
may be altered receptivity of the uterus, due to plications. Among males, obesity has been directly
disturbed endometrial function [10, 12]. Even and indirectly related to biological changes that
studies limited to obese women using donor could reduce fertility. Obese women undergoing
oocytes, eliminating the potential effect of older in vitro fertilization require higher dosages of
maternal age on lower quality of the embryos, gonadotropins, have a lower chance of pregnancy,
have reported significantly reduced implantation and a greater miscarriage rate. Weight loss should
and pregnancy rates and higher abortion rates be the primary therapy for overweight and obese
[11, 12, 91, 92]. A recent national US study of men and women considering pregnancy, along
ART reported reduced clinical pregnancy rate with changes in lifestyle factors such as smoking,
with increasing BMI with autologous but not drinking, and stress management.
donor oocytes and reduced live birth rate with
increasing BMI regardless of oocyte source
[91–93]. These findings are in accord with prior
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Endometrial Receptivity in Natural
and Controlled Ovarian-Stimulated 5
Cycles
José A. Horcajadas, José A. Martínez-Conejero,
and Carlos Simón
Abstract
The development of endometrial receptivity is a prerequisite for successful
embryonic implantation in mammals. The receptive status is reached only
during a short period of time in the midluteal phase, this being maximal
7 days after the endogenous peak of LH (LH+7), named as the window of
implantation (WOI). At this time, the endometrial epithelium acquires the
functional ability to support blastocyst adhesion. In ART, controlled ovar-
ian stimulation (COS) induces lower implantation rates per embryo trans-
ferred than natural or ovum donation cycles, suggesting suboptimal
endometrial development due to the abnormal endocrine/paracrine milieu.
Researchers have investigated the functional genomics of endometrial
receptivity in natural cycles and the impact of COS on the gene expression
pattern of the endometrium, even with the use of different drugs such as
gonadotrophin-releasing hormone (GnRH) agonists or antagonists. This
paper reviews results obtained in different studies to elucidate the changes
induced by the different clinical protocols with the objective to introduce
a new molecular objective tool to analyze the endometrial receptivity sta-
tus in natural cycles and to understand the impact of COS in the human
endometrium.
Keywords
Endometrial receptivity • Gene expression • Controlled ovarian stimulation
• Microarray
C. Simón (*)
Fundación IVI-Instituto Universitario IVI-University
of Valencia, Valencia 46015, Spain
and
Valencia Stem Cell Bank, Centro de Investigaciones
Príncipe Felipe, Valencia 46012, Spain

44 J.A. Horcajadas et al.
Others researchers have elucidated biochemical

5.1 Introduction changes in the endometrium induced by ovarian
stimulation. In this context, down-regulation of
Human endometrium is a dynamic tissue that the endometrial estrogen and progesterone
undergoes well-defined cycles of proliferation, receptors [14] and biochemical changes in the
differentiation, and shedding (menstruation) in endometrial fluid [15] have been described. As
response to the prevailing endocrine and para- has been mentioned before, this is not surpris-
crine environment, the main objective being to ing, considering that the aim of COS is to recruit
reach the receptive status. In the natural cycle, a sufficient number of oocytes, having as a side-
the ovary releases one oocyte in a standard ovu- effect supraphysiological concentrations of ste-
lation, approximately 14 days before the next roid hormones and paracrine mediators that
menstruation. If the oocyte is fertilized, the might target the endometrium. The impact of
resulting embryo will arrive at the endometrium some specific molecules on the development of
at the morula stage (days 4–5). At this time, endometrial receptivity has been reported from
receptiveness of the endometrium will occur, in a different perspectives, encompassing the impact
synchronized manner with the development of of progesterone on endometrial stromal cell
the embryo. development in vitro [16] such as interleukin
In assisted reproduction technologies (ART), (IL)-1b [17] or IL-11 [18]. More recently, the
the main goal of ovarian stimulation is to trigger in vitro effects of steroids on freshly isolated
oocyte maturation of an appropriate number of endometrial endothelial cells have also been
follicles to obtain 6–8 mature oocytes. However, reported [19].
lower implantation rates per transferred embryo However, the most attractive strategy to investi-
than those in natural cycles remain a major prob- gate the genomic profile of the endometrium and
lem that is compensated by increasing the num- the impact of COS protocols on endometrial recep-
ber of transferred embryos [1] at the cost of tivity has been the use of microarray technology,
increased numbers of multiple pregnancies. which allows studying of the entire transcriptome
Furthermore, implantation rates obtained in ovum of a given tissue in a single experiment.
donation programs are higher than in patients
under controlled ovarian stimulation (COS), sug-
gesting that the low rates observed when the ova-
ries are stimulated are related to a detrimental 5.2 The Endometrium as a Tissue
effect on the endometrium. This issue remains
more evident in high responders patients where The lining of the human uterus is a complex
supraphysiological concentrations of estradiol mucosa composed of a basal layer (basalis), which
on the day of human chorionic gonadotrophin persists from cycle to cycle, and a transient super-
(hCG) administration are deleterious to embry- ficial layer (functionalis). The function of the
onic implantation [2–5]. Furthermore, it has been latter is to accommodate the implanting blasto-
demonstrated in animal models that while low cyst and provide the maternal component that will
doses of estradiol maintain the uterus in a recep- interact with the placenta. The tissue components
tive state, high doses cause it to become refrac- of the endometrium are a luminal and glandular
tory [6]. Uterine receptivity is diminished during epithelium with a connective stroma in which is
ovarian stimulation compared with natural embedded an elaborate vascular tree. Endometrial
cycles [7]. A substantial number of endometrial components features change along the menstrual
alterations inducing have been documented in cycle [20]. Cyclic changes of the endometrium
COS cycles using morphological methods. An have been well described at the light microscopy
advancement in the early luteal phase using level [21]. Although some authors prefer to sim-
histology [8–11] and scanning electron micro plify the menstrual cycle and divide it into two
scopy [12, 13] has been previously demonstrated. main phases, proliferative and secretory, others
5 Endometrial Receptivity in Natural and Controlled Ovarian-Stimulated Cycles 45
prefer to consider three different phases: the confined to the perivascular regions, but may also
proliferative phase (days 5–14), the secretory extend to adjacent glands and there is lympho-
phase [14–28], and menses (days 1–4) if no cytic infiltration. By day LH+12, the predecidual
implantation occurs. reaction extends beneath the luminal epithelium.
For more than 50 years, histological evalua- There is an increase in the lymphocyte number.
tion of the endometrium has been the gold stan- The predecidual reaction is extensive on days
dard for clinical diagnosis set on the basis of LH+13/14, with a sheet-like formation in the
the morphological observations of Noyes et al. stroma. Stroma disintegration and extravasation
[22, 23]. These authors described “specific” of erythrocytes are evident. If no implantation
morphological features of the different endo- occurs, shedding of the functionalis layer of the
metrial compartments throughout the menstrual endometrium ensues [20].
cycle. The early proliferative phase (days 5–7)
is characterized by straight, fairly undifferenti-
ated glands with circular cross-section lined by a
columnar epithelium with basally located nuclei. 5.3 Hormonal Regulation
Their luminal diameter (below 50 mm) changes of The Endometrium
little in the proliferative and the height of the cells
remains fairly constant (around 21 mm). Few The human endometrium undergoes cyclical
mitotic figures can be seen. By the midprolifera- variation in each menstrual cycle during the
tive phase (days 8–10), the endometrial glands reproductive life. Endometrial changes are driven
are longer with slight tortuosity. Mitotic figures by the ovarian steroid hormones. Estradiol peaks
are prominent and cells appear pseudostratified. at the end of the proliferative phase and proges-
In the late proliferative phase [11–14], the glands terone increases at the beginning of the secretory
appear with a marked tortuosity and wider lumena. phase, reaching the maximum 7 days later
Pseudostratification increases and stromal edema (Fig. 5.1). Esteroid hormones elicit their actions
starts to be evident. During secretory phase by binding to specific high-affinity nuclear recep-
(LH+2/3), there is still a moderate degree of glan- tors, which modulate the transcription of a large
dular and stromal mitosis. The cells appear taller variety of genes. Global gene expression analy-
and less pseudostratified than before. At LH+4, ses performed along the menstrual cycle have
only occasional mitoses can be seen. Sub- and revealed the relationship between molecular pro-
supranuclear vacuoles within the gland cells are filing and hormonal regulation.
maximal on this day. Gland cell size is also maxi- Both estrogen receptor a (ERa) and b (ERb)
mal on this day. At LH+5, mitosis activity has are expressed in the endometrium, being ERa
ceased absolutely in glands although can be visu- dominant. This receptor is present in both epithe-
alized in stroma. Around 25% of the endome- lial glands and stroma of the functionalis, being its
trium is occupied by glands in this phase. At expression maximal during the proliferative phase
LH+7, the gland cells contain little secretor mate- and declining during the secretory phase. Epithelial
rial and have acquired a low columnar to cuboi- ERb also decreases during the secretory phase,
dal appearance. This is the point of maximal but it is not detected in stroma. Progesterone
receptivity for clinical researchers. The amounts receptor A and B are coexpressed in endometrium
of secretory product within the glands and stromal and are stimulated by estrogen during the prolif-
edema are both maximal by day LH+8. In the erative phase and downregulated by progesterone
last week of the secretory phase are few changes in the secretory phase.
and they mainly occur in stroma and blood ves- The coordinated action of steroids, through
sels. The late secretory phase is characterized by their nuclear receptors in the endometrial cells,
regression and glandular involution. At day promotes the gene regulation of hundreds of
LH+10, stromal edema has decreased, and at genes, inducing the formation of a receptive
LH+11, the stromal predecidual reaction is mainly phenotype. Endometrial cells undergo specific
Fig. 5.1 Profile of expression of the hormones during the menstrual cycle
structural and functional changes that allow determine how, when, and where they are
adhesiveness for the trophoectoderm. Detailed expressed; and bioinformatics, although not
analyses, phase by phase, by using microarray graced with the -omics suffix, remains a key ele-
technology are published by Ponnampalam et al. ment in collection, management, and analysis of
[24] and Talbi et al. [25]. large-scale data sets that are generated by the
approaches described here. These technologies
allow the analysis of thousands of molecules in a
single experiment that ultimately makes possible
5.4 Functional Genomics Studies a global view of the molecular profile of a bio-
of the Human Endometrium logical sample. One of these newly developed
in Natural Cycles tools is microarray technology, initially described
in 1995 [26]. In the field of human reproduction,
Following completion of the human genome most of the studies performed have been func-
sequence, the main goal of researchers has been tional genomics analyses, directed mainly to
to identify the genes involved in the physiologi- deepen the molecular knowledge of human endo-
cal and pathological processes of their particular metrial physiology. Studies with human endome-
fields of expertise. Parallelly, the development of trial cells include cDNA and oligonucleotide
new technologies and the bioinformatics have analyses of endometrial stromal cell decidualiza-
made possible to take the best from the information stimulated by cAMP ± progesterone [27–29],
tion coming from the Human Genome. The suc- compared with nondecidualized endometrial
cess of this project has generated a burst of the stromal cells. Some of these were time-courses
“-omics” sciences: genomics is the study of and have resulted in important insights into bio-
genomes and the complete collection of genes chemical pathways participating in the process of
that they contain; functional genomics, also endometrial stromal decidualization, with new
known as transcriptomics, attempts to analyze players being involved.
patterns of gene expression and to relate this to Much information has been gained, in a whole
function; metabolomics is a large-scale approach functional genomics context, by employing endo-
to monitoring as many as possible of the com- metrial biopsies. Some authors have analyzed
pounds involved in cellular processes in a single the gene expression profile of the endometrium
assay to derive metabolic profiles; proteomic throughout the menstrual cycle [24, 25]. One of
approaches examine the collection of proteins to them concluded that it is possible to classify
endometria precisely according to their transcrip- by microarray technology have to be added to

tional profile, regardless of the morphological other findings obtained by other methods. Some
appearance. More importantly, they established authors have assigned separately essential candi-
the existence of clusters of genes characteristic of dates to the different phases of embryonic implan-
the different phases of the cycle, highlighting the tation. MUC1 (also with a role in first attachment),
potential of gene expression profiling for the MUC4, MUC6, and MUC16 which form the
development of molecular tools in the evaluation epithelial glycocalyx are necessary for epithelial
of endometrial status [24]. This study has been polarity, a key characteristic of the epithelial
confirmed and extended by the work of Linda surface. A brief review of the relevance of candi-
Giudice and her group, who have dissected the date adhesion systems for the second phase
molecular phenotyping of human endometrium attachment, such as basigin (CD147), CD44,
throughout the menstrual cycle phases underlying osteopontin, several integrin subfamilies, trophinin,
its biological processes in normo-ovulatory and CD9, is given by Aplin [37].
women [25]. The race for finding is still continuing, every
Other studies have focused on the receptive year many papers are published with the discov-
endometrium and have defined the gene expres- ery of new biomarkers of endometrial receptivity.
sion profile of this tissue during the WOI [30–34]. In any case, the role of these genes should be
Although only one gene, osteopontin, was con- tested by functional analysis in animal or in
sistently upregulated in all five studies, there are in vitro models in the near future.
several important molecules that have been high-
lighted by their presence in four of the five papers.
Some of them are proteins previously identified 5.5 Functional Genomics Studies
in the endometrium, with or without a described of the Human Endometrium
function. Genes are involved in lipid metabolism in Stimulated Cycles
(apolipoprotein D), immune response (decay
accelerating factor for complement, serine or Following the studies performed in natural
cysteine proteinase, interleukin [IL]-15), regula- cycles, efforts were concentrated on the genomic
tion of cell cycle (growth arrest and DNA-damage impact of COS on the human endometrium dur-
inducible, alpha), ion binding (annexin IV), or ing ART treatments. The aim of the first study
enzymes with different functions in different tis- was to investigate the impact of ovarian stimula-
sues (monoamine oxidase A). More detailed tion using urinary gonadotrophins in a long pro-
reviews of these studies can be found in Giudice tocol with gonadotrophin-releasing hormone
[13] and Horcajadas et al. [35]. (GnRH) agonists without progesterone supple-
The results obtained by these laboratories, mentation (similar to the natural cycle) on endo-
taken together, have demonstrated that endome- metrial gene expression profiles during the WOI
trial receptivity is an equilibrated, complex, and by comparing the endometrial transcriptomic
active process involving hundreds of up- and profiles at hCG+7 of COS cycles vs. LH+7 of a
downregulated genes. These results also indicate previous natural cycle in the same women. For
that a key molecule with the capacity to regulate this purpose, microarray technology by
endometrial receptiveness by itself does not exist. Affymetrix (GeneChip HG_U133A, USA) was
Rather, an endometrial gene signature composed used, which contained more than 22,000 genes to
of 25 genes is prevalent in the development of be tested simultaneously [38]. Results were vali-
receptiveness in natural cycles and dysregulated dated by semiquantitative polymerase chain
in nonoptimal conditions [36]. reaction (PCR) and quantitative PCR experi-
One of the main drawbacks of the previous ments. It was found that more than 558 genes
studies is the inability to differentiate among the showed a differential expression of more than
different cell types present in the endometrium. twofold when ovarian stimulation and normal
Therefore, the findings described above obtained cycles were compared at hCG+7 vs. LH+7.
Analyzing this list of genes, a surprisingly high terns were compared, and that ovarian stimulation
number of genes involved in endometrial recep- may therefore not have a major impact on endo-
tivity (WOI genes) were found to be aberrantly metrial receptivity. They also concluded that
expressed in endometria following ovarian stim- significant changes were found when comparing
ulation (342 genes), showing the expression lev- cycles using GnRH agonist vs. GnRH antagonist
els to be more similar to those in a nonreceptive (13 genes significantly different) [39]. In our lab-
endometrium. It clearly showed that endometrial oratory, a second study was performed to evalu-
development is hampered and delayed under ate the impact of standard and high doses of a
these conditions, as other authors previously had GnRH antagonist (ganirelix) in stimulated cycles
suggested [38]. This study simultaneously reana- compared with GnRH agonist (buserelin); both
lyzed the LH+2 vs. LH+7 endometrial gene protocols were supplemented with progesterone.
expression profiles in previous natural cycles in All the groups were initiated with a fixed dose of
the same subject using a specific GeneChip, and rFSH, and endometrial biopsies were collected at
the results obtained were consistent with previ- hCG+2 and hCG+7 in ovarian stimulation cycles.
ously published results [33]. Endometrial collection at LH+2 and LH+7 from
the previous natural cycle was included as a con-
trol. At day hCG+2, endometrial dating, estro-
gen and progesterone receptors, and pinopode
5.6 Functional Genomics Studies appearance were comparable in all the groups,
of the Human Endometrium including the natural cycle. At hCG+7, endome-
in Ovarian Stimulation Cycles: trial dating, steroid receptors, and the presence of
Agonists vs. Antagonists pinopodes were comparable in both GnRH
antagonist groups and the natural cycles. In the
In 2004, Mirkin et al. compared the gene expres- protocol employing a GnRH agonist, however,
sion profile in the peri-implantation endometrium endometrial dating and pinopode expression sug-
in natural vs. gonadotrophin-stimulated cycles gested an arrested endometrial development
using recombinant FSH (rFSH), with either compared with the other regimens. Gene expres-
GnRH agonist or GnRH antagonist, with or with- sion profiles of the treatment cycles were largely
out progesterone supplementation of the luteal comparable with that of the natural cycle at
phase [39]. Endometrial biopsies were collected LH+2 [40].
in the previous natural cycle LH+8 and 9 days For WOI genes, expression patterns were closer
after hCG administration (hCG+9) in the next to those in the natural cycle following standard (50
ovarian stimulation cycle. Analysis was per- genes dysregulated) or high-dose ganirelix (23
formed with high-density oligonucleotide dysregulated) administration compared with
microarrays (GeneChip HG_U95Av2 Array; buserelin administration (85 dysregulated) [40].
Affymetrix), containing more than 12,000 gene To reflect clinical practice, progesterone supple-
targets. Other structural and functional features mentation was given in the luteal phase in all three
of the endometrium were also investigated. The arms of the study. Under this homogenous condi-
observations made corroborated the morphologi- tion, in each of the treatment groups, expression of
cal changes previously described by other authors. about 100 genes was different compared to the
However, those changes were associated with natural cycle. This suggests that endometrial gene
significant, albeit small, variations in gene expres- dysregulation under COS is affected in a global
sion (18 genes per expressed sequence tag, with a manner, and as a result, a different endometrial
fold change ranging between 1.55 and 3.40). profile arises. The endometrial genomic profile
Mirkin et al. concluded that although ovarian after daily treatment with standard or high-dose
stimulation causes structural and functional GnRH antagonist in women undergoing ovarian
changes compared with natural cycles, small stimulation mimics more closely the natural cycle
changes were found when gene expression pat- as compared with GnRH agonist.
Table 5.1 Different studies of the gene expression profile of the endometrium performed at the time of implantation
comparing natural and stimulated cycles in human using wide functional genomics analysis [45] (with permission)
Day in Day in stimulated Paired # Genes

Study # Samples natural cycle cycle analysis FC Up Down
Mirkin et al. [39] 13 LH+8 (n = 5) hCG+9 (n = 5) Atg Partially >1.19 6 6
LH+8 (n = 5) hCG+9 (n = 5) Ag >1.2 5 1
Horcajadas 19 LH+7 (n = 14) hCG+7 (n = 5) No >3 281 277
et al. [38]
Simón et al. [40] 28 LH+7 (n = 14) hCG+7 (n = 4) No 22 69
Atg standard dose
LH+7 (n = 14) hCG+7 (n = 5) >2 88 24
Atg high dose
LH+7 (n = 14) hCG+7 (n = 5) Ag 22 100
Horcajadas 49 LH+1 (n = 5) hCG+1 (n = 5) No 0 0
et al. [45] LH+2 (n = 5) hCG+2 (n = 5) 0 0
LH+3 (n = 5) hCG+3 (n = 5) 0 0
LH+5 (n = 5) hCG+5 (n = 4) 0 0
LH+7 (n = 5) hCG+7 (n = 5) 69 0
LH+9 (n = 5) hCG+9 (n = 5) – 0 73
Liu et al. [42] 13 LH+7 (n = 5) hCG+7 (n = 4) No >2 244 159
high-serum E2 levels
LH+7 (n = 5) hCG+7 (n = 4) 5 2
low serum E2 levels
Haouzi et al. [43] 84 LH+2 (n = 21) hCG+2 (n = 21) Yes 321 4
LH+7 (n = 21) hCG+5 (n = 21) >2 657 0
FC: fold change
Many groups have continued the investiga- cycles compared to natural cycles in the same
tions about the impact of the ovarian stimulation patients [43, 44]. These works, with similar
protocols in the gene expression profile of the approach to ours in 2005 [38], showed similar
endometrium. Some of them analyzed the expres- results emphasizing the concept that GnRH
sion of molecules of interest such as angio antagonist protocol is more similar to the natural
poitins or vascular factors by using classical cycle receptivity than under the GnRH agonist
techniques of immunohistochemistry, quantita- protocol [44]. A brief summary of the results is
tive PCR, or western blot [41] to conclude that represented in Table 5.1.
there is an advanced endometrial angiogenesis
after gonadotrophin stimulation. But the most
interesting for us are those that have continued 5.7 Endometrial Receptivity
analyzing the endometrium in a global manner as a Global Process
using microarray technology. Liu et al. presented
in 2008 a work where they analyzed the effect of The significant histological, biological, and phys-
high-serum estradiol levels in gonadotrophin- iological features that occur in the endometrium
stimulated cycles at hCG+7 comparing the results throughout the menstrual cycle are ultimately the
to natural cycle at LH+7 [42]. They found 441 result of changes at the gene transcription level,
genes differentially expressed among the three together with the posttranscriptional modifica-
different groups (natural cycle, moderate- tions and epigenetic changes. Most of the labora-
responder, and high-responder). In 2009, the tories have their favorite protein or molecule and
group of Dr. Hamamah has published informa- have tried to elaborate its function in endometrial
tion analyzing the impact of different stimulation receptivity. But, at the moment, functional studies
have not demonstrated the existence of a magic prereceptive (unable to accept the adhesion of the
bullet for human endometrial receptivity as we human blastocysts) to the receptive endometrium
have mentioned previously. Probably, we will (LH+7), which is comparable among the differ-
never be able to understand this complex process ent subjects investigated (Fig. 5.2). In stimulated
with the narrow focus of one gene, because it is cycles, the endometrial gene expression pattern
the result of an equilibrated expression of many was very similar to natural cycles during the WOI
genes involved in specific pathways. For this rea- in the prereceptive phase from hCG-1 to hCG-5
son, our laboratory analyzed the transcriptomics (Fig. 5.2). This observation was confirmed using
of the early and midsecretory phase day by day hierarchical clustering. However, the gene expres-
and compared natural vs. COH cycles [45]. Data sion profile of the receptive endometrium in the
obtained from the microarray analyses of 50 COS cycle at hCG-7 showed significant statisti-
endometrial biopsies were analyzed using differ- cal differences compared with the natural cycle at
ent methods such as sample and gene clustering, LH-7 (Fig. 5.2).
biological processes, or selection of differentially In order to understand how cellular functional-
expressed genes, as implemented in several ities are activated and deactivated along the WOI
microarray data analysis platforms [45]. The first in natural and stimulated cycles, we analyzed
conclusion that could be drawn from that work their corresponding temporal functional profiles.
was that the development of the human endome- For that end, we used the first day as reference
trium in natural cycle follows a genetic program and we compared each subsequent day to this ref-
with a well-defined molecular transition from the erence time by a gene set enrichment analysis,
Fig. 5.2 Principal
Component Analysis (PCA)
of human endometrium
throughout the development
of the secretory phase (after
the endogenous peak of
LH) in natural cycle and
after hCG injection in
stimulated cycle [45]
(with permission)
as implemented in the FatiScan tool of Babelomics study of endometrial development in health and
[46]. Many overrepresented biological terms were disease [49, 50].
shared in both natural and COS categories, par- During the last decade, several groups have
ticularly on days +3 and +5, suggesting a similar attempted to create an objective and modern tool
development on the first days of the WOI. On day for endometrial evaluation. However, at the pre
+7, however, the natural cycle showed a higher sent, commercially available kits have demon-
number of overrepresented biological terms, such strated not to be strong enough for clinical use.
as localization, response to external stimulus, This year, our laboratory has presented the
locomotion, response to biotic stimulus, and Endometrial Receptivity Array (ERA) [51].
others [45]. Interestingly, most of these Gene During the last 5 years, we have analyzed the dif-
Ontology (GO) terms are not present in the tran- ferential gene expression profile of endometria at
sition from day hCG+5 to hCG+7 in COS cycles. LH+1, LH+3, LH+5 (prereceptive phase) vs.
Only two GO terms are conserved in the transition LH+7 (receptive phase) by a T-test. A list of 569
from the prereceptive to receptive state in natural probes representing 238 genes was selected to
and COS cycles; these terms are the response to create our ERA according to very strict criteria.
the stress and cellular physiological process. This molecular tool can be classified as receptive
We also found similarities in the biological or nonreceptive endometrial biopsies from differ-
terms underrepresented in the prereceptive endo- ent phases of the menstrual cycle (Fig. 5.3). The
metrium, except on day LH+7 when more differ- functional sense of these genes was assessed by
ences were observed. On this specific day (LH+7), FATIGO-GEPAS [46]. A significant number of
no common biological term was identified in these genes are implicated in the response to
natural and COS cycles. Furthermore, some terms stress, defense response, and cell adhesion. This
appeared to be underrepresented in hCG+7 of molecular method that contents a gene selection
COS cycles, such as response to external stimu- for endometrial receptivity offers a new objective
lus or organismal physiological process, which tool for endometrial diagnosis. We are now in the
are overrepresented in LH+7 of natural cycles [45]. functional validation of this array using endome-
These results show us that we can consider a trial samples with specific pathologies such as
function or a dysfunction taking into account a implantation failure, endometriosis, and others.
gene, a couple of genes, or a short number of In addition to this method, the future of endome-
genes. Endometrial receptivity is a complex pro- trial evaluation has to be directed to noninvasive
cess in which every regulated gene contributes to methods such as endometrial fluids or serum
the global process in a particular manner. markers. Researchers are now working in these
two lines of investigation to provide noninvasive
diagnostic tools.
An alternative approach to study endometrial
5.8 New Methods for Endometrial receptivity and also embryonic implantation has
Receptivity Studies been culture models. We can divide these models
in: explants, monolayer cultures, coculture, and
The most widely used techniques to study the three-dimensional (3D) cultures. Organ explants
receptive endometrium included microscopy would appear to provide perfect models for mim-
[47], quantitative PCR, in situ hybridization and icking the in vivo environment, as the 3D struc-
gene expression microarrays [36], and proteom- ture and integrity of the endometrium are preserved
ics and metabolomics of endometrial flushings or and all layers of the endometrium are included.
secretions [48]. It is evident that evaluation of Landgren et al. [52] developed a model using
endometrial function cannot be aside of new endometrial biopsies taken 4, 5, and 6 days after
technologies. The histological studies suggest the LH peak from healthy women with normal
that new technologies should be added for objec- regular menstrual cycles. It was used for placing
tive identification of biological samples and the embryos on the lining epithelium of the explant
Fig. 5.3 Hierarchical
clustering of the 68
samples of menstrual
cycle. Samples are
represented in vertical and
values of gene expression
in horizontal. Color
indicates gene expression
value intensities
(blue low; red high)
within 3h of the biopsy being taken. Monolayer many molecules whose expressions are directly
culture consists in single cultures of endometrial related with the receptive status and are altered
epithelial cells in flasks and wells. These cultures during COS cycles [59]. Gene-by-gene analyses
can be performed using primary cell culture com- and microarray technology have produced huge
ing from endometrial biopsies or established amount of data. However, it has been demon-
endometrial epithelial cell lines. These cultures strated that endometrial receptivity does not
have been used mainly for studying the response depend on a single molecule. All the functional
to drugs and for embryo adhesion assays [53]. genomics studies have shown that endometrial
Coculture consists in a separated but communi- receptivity is a very complex process, in which
cated culture of epithelial and stromal endometrial an uncountable number of genes are involved.
cells. This has been used to get high rates of blas- Now it is the time to learn about what the genomic
tocyst formation in clinic, especially as a salvage era can add to our understanding of human endo-
treatment option in couples with repeated implan- metrial receptivity. In this sense, the use of spe-
tation failures [54, 55]. The ultimate in vitro model cific customized microarray such as the ERA
to study the endometrial receptivity and the [51] could help to evaluate the endometrium in an
embryonic implantation therefore would contain objective manner. Future directions in endome-
all the cell types of the endometrium (epithelium, trial receptivity studies will also require comple-
stroma, endothelial, and immune cells) so that the mentarily with proteomics and functionomics.
complex interactions between the maternal tissue
and the blastocyst could be characterized. However,
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Cytokine profiling in endometrial secretions: a the endometrium? RBM Online. 2007;15:45–50.
Current Understanding of
Mullerian-Inhibiting Substance 6
Antonio La Marca, Giovanna Sighinolfi,
and Annibale Volpe
Abstract
In the ovary, Mullerian-Inhibiting Substance (MIS) is produced by the

granulosa cells of early developing follicles and inhibits the transition
from the primordial to the primary follicular stage. MIS levels can be
measured in serum and have been shown to be proportional to the number
of small antral follicles. In women, serum MIS levels decrease with age
and are undetectable in the postmenopausal period. In patients with pre-
mature ovarian failure, MIS is undetectable or greatly reduced depending
on the number of antral follicles in the ovaries. In contrast, MIS levels
have been shown to be increased in women with PCOS. MIS levels appear
to represent the quantity of the ovarian follicle pool and may become a
useful marker of ovarian reserve. In IVF, MIS may permit the identifica-
tion of both the extremes of ovarian stimulation; a possible role for its
measurement may be in the individualization of treatment strategies in
order to reduce the clinical risk of ART along with optimized treatment
burden.
While MIS has the potential to increase our understanding of ovarian
pathophysiology, and to guide clinical management in a broad range of
conditions, a number of important questions relating to both the basic
physiology of MIS and its clinical implications need to be answered.
Keywords
MIS • Folliculogenesis • POI • PCOS • Ovarian reserve • Ovarian ageing •
IVF • Poor response • OHSS
A. La Marca ()
Mother-Infant Department, Section of Obstetrics and
Gynecology, University of Modena and Reggio Emilia,
Modena, Italy

58 A. La Marca et al.
causes of Persistent Müllerian Duct Syndrome in

6.1 The Mullerian-Inhibiting humans [9]. In the ovary of rats, MIS type II
Substance (MIS) and MIS receptor is expressed both in granulosa and theca
Receptors cells [10].
The identity of MIS type I receptor(s) is not
MIS (also called anti-Mullerian hormone, AMH) yet clear, particularly in gonads. At least three
is a member of the transforming growth factor- type I receptors have been extensively studied
beta (TGF-beta) superfamily [1]. MIS is a [11]. It has been hypothesized that MIS could
homodimeric disulfide-linked glycoprotein with share a type I receptor with another member of
a molecular weight of 140 kD. The gene is located the TGF-family. The first identified MIS type I
on the short arm of chromosome 19 in humans, receptor [12], Alk6, was singled out from the six
band 19p 13.3 [2]. The MIS gene is 2,750 bp type I receptors of the TGF-b family because of
long, and it is divided into five exons. The 3¢ part its ability to interact in a ligand-dependent man-
of the fifth exon codes for the bioactive part of the ner with MISRII in CHO cells, permanently
molecule and is extremely GC rich. MIS is expressing human MISRII (CHO-3W) [18]. Alk
strongly expressed in Sertoli cell from testicular 2 and Alk 3 have been successively proposed as
differentiation up to puberty and to a much lesser alternative possible MIS type I receptor [11].
degree in granulosa cells from birth up to meno- Alk6 and Alk 2 [13, 14] may mediate MIS action
pause [3, 4] in other target cells, whereas Alk 3 is the only
MIS seems to act only in the reproductive one to have been clearly shown to mediate MIS
organs [4]. The most striking effect of MIS is its action upon Müllerian ducts [15].
capacity to induce regression of the Müllerian
ducts, the anlage of the female internal reproduc-
tive organs. In the absence of MIS, Müllerian 6.2 The Role of MIS in Ovarian
ducts of both sexes develop into uterus, Fallopian Folliculogenesis
tubes and the upper part of the vagina [5, 6]. MIS
is expressed also in the ovarian granulosa cells.
MIS expression in the ovaries has been observed In primordial follicles, MIS expression seems to
as early as 36-week gestation in humans [7]. be absent. MIS immunostaining can first be
MIS employs a heteromeric receptor system observed in granulosa cells of follicles at the pri-
consisting of single membrane spanning serine mary stage of development. In one study, approx-
threonine kinase receptors called types I and II. imately 75% of secondary follicles were positive
The type II receptor imparts ligand-binding spec- for MIS immunostaining. The strongest staining
ificity and the type I receptor mediates down- was observed in preantral and small antral folli-
stream signalling when activated by the type II cles [16].
receptor. MIS continues to be expressed in the growing
The human gene for MIS type II receptor was follicles in the ovary until they have reached the
isolated in 1995 [8]. It is located on chromosome size and differentiation state at which they may
12 and constituted by 11 exons spread over more be selected for dominance. In the mouse, this
than 8 kbp. The MISRII messenger is expressed occurs at the early antral stage in small growing
by MIS target organs, namely the Müllerian follicles, whereas in the human it is evident in
ducts, and the gonads. MIS type II receptor is antral follicles 4–6 mm in diameter [16]. Thus,
localized to the mesenchyme around the MIS is expressed in follicles that have undergone
Müllerian duct in the urogenital ridge of both the recruitment from the primordial follicle pool, but
male and female rat and mouse. It is interesting have not been selected for dominance. MIS is not
to note that loss of function mutations in the type expressed in atretic follicles or theca cells
II receptor as well as the MIS ligand itself are [17–19]
6 Current Understanding of Mullerian-Inhibiting Substance 59
It has recently been demonstrated that oocytes of primordial follicles compared to their wild-type
from early preantral, late preantral and preovula- littermates [25]. The increased rate of recruit-
tory follicles up-regulate MIS mRNA levels in ment from the primordial pool observed in the
granulosa cells, in a fashion that is dependent MIS null-mice was already evident before the
upon the developmental stage of the oocyte. initiation of the oestrous cycle. These studies
These findings therefore suggest that oocyte reg- confirmed the concept that in the absence of MIS,
ulation of granulosa cell gene expression occurs primordial follicles are recruited at a faster rate,
during extended periods of follicle development resulting in premature exhaustion of the primor-
and that oocyte regulation of MIS expression dial follicle pool [25]. Since MIS null-mice have
may play a role in intra- and inter-follicular coor- low levels of FSH, with increased numbers of
dination of follicle development [20] growing follicles, it has been hypothesized that
The main physiological role of MIS in the follicles are more sensitive to FSH in absence of
mouse ovary seems to be limited to the inhibition MIS. The possible inhibitory effect of MIS on
of the early stages of follicular development [21, follicular sensitivity to FSH could play a role in
22], since both in vivo and in vitro experiments the process of follicular selection [25]. Diminished
have indicated that the transition from primordial expression of MIS within the follicles would
into growing follicles becomes enhanced in the reduce the threshold level for FSH, allowing fol-
absence of MIS, leading to early exhaustion of licles to continue growing and to ovulate in the
the primordial follicle pool [23] (Fig. 6.1). next estrous cycle [23, 26].
In one study, the ovaries of 4-month-old MIS Very recently, ovaries from rats placed in
knockout mice contained 3 times as many small organ culture and incubated in the absence and
non-atretic growing follicles and a reduced number presence of MIS permitted to show that MIS
Fig. 6.1 In women, MIS expression can first be observed recruitment and in the selection of the dominant follicle
in primary follicles, and is strongest in preantral and small (from ref [24] with permission)
antral follicles. MIS may play an inhibiting role in initial
alters the expression of several hundred genes levels as low as 2 ng/mL was developed 9 years
[27]. The overall effects of MIS exposure were to ago [30]. However, recently new commercial
decrease the expression of stimulatory factors, ultrasensitive sandwich ELISA assays have been
increase the expression of inhibitory factors and developed capable of detecting concentrations
regulate cellular pathways that result in the inhi- less than 0.1 ng/mL. The increased sensitivity
bition of primordial follicle development [27]. and availability of different assays have high-
At present, however, these views remain largely lighted the urgent need to agree on the standard
speculative as few in vitro or in vivo studies have preparations used, in order to avoid confusion in
been conducted which address physiological role reported levels and interpretation.
of MIS in the human ovary. At present, there are two highly sensitive sand-
Current theories also suggest a role for MIS as wich ELISA assays available: the diagnostic
a co-regulator of steroidogenesis in granulosa systems laboratories (DSL) and the Immunotech-
cells, as MIS levels appear to be related to oestra- Beckman assay. The sensitivity of the DSL is
diol levels in follicular fluid (FF) from small reported to be 0.025 ng/mL compared with
antral follicles [28]. This was confirmed by a 0.07 ng/mL for the Immunotech-Beckman assay,
recent study which showed that polymorphisms although this difference was not confirmed in a
in the gene for MIS or MIS receptor type II seem recent clinical study [31]. The intra- and inter-
to be related to follicular phase oestradiol levels, assay variations of the two assays are similar (<7
suggesting a role for MIS in the FSH-induced and <5%, respectively). The DSL assay is not spe-
steroidogenesis in the human ovary [29]. cies-specific, a feature which can be advantageous
Although MIS has been shown to have mainly for research laboratories using rodent models.
autocrine and paracrine actions in follicle develop- Initial studies comparing the two assays have
ment, the protein is also measurable in serum. shown that MIS levels appear to be four to five-
Antral follicles are considered the primary source fold lower with the DSL assay compared to the
of circulating MIS as they contain a large number Immunotech-Beckman assay [32, 33]. In their
of granulosa cells. A body of clinical data suggests report, Bersinger and coll. [33] alluded to prob-
that MIS is preferentially and constantly secreted lems inherent to MIS measurements which stem
by small rather than large antral follicles. The from residual matrix effects and instabilities of
amount and the rate of MIS production by a single certain antigenic determinants. However,
antral follicle should be investigated and in although developed independently, these assays
Granulosa cells secrete MIS into both the are now both produced by a single company
bloodstream and FF, although concentrations are (Beckman-Coulter), and cross-referencing has
very much higher in the latter. However, the exact shown that the correlation between the two assays
role of MIS in this compartment has not been is >0.9 (personal communication from Beckman-
elucidated. Coulter representative). While a uniform conver-
sion factor has never been published, the
manufacturers suggest that 1.5 may be a good fig-
6.3 Circulating MIS in Women ure for this purpose (personal communication).
Both kits are likely to remain in production
6.3.1 Current Assays over the next few years as approximately half of
researchers are using the DSL assay and the
other half the Immunotech-Beckman product.
Until recently, MIS assays were only available in However, it is anticipated that within 2 years, an
a few laboratories around the world. The lack of automated system for MIS measurement will
access to a single reliable and standardized probably become available, and industry sources
commercial assay has hindered the development indicate that it is likely that this will be cali-
of MIS as a clinical marker of ovarian reserve. brated to the Immunotech-Beckman kit (personal
A sensitive ELISA assay capable of detecting communication ).
6.3.2 Factors Modulating Serum therefore, probably of minor significance when

MIS Levels interpreting data for clinical purposes.
Furthermore, MIS levels appear to be unmodi-
fied in conditions under which endogenous
In women, MIS levels are almost undetectable at gonadotrophin release is substantially dimin-
birth. After a slight increase in the weeks after ished, such as during pregnancy [48], under
birth, MIS levels reach the highest values during GnRH agonist pituitary downregulation [49] and
late puberty [34] and then show a progressive oral contraceptive administration [45, 50, 51].
decline along reproductive life as the follicular This indicates that non-cyclic FSH-independent
reserve becomes depleted [34, 35], becoming ovarian activity persists even when pituitary FSH
undetectable after menopause [36, 37]. Further secretion is suppressed. These findings are con-
evidence that circulating MIS appears to be solely sistent with the concept that MIS levels reflect the
of ovarian origin comes from a study in which continuous FSH-independent non cyclic growth
MIS was undetectable 3–5 days following bilat- of small follicles in the ovary (Fig. 6.2)
eral ovariectomy [37]. As MIS levels essentially Obesity has been associated to reduced fertil-
reflect the follicular ovarian pool, reduction in the ity even in the presence of ovulatory menstrual
number of small growing follicles may be fol- cycles and to increased probability of miscarriage
lowed by a reduction in circulating MIS. The than normal weight women [52, 53]. Non-PCOS
reduction in ovarian reserve is a physiological obese women show reduced levels of inhibin B
process occurring in the late reproductive period and MIS [54, 55], suggesting that obesity may be
and consistently associated to a decrease in MIS associated to impaired ovarian reserve. However,
levels [38, 39]. The strong correlation existing a recent study [56] examined the correlation of
between MIS levels and the resting pool of folli- obesity with hormonal and ultrasound-derived
cles has recently been highlighted by some papers markers of ovarian reserve and found that serum
showing that MIS measurement may be used to MIS levels are lower in obese women compared
predict the occurrence of menopause [40, 41]. with age-matched women of normal weight,
MIS may constitute a unique endocrine param- despite similar antral follicular count. This sug-
eter for the investigation of ovarian function, gests that MIS levels in obese women may be
since several studies have demonstrated that, in lower for physiological reasons related to obesity
contrast to sex steroids, gonadotropins and pep- itself and may not be necessarily indicative of
tides such as inhibin Band MIS serum levels do impaired ovarian reserve [56]. Other factors
not significantly change throughout the menstrual related to reduced MIS levels are smoking [57],
cycle [42–45]. However, others have reported alcohol use [58] and race or ethnicity [59].
significant cyclical fluctuations in MIS levels
with a rapid decrease in MIS levels in the early
luteal phase [46, 47]. Excursions from mean lev- 6.4 The Role of MIS in Investigating
els of +3% and to −19% have been reported [46, Ovarian Dysfunction
47]. These variations are similar to reported inter-
cycle fluctuations for MIS [45]. In the clinical
setting, the inter- and intra-cycle variability in The observed relationship between the follicular
serum MIS levels may be considered to be low ovarian pool and serum MIS levels indicates that
enough to permit random timing of MIS mea- serum levels could provide additional informa-
surements during the menstrual cycle. Of course, tion (linked to the follicle dynamics) during the
further studies on a large sample of patients, diagnostic evaluation of hypogonadism. MIS
based on daily blood samples, are needed to serum levels have been found to be normal in
clarify whether MIS levels vary significantly women with hypogonadotropic amenorrhea,
during the menstrual cycle. Up to now, reported indicating that initial follicle recruitment is not
fluctuations appear to be of small amplitude, and abolished in hypogonadotropic hypogonadism [60].
Fig. 6.2 In contrast to the other known ovarian reserve of antral follicles. The E2 levels are less a reflection of the
markers, AMH may not only reflect the number of early and number of antral follicles, but rather of their growth activity
developing antral follicles, but also earlier stages of follicle during the follicular phase. On the basis of this reason, high-
development. The FSH, estradiol, and inhibin secretions are est biological plausibility as marker of ovarian reserve is to
mutually connected by a negative feedback. Therefore, their be attributed to AMH, followed by inhibin B, FSH, and E2
circulating levels are only an indirect reflection of the number (from ref [24] with permission)
This finding has been recently confirmed in with SCA-POI and in 4/66 (6%) with iPOI. Eight
young women with anorexia nervosa-related of 12 (67%) women with SCA-POI with less than
amenorrhea [61]. In contrast, in women with 5 years and 1/14 (7%) with longer disease dura-
hypergonadotropic amenorrhea (premature ovar- tion had MIS concentrations within the normal
ian insufficiency, POI), serum MIS levels are range. These findings indicate that women with
very low or undetectable. In a recent study in POI SCA-POI, differently from those with iPOI, pres-
patients, the number of MIS immunopositive fol- ent a preserved ovarian follicle pool for several
licles present in ovarian biopsy material was years after diagnosis of ovarian insufficiency.
closely correlated with serum MIS levels [62], The results of our study on women with SCA-
suggesting a diagnostic role for MIS in the evalu- POI may be of high relevance for future clinical
ation of hypogonadism. studies aimed at modulating the autoimmune
In a recent study, we measured serum concen- process and at preserving the residual functional
trations of MIS in 26 women with POI due to ste- tissue and/or delay the progression of the destructive
roidogenic cell autoimmunity (SCA-POI) and 66 autoimmune process [63] (Figs. 6.3 and 6.4).
with non-autoimmune idiopathic POI (iPOI) [63] Serum MIS measurement may also have a role
and found significantly higher MIS levels in in identifying incipient ovarian failure in young
women with SCA-POI than in women with iPOI. eumenorrheic women with moderate hypergonado-
MIS was detected in 11/26 (42%) women with tropism. Incipient ovarian failure [64] may pre-
SCA-POI and in 7/66 (11%) with iPOI. Serum cede the onset of cycle irregularity (transitional
concentrations above the fifth percentile of the ovarian failure) and hence the menopausal transi-
normal range were detected in 9/26 (35%) women tion by 3–10 years .In a recent study, serum levels
of MIS were able to discriminate between incipi-

ent and transitional ovarian failure, defined as
elevated follicular phase FSH levels along with a
regular menstrual cycle or cycle disturbances,
respectively. In women with incipient failure,
levels were below the fifth percentile of normo-
ovulatory women in 25% and undetectable in
7%, compared with 66 and 52% in women with
transitional ovarian failure [65]. This finding
indicates that MIS provides an accurate assess-
ment of ovarian follicle pool in young hypergo-
nadotropic patients especially in the clinically
challenging subgroups of patients with elevated
FSH who do not fulfil the strict definition of
POF [65].
Moreover, young sisters or daughters of
Fig. 6.3 Box-and-whisker plot of AMH serum concentra-
women affected by POF, patients undergoing
tions in patients with POI due to steroidogenic cell autoim-
munity (SCA-POI), in patients with idiopathic POI, in ovarian surgery, and patients affected by Turner
post-menopausal women and in healthy control women. syndrome may all benefit from the information
Boxes represent median (thick line in the middle of the boxes) which MIS levels might provide in this context.
and interquartile ranges (25th and 75th percentile; thick
This is illustrated in a recent report on the fertility
lines at the bottom and the top of boxes). Error bars represent
10th and 90th percentiles. Open square, p = 0.03 SCA-POI preservation in girls with Turner Syndrome [66].
vs. idiopathic POI; closed circle, p = 0.018 SCA-POI vs. natu- Forty-seven young girls with Turner Syndrome
ral menopause; open circle, p < 0.0001 SCA-POI vs. healthy underwent laparoscopy for ovarian tissue cryo-
controls (Bonferroni’s corrected p values) (from ref [63],
preservation and 15 of them had follicles in the
with permission)
tissue piece analysed. When investigating which
factors had the highest predictive value for find-
ing follicles, the most powerful were the presence
of 46XX/XO chromosomal mosaicism, serum
FSH levels below 11 IU/L and serum MIS levels
greater than 0.28 ng/mL [66]. MIS may therefore
have a role in the diagnostic work-up and fertility
counselling of patients with Turner Syndrome.
A particularly promising field for further
research is assessing the value of MIS as a poten-
tial marker of ovarian function in women who
underwent chemotherapy or radiotherapy for
malignant disease. Longitudinal studies have dem-
onstrated that MIS measurement can be used as a
reliable and early marker of ovarian damage, and
that a decrease in MIS precedes alterations in other
markers [67–71]. The ability to predict the impact
of these treatments on ovarian follicular pool
(including future fertility) in individual cases may
Fig. 6.4 Distribution of FSH serum concentration in be of considerable value in guiding clinicians and
women with SCA-POI, according to AMH serum concen-
tration (n = 11 women with detectable (+) AMH and n = 15
patients when considering whether or not fertility
women with undetectable (−) AMH). Bars represent mean preservation strategies should be employed, and if
values (from ref [63], with permission) so, which strategy is most appropriate. At present,
care is guided largely by the nature of the interven- women with PCOS since high MIS levels may
tion and the age of the woman. inhibit the primordial follicle pool depletion [78].
A number of studies have shown serum MIS MIS measurement has been found to offer a
levels to be increased in women with PCOS com- relatively high specificity and sensitivity (92 and
pared to controls [72–77]. This is thought to be 67%, respectively) as a diagnostic marker for
the result of increased granulosa cells synthesis PCOS [86]. On this basis, it has been proposed
and secretion of MIS in the polycystic ovaries that in situations where accurate ultrasound data
[78]. Indeed, levels of MIS are on average 75 are not available, MIS could be used instead of
times higher in granulosa cells from polycystic the follicle count as a diagnostic criterion for
ovaries, compared with levels in normal ovaries PCOS [86].
[79]. In addition, increased MIS levels in PCOS Other than for diagnostic evaluation, MIS mea-
may be due to the disruption in folliculogenesis surement may also be useful in the therapeutic
leading to an excess accumulation of preantral approach of PCOS patients. Indeed, overweight
and small antral follicles [80]. women with PCOS who respond to weight loss
Increased MIS levels have also been found in with menstrual improvements have significantly
prepubertal [81] and peripubertal [82] daughters reduced preweight-loss MIS levels, indicating
of PCOS women as well as in adolescent PCOS that baseline MIS may provide a potential clinical
girls with normal menstrual cycles [83], suggest- predictor of menstrual improvements with weight
ing that altered follicle development is already loss in PCOS [87]. Similarly, basal MIS levels
present during infancy and early adulthood before evaluation may be useful in the prediction of ovar-
the clinical phenotype of ovarian dysfunction is ian response to clomiphene citrate [88]. Finally, it
present. has been shown that metformin administration in
MIS levels appear to be related to the severity women affected by PCOS is associated with a
of the syndrome since levels have been observed reduction in both MIS serum levels and antral fol-
to be higher in insulin-resistant PCOS women than licles, suggesting that the measurement of MIS
in patients with normal insulin sensitivity [84]. could be used to evaluate the treatment efficacy
Similarly, MIS is higher in amenorrheic compared with insulin sensitizers [89].
to oligomenorrheic women with PCOS [75], which
could indicate a role for MIS in the pathogenesis
of PCOS-related anovulation. Alternatively, high 6.5 MIS as a Marker for Ovarian
MIS values could reflect more impaired disruption Ageing
in folliculogenesis and granulosa cell function in
the ovary of amenorrheic compared to oligomen-
orrheic PCOS women [75]. The age-related decline in female reproductive
In a recent longitudinal study, serum MIS lev- function due to the reduction of the ovarian fol-
els were measured in 98 women with PCOS and licle pool and the quality of oocytes has been well
41 controls at 2 time points (interval between the established. A reliable marker for the age at
visits: 0.3–9 years). Although serum MIS levels which subfertility will occur would have great
declined over time in both groups, the reduction potential value as a predictor of future reproduc-
observed in PCOS patients was less than that in tive lifespan. The ideal marker would show a sig-
controls. The authors of this study postulated that nificant change in levels from adolescence to the
this may indicate a longer reproductive life span late reproductive period. Increased basal levels of
in PCOS patients [78]. On histological examina- FSH and a decrease in inhibin B and in the antral
tion, polycystic ovaries exhibit the same number follicle count (AFC) on ultrasound examination
of primordial follicles, whereas the number of are widely taken to indicate a reduced ovarian
developing follicles is doubled compared with reserve.
normal ovaries [85]. Hence, it may be proposed Recent studies have indicated that MIS may
that the process of ovarian ageing is delayed in constitute an important novel measure of ovarian
reserve. Evidence for this come from studies mean longitudinal decline over time both in
demonstrating that serum MIS levels fall through- younger women (<35 years) and in women over
out reproductive life [90], with levels becoming 40 years [36].
undetectable after spontaneous menopause [34, Recently, MIS levels were measured in 144
36] (Figs. 6.5 and 6.6). fertile normal volunteers and used to determine
Serum levels on day 3 of the menstrual cycle an estimate of mean MIS as a function of age
show a progressive decrease with age and appear [41]. There was good conformity between the
to correlate well with AFCs age and FSH [90]. In observed distribution of age at menopause and
an interesting prospective study, a group of that predicted from declining MIS levels, further
women was followed longitudinally, with an supporting the hypothesis that MIS levels may
interval between the two visits ranging from 1.1 predict the age of onset of menopause. Other
to 7.3 years. Although the number of antral folli- studies have recently confirmed that a single MIS
cles and the levels of FSH and inhibin B did not measurement may be a good predictor for the
change, a reduction in mean MIS levels of about onset of menopause in ageing women [40, 92].
38% was observed [90]. In conclusion, compared to other known mark-
The same group prospectively studied 81 ers, MIS seems to better reflect the continuous
women for 4 years (mean age 39.6 and 43.6 at the decline of the oocyte / follicle pool with age [36].
beginning and at the end of the study, respec- The decrease in MIS with advancing age may be
tively). Although the AFCs did not change over present before changes in currently known age-
this time period, MIS, FSH and inhibin B all ing-related variables, suggesting that serum MIS
demonstrated significant changes. However, MIS levels may be the best marker of ovarian ageing
was the only marker of ovarian reserve showing a and menopausal transition.
Fig. 6.5 Correlation between logAMH and age of women. age−0.0015 age2). Ninety five percent confidence interval is
Regression analysis revealed that age-related changes were shown (R2 = 0.24; P < 0.001). X axis: age (years); Y axis:
bestfittedbyapolynomialfunction(logAMH = 1.0547 + 0.0546 logAMH (from ref [91] with permission)
Fig. 6.6 AMH levels throughout the reproductive period. limits are reported. Box indicates 25th and 75th centiles
Median, lower (2.5th centile) and upper (97.5th centile) (from ref [91] with permission)
6.6 MIS as Predictive Marker in IVF with a greater number of retrieved oocytes. In

particular, MIS levels were 2.5-fold higher in
patients with at least 11 oocytes compared with
Recently, Anti-Mullerian Hormone (MIS) has those with 6 oocytes or fewer retrieved. Results
been evaluated by several groups as a potential from this study were successively confirmed by
novel clinical marker of ovarian reserve and several retrospective and prospective studies by
response to gonadotropins (see ref [93] for different independent groups.
review). In particular, in the last few years several In Table 6.1, all retrospective and prospective
large prospective studies have been published studies that have found a correlation between
reporting extremely interesting new data on the the number of retrieved oocytes and MIS levels
possible clinical application of MIS measurement have been summarized. The majority of authors
in the prediction of quantitative and qualitative have compared MIS with age and other hormonal
ovarian response in ART. markers (FSH, estradiol and Inhibin B) and only
few of them also with ultrasound markers of
ovarian reserve. The balance of the published
6.6.1 Prediction of Quantitative studies seems to indicate that MIS is a better
Ovarian Response marker in predicting ovarian response to COS
than age of the patient, day 3 FSH, oestradiol and
inhibin B.
Much data show a strong and positive correlation With high probability, AFC and MIS perform
between basal MIS serum levels and the number with similar power in the prediction of the num-
of retrieved oocytes in women undergoing ovar- ber of retrieved oocytes. This is confirmed by a
ian stimulation. In the evaluation of MIS as a recent meta-analysis in which the value of serum
marker of ovarian response to FSH, the first paper MIS levels as a test to predict ovarian response in
reporting an association between circulating MIS IVF in comparison to the performance of the
and ovarian response to gonadotropin was by AFC has been assessed [115]. A total of thirteen
Seifer and colleagues, 7 years ago [94]. The author studies were analysed reporting on MIS and seven-
observed that higher day 3 MIS was associated teen on AFC. The ROC curves for the prediction of
Table 6.1 Studies on AMH as marker of ovarian response to COH

R with AMH better than
References n oocytes AFC Ov. Vol d3 FSH d3 E2 d3 inhB Age
Seifer et al. [94] 107 0.48 √ √
Van Rooij et al. [95] 130 0.57 = √ √ √ √
Fanchin et al. [96] 93 0.43
Muttukrishna et al. [97] 69 0.69 √ √
Hazout et al. [98] 109 0.38 √ √ √ √
Muttukrishna et al. [99] 108 0.5 = √
Elder Geva et al. [100] 56 0.64 X √ √
Silberstein et al. [101] 257 0.33 √
Ficicioglu et al. [102] 50 0.56 √ √ √ √
Lekamge et al. [103] 126 0.34 =
La Marca et al. [104] 48 0.7
Kwee et al. [105] 110 0.63 X √ √ √
Nakhuda et al. [106] 77 0.63 √
McIlveen et al. [107] 84 0.78 √ √ √ = √
Nelson et al. [108] 340 0.71 √ √
Elgindy et al. [109] 33 0.88 = √ √
Lie Fong et al. [110] 125 0.47
Jee et al. [111] 59 0.53 X
Jayaprakasan et al. [112] 135 0.47 = √ √ √ √
Wunder et al. [113] 276 0.35 √ X
Nelson et al. [114] 538 0.64
Comparison with other predictors (from ref [93])
ovarian response indicated no significant dif- retrieved oocytes (reviewed in ref [117]). More
ference between the performances of MIS and logically, poor response is generally considered
AFC. Hence, it may be concluded that at present to have occurred if the cycle is cancelled due to
MIS appears to offer at least the same level of an inadequate ovarian response to stimulation.
accuracy and clinical value for the prediction of Whatever definition is used, poor responders
ovarian response as AFC [115]. have definitely lower pregnancy rates compared
to normal responders of similar age [117].
6.6.1.1 Prediction of Poor Response and In the clinical setting, it may be useful to cor-
Cycle Cancellation rectly predict the occurrence of poor response as
this may lead to avoiding treatment for women
A proportion of women (2–30%) undergoing destined not to respond to COS thus contributing
COS experience poor response [116] for which to reducing the cycle cancellation rate, the treat-
there is no universally accepted definition. ment costs and psychological stress for the cou-
Numerous criteria have been used to characterize ple. Finally, an improved counselling for the
poor response. The number of developed follicles prediction of poor response may ameliorate dis-
and the number of retrieved oocytes are two of appointment and distress.
the most important criteria for defining poor A large number of clinical parameters have
response. The proposed number varies among been shown to predict the poor ovarian response
different authors and ranges from less than three to stimulation with exogenous gonadotropins and
to less than five dominant follicles on the day of have been introduced in the clinical practice.
hCG and from less than three to less than five These include age, basal serum FSH and inhibin
B levels, AFC, ovarian volume, a number of the aim of refraining bad prognosis couples from
dynamic tests and more recently MIS. IVF, then in order to have a low number of false
Several authors investigated the utility of MIS positive results, specificity more than sensitivity
in the prediction of poor response to FSH. should be taken into consideration. On this basis,
Reported sensitivity and specificity ranged it should be highlighted that half of the studies on
between 44–97 and 41–100%, respectively MIS have reported a specificity higher than 0.85.
(Table 6.2). It is clear that not all studies found One of the main advantages of MIS with
for MIS an optimal sensitivity (>0.75) and speci- respect to the other hormonal markers of ovarian
ficity (>0.85). However, if MIS is measured with reserve is the possibility to be used as a menstrual
Table 6.2 Sensitivity and specificity of AMH for the prediction of poor response to gonadotropin stimulation (from
ref [93])
Study CUT-OFF Sens Spec Definition of
References n design value (%) (%) poor response AMH assay
Van Rooij 119 Prosp 0.3 mg/L 60 89 <4 oocytes Immunotech-Beckman-Coulter
et al. [95]
Muttukrishna 69 Prosp 0.1 ng/mL 87.5a 72.2a <4 oocytes or Immunotech-Beckman-Coulter
et al. [97] cancellation
Muttukrishna 108 Retro 0.2 ng/mL 87 64 £4 oocytes Immunotech-Beckman-Coulter
et al. [99]
Tremellen 75 Prosp 8.1 pmol/L 80 85 £4 oocytes Immunotech-Beckman-Coulter
et al. [118]
Panarrubia 80 Prosp 4.9 pmol/L 53a 96a Cancellation Immunotech-Beckman-Coulter
et al. [119]
Ebner 141 Prosp 1.66 ng/mL 69 86 <4 oocytes Immunotech-Beckman-Coulter
et al. [120]
Ficicioglu 50 Prosp 0.25 pg/mL 90.9 90.9 <5 oocytes Diagnostic system laboratories
et al. [102]
La Marca 48 Prosp 0.75 ng/mL 80 93 <4 oocytes or Immunotech-Beckman-Coulter
Freour 69 Prosp 1.3 mg/L 44 100 <6 oocytes Immunotech-Beckman-Coulter
et al. [121]
Smeenk 80 Prosp 1.4 mg/L 62 73 £4 oocytes Immunotech-Beckman-Coulter
et al. [122]
McIlveen 84 Prosp 1.25 ng/mL 58 75 £4 oocytes Immunotech-Beckman-Coulter
et al. [107]
Kwee 110 Prosp 1.4 mg/L 76 86 <6 oocytes Diagnostic system laboratories
et al. [105]
Nakhuda 77 Prosp 0.35 ng/mL 90.1a 81.8a Cancellation Diagnostic system laboratories
et al. [106]
Lekamge 126 Retro 14 pmol/L 73 73 £4 oocytes Immunotech-Beckman-Coulter
et al. [103]
Nelson 340 Prosp 5 pmol/L 75b £2 oocytes Diagnostic system laboratories
et al. [108]
Gnoth 132 Prosp 1.26 ng/mL 97 41 £4 oocytes Diagnostic system laboratories
et al. [123]
Nardo 165 Prosp 1.0 ng/mL 87 67 £4 follicles on Diagnostic system laboratories
et al. [124] day 8 of COH
Jayaprakasan 135 Prosp 0.99 ng/mL 100 73 <4 oocytes or Diagnostic system laboratories
Retro retrospective study; Prosp prospective study
a
For cycle cancellation identification
b
Percentage of correctly classified poor responder patients
cycle-independent marker since MIS seems to be potentially life-threatening condition. Mild and
stable and to have very low inter- and intra-cycle moderate forms of OHSS may occur in 15–20%
variability. In the first published study based on a of all ovarian stimulation cycles; however, the
single random measurement of MIS, a sensitivity severe form of the syndrome has been reported
of 80% and specificity of 93% has been calcu- as frequently as 1–3% [125].
lated for the prediction of poor response [104]. The specific risk factors for OHSS include
Variable predictive performance for MIS was young age, low BMI, signs of PCOS, previous
reported in the various studies and this has been history of OHSS and high estradiol on the day of
considered by some authors to be partly due to hCG [125]. The key to preventing OHSS is the
the use of different variants of MIS assay. recognition of risk factors for OHSS leading to an
Most importantly, the performance of any test individualization of gonadotropin starting dose
of ovarian reserve, including MIS, is strictly which should be the minimum dose necessary to
dependent on the prevalence of the disease (poor achieve the therapeutical goal. However, the accu-
response) we want to identify. Throughout the rate prediction of OHSS in an individual IVF
published studies, the prevalence of poor response cycle remains a difficult task. Indeed, PCOS (the
may vary on the basis of the percentage of older main risk factor used in the prediction of OHSS)
(high incidence of poor response) and younger is present only in 20% of women undergoing
(low incidence of poor response) patients included COH and in less than 20% of patients developing
in the study and of course on the basis of the symptoms of impending OHSS [126].
adopted definition for poor response. As a conse- The recognition of a dose-response relation-
quence, the same test, measured at the same labo- ship between MIS and ovarian response to FSH
ratory, will have different predictive performance leads to the hypothesis that hyper-response to
if the proportion of older patient and the defini- ovulation induction might result from high MIS.
tion of poor response will change. In this context, high basal MIS may be associated
In conclusion, the balance of all the clinical with an increased risk of developing OHSS.
studies on MIS seems to suggest that MIS mea- At present, few studies have been published
surement prior to gonadotropin secretion may be reporting on this issue (Table 6.3). However, it
useful in the prediction of women at risk for poor seems that hyper-response and OHSS may be
response or no response to gonadotropins. associated to significantly higher mean basal MIS
Moreover, the absence of modifications in serum levels. In the last years, four prospective studies
MIS levels throughout the menstrual cycle permits performed on large number of subjects have been
clinicians to have a reliable serum marker of published reporting relevant value for MIS for
ovarian reserve that can be measured indepen- the prediction of hyper-response and OHSS
dently of the day of the cycle. (Table 6.4).
Particularly, the studies by Lee et al. [128] and
6.6.1.2 Prediction of Hyper-Response Nardo et al. [124] have independently calculated
and OHSS a similar performance of MIS for the prediction
of hyper-response and OHSS. The reported cut-
Ovarian hyper-response is the opposite side of off value is of about 3.5 ng/mL, above which
the spectrum of ovarian reserve and might lead to hyper-response/OHSS may be anticipated. In the
a potentially life-threatening condition, the ovarian study by Lee et al. [128], a cohort of 262 IVF
hyperstimulation syndrome (OHSS). cycles was investigated, in order to evaluate the
OHSS refers to an exaggerated ovarian predictive value for OHSS by means of age, BMI,
response to gonadotropin treatment. The syn- estradiol and MIS levels. Authors found that the
drome has a broad spectrum of clinical mani- ROC of the basal MIS was larger than age and
festations, from mild illness needing only BMI and works as well as the number of follicles
careful observation to severe illness requir- and estradiol levels on the day of hCG. Basal
ing hospitalization and intensive care being a MIS levels predicted OHSS with a sensitivity of
Table 6.3 Basal AMH levels in women with normal and hyper-response to COH and OHSS (from ref [93])
Mean AMH levels (ng/mL)
Excessive response
References Design n Normal response (>20 oocytes) OHSS
Tremellen et al. [118] Prosp 75 2.1 2.95
Eldar Geva et al. [100] Prosp 56 2 5.3
Nakhuda et al. [127] Retro 30 0.63 3.6
Nelson et al. [108] Prosp 340 1.4 3.9
Nardo et al. [124] Prosp 165 3.04 5.56
Retro retrospective study; Prosp prospective study
Table 6.4 AMH cut-off values for the prediction of hyper-response to COS and OHSS (from ref [93])
Study CUT-OFF Prediction of Prediction
References n design value Sens (%) Spec (%) hyper-response of OHSS
Kwee et al. [105] 110 Prosp 5 mg/L 53 91 Öa
Nelson et al. [108] 340 Prosp 25 pmol/L 60 94.9 Öb
Lee et al. [128] 262 Prosp 3.36 ng/mL 90.5 81.3 Ö
Nardo et al. [124] 165 Prosp 3.5 ng/mL 88 70 Öa
Prosp prospective study
a
Excessive response if >20 oocytes retrieved
b
Excessive response if ³21 oocytes retrieved
90.5% and specificity of 81.3%. Interestingly, the the application of mild patient friendly stimula-
cut-off value calculated (3.36 ng/mL) corre- tion protocols in order to avoid OHSS.
sponded to the highest quartile of the MIS values
in their population, suggesting that hyper-
response and OHSS may be caused by gonado- 6.6.2 Prediction of Qualitative Ovarian
tropin administration to women with “enhanced Response
ovarian reserve” [128]. This was also evident in a
previous study by our group [104] in which all
cases with ovarian hyper-response to COS were It is extensively recognized that pregnancy in
in the group of patients with basal MIS levels in ART is mostly related to the qualitative than
the highest MIS quartile. Considering that PCOS quantitative aspects of IVF. As the status of the
has been associated to high MIS levels, it is logi- ovarian reserve includes both the quantity and
cal to conclude that the prevalence of PCOS quality of ovarian follicle pool, MIS may reflect
patients among women with MIS levels in the not only quantitative but also qualitative ovarian
highest MIS quartile may be increased thus in responsiveness. Indeed, several authors have
part explaining the observed high rate of OHSS found a significant positive correlation between
in this group of women. MIS levels, oocyte quality [98, 101, 103, 120,
In conclusion, according to published papers, 129, 130] and embryo morphology [101].
MIS measurement prior to gonadotropin stimula- However, this relationship has not been confirmed
tion could provide useful information to direct by others [110, 122]. In order to clarify the
complex relationship between MIS and oocyte While studies on FF seem to indicate that MIS
quality, embryo quality and implantation and may be useful in the prediction of oocyte and
pregnancy rate, we should separately comment embryo quality and finally pregnancy, the same
studies on MIS in the FF and in serum. could not be said for circulating MIS. At present,
In an elegant study, MIS was measured in the only few studies concluded that serum MIS mea-
FF obtained from both small and large follicles surement may be able to give relevant informa-
on the day of oocyte retrieval [131]. MIS levels in tion on gametes and embryo quality and on the
FF were found to be roughly 3 times higher in outcome of the treatment cycle.
small than in large follicles confirming the Silberstein and coll [101] found that serum
hypothesis that MIS production by granulosa MIS measured on the day of hCG correlated with
cells probably declines during final follicular the quality of embryos obtained permitting to
maturation. Moreover, in both small and large discriminate between embryos with high and low
follicles, FF MIS levels correlated positively with implantation potential. Consequently, implanta-
the number of early antral follicles on cycle day 3 tion rate but not pregnancy rate was higher in the
before COS, growing follicles on the day of hCG group with high basal MIS levels [101]. However,
administration and oocytes retrieved. This inter- the lack of a consistent correlation between serum
esting finding may indicate that peripheral MIS MIS and embryo morphology and embryo aneu-
levels are not exclusively dependent on the num- ploidy rate, which is not in favour of a direct rela-
ber of follicles; they are also modulated by indi- tionship between oocyte quantity and embryo
vidual follicular ability to produce MIS. Hence, quality, has been clearly demonstrated [110].
elevated peripheral MIS levels indicate not only Hence, serum MIS seems not to be an adequate
that the number of antral follicles is increased, marker for embryo quality.
but also that each follicle probably produces more The vast majority of the studies investigating
MIS individually. This offers us a new under- the performance of serum MIS in the prediction
standing of the reported association between of pregnancy occurrence following IVF reported
peripheral MIS levels and the ovarian fertility that MIS measurement is not useful in the predic-
potential and leads the author to speculate that tion of success. Until present, only one study has
serum MIS measurement could reflect not only been published relating serum MIS levels to the
quantitative but also qualitative ovarian respon- live birth rate following IVF [108]. In this large
siveness to COS [131]. prospective study of 340 patients, it was demon-
In a successive study by the same group [130], strated that the live birth rate dramatically
118 monodominant follicle cycles were prospec- increased with increasing basal MIS values.
tively studied. MIS was measured in the FF and However, this was valid only for women with
the fate of oocytes and embryos generated was basal levels less than 7.8 pmol/L. Above this
observed. It was found that embryo implantation, value, there was no discrimination for the live
clinical pregnancy and ongoing pregnancy rate birth. Basal MIS seems not able to predict preg-
increase dramatically from the low to the high FF nancy or non-pregnancy, but simply enables
MIS groups. The embryo morphology was simi- patients to be identified as being at a low or high
lar within the groups so indicating that MIS in FF probability of pregnancy after IVF. As concluded
may be an additional factor in the selection of the by the same author, this finding may at least in
oocyte [130]. This is particularly relevant in part be explained by the very good correlation
countries with restrictive law limiting the number existing between basal MIS and the number of
of oocytes that may be inseminated. A recent retrieved oocytes [108].
study on a large number of subjects (n = 276) con- In conclusion, the possible prediction of quali-
firmed the previous finding that levels of MIS in tative aspects of ART programs by serum MIS
FF were significantly increased in women who measurement remains largely controversial.
became pregnant in the respective IVF/ICSI treat- Evidence suggests that this relationship may only
ment cycle [113]. be indirect and related to the strong correlation
existing between serum MIS and the quantitative Although MIS measurement is of course more
ovarian response to COS. expensive than age evaluation as a single marker
of ovarian reserve, it clearly performs better in
the prediction of both poor and hyper-response to
6.7 Conclusions COS (Table 6.5). Furthermore, MIS ease of mea-
surement confers a relevant advantage to FSH
which is cycle-dependent and less powerful. MIS
In summary, the currently available data indicate may be informative on ovarian reserve also in
that MIS is produced by growing preantral and women during GnRH agonist treatment or hor-
early antral follicles. It has been demonstrated in monal contraception that consequently exhibits
mice that MIS inhibits initial follicle recruitment suppressed FSH levels. Finally, it seems that poor
from the resting primordial stage. In addition, response may be predicted by MIS with a perfor-
MIS may affect FSH-dependent growth of more mance which is similar to the AFC. Conversely,
mature follicles. However, the precise nature of MIS seems superior to AFC in the prediction of
the function of MIS within the human, ovary, and hyper-response [124]. While AFC is a very
in particular the paracrine role of MIS on ovarian common and useful measurement, it may be
folliculogenesis and steroidogenesis in the human sometimes technically challenging and operator-
remains largely speculative. dependent. Considering all these peculiar charac-
As MIS is related to the ovarian follicular sta- teristics, it may be concluded that MIS is a
tus, circulating MIS measurement may provide candidate to be proposed as the ideal test for the
useful information in women with ovarian dys- ovarian reserve evaluation [132] (Table 6.5).
function. Circulating MIS levels are increased in One new interesting field of application for
women with PCOS and its use as a clinical diag- MIS measurement, may be its use in the indi-
nostic marker for the syndrome has been pro- vidualization of ovarian stimulation regimens.
posed. It is still unclear whether MIS levels may In many centres, the starting FSH dose for
reflect the severity of ovarian function disruption the first IVF is often selected on the basis of age
or have a role in predicting the outcome of indi- and possibly also BMI of the patient. Some
vidual treatment regimens. authors have recently proposed adjusting the
MIS could be a valuable marker of ovarian treatment strategy on the basis of MIS levels
reserve in the general population, which may [108, 114, 123].
facilitate reproductive life planning for women. As low and high MIS values are predictive of
However, longitudinal data on MIS values during poor- and high-response to gonadotropins, respec-
the reproductive life span are not available, and it tively, it has been proposed to administer the FSH
remains unknown whether MIS levels may enable daily dose according to the pre-IVF MIS levels and
age-independent prediction of an individuals independently of the age and BMI of the patient
reproductive lifespan and spontaneous pregnancy [108, 114, 123].
in the general population. At present, the applica- In summary, published studies indicate a rele-
tion of the MIS measurement for fertility assess- vant role for MIS measurement in the identifica-
ment in the general population outwit the context tion of both the extremes of ovarian response to
of research studies, which is inappropriate. stimulation and probably in the consequent indi-
For women who desire to become pregnant by vidualization of treatment strategies in order to
means of ART, it is important to offer counselling possibly reduce the incidence of cycle cancella-
about the optimal balance between benefit and tion and OHSS. It still remains to clarify the cost/
risk. Since these outcomes are highly dependent benefit of its use as a single assay before begin-
on ovarian reserve, much effort has been put into ning an IVF cycle and whether the MIS-
identifying good clinical markers of ovarian determined strategy of COS for assisted
reserve regarding individual prognosis for success conception may be associated to improved live
and to design appropriate stimulation protocols. birth rate.
Table 6.5 Comparison of characteristics of the most widely used markers of ovarian

reserve (from ref [93])
Characteristics for a good marker Age AMH FSH AFC
Prediction of poor response + +++ ++ +++
Prediction of hyper-response + +++ − +++
Low inter-cycle variability +++ ++ − +
Low intra-cycle variability +++ ++ − +
Blinded to the operator +++ +++ +++ −
Applicable to all patientsa +++ +++ + ++
Cheapness +++ − − −
a
FSH and AFC are not informative in patients on hormonal contraception or GnRH
agonist treatment. Moreover, the count of antral follicles may be difficult in women
with ovarian cysts or with previous pelvic surgery
10. Ingraham HA, Hirokawa Y, Roberts LM, Mellon SH,

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Evidence-Based Use of Progesterone
During IVF 7
Elena H. Yanushpolsky
Abstract
It has been well demonstrated that luteal phase physiology is disrupted in
IVF cycles conducted with either GnRH agonists or antagonists, and that
supplementation of the luteal phase with exogenous progesterone is neces-
sary to optimize IVF cycle outcomes. There is now sufficient evidence to
state that intravaginal progesterone preparations in therapeutic doses are
equally efficacious and better tolerated by patients compared to traditional
intramuscular progesterone preparations. There is no need to monitor
serum progesterone levels with vaginal supplementation as adequate endo-
metrial maturation is achieved at relatively low serum progesterone levels
because of superior local absorption.
In this chapter, we examine the evidence for efficacy, dosing, and tim-
ing of several progesterone preparations and also discuss as yet unclear
issues of the ideal duration of progesterone support in early pregnancy and
progesterone replacement in frozen embryo transfer cycles and donor/egg
recipient cycles.
Keywords
Corpus luteum • Luteal phase support • GnRH agonists • GnRH antago-
nists • IVF • Intramuscular progesterone • Vaginal progesterone
• Intravaginal progesterone • Oral progesterone • Pregnancy rates • Serum
progesterone levels • Early pregnancy progesterone support
E.H. Yanushpolsky (*)

Brigham and Women’s Hospital, Boston, MA , USA

80 E.H. Yanushpolsky
preparation, which is the most convenient and

7.1 Introduction easy for patients to self-administer has been
shown to lack adequate efficacy [8, 9]. The ques-
Establishment of successful pregnancy in natural
tion of the best parenteral progesterone prepara-
ovulatory cycles depends in large part on ade-
tion with respect to efficacy, convenience, and
quate endometrial development, which is induced
tolerability has been the focus of intense investi-
by sequential and combined exposures to estro-
gation in recent years.
gen and progesterone, which are produced by the
developing follicle prior to ovulation and by cor-
pus luteum after ovulation. Both follicular estro-
gen production and corpus luteum function are 7.2 What is the Evidence and
governed by pulsatile GnRH output from the How Should We Interpret
hypothalamus [1]. Stimulated in vitro fertiliza- it for Our Practice?
tion (IVF) cycles are different from natural cycles
in supraphysiological estrogen levels in the folli- Practice of medicine in any field has not always
cular phase and rapid changes in the estrogen and been and is not always based on solid scientific
progesterone levels in the luteal phase after folli- data. This has not been for the lack of desire on
cle aspiration. Removal of granulosa cells in the the part of physicians to base their decisions on
process of oocyte retrieval leads to depletion of the best scientific methods and evidence, but
the cell pool needed to produce progesterone in rather because of the paucity of data that are
the luteal phase. In addition, since most IVF derived from rigorously designed and properly
cycles currently involve administration of GnRH powered studies. The strongest evidence is
modulators, either agonists or antagonists, physi- derived from prospective, randomized, double-
ologic pulsatility necessary for adequate corpus blind, placebo-controlled trials involving homo-
luteum function and progesterone production is geneous patient populations with adequate
disrupted leading to well-described luteal phase numbers to have the power to detect real differ-
foreshortening and dysfunction [2–4]. Exogenous ences in outcomes. Under ideal circumstances,
supplementation of the luteal phase of IVF cycles confirming results from more than one such study
with either progesterone or human chorionic should provide the best evidence for changing or
gonadotropin (hCG) has been shown to result in adjusting clinical practice [10]. Prospective, ran-
better pregnancy rates and outcomes compared domized, adequately powered studies require
to no supplementation [5, 6]. However, data rela- substantial time and resources to complete, and
tive to the best method of luteal phase support in the nature of some investigations often precludes
IVF have been insufficient until recently [7]. The the double-blinded design. Smaller prospective,
best method for luteal phase support in IVF randomized studies but with lower powers to
should be optimally efficacious, with minimal detect effect of interest may be combined into
side effects, and with ease and convenience of meta-analyses in order to increase the overall
administration. A 2004 Cochrane systematic validity of evidence. However, even results of the
review of 59 studies comparing intravaginal pro- best designed studies and meta-analyses must be
gesterone supplementation, intramuscular pro- evaluated critically with respect to potential con-
gesterone supplementation, and supplementation founders, applicability to specific populations,
with hCG injections reported similar IVF preg- and publication bias when clinical practice
nancy rates, but the odds for ovarian hyperstimu- changes are in question [11]. Although retrospec-
lation (OHSS) complication were 20-fold higher tive studies are relatively inexpensive and easy to
with hCG than with progesterone supplementa- complete, we should exercise great caution in
tion [6]. They concluded that progesterone should interpreting data derived from retrospective
be the preferred form of luteal phase support in reviews and studies using nonconcurrent controls
IVF over hCG injections, yet oral progesterone because bias and confounding that are inherent in
7 Evidence-Based Use of Progesterone During IVF 81
such studies produce results with no internal or cycles supplemented with either intramuscular
external validity [12]. A good example of such progesterone in oil or with micronized progester-
retrospective studies on the subject of progesterone in vaginal capsules. They randomized 262
one support in IVF with either intramuscular or IVF, GIFT, and ZIFT patients into two groups to
intravaginal progesterone preparations producing receive either intramuscular progesterone injec-
diametrically opposite conclusions are those of tions 50 mg daily, or micronized progesterone in
Papaleo et al. [13] and Ho et al. [14]. capsules vaginally 600 mg in three divided doses
With the issues of proper methodology in starting on the day before oocyte retrieval. They
mind, we examine the current evidence on the also measured luteal phase and early pregnancy
parenteral progesterone support in IVF-ET cycles serum progesterone levels in pregnant and non-
with the main focus on prospective, randomized pregnant patients and found that, despite signifi-
studies and meta-analyses. cantly higher serum progesterone levels observed
in patients receiving intramuscular progesterone,
those receiving intravaginal progesterone had a
7.3 Evolution and Accumulation higher overall pregnancy rate as well as implanta-
of Evidence on Progesterone tion rate.
Support in IVF Although original histopathologic studies
[20, 22] demonstrating adequate secretory endo-
The most common form of progesterone that has metrial maturation utilized micronized progester-
been used for luteal phase support in IVF has one capsules at doses between 300 and 600 mg
been progesterone in oil administered as intra- daily, several randomized studies comparing
muscular injections (IMP) at dose of 50–100 mg intravaginal and intramuscular progesterone sup-
daily. While reasonably effective, IMP are pain- plementation in IVF in the mid-to-late 1990s
ful for the patients, inconvenient to administer, chose to use lower doses (100–200 mg daily) and
usually requiring another person to help with in different and nonstandardized compounded
administration, and associated with severe side preparations such as creams and suppositories
effects, such as welts, infections, abscesses, aller- [23–25]. Such doses and preparations are cur-
gic reactions, and even pulmonary complications rently recognized as insufficient and inappropri-
requiring hospital admissions [15–17]. Not sur- ate for luteal phase support in IVF.
prisingly, clinicians were interested in identify- An earlier meta-analysis of luteal phase sup-
ing other forms of progesterone support in IVF port in infertility treatment that addressed
that will be at least equally efficacious and better comparison of intravaginal vs. intramuscular
tolerated by patients. Belgian researches led the progesterone [5] preparations included five pro-
way in investigations of luteal phase physiology, spective studies, two of which [23, 24] contained
pathology, and corrective supplementation options the majority of patients included in the meta-
related to optimal endometrial maturation and analysis, all of whom used intravaginal proges-
improved implantation in IVF cycles in the early terone preparations of 100–200 mg daily. The
1990s [18, 19]. Using histopathological evalua- authors’ conclusion was that intramuscular pro-
tions, they demonstrated that similar and appro- gesterone conferred the most benefit compared
priate luteal endometrial maturation can be to intravaginal use. It is implicitly understood
achieved with either intramuscular progesterone that the value of conclusions that can be drawn
100 mg daily injections or with intravaginal from any meta-analysis is dependent on the size
micronized progesterone administration at doses and study design and quality of its individual
between 300 and 600 mg daily. Oral micronized components [10]. It is not surprising therefore
progesterone was ineffective in inducing secretory that a meta-analysis that included the majority of
endometrial changes [20]. With this information patients receiving insufficient amounts of vaginal
on hand, Smitz et al. [21] set out a first prospective progesterone for luteal support would come to an
randomized study comparing pregnancy rates in IVF unfavorable conclusion.
By the time Daya and Gunby [6] published t issue levels achieved with Crinone 8% as
their meta-analysis in 2004, five additional pro- compared with intramuscular progesterone were
spective studies comparing intravaginal and well demonstrated in several prior studies
intramuscular progesterone preparations were [29–31]. Dal Prato et al. [27] presented a nonin-
eligible for inclusion. The authors admitted that feriority study design for three luteal phase treat-
methodological quality of the majority of the ment arms in IVF patients – intramuscular
studies was only fair with an average validity progesterone 50 mg/day, Crinone 8% (90 mg)
score of only 51%. Three of the ten included vaginal gel once/day, and Crinone 8% vaginal gel
studies utilized subtherapeutic vaginal progester- twice/day starting the day after oocyte retrieval.
one doses of 100–200 mg daily [23–25] as They achieved their precalculated necessary sam-
described above. The authors concluded that ple size with 138 patients in the intramuscular
there was significant statistical heterogeneity for progesterone group, 137 patients in Crinone 8%
the outcomes of clinical pregnancy, miscarriage, once-daily group, and 137 patients in the Crinone
and ongoing pregnancy. Only two out of ten 8% twice-daily administration group. No statisti-
studies reported live births as an outcome, and cal differences were found between the three
statistical significance was reached in favor of groups with respect to pregnancy rates (hCG
intramuscular progesterone administration for >5 mIU/mL), implantation rates, clinical preg-
the live birth parameter. However, authors of one nancy rates, miscarriages, and delivery rates. The
of those two studies admitted to methodological authors concluded that vaginal gel can be suc-
flaws related to lack of stratification by age lead- cessfully used as an alternative to intramuscular
ing to a greater number of patients >40 years old progesterone in luteal support in IVF, and that
in the intravaginal progesterone group, thus bias- once-daily dose was sufficient for optimal results.
ing results by the most important confounders of The “Correspondence” publication by
IVF outcome – age and ovarian reserve [26]. Yanushpolsky et al. [28] was a report of an interim
Given continued uncertainly of the compara- analysis of a larger study powered at 400 patients
tive efficacy of intramuscular vs. intravaginal in two arms of either intramuscular progesterone
progesterone supplementation in IVF, there was a supplementation starting the day after oocyte
great need for rigorously designed and properly retrieval or Crinone 8% vaginal gel starting 2
powered prospective randomized studies using days after oocyte retrieval. The rationale for a
proper therapeutic progesterone doses to resolve later start of Crinone was based on the known
the issue. Two studies that fulfilled these require- superior absorption data [29–31] as well as favor-
ments were published in 2008 [27, 28]. Both able results reported in preliminary studies by
studies aimed at elimination of important con- Schoolcraft et al. [32]. Similar results were
founders such as age and ovarian reserve by observed between intramuscular progesterone
excluding patients over age 40, and those with and intravaginal progesterone arms with respect
day 3 FSH levels >15 mIU/mL. Dal Prato et al. to positive beta-hCG rates, implantation rates,
[27] restricted enrollment to women <37 years clinical and ongoing/delivered pregnancy rates,
old, and Yanushpolsky et al. [28] stratified women as well as failed pregnancies. In follow-up ques-
by age <35 years old and 35–39 years old prior to tionnaires, patients expressed significantly greater
randomization to insure equal age distribution satisfaction with intravaginal route of progester-
between treatment groups. In 1997, a new micron- one administration [28].
ized vaginal progesterone bioadhesive gel prepa- The most recent and rigorous meta-analysis on
ration (Crinone 8% (90 mg) vaginal gel) became the route of luteal progesterone administration in
commercially available and FDA approved in the IVF [33] included both Dal Prato et al. [27] and
Unites States for progesterone supplementation Yanushpolsky et al. [28] studies with proper meth-
at once-daily dose and for replacement at twice odology and dosing and excluded three studies
per day dosing in IVF treatments. Excellent trans- with subtherapeutic progesterone doses [23–25].
vaginal absorption and superior progesterone A total of nine studies met inclusion criteria with
seven of nine using Crinone 8% vaginal gel and rates, or failed pregnancy rates between the
two using micronized progesterone capsules groups. Ninety percent of enrolled patients were
600 mg (200 mg 3 times a day). All studies used available to answer a questionnaire regarding
intramuscular progesterone in oil 50 mg daily. product satisfaction with significantly higher sat-
The endpoints of analysis were clinical pregnancy isfaction scores reported by those in the vaginal
and ongoing pregnancy rates. The Odds Ratio for progesterone arm.
these endpoints were 0.91 (95% CI 0.74–1.13) The other study by Kahraman et al. [35]
and 0.94 (95% CI 0.71–1.26), respectively. The included only those patients treated with the
authors concluded that daily administration of GnRH antagonist protocol, and it is the first pro-
vaginal progesterone (90 mg bioadhesive gel or spective, randomized study of progesterone for-
600 mg [200 mg 3 times a day] progesterone cap- mulations for luteal support in IVF patients on
sules) is comparable to daily administration of antagonist protocols. With respect to methodol-
50 mg intramuscular progesterone in oil for luteal ogy, this study was adequately powered at 209
phase support in IVF. It should be noted that and 217 patients in each of the treatment arms
all publications included in the meta-analysis and important confounders were properly
reported use of “long” GnRH downregulation excluded. Older patients (>37 years old), those
regimens for patients enrolled in the studies. with diminished ovarian reserve according to
As we have discussed earlier in the chapter, their basal antral follicle counts and FSH levels,
even the best intended, most rigorous meta- as well as patients who failed in at least three
analyses have limitations because of nonhomoge- prior IVF cycle were excluded from enrollment
neity of data in the studies on which meta-analyses to minimize confounders. Eligible patients were
are based. Therefore large, well-designed, ade- randomized into two groups to receive either
quately powered, prospective, randomized stud- intramuscular progesterone 100 mg daily, or
ies remain the gold standard evidence on which Crinone 8% (90 mg) gel twice daily on the day
medical decisions should rely. Two such studies after oocyte retrieval. Progesterone supplemen-
on the comparison of efficacy and patient tolera- tation was extended up to the evidence of fetal
bility of intramuscular progesterone and intrav- heart activity on ultrasound examination.
aginal gel for luteal phase support in IVF have Investigators found no statistical differences
become available in the literature in 2010. One of between two arms relative to demographic and
them by Yanushpolsky et al. [34] is a report of a clinical parameters, and similar implantation,
completed, fully powered, prospective random- pregnancy, clinical and ongoing pregnancy rates
ized study, the interim results of which at half as well as biochemical and miscarriage rates
point enrollment were included in the meta-anal- were observed in both arms.
ysis by Zarutski and Phillips [33]. All 407 patients The authors admitted that the rationale for
in that study were on “long” GnRH downregula- using vaginal gel twice daily was not based on
tion protocol and were randomized to receive solid data, as they were aware of the data demon-
either intramuscular progesterone 50 mg daily strating equal efficacy of once-daily vs. twice-
starting the day after oocyte retrieval or Crinone daily regimens [27], but more out of desire to
8% (90 mg) intravaginal gel starting 2 days after reduce possible patient anxiety.
oocyte retrieval. All pregnant patients were sup- At this time in 2010, we can state with confi-
plemented with Crinone 8% gel daily until 10 dence that the body of scientific evidence con-
weeks gestation. firms equal efficacy of intramuscular and
There were no differences between the treat- intravaginal progesterone preparations in thera-
ment arms with respect to patients’ demographic peutic doses for luteal phase support. Intravaginal
and clinical characteristics. Likewise, there were progesterone preparations are more convenient
no statistical differences with respect to preg- and better tolerated by patients than intramuscu-
nancy rates (beta hCG >5 IU/mL), implantation lar preparations and therefore should be the first
rates, clinical pregnancy and ongoing/delivered line of progesterone supplementation in IVF.
Several other aspects of vaginal progesterone daily dose (90 mg) was approved by FDA for
support deserve discussion: progesterone supplementation in ART, while
1. What is the optimal dose? twice-daily dose was recommended for replace-
2. How do various vaginal progesterone prepara- ment in postmenopausal or agonadal women
tions compare with respect to efficacy and [36, 37]. Among prospective randomized studies
acceptance by patients? comparing intramuscular progesterone to Crinone
3. What is the optimal timing of progesterone gel and demonstrating similar results, some
initiation within the IVF cycle? involved once-daily gel administration, while
4. How long should progesterone be continued others involved twice-daily doses [28, 35].
in early pregnancy and should we monitor A study by Dal Prato el al contained three arms
serum progesterone levels? comparing intramuscular progesterone 50 mg
5. What about progesterone replacement in daily, Crinone gel once a day, and Crinone gel
Frozen embryo transfer cycles and donor twice a day. All three arms had similar results
oocyte recipient cycles? with respect to IVF outcomes [27].
We will address each of these questions sepa- We conclude that a safely effective dose of mic
rately with respect to the evidence available at ronized progesterone in oil capsules is 600 mg
this time. daily (200 mg 3 times a day), while both single-
dose as well as twice-daily dose of Crinone 8%
gels are efficacious for luteal phase support.
With respect to convenience, single-dose
7.4 What is the Optimal Dose medications are preferred by patients. Total
of Vaginal Progesterone costs have to be compared between preparations
Supplementation? and weighed against convenience of administra-
tion. In most cases, these will be individual
An optimal dose of any medication for any indi- decisions that each patient will have to make for
cation should be the dose that will be optimally herself.
effective, most convenient to administer, as well
as cost effective. The body of literature that we
have reviewed so far focused on comparison of
7.5 How Do Various Vaginal
intramuscular progesterone preparations from 50
to 100 mg daily with intravaginal preparations of Progesterone Preparations
micronized progesterone in oil capsules ranging Compare with Respect
from 100 to 600 mg daily in three divided doses, to Efficacy and Acceptance
or micronized progesterone bioadhesive gel by Patients?
(Crinone 8% [90 mg]) inserts once or twice daily.
It has been well demonstrated in histopathologi- There are currently three standardized vaginal
cal studies [22], as well as clinical studies [21], progesterone formulations commercially avail-
that progesterone capsules in doses less than able for use in IVF. Micronized progesterone
300 mg/day are inferior in efficacy to intramus- capsules (Utrogestan/Prometrium) are available
cular progesterone. Most studies demonstrating and widely used in Europe and South America,
equal efficacy involved micronized progesterone but not FDA approved for use in IVF/ART in the
capsules 600 mg (200 mg 3 times a day). United States. The two progesterone formula-
Crinone vaginal gel has been shown to have an tions approved by FDA for use in IVF are Crinone
excellent coefficient of absorption [29–31] into 8% vaginal gel, and more recently – Endometrin
endometrial tissues and capable of inducing 100 mg (natural progesterone in capsules). The
appropriate secretory endometrial changes nec- unique feature of Crinone bioadhesive gel is its
essary for luteal phase support [32]. A single property of sustained release delivery over time
allowing for once-daily administration. Other

formulations require multiple daily doses for
7.6 What is the Optimal
optimal efficacy. Timing of Progesterone
While both Utrogestan and Crinone have been Initiation Within the
compared in prospective, randomized trials to IVF Cycle?
intramuscular progesterone and have been shown
to be of equal efficacy in appropriate doses (as In stimulated IVF cycles, there is substantial
discussed above), no such comparison exists for endogenous progesterone production starting
Endometrin. Instead, Endometrin at twice-daily immediately after, and possibly even slightly
and 3 times daily doses has been compared to before the hCG trigger. While progesterone sup-
Crinone once-daily dose for luteal support in IVF plementation in the luteal phase is important, it is
in a prospective, randomized multicenter trial similarly important not to advance endometrial
with noninferiority design [38]. The study had maturation out of sink with embryo develop-
enough power to demonstrate noninferiority of ment. It has been demonstrated that administra-
Endometrin at twice a day and 3 times a day doses tion of intramuscular progesterone early in the
in women <35 years old with FSH <10 mIU/mL cycle, i.e., prior to oocyte retrieval compared to
compared to Crinone 8% gel once daily. The within 24 h after oocyte retrieval, will result in
authors stated that the study did not reach enough lower pregnancy rates [43]. Mochtar et al. [44]
power for meaningful conclusions for women examined effects of timing of vaginal progester-
over age 35. one initiation (Micronized progesterone 400 mg
Three other prospective studies addressed twice daily) in a prospective randomized design
comparison of Micronized progesterone capsules (n = 385). Progesterone was initiated either on
(Utrogestan 600 mg) in two or three doses a day the day of HCG trigger, or on the day of oocyte
with Crinone gel once daily [39–41] and Simunic retrieval, or on the day of embryos transfer (day 3).
et al. [39] also assessed patient preference for While ongoing pregnancy rate was lower in
either of the preparations. All three studies con- the hCG group compared to two others, the dif-
cluded that pregnancy rates were similar between ference was not statistically significant. If start-
Utrogestan and Crinone groups, but patients ing progesterone supplementation too early in
expressed superior tolerability and acceptability the cycle has a negative effect on the outcome,
of Crinone in Simunic’s study. starting progesterone too late could be equally
A recent meta-analysis [42] pooled together detrimental. A prospective, randomized study by
results of seven randomized trials comparing Williams et al. [45] comparing progesterone start
once-daily or twice-daily Crinone vaginal gel with 3 vs. 6 days after retrieval found higher preg-
either twice or three times daily Endometrin, or 3 nancy rates with earlier start. There was hetero-
times daily Utrogestan. The authors concluded geneity with the exact timing of progesterone
that all vaginal progesterone preparations in ade- start in prospective studies that we examined ear-
quate doses were equally efficacious for luteal lier in this chapter. Dal Prato et al. [27], Kahraman
support in IVF cycles with respect to clinical et al. [35], and Doody et al. [38] started supple-
pregnancy rates. mentation on the day after retrieval, while
We conclude that all three available vaginal Yanushpolsky et al. [28] and a smaller trial by
progesterone preparations – Crinone gel once a Schoolcraft et al. [32] started supplementation
day, Utrogestan 200 mg 3 times a day, and with vaginal progesterone 2 days after retrieval,
Endometrin 100 mg twice or three times a day – but all had similar results.
are equally effective for luteal phase support in We conclude that there is an acceptable win-
IVF cycles. However, data are insufficient for dow of time – 24–48 h after oocyte retrieval for
definite conclusion on the efficacy of Endometrin initiation of vaginal progesterone supplementa-
in women over age 35. tion with optimal cycle results.
there has been little reason to be concerned with

7.7 How Long Should serum progesterone level monitoring. In addition,
Progesterone Be Continued absorption and pharmacokinetic studies by
in Early Pregnancy and Do Bulletti [29] and Ciccinelli [30] confirmed that
We Need to Monitor Serum there was no correlation between the desired
Progesterone Levels? endometrial maturation effect and serum proges-
terone levels, and that there is no reason to sub-
While it has long been established that luteal ject patients to unnecessary blood drawings and
phase support with progesterone in IVF cycles potentially unsubstantiated reasons for anxiety
leads to improved outcomes, and there is cur- involved with serum level monitoring. None of
rently sufficient evidence to state that vaginal the major randomized studies that demonstrated
progesterone is equally efficacious and better tol- equal efficacy of intravaginal and intramuscular
erated form of luteal supplementation, there is preparations reported serum progesterone levels
still limited data and little consensus on the nec- because they were felt to be irrelevant to the out-
essary duration for progesterone supplementation comes [27, 28, 35].
in early pregnancy, i.e., beyond first positive preg- There is no scientific evidence for monitoring
nancy test. Andersen et al. [46] conducted a pro- serum progesterone levels during supplemented
spective study in which patients enrolled in luteal phase of stimulated IVF cycles.
stimulated IVF cycles and receiving micronized
progesterone 600 mg in the luteal phase were ran-
domized to stopping progesterone support after
7.8 What About Progesterone
first diagnosis of pregnancy vs. continuing for
3 more weeks (n = 303). They found no differ- Replacement in Frozen
ences in miscarriage rates and argued that proges- Embryo Transfer Cycles
terone supplementation can be safely withdrawn and Donor Oocyte Recipient
after the first positive beta-hCG result. Cycles?
Aboulghar et al. [47] conducted a survey of 21
IVF programs with respect to progesterone sup- Frozen embryo transfer cycles and Donor Oocyte
port in early pregnancy after IVF. They found no recipient cycles are different from stimulated IVF
uniform pattern or consensus, and then set out a cycle in that there is no endogenous progesterone
prospective study where they randomized patients production, and therefore, instead of luteal phase
to either continuation or discontinuation of pro- supplementation, there is a need for luteal phase
gesterone support on the day of first ultrasound “creation” or replacement.
demonstrating positive fetal heart activity Just as with stimulated cycles, intramuscular
(n = 257). They found no significant differences progesterone in doses of 50–100 mg daily has
in miscarriage rates or bleeding in early preg- been the most common progesterone preparation
nancy rates between the groups. They concluded used in these replacement cycles, and just as with
that there was no advantage to continuing proges- stimulated cycles, there have been adverse reac-
terone support beyond the time of first ultrasound tions, complications, and inconveniences for the
viability study. They also called for large, pro- patients related to intramuscular progesterone
spective, randomized trials to settle the issue of administration; there is a need to find more toler-
timing of progesterone support in early IVF able but equally efficacious progesterone formu-
pregnancies. lations for the replacement cycles. Unfortunately,
Since the first group of studies by Smitz et al. there is a paucity of data addressing comparison
[21] showing lower serum progesterone levels of intramuscular and intravaginal progesterone
but adequate endometrial maturation and similar preparations in frozen embryo transfer and donor
pregnancy rates with vaginal progesterone prepa- egg/recipient cycles, and no definite conclusions
rations compared to intramuscular preparations, can be drawn at this time. Crinone vaginal gel has
been approved by FDA for supplementation at review and meta-analysis of four prospective
once-daily dose and for replacement at twice a randomized studies (n = 587 patients) addressing
day dose. There are only two small prospective IVF cycle outcomes of patients who received
studies comparing intramuscular progesterone to estradiol supplementation in the luteal phase and
Crinone vaginal gel twice a day and once a day in those who did not. They concluded that the evi-
donor egg/recipient cycles with similar results dence available at the time suggested that the
[37, 48]. Three other reports by Berger and addition of estrogen to progesterone for luteal
Phillips [49, 50] and Williams et al. [51] are ret- phase support does not increase the probability
rospective studies also showing equivalent preg- of pregnancy in IVF. A more recent meta-analysis
nancy outcomes for frozen and donor egg/ of nine randomized studies was presented by
recipient cycles in intramuscular progesterone Jee et al. [55] in January 2010. No statistically
and Crinone gel groups at twice a day doses as significant differences in IVF pregnancy rates
well as once a day doses. As all retrospective were observed between the groups of patients
studies, these cannot be relied upon as definite who received additional estradiol in the luteal
evidence of efficacy. Large and properly designed phase and those who received progesterone
prospective, randomized trials are much needed supplementation alone. Authors of both meta-
to establish efficacy of vaginal progesterone analyses called for large, prospective randomized
replacement in Frozen embryo transfer cycles studies in order to confirm their findings. The
and donor egg/recipient cycles. largest prospective randomized study so far was
published by Elgindy et al. [56] in May 2010. It
included 270 patients undergoing ICSI cycles in
long agonist protocols in three arms. All patients
received intramuscular progesterone (100 mg
7.9 Is There Benefit of Adding daily). Patients in Group A received no addi-
Estradiol To Progesterone for tional estrogen, while patients in Group B
Support of the Luteal Phase received additional oral Estradiol valerate 6 mg
in the Ivf Cycles? daily, and patients in Group C received daily
Estradiol valerate 6 mg per vagina. There were
The pattern of estrogen and progesterone decline no differences in progesterone levels in the luteal
in the luteal phase of IVF cycles has been well phase among all three groups, and luteal estradiol
described in the literature and lower pregnancy levels were similar between Groups B and C.
rates have been observed with lower luteal phase Likewise, there were no differences in pregnancy
estradiol to progesterone ratios [52, 53]. The rates between progesterone only group
question of causality vs. association, i.e., do the (Group A), and oral Estradiol supplemented
lower estradiol levels in the luteal phase cause group (Group B), but higher pregnancy rates
lower pregnancy rates, or are lower estradiol lev- were observed in patients supplemented with
els the result of lack of implantation, could not vaginal Estradiol valerate 6 mg daily. The authors
be resolved without properly designed studies. concluded that pregnancy rates in patients under-
Several small prospective randomized studies going ICSI in long agonist protocols may be
addressing addition of estradiol supplementation improved by addition of vaginal estradiol in the
in doses between 2 mg and 6 mg/day suggested luteal phase.
a benefit, while almost a similar number of small While this prospective randomized study does
prospective randomized studies demonstrated not answer the question regarding benefits of
no improvement in pregnancy rates with estra- luteal estradiol support definitively, it raises the
diol supplementation [7]. A meta-analysis possibility of greater benefit of vaginal estradiol
assessment of the best available data was neces- supplementation over oral supplementation.
sary for understanding of this issue. In 2008, Large prospective randomized studies will be
Kolibianakis et al. [54] published a systematic needed to answer the question regarding potential
benefits of vaginal estradiol supplementation for Benefits of adding estradiol to progesterone

all IVF cycles. supplementation in the luteal phase of IVF cycles
have not been demonstrated in the published
meta-analyses of the data. However, the most
recent prospective randomized study suggested
7.10 Conclusions potential benefit of vaginal, but not oral estrogen
supplementation in long protocol ICSI cycles.
Progesterone supplementation of luteal phase of More studies are needed to evaluate potential
stimulated IVF cycles leads to higher pregnancy benefits of vaginal estradiol supplementation.
rates and is necessary for optimal results. Vaginal
progesterone supplementation is equally effec-
tive and better tolerated by patients than intra-
muscular preparations. There exists sufficient References
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Monozygotic Twinning
and Perinatal Outcomes 8
Kenneth J. Moise jr. and Ramesh Papanna
Abstract
The choice made by women to delay childbearing until later in their repro-
ductive life coupled with the more widespread use of artificial reproduc-
tive techniques has contributed to an increasing incidence of multifetal
gestations. When compared to dichorionic twins, monochorionic twins are
at an increased risk for congenital anomalies as well as unique conditions
including twin–twin transfusion syndrome (TTTS), selective intrauterine
growth restriction (sIUGR), and twin reversed arterial perfusion (TRAP)
sequence. Chorionicity can be accurately determined in the late first tri-
mester through ultrasound examination of the interface between the inter-
vening twin membrane and its attachment to the placenta. In the case of
the monochorionic gestation, frequent ultrasound surveillance beginning
at 16 weeks’ gestation is important for the timely management of perinatal
complications. The current standard treatment for severe TTTS is laser
photocoagulation of placental anastomoses. Selective reduction is an
option in an anomalous cotwin, previable sIUGR, or TRAP sequence in
monochorionic twin gestations.
Keywords
Twin–twin transfusion syndrome • TTTS • Monochorionic twins • Twin
reversed arterial perfusion sequence • TRAP • Acardiac twinning
• Selective IUGR • Monoamniotic twins • Multifetal pregnancies
K.J. Moise Jr. ()

Division of Maternal-Fetal Medicine, Baylor College
of Medicine and the Texas Children’s Fetal Center,
Texas Children’s Hospital, Houston, TX, USA

92 K.J. Moise Jr. and R. Papanna
usually monochorionic, diamniotic (MC/DA),

8.1 Background has been associated with assisted hatching, intra-
cytoplasmic sperm injection, and the late transfer
Although the incidence of spontaneous twinning
of cultured blastocysts [3].
has been previously reported to be 1 in 80 preg-
nancies, the growing trend for women to post-
pone childbearing until later in their reproductive
life along with the increased use of assisted repro- 8.2 Embryology
ductive technologies (ART) has led to a rise in
the incidence of twinning. Data from the Centers A dizygotic twin pregnancy results from the release
for Disease Control from 2006 indicate that of two zygotes within the same menstrual cycle
3.25% of livebirths were twins (Fig. 8.1) [1]. The which are each fertilized by separate spermatozoa.
incidence of triplet and higher order gestations This process results in a dichorionic, diamniotic
peaked in 2003 at 187/100,000 livebirths with a (DC/DA) twin pregnancy. Monozygotic twinning
notable decreasing in the incidence since that occurs when a single fertilized zygote divides into
time (Fig. 8.2). Voluntary guidelines for the num- two embryos within the first 12 days after concep-
ber of embryos transferred at the time of in vitro tion. If the embryo divides before the third day of
fertilization have probably contributed to this life, DC/DA twins result, comprising 25% of
change. However, although approximately 1% of monozygotic twins. Division of the embryo
all infants born in the United States are a product between the fourth and eighth day of life results in
of ART, these infants account for 18% of all mul- MC/DA twins which comprise 75% of monozy-
tiple births. In addition, these pregnancies con- gotic twins. Division of the blastocyst into two
tinue to be a contributing factor to perinatal consepctuses between the ninth and the twelfth day
morbidity with 14% of ART singletons, 65% of will result in monochorionic, monoamniotic (MC/
ART twins and 97% of ART triplets, and higher MA) twins. These represent <1% of monozygotic
order multiples born prematurely [2]. Finally, twins. Division after the twelfth day and up to six-
a threefold increase in monozygotic twinning, teenth day of life will result in conjoined twins.
Fig. 8.1 Incidence of
twin gestations in the
United States between
1996 and 2006 [1]
8 Monozygotic Twinning and Perinatal Outcomes 93
Fig. 8.2 Incidence of
triplet and higher order
multiple gestations in the
United States between
1996 and 2006 [1]
Higher order multiples can result from fertilization passed from the dead fetus to its sibling, the rare
of multiple ova, whether from spontaneous ovula- ultrasound observation of the acute death of one
tion or ovulation induction, the transfer of multiple monochorionic twin has helped to elucidate
embryos in IVF, or the rare natural cleavage of a the true pathophysiology of the consequences of
single fertilized conceptus. the death of one of the fetuses in a monochori-
onic twin gestation. As the compromised twin
begins to die, it experiences hypotension. This
8.3 Perinatal Risks leads to exsanguination of the normal cotwin into
the “sink” of the abnormal twin’s circulation
Identical twins are associated with a higher inci- through placental vascular anastomoses [5].
dence of complications as compared to singleton Acute anemia and hypotension then follow in the
pregnancies and even dizygotic twins. normal twin. This leads to the death of the normal
Structural fetal anomalies are reported to twin in 12% of cases, while major neurologic
occur in 0.6% of singletons, 1% of dizygotic deficit occurs in an additional 18% of cases when
twins, and 2.7% of monozygotic twins [4]. Even this twin survives [6].
though the two embryos share a common genome,
82% of identical twins are discordant for struc-
tural abnormalities. 8.4 Determining Amnionicity/
The shared placenta of monochorionic twins Chorionicity
contains anastomotic vessels between the two
fetal circulations in virtually all cases. These Historically, chorionicity was determined at the
anastomoses lead to a unique situation where the time of delivery by obtaining a histological cross-
death of one twin can result in compromise of its section of the intertwin membrane at its insertion
sibling. This can occur in cases of life-threatening into the placenta. This late diagnosis of chorio-
congenital anomalies, twin–twin transfusion, or nicity provided information to the parents as to
severe selective intrauterine growth restriction whether their twins were “identical;” however,
(sIUGR) (see below). Although originally thought this practice did not allow for stratification of
to be the consequence of “bad humors” that perinatal risks early in gestation.
The modern management of a twin gestation

involves the diagnosis of chorionicity early in
gestation. Current guidelines from the American
College of Radiology and the American Institute
of Ultrasound in Medicine recommend that
chorionicity be determined and documented in
all multiple gestations [7]. In late 2008, the Royal
College of Obstetricians and Gynaecologists
issued a green top guideline indicating that
chorionicity should be determined in multiple
gestations in the first trimester [8]. Similarly, the
National French College in Obstetrics and Gyne
cology has made a similar recommendation [9]. Fig. 8.3 Ultrasound detection of chorionicity in a triplet
gestation at 12 weeks’ gestation. Smaller arrow indicates a
Amnionicity of a twin gestation can usually be “T sign” indicative of a monochorionic, dichorionic inter-
determined as early as 8 weeks of gestation by vening membrane between Twin #1 and Twin #2. Larger
the presence of an intervening twin membrane arrow indicates a “lambda sign” between the Twin #2 and
identified by ultrasound. Although the presence the Singleton gestation. This gestation would therefore be
classified as a dichorionic, triamniotic triplet gestation
of two separate yolks sacs is a good predictor of
diamnionicity, a single yolk sac with two fetal
poles may still result in a diamniotic pregnancy suggests a monochorionic gestation, while
in 5–15% of cases [10]. >2 mm suggests dichorionicity.
Chorionicity can be determined at the time of
a first trimester ultrasound at 10–13 weeks’ gesta-
tion with a sensitivity and specificity of 90 and 8.5 Dichorionic, Diamniotic
100%, respectively, with both negative and posi- (DC/DA) Twins
tive predictive values of about 98% [11]. The
presence of placental tissue between the com- More than two thirds of all spontaneous twins are
bined chorion/amnion layers of the intervening DC/DA. Although one third of these are the result
twin membrane at its insertion into the placenta is of early cleavage of a single zygote. The placen-
indicative of a DC/DA gestation. This ultrasound tas of these identical twins lack vascular anasto-
finding is known as the “lambda” or “twin peak” moses; therefore all DC/DA twins are considered
sign. The absence of intervening placental tissue low risk for perinatal complications when com-
between the membranes is known as a “T sign” pared to MC/DA twins. DC/DA twins, however,
and indicates the presence of a single chorion – a are associated with slightly higher risks of con-
MC/DA twin gestation (Fig. 8.3). genital anomalies and selective growth restric-
During the second trimester, determining cho- tion when compared to singleton gestations.
rionicity by ultrasound is less reliable than in the
first trimester with a sensitivity, specificity, posi-
tive predictive value, and negative predictive 8.5.1 Overall Clinical Management
value of 88, 95, 88, and 95%, respectively [12].
The presence of an intertwin membrane is first Routine comprehensive ultrasound for anatomi-
confirmed. The next step involves the determina- cal assessment should be undertaken at 18–20
tion of fetal gender – dichorionicity is confirmed weeks of gestation and serial scans for fetal
if a male and a female fetus are both present. The growth continued every 3–4 weeks until delivery.
presence of two separate placental masses also Delivery is generally recommended between
confirms dichorionicity. Finally, the thickness of 38 and 39 weeks of gestation with 40 weeks gen-
the intervening membrane can be used if the erally considered by most obstetrical experts as
twins are like sex and there is a single placenta “postdatism.” Growth restriction can occur in one
mass. A membrane thickness of <2 mm thickness or both fetuses and is usually related to a
differential uterine blood flow to the separate pla- the development of TTTS [14]. In another large
cental masses. This is defined as a difference in series of 172 MC/DA twins, an absent or reversed
estimated fetal weight by ultrasound of greater wave in the ductus venosus in the first trimester
than 20% between the twin fetuses (larger twin’s proved to be a better predictor of TTTS than
estimated weight minus the smaller twin’s esti- either a discrepancy in the nuchal translucency
mated weight divided by the larger twin’s weight). or the crown-rump length [15]. Finally, in the
However, this rule may not apply to all twin pairs second trimester, composite poor perinatal out-
as one twin may have an accelerated growth come in MC/DA twins was calculated using a
curve while the other grows normally. This has multiple logistic regression. A threshold of 50%
led some authorities to recommend that each chance for complications had a sensitivity of 48%
fetus be assessed on its own growth curve with and a positive predictive value of 73% [13]. These
serial ultrasounds. A fetus found to have an esti- investigations would suggest that perinatal risks
mated fetal weight of less than the 10% (usually in the MC/DA twin gestation can be further refined
using singleton ultrasound growth curves) is con- with specific ultrasound parameters obtained in
sidered growth restricted. Antenatal testing using the late first and early second trimester. The clini-
nonstress testing or biophysical profiles should cal use of such data will await further trials to
be initiated by 30–32 weeks of gestation with determine if these parameters can be incorporated
delivery of the pregnancy planned if there is evi- into specific algorithms for subsequent ultrasound
dence of fetal compromise in the affected twin. surveillance of these pregnancies.
8.6 Monochorionic, Diamniotic 8.6.2 Overall Clinical Management

(MC/DA) Twins
Once monochorionicity is established, a limited
Approximately one third of spontaneous twin ultrasound should be undertaken at 16 weeks’
pregnancies are monozygotic, of which 75% are gestation to assess for discordance in fetal size
MC/DA. As mentioned earlier, these twin gesta- and amniotic fluid volumes. A routine compre-
tions should be stratified as high risk when com- hensive ultrasound for anatomical assessment
pared to DC/DA twins. should then be performed at 18 weeks of gesta-
tion. Thereafter, limited ultrasound scans should
be alternated with complete growth assessments
8.6.1 Risk Assessment every 2 weeks for the remainder of the pregnancy.
A recent large Dutch trial of twin gestations with
Several authors have attempted to stratify the known chorionicity found that MC twins were at
MC/DA twin gestation into high risk and low risk an 8.8-fold (95% CI: 2.7–28.9) increased risk for
groups based on ultrasound parameters. Lewi unexplained intrauterine fetal demise late in ges-
et al. [13] prospectively followed 202 sets of MC/ tation [16]. Based on these data, the authors rec-
MA twins and attempted to predict a composite ommended consideration for delivery by 37
perinatal outcome of twin–twin transfusion syn- completed weeks of gestation.
drome (TTTS), sIUGR, and fetal death. In the
first trimester, a discordance in the crown-rump
length and/or amniotic fluid volume was associ- 8.6.3 Twin–Twin Transfusion
ated with a worsening prognosis. Using a multiple Syndrome (TTTS)
logistic regression model and a threshold for
complications of 50%, the sensitivity of their TTTS complicates approximately 1 in 40–65
model was 29% with a positive predictive value twin pregnancies so that approximately 2,500
of 70%. Similarly, a difference in the nuchal cases occur in the U.S. each year. About 9–15%
translucency in the first trimester of more than of MC/DA twin pregnancies ultimately develop
20% has been associated with a >50% chance for TTTS [13].
The initial diagnostic criteria for “twin-twin (atypical stage III). In addition, although higher
transfusion” were based on pediatric parameters Quintero stages are generally associated with a
including discordance in birthweights and neona- worsening perinatal prognosis, the clinical presen-
tal hemoglobin [17]. Many of these cases involved tation of a particular case does not always follow
the acute transfusion of red cells through intrapla- an orderly progression of stages. As an example, a
cental anastomoses at the time of delivery. With stage I case may progress rapidly over several days
the advent of routine prenatal ultrasound, these to stage III. In addition, regression of disease can
criteria have been abandoned in lieu of ultrasound occur in as many as 41% of stage I cases [19].
parameters. True TTTS is now considered a sec- The Quintero staging system has been criti-
ond trimester entity with severe perinatal compli- cized for its lack of ability to prognosticate peri-
cations. In general, the smaller twin (usually with natal survival after therapy. Other centers have
less amniotic fluid) is called the donor twin and proposed staging systems based on echocardio-
the larger twin (usually with the greater amount graphic abnormalities of the fetal heart (more
of amniotic fluid) is referred to as the recipient. often seen in the recipient fetus) [20, 21]. However,
The following ultrasound criteria are gener- a recent working group has recommended that the
ally accepted for the diagnosis of TTTS: Quintero staging system continue to be used in
• Polyhydramnios in the amniotic fluid com- the community due to its simplicity [17, 22].
partment of the recipient twin: >8 cm maxi-
mum vertical pocket at <20 weeks’ gestation 8.6.3.1 Pathophysiology
or >10 cm vertical pocket at ³20 weeks. There are four types of vascular connections in
• Oligohydramnios in the amniotic fluid com- the monochorionic placenta. Arterio-venous (AV)
partment of the donor twin: <2 cm maximum and veno-arterial (VA) anastomoses consist of
vertical pocket. feeder vessels on the surface of the chorionic
Five stages of TTTS have been proposed by plate that descend into a common cotyledon
Quintero et al. [18]. Stage I disease involves a dis- capillary network. By convention, the anastomo-
cordance in amniotic fluid volumes as noted ses are named based on their directional flow
above. Stage II disease is defined as the absence from the donor to the recipient twin. In contrast,
of a visible fetal bladder by ultrasound in the arterio-arterial (AA) and veno-venous (VV) anas
donor twin. Stage III disease is present when tomoses are seen exclusively on the surface of
Doppler ultrasound assessment of flow in the the placenta (Fig. 8.4). Flow in these latter two
umbilical artery, umbilical vein, or ductus veno- types of connections is bidirectional and net flow
sus of either twin is abnormal. Stage III disease is related to opposing hydrostatic pressures of
encompasses a wide spectrum of presentations each fetus. Computer modeling has demonstrated
with more than 64 variations. In general, the donor that if the net number and diameter of connec-
twin in stage III exhibits Doppler abnormalities of tions are unbalanced, i.e., there are more AV con-
absent or reversed diastolic flow in the umbilical nections between donor and recipient than there
artery. In the recipient twin, abnormalities of flow are VA connections between recipient and donor,
in the ductus venosus (absent or reversed “A” then increasing hydrostatic and osmotic forces
wave) or umbilical venous pulsations are seen. In will result in the TTTS phenotype [23]. In con-
stage IV disease, hydrops fetalis – the collection trast, if the connections are balanced, i.e., equal
of fluid in extracellular compartments such as numbers of bidirectional anastomoses, then TTTS
ascites and pleural effusions – is typically seen in does not result. Postdelivery injection studies of
the recipient twin. Finally, in stage V disease, fetal the placenta have indicated that AA anastomoses
death has occurred in one or both twins. tend to be protective against the development of
Atypical presentations can occur such as an TTTS [24].
abnormal umbilical Doppler flow in the donor This imbalance in vascular volume results in
twin with a normal bladder seen on ultrasound multiple endocrine and cardiovascular changes in
to be experimental. In 2004, the Eurofetus

consortium randomized 142 patients between
laser therapy and the gold standard for treatment,
amnioreduction. The trial was stopped at 50% of
the proposed sample size when the interim analy-
sis indicated significant improvements in overall
perinatal survival, gestational age at delivery, and
6-month survival without neurologic sequelae
[30]. Since that time, both the Cochran library
and a subsequent meta-analysis have reported
laser to be more beneficial than amnioreduction
in the treatment of TTTS [31, 32].
Fetoscopic laser ablation of placenta anasto-
Fig. 8.4 Monochorionic twin placenta with the arterial
system of both fetuses injected with green dye and the
moses is now accepted around the world as the
venous system of both fetuses injected with orange dye. standard of care for the treatment of TTTS. In the
Arrows indicate (top to bottom) – AA arterio-arterial; AV U.S., patients with extreme symptoms related to
arterio-venous; VA veno-arterial anastomoses extensive polyhydramnios in stage I disease, or
patients with stage II–IV disease, are considered
both fetuses. Relative hypovolemia occurs in the candidates for therapy between 16 and 26 weeks
donor twin leading to anuria and eventual anhy- of gestation. Today, a selective sequential abla-
dramnios (Quintero stage II). Relative hyperv- tion technique is used in which AV then VA then
olemia in the recipient fetus leads to cardiomegaly AA anastomoses are coagulated. In some centers,
with increased production of natriuretic hormones; a “Solemnization” technique is then employed to
subsequent polyuria leads to polyhydramnios in coagulate placental tissue between these targeted
the recipient’s amniotic cavity [25]. As the dis- vessels to prevent the patency of small anastomo-
ease progresses, the donor twin compensates by ses that can be difficult to visualize. Typically, an
an up-regulation of its renin-angiotensin system amnioreduction is performed at the end of the
leading to a shunting of these vasoactive sub- procedure to normalize the amount of amniotic
stances through placental anastomoses to the fluid in the recipient’s sac.
recipient [26]. Recipient hypertension then domi- Preterm premature rupture of the membranes
nates the clinical scenario leading to congestive complicates as many as 30% of laser cases.
heart failure (Quintero stage III) and eventually Preterm delivery is common with an average ges-
fetal hydrops (Quintero stage IV). Ultimately, tational age at delivery of 30–32 weeks in most
fetal death occurs in one or both fetuses (Quintero U.S. studies. In experienced laser centers, sur-
stage V). vival of both fetuses occurs in approximately
70% of cases; survival of at least one fetus occurs
8.6.3.2 Clinical Management in 90% of cases. In a 2-year study from Holland,
Historically, the overall perinatal survival rate of greater than 80% of survivors were without neu-
untreated TTTS was 30% [27]. Attempts at rologic deficits [33].
amnioreduction to reduce the symptoms of poly-
hydramnios resulted in some increase in survival
(78% at birth and 60% at 4 weeks of age); how- 8.6.4 Selective Intrauterine Growth
ever, neurologic injury was evident in 25% of Restriction (sIUGR)
survivors [28]. Fetoscopic-guided laser photoco-
agulation of the problematic placental anastomo- Growth discordance in twins is usually defined as
ses was first proposed by De Lia [29]. Many greater than 20% discordance in the estimated
perinatologists initially considered the therapy fetal weight by ultrasound parameters. When one
twin is noted to be at <10% estimated fetal 8.6.5 Twin Reversed Arterial Perfusion
weight, selective IUGR is diagnosed. This entity (TRAP) Sequence
is as common as TTTS, complicating 10–15% of
MC/DA twin gestations. The etiology is unclear The incidence of twin reversed arterial perfusion
but proposed to result from an unequal division (TRAP) sequence (acardiac twinning) is approx-
of the inner cell mass early in embryonic life. imately 1 in 30,000 pregnancies or 1% of mono-
This may then result in a decreased potential for chorionic twins [37]. Often the acardiac fetus is
fetal growth in the affected fetus. Alternatively, thought to represent a “vanishing twin” during a
an unequal split in the cytotrophoblast that will first trimester ultrasound only to find an amor-
ultimately form the placenta can result in dispro- phic mass that has increased in size at the time of
portionate placental sharing. Recently, Gratacos a second trimester ultrasound. There are two
et al. [34] classified the sIUGR in MC/DA twins important ultrasound findings that are consis-
based on the characteristics of the umbilical arte- tently noted in TRAP sequence: one of the two
rial Doppler in the smaller fetus. Type I (29%) fetal masses has a rudimentary or absent heart
had end-diastolic flow present on Doppler wave- and there is reversed flow by Doppler in the
forms, type II (22%) had absent or reversed dia- umbilical artery into this fetal mass. Two thirds
stolic flow, and type III (49%) had intermittent of cases are MC/DA, while the remainder are
absent or reversed end-diastolic flow. Type III MC/MA. Umbilical cord insertions into the pla-
sIUGR was found to be associated with a greater centa are immediately proximate to one another.
number of large AA placental anastomoses when The most common phenotype is preservation of
compared to the other two types of sIUGR and the lower fetal extremity structures with abnor-
controls. There was also a higher incidence of mal or absent structures in the upper half of the
fetal demise of the smaller twin fetus in conjunc- acardiac fetus (Fig. 8.5). This is thought to be
tion with cerebral lesions on neonatal head ultra- related to perfusion with deoxygenated blood
sound in the surviving cotwin. from the normal, or pump twin, to the acardiac
twin via the umbilical arteries which course first
8.6.4.1 Clinical Management through the lower portion of the fetus before
The management of discordant growth depends flowing cephalad. Proposed theories for the
on the gestational age at diagnosis. If detected at development of TRAP sequence include abnor-
a previable gestational age, selective reduction mal cardiogenesis in the acardiac fetus and early
should be considered for the premoribund sIUGR reversal of flow through a large placental AA
fetus in an attempt to prevent sequelae in the anastomosis leading to underdevelopment of the
normally grown cotwin. This can be performed heart in the acardiac twin [38].
by occlusion of the umbilical cord through ultra- Progressive myocardial demand on the pump
sound-directed bipolar cautery or radiofrequency twin to perfuse its own circulation as well as that of
ablation [35]. Laser ablation of placental anasto- the acardiac twin results in cardiac failure and
moses for the treatment of sIUGR is offered by polyhydramnios. Left untreated, fetal demise of the
some centers in an effort to protect the normal pump twin occurs in more than 55% of cases [39].
twin from the complications of death of the
growth-restricted twin. However, in one study, 8.6.5.1 Clinical Management
survival of both twins was 28% in the laser group Antenatal management of TRAP sequence
compared to 81% in the observation group [36]. involves a thorough ultrasound examination of the
If discordant growth in a MC/DA pregnancy is pump twin to exclude anatomical abnormalities.
detected after a viable gestational age has been Earlier postnatal studies indicated that if the
attained, consideration for steroid administration weight of the acardiac fetus was less than 50% of
to enhance fetal lung maturity, antenatal surveil- that of the normal fetus, the incidence of preterm
lance, and delivery should be undertaken. delivery was only 35% and none of the pump
fetus, this is best accomplished by the use of

a specialized radiofrequency ablation needle
directed by ultrasound guidance to target the
umbilical cord insertion into the abnormal fetal
mass. A survival rate of greater than 85% can be
expected in the pump twin, although PPROM and
preterm delivery are still a risk factor [40].
8.6.6 Anomalous Cofetus
Discordance for a chromosomal or structural

fetal abnormality can occur in both monochori-
onic and dichorionic twins. This discordance is
not unexpected in the dichorionic gestation, given
that the majority of these pregnancies are dizy-
gotic. In the monozygotic gestation, however,
this dissimilarity is thought to be the result of
aberrancies after early cleavage of the embryo to
form two separate cell lines. In the typical case of
a heterokaryotypic defect, one embryo loses an X
chromosome resulting in the usual phenotype of
Turner’s syndrome – cystic hygroma or hydrops.
The cotwin appears normal on ultrasound.
Assessment of the karyotype in the normal
Fig. 8.5 Acardiac twin fetus appearing twin is essential in these cases.
twins developed congestive heart failure [39]. 8.6.6.1 Clinical Management

Moore et al. went on to propose a formula for cal- Selective feticide for discordant abnormalities is
culating the weight of the acardiac fetus based on usually undertaken in the dichorionic multiple
its length. Many authorities, however, have aban- gestation by injection of potassium chloride into
doned this concept for deciding if the pump fetus the pericardial space of the anomalous twin.
is at risk. Wong and Sepulveda [38] have proposed However, in the monochorionic twin gestation,
that an abdominal circumference ratio of <50% this technique cannot be used as placental anasto-
should be considered indicative of a small to moses will allow for passage of the noxious agent
medium-sized acardiac fetus with little risk for to the normal fetus resulting in a dual demise
compromise in the pump twin. A ratio of >50% [41]. In these cases, selective feticide is performed
warrants consideration of intervention particularly using ultrasound-guided methods for umbilical
if there are signs of cardiac failure in the pump cord ligation of the affected fetus. Originally
twin such as cardiomegaly or tricuspid regurgita- undertaken using bipolar energy delivered by dis-
tion. Doppler ultrasound can also be used to assess posable forceps, this method has been replaced in
the vascularity of the acardiac mass. A large most centers by radiofrequency energy delivered
amount of flow seen using power Doppler in con- through special engineered needles. The latter
junction with a low resistance to the arterial flow technique offers the advantage of a smaller punc-
in the umbilical cord to the acardiac are also sug- ture of the amniotic cavity (17-gauge device vs.
gestive that it will increase in size. Should a deci- 3.3 mm cannula) and has been associated with a
sion be made to occlude the flow to the acardiac lower incidence of preterm premature rupture of
the membranes [42]. Survival rates in the normal obstetrician agree that a gestational age of fetal
cofetus have been reported to be 85%. viability has been reached. In a series of 96 cases
of MC/MA twins, outpatient fetal monitoring
was associated with a perinatal mortality of 15%
8.7 Monochorionic, Monoamniotic as compared to no perinatal losses when patients
(MC/MA) Twins were hospitalized for fetal surveillance [44].
Antenatal steroids to enhance fetal lung maturity
MC/MA twins account for about 1 in 8,000 preg- are indicated. Delivery by cesarean section at
nancies and 5% of monochorionic twins [43]. 32–34 weeks is generally accepted since approxi-
Although the majority of monoamniotic twins mately 1 in 25 MC/MA twins can be lost after
occur naturally, iatrogenic or spontaneous rupture this gestation [45].
of the intervening membrane in a MC/DA preg-
nancy can also lead to a monoamniotic condition.
MC/MA twins are at risk for developing compli- 8.8 Conclusion
cations similar to MC/DA twins, although the
incidence of TTTS is thought to be reduced by A recent lead editorial in the obstetrical literature
threefold due to the usual presence of large AA was entitled “There is NO diagnosis of twins”
anastomoses [44]. The major risk for perinatal [46]. The authors went on to advise that there are
loss, however, is related to entanglement of the only monochorionic or dichorionic twins.
umbilical cords, which is seen in virtually all Clearly, monochorionic multiple gestations are
cases (Fig. 8.6). The perinatal mortality associ- at significant risk for unique complications not
ated with cord compromise in these pregnancies seen in singleton pregnancies or dichorionic
was approximately 50% in early studies; however, multiple gestations. Because there are now effec-
a more recent series suggest that the perinatal tive treatments for these conditions, ultrasound
mortality is ~15% after 20 weeks’ gestation [45]. determination of chorionicity by reproductive
endocrinologists and obstetrical providers should
be part of routine prenatal care in multiple
8.7.1 Clinical Management
gestations.
In general, most centers in the U.S. now offer in-
patient admission for intensive fetal monitoring
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Multiple Pregnancy Vanishing Twin
Syndrome 9
Gabriel de la Fuente, Jose Manuel Puente,
Juan A. García-Velasco, and Antonio Pellicer
Abstract
The vanishing twin syndrome is a relatively frequent finding, especially
since the widespread use of assisted reproductive technology (ART). Both
an earlier follow-up of pregnancy and the use of transvaginal ultrasound,
providing a better ultrasound resolution, have brought us closer to the real
incidence of this phenomenon, which was underestimated prior to the
introduction of these techniques. Thanks to this, we can establish the real
incidence of early embryonic loss, from its early ultrasound identification,
as well as study the incidence of complications, clinical management, and
perinatal prognosis associated with these pregnancies.
Keywords
Vanishing twin syndrome • Chorion • Amnion • Ultrasound • Zygosity
These data, however, refer to spontaneous preg-

9.1 Incidence of Multiple nancies and not to the influence of assisted repro-
Pregnancy ductive technology (ART). Said treatments have
brought about a considerable increase in the
9.1.1 Incidence in Assisted number of both dichorionic and monochorionic
Reproductive Technology (ART) twin pregnancies, as well as that of triplet and
and the Importance of higher-order pregnancies. The reason lies both in
Chorionicity and Amnionicity ovulation induction treatments, leading to almost
30% of multiple pregnancies, and in the transfer
Spontaneous twin pregnancies represent about of more than one embryo during in vitro fertiliza-
1–2% of all pregnancies. For practical purposes, tion (IVF) treatments. Scientific associations are
the probabilities of twin, triplet and quadruplet currently committed to reducing these figures in
pregnancies can be estimated at 1/80, 1/80 [1] view of the high number of perinatal complica-
(1/6,400), and 1/80 [2] (1/512,000), respectively. tions entailed in comparison to singleton preg-
nancy, especially in relation to increased preterm
birth; recommendation guides based on age and
G. de la Fuente (*)
on the technique used have been prepared [3].
IVI Madrid & Rey Juan Carlos University,
Madrid, Spain In cases of monozygotic pregnancy only, the
e-mail: [email protected] influence of assisted reproduction treatments

104 G. de la Fuente et al.
with IVF seems clear, with an estimated twofold of division, they can be completely independent,
increase in the relative risk compared to sponta- or share the placenta, amniotic sac, and even fetal
neous pregnancy, and where the blastocyst trans- structures in cases of late division.
fer (RR = 4.25) and intracytoplasmic sperm From a practical standpoint, however, it is
injection (RR = 2.25) are the main associated most important to establish chorionicity and
factors [1]. amnionicity, given that the probability of fetal
All twin pregnancies in general entail increased complications in monochorionic pregnancies is
complications. The most frequent are related to considerably higher in view of the presence in all
preterm birth (50% in twin pregnancies and 90% cases of vascular anastomosis between the circu-
in triplet pregnancies). Preterm delivery, whether lations of both twins. In cases of monoamniotic
spontaneous or induced by pregnancy complica- pregnancy, the risk of mortality is very high due
tions, accounts for the approximately fivefold to the possibility of intertwining of both umbili-
increase in perinatal mortality associated with cal cords, leading to a mechanical interruption of
multiple pregnancies in comparison to singleton flow through the cords. A relevant fact in these
pregnancies. Moreover, other complications such types of pregnancies is that death after the first
as miscarriage or intrauterine loss of one of the trimester of one of the fetuses in a monochorionic
twins, growth disorders, and a higher incidence pregnancy is associated with a high probability of
of congenital abnormalities should be considered. sudden death or severe neurological damage of
Nevertheless, it is essential to differentiate cho- the surviving twin; this should always be taken
rionicity of twin pregnancies, as monochorionic into account as it entails an important implication
twin pregnancies show an even higher risk of for the treatment of complications.
complications due to the potential blood shunting It should be noted that ultrasound diagnosis
via intertwin vascular anastomoses that are pres- enables to establish the chorionicity of a twin preg-
ent in all monochorionic placentas. This makes nancy as of the fifth week of amenorrhea. In these
the follow-up protocol completely different in one early phases of gestation, transvaginal ultrasound
and the other type of pregnancy and therefore is easier and more reliable, enabling to readily dis-
extreme care should be taken in differentiating tinguish dichorionic twins through the presence of
between both entities. The early and precise diag- a thick septum between the sacs, with sensitivities
nosis of chorionicity and amnionicity is therefore and specificities close to 100% [2]. This septum,
an essential priority objective of the first trimes- formed by two sheets of corium and two of amnion,
ter ultrasound examination, in order to establish becomes thinner as pregnancy progresses, but
an exhaustive action and follow-up protocol of remains thicker and easier to identify at the base of
these pregnant women leading to an early diag- the membrane, as an inverted “V” or lambda-
nosis of potential complications and eventually to shaped (twin peak sign) [4, 5] triangular projection
appropriate therapies aimed at reducing morbid- of tissue. In monochorionic-diamniotic pregnan-
ity and mortality. cies, the separation membrane is made up of the
Zygosity is determined by the genetic origin fusion of two amnion membranes only. It is there-
of the twins. Twins coming from fertilization of fore thinner and forms an image similar to a “T” at
two separate oocytes are called dizygotic, and its insertion to the placenta.
gestation will have two placentas (dichorionic) Similarly, we may resort to the determination
and two amniotic sacs (diamniotic); they can be of the intertwin membrane thickness. However,
of the same or different sex, since they are geneti- the latter is less reliable due to its variability and
cally different. On the other hand, twins coming the inter-/intraobserver differences during assess-
from one single fertilized oocyte that divides into ment [5].
two embryos are called monozygotic and will be In ART pregnancies, ultrasound is conducted
genetically identical. Monozygotic twins repre- either 24 days after oocyte pick up or after insem-
sent approximately 30% of twin pregnancies; ination, or 21–23 days after embryo transfer,
they are of the same sex, and depending on the time meaning in all cases a gestational age of about
9 Multiple Pregnancy Vanishing Twin Syndrome 105
5 weeks and 3 days. At that gestational age, it is miscarriage rate is higher in aged patients, mainly
simple to visualize both independent gestational due to a higher frequency of chromosomal abnor-
sacs and the thick septum that separates them, in malities. Similarly, the miscarriage rate is higher
cases of dichorionic pregnancy. in obese patients, with several associated mecha-
nisms. On the whole, total fertility of obese
Amnionicity: Every dichorionic pregnancy is patients (BMI above 25 and particularly above
diamniotic. The number of sacs in monochorionic 30) is reduced, due to affected oocyte quality,
pregnancies can be determined as of the eighth endometrial receptivity, and early and late embry-
week of gestation, since before that, visualization onic loss.
is more difficult because the amnion and the Regarding multiple pregnancies, several authors
embryo are very close to each other. Proximity of claim a lower pregnancy loss rate for multiple
both cords at their placental insertion and the pregnancy than for singleton pregnancy. In this
visualization of intertwined cords are typical signs sense, Tummers et al. [20] report a 23% miscar-
of monochorionic monoamniotic pregnancies. riage rate for singleton pregnancies, 12% mis-
carriage of one of the two embryos, and 5% cases
of total embryonic loss. Glujovsky et al. [21]
studying the rate of early spontaneous embry-
9.2 Early Embryonic Loss onic loss (before the ninth postmenstrual week)
Associated with Multiple both in singleton and multiple pregnancies con-
Pregnancy ceived by ART observed a lower rate of sponta-
neous loss for twin pregnancies (OR = 0.6 with
9.2.1 Incidence and Associated 95% confidence intervals in 0.50–0.79).
Mechanisms Singleton pregnancies showed a 23.7% miscar-
riage rate, whereas in 86.5% of the cases where
The incidence of embryonic loss is high and is two sacs were observed in the first ultrasound,
estimated by some authors at ca. 40% [6, 7]. pregnancy proceeded beyond the eighth week
There are numerous causes for this early (period after which the patient was discharged);
embryonic loss, with associated factors such as in 9.2% of the cases, a single viable embryo was
age [8–10], a high body mass index [11–15], and observed, whereas in 4.2%, there was total
the occurrence of a multiple pregnancy. embryonic loss. These figures remained the same
With regard to recurrent embryonic loss, chro- for pregnancies with more than two embryos.
mosomal and genetic causes [16] are the main These results cannot be explained using only a
etiological factors involved. The presence of mathematical model of embryo implantation
antiphospholipid antibodies (lupus anticoagulant prediction; therefore, these and other authors
and anticardiolipin antibodies) has also been claim the existence of an embryonic synergism
demonstrated as causes of recurrent pregnancy in multiple pregnancies that would improve the
loss as well as some uterine abnormalities such as local environment for implantation [22–24].
a septate uterus [17]. The importance of proper Matorras et al. [23] estimated that the probabil-
development of the endometrium and its correct ity of successful implantation of an embryo
decidualization, which would enable selection of increases by 22% for every additional embryo
the competent embryo, has recently become clear transferred. Furthermore, the coexistence of
[18]. Some authors believe that endometrial other embryos improves not only the chances of
stromal cells would therefore become “sensors” implantation, but may also promote a more
capable of detecting compromised embryos with favorable environment for embryonic mainte-
severe chromosomal defects and would somehow nance and development. Other variables associ-
prevent their implantation [19]. ated with multiple implantation were a young
Maternal age is a widely studied factor, and maternal age, a greater endometrial thickness,
the extensive experience gained shows that the a male sterility, and a high embryo score.
Lambers et al. [25] described an ongoing preg- authors. Either an empty gestational sac where no
nancy rate for singleton and multiple pregnancies embryo grows, or an embryo without cardiac
of 81.5 and 97.5% and the risk of loss per activity can be observed, while the other twin
implanted gestational sac was 18.5 and 11.46% develops correctly. Since hematomas are frequent
(P < 0.001), respectively. This lower rate of loss at early ages of pregnancy, we shall take care in
per gestational sac found in multiple pregnancies differentiating them, considering that in early
represents a combination of both genetic and pregnancy there will be already the yolk sac and
embryonic development factors and of an appro- chorionic ring (Figs. 9.1 and 9.2).
priate uterine environment. The occurrence of
multiple implantation is predominantly dependent
on (morphological) embryo quality, but the con-
tinuation of pregnancy seems to be more depen
9.3.2 Incidence in ART
dent on the combination of genetic (younger
The incidence of VT is variable, since neither the
maternal age) and developmental potential of the
definition of the syndrome nor the population
embryo.
included in the different studies is homogeneous.
Some authors report rates of 71% loss of at least
one of the two twins in spontaneous pregnancies
9.3 “Vanishing Twin” Syndrome before the tenth week of gestation [26]. Others
estimate the loss rate during the first trimester at
9.3.1 Definition 53% [27].
Data obtained from patients who became
The vanishing twin (VT) syndrome refers to the pregnant after assisted reproductive techniques
loss of one of the twins during the first trimester. offer the advantage that pregnancy is monitored
Definition of the syndrome varies among different from very early stages, allowing embryonic loss
Fig. 9.1 Vanishing twin. Smaller sac without embryo development

Fig. 9.2 Vanishing twin. Smaller sac without embryo development (3D Image)
to be diagnosed with considerable reliability. The real hematoma is located between the corium
However, these patients are usually older, and in and the myometrium. In that sense, follow-up of
some cases, show disorders that could favor early patients conceiving pregnancy after ART is an
pregnancy loss, resulting in values that could advantage, as it is conducted from earlier stages.
theoretically exceed those of the general popula- At IVI, we perform a first ultrasound exami-
tion. Incidences reported by different authors are nation either 24 days after oocyte retrieval or
quite inconsistent; in general, approximate values insemination, or 21–23 days after embryo trans-
can be estimated between 9% [28] and 20% [29], fer, meaning in all cases a gestational age of
but could reach 50% in some cases of monocho- about 5 weeks and 3 days; this study shows the
rionic twins [30]. High loss rates of 25–30% have gestational sac as an anechoic structure surrounded
been reported when the pregnancy is achieved by a double hyperechogenic ring (double decid-
through egg donation [31]. Hence, and in spite of ual sign). Its location is always eccentric,
the disparity found, we can conclude that for immersed in the decidua. The anechoic ultrasound
pregnancies conceived after ART, one every 8–10 image actually corresponds to the gestational
singleton pregnancies originated initially from a sac’s liquid content, which contains from the
twin pregnancy. beginning the amnion, the yolk sac, and the
embryonic pole. However, initially they are so
small that visualization is impossible. The pres-
9.3.3 Ultrasound Diagnosis ence of the yolk sac is key to make sure it is a
gestational sac and not a pseudosac. The yolk
As mentioned before, methodology is essential sac is visible for mean sac diameters (MSD)
for appropriate diagnosis of a vanishing embryo above 6 mm, but is always observed with a MSD
(VE) and to avoid confusion with fluid collections of 10 mm. It corresponds to the secondary or
located in the decidua or with hematomas, which definitive vitelline sac. It grows approximately
are not uncommon in these stages of pregnancy. 1 mm/week and its values range from 2 mm
(week 5) to 6 mm (week 10). Nomograms of 9.3.4 Symptoms

diameter and volume growth have been deter-
mined with considerable precision. As of week The main symptom of the VT syndrome is bleeding,
12, it is usually no longer visible. Visualization with a bleeding rate during the first trimester of
of the yolk sac is key to early pregnancy diagno- up to 52.8% compared to 15.9% [33] in the preg-
sis and its clear distinction from other entities nant control population after ART. Other authors
such as fluid collections (pseudosacs), small [34] do not report any higher bleeding rates,
adenomyotic foci, etc. Size and echogenicity of whereas the literature refers to a great disparity in
the yolk sac are also early prognostic factors of values that range from 7.8 to 76.5% [35–39].
embryo health.
10 days later, we perform a second ultra-
sound examination aimed at assesing: (a) 9.3.5 Perinatal Results
Pregnancy viability; and (b) Embryonic cardiac
activity. Given that the presence of a VE is a frequent
Identification of bad prognostic factors: event in pregnancies after ART, it is important to
• Small gestational sac advise the couple on the prognosis of these preg-
• Early oligohydramnios nancies, and if possible, to establish measures
• Embryo crown-rump length (CRL) 1 week, aimed at minimizing possible harmful effects.
lower than expected A general increase in the number of perinatal
• Yolk sac size <3 or >7 mm complications in ART pregnancies has been
• Embryonic bradycardia (<85 bpm) reported, even when distinguishing between sin-
The identification of bad prognostic factors gleton and multiple pregnancies. The cause may
may raise suspicion of upcoming embryonic loss. be, on occasions, age or the maternal pathology
A weekly ultrasound follow-up may be important sometimes associated with these types of patients,
in these cases in order to diagnose the time of but it is also true that on many occasions, the
embryonic death, as death after the ninth week presence of a VE, whether spontaneous or prod-
leads to a worse prognosis and may influence the uct of embryo reduction (ER), may entail adverse
results of the biochemical screening for aneu- consequences for both the duration and growth of
ploidy [32]. In applying these bad prognostic the surviving fetus(es) [40–45].
markers, it should be noted that for pregnancies Regarding early complications of pregnancies
conceived through ART and being the conception with VE, there seems to be no higher miscarriage
date known, the limits of deviation from normal- rate for these pregnancies (twin converted to sin-
ity become significantly narrower. However, gleton) compared to initially singleton pregnan-
some variations – even in cases of good prognosis cies [46]. For some authors as already shown, the
– attributed to variations in embryonic growth miscarriage rate after the diagnosis of a VE might
rate and/or possible intra- and interobserver mea- even be lower (5% as opposed to 20%) [27].
surement bias are observed. Finally, we perform Regarding the course of pregnancy, we have
a last ultrasound examination between menstrual found in our group a lower risk of preeclampsia
weeks 8 and 9 where we assess early embryonic and a higher rate of premature rupture of mem-
and placental development, and if this is ade- branes (PROM), both preterm and term. The
quate, we discharge the patient to the obstetri- lower frequency of preeclampsia may be attrib-
cian’s care. This protocol for early care of uted to the immune tolerance induced by the twin
pregnancy based on three ultrasound controls that later stops developing, whereas the rupture
allows us to diagnose all pregnancies with early of membranes may be attributed to the release
VE (before 9 weeks) as well as to exclude the into the bloodstream of proinflammatory sub-
common complications of ART pregnancies stances that would facilitate medium- and long-
(threatened miscarriage, miscarriage, and ectopic term PROM. Although not statistical significant,
pregnancy). Pinborg et al. [34] observed a slightly higher risk
of preeclampsia in the VT survivors (OR 0.77 for were associated with a 47% increased odds of
singleton pregnancies compared to OR 1 for low birthweight, as well as a 33% increased odds
pregnancies with VE). This same study found a of preterm low birthweight and a 57% increased
1.4% rate of abruptio placentae in the survivor odds of term low birthweight. Therefore, early
cohort, twice that of singleton pregnancies, which fetal loss in ART pregnancies that result in a twin
also affected the cases of early VE, although live birth was associated with significantly
again statistical significance was not attained. increased risks for lowered birthweight and short-
However, this information should be considered ened gestation.
insofar as recommendations and guidelines to These authors do not specify the time of
reduce its incidence can be established. embryonic or fetal death, which, as mentioned
A lower birthweight for the survivors of the before, is important for the prognosis [34].
VT syndrome has also been observed; the higher Almong [49] also finds a higher rate both for
the gestational age at the time of vanishing, the very low weight (<1,500 g) 3.5 vs. 0.6% and for
higher the risk of the surviving newborn being delivery before 28 weeks of pregnancy that
small for gestational age. reached 7% in pregnancies with VE and 1.2% in
In an extensive retrospective study of 642 the remaining pregnancies. The study population
cases of VT, 5,237 singleton pregnancies, and was composed of a total of 228 patients, 57 with
3,678 twin pregnancies after ART, the difference VE and 171 controls. However, these data were
between the early survivor and the singleton not confirmed by La Sala et al. [50] in a study
cohort in mean birthweight was 77 g. conducted on 322 singleton pregnancies after
Early survivors had a better outcome than IVF vs. 44 cases of VE during the first trimester
intermediate and late survivors; however, they and 320 singleton pregnancies after ICSI vs. 44
still had poorer birthweight outcome than single- cases of VE during the first trimester. No signifi-
tons [34]. cant statistical differences were found in the per-
The rate of intrauterine growth restriction centage of births before 28 weeks (no cases found
(IUGR) in singletons originating from a twin in the VE group), nor in birthweight <1,500 g,
gestation was 3.8% compared to 3.6% in single- nor in the IVF group or the ICSI group. Hence, the
ton pregnancies. These data are very important VT syndrome per se, according to La Sala, would
for reassuring the patient suffering from VE not change the outcome of the survivors.
within the first 8 weeks. However, the rate of Based on 46 cases of VE and 96 controls,
IUGR rose to 7.7% in cases of death between Shebl et al. [51] also found that the frequency of
weeks 8 and 22, and to 12.5% as of week 22. The low birthweight (26.1 vs. 12.0%) and being small
causes of this small difference in weight and for gestational age (32.6 vs. 16.3%) was signifi-
growth in cases of early VE are difficult to cantly higher in the VE group.
explain, but possible hypotheses include inade- However, no significantly higher rate of pre-
quate placentation, the effect of embryo degrada- term birth was found. The author insisted on
tion products, or increased bleeding that could informing the patients about the associated risks
act as an independent factor [47]. when transferring more than one embryo.
Luke et al. [48] compared pregnancies con- Hence, there seems to be a clear influence of
ceived after ART where early ultrasound showed VE on the perinatal result of pregnancy in terms of
initially a higher number of gestational sacs with weight and gestational age. It should be noted,
positive cardiac activity, but ending in singleton however, that almost 2/3 [34] and in other studies
and twin pregnancies, with initially singleton and 80% [33] of VE occurred during the first trimester;
twin pregnancies. The presence of three fetal therefore, the influence on weight and gestational
heartbeats was associated with a 47% increased age at birth, if any, is significantly lower. Moreover,
odds of early preterm birth, a 28% increased risk as pointed out by La Sala et al. [50], VE is sensu
of moderate preterm birth, and a 26% lower odds stricto a phenomenon confined to the first trimes-
of a term birth. Likewise, three fetal heartbeats ter and we should therefore avoid increasing
maternal anxiety of a patient experiencing a VE that maternal age in pregnancies after frozen
with pessimistic information about increased risk embryo transfer is that of the freezing date, and
of preterm delivery or low birthweight. maternal age after oocyte donation is the age of
Furthermore, some publications have shown the donor.
concern for the neurological future of children In the biochemical screening of the second
born after a VE-complicated pregnancy. Although trimester, serum levels of free b-hCG, alpha-
in many studies, small samples do not allow for fetoprotein, and oestriol appear to be modified in
definitive conclusions, the development of chil- pregnancies conceived after ART; this could
dren born after an initially twin pregnancy with increase the trisomy 21 false positive rate for
VE does not differ in general from that of chil- these patients [56, 57], although this information
dren born after initially singleton pregnancies has not been corroborated by other authors [58].
[52]. However, these same authors [53] later The presence of a VT could alter the results of
found developmental disorders in some cases of the first trimester biochemical screening, given
VE. Of great interest is the work published by that production of free beta HCG and PAAP-A
Pinborg in 2005 where the incidence of cerebral by the placenta of the embryo that stops develop-
palsy, neurological sequelae, and developmental ing could alter the results for the surviving twin,
disorders observed between the early VE preg- particularly in occasions where the biochemical
nancies and the singleton pregnancies was not determination is conducted in early stages (8–9
statistically significant. They observed that the weeks). Moreover, if ultrasound control is not
number of neurological deficits grew as the ges- conducted at this early stage, the time of embry-
tational age at which the VE occurred increased. onic death will remain unknown (early or late);
A possible explanation of this finding is the fail- hence, the importance of conducting serial con-
ure to establish the number of deaths in mono- or trols in this type of pregnancies.
dichorionic pregnancies, each with very different However, results found in literature [33] do
neurological prognosis, being the case of mono- not seem to identify significant differences
chorionicity the most unfavorable. between free beta HCG and PAAP-A levels in
VT pregnancies before 9 weeks compared to the
control group in singleton pregnancies; authors
9.3.6 Trisomy 21 Screening expressed their doubt in cases of death at a later
and Vanishing Twin stage. In these cases, the screening recommended
would use only maternal age and the value of the
Nowadays, there are numerous trisomy 21 screen- nuchal translucency.
ing strategies, both in the first and the second tri-
mesters, with detection rates of up to 85–90%
and 5% false positive rate [54]. The most widely 9.3.7 Induced Vanishing Twin Embryo
accepted method in Europe is the one that com- Reduction
bines maternal age with assessment of the free
beta HCG and PAAP-A obtained between week 8 ER consists in causing the death of one or several
and 12 + 6 menstrual weeks, and nuchal translu- embryos or fetuses with the purpose of prevent-
cency measurement obtained between weeks ing the more severe consequences associated
11 – 13 + 6. CRL values should range between 45 with multiple pregnancies: severe preterm birth
and 84 mm. and its associated neurological sequelae.
The results of combined biochemical-ultra- Fortunately, the use of this technique has fallen
sound screening in pregnant women after ART steadily since the gradual implementation of a
(IUI, IVF, frozen embryo transfer, and oocyte policy to reduce the number of transferred
donation) show no differences with results for the embryos. In our center, the mean number of
general population [55]; therefore, there is no embryos transferred is lower than two, and we
need to make any adjustments, only to remember believe it is the right thing to do in order to reduce
the rate of twin pregnancies and, what is worse, with hematomas or other type of fluid collections.
of triplet and higher-order pregnancies. Also, bad prognostic factors of pregnancy
Among several possible ER situations, the most such as embryonic bradycardia, small gesta-
common is that after a triplet pregnancy, ER of one tional sac, and early oligohydramnios should
of the embryos is performed, resulting in the end be identified. Given that in general the prog-
in a twin pregnancy. Excluding early complica- nosis worsens as gestational age increases, it
tions derived from the procedure, these cases have is very important to determine adequately the
shown that the medium- and long-term prognosis probable date of VE in order to provide the
is worse than that of a twin pregnancy, as the abor- patient with appropriate information regard-
tion rate [59] increases to 8% and the growth of the ing the perinatal prognosis and possible
both surviving fetuses is also lower than in twin medium- and long-term impact.
pregnancies, since after an ER, the fetal placentas 4. Patients with VE show an increased bleeding
of initially triplet twins are smaller than in the case frequency during the first trimester. However,
of initially twins. The explanation of a higher abor- the abortion rate does not increase and may
tion rate is given by the release of proinflammatory be even lower according to some authors.
agents by the twin subject to ER, primarily cytok- 5. Some studies show a higher rate of PROM
ines that stimulate the production of prostaglan- attributed to the release into the bloodstream
dins; whereas the lower weight could be attributed of proinflammatory substances that would
to an inadequate placentation derived from the facilitate medium- and long-term PROM.
higher number of gestational sacs, which does not 6. An increase in the rate of weight <1,500 g
seem to improve after an ER. and of delivery before 28 and 32 weeks is
The model that best describes the most com- reported, although these findings are primar-
mon VE phenomenon is probably the few cases of ily due to cases of VE beyond 8 weeks, which
ER from 2 to 1 fetus or from 3 to 1 fetus. Few lit- represent a minority of total VEs.
erature cases allow for definitive conclusions, 7. In cases of VE before 9 weeks, the possibili-
given that in this type of patients the ER is very ties of having weights <1,500 g and deliver-
often indicated due to a uterine problem (mulle- ies before 28 and 32 weeks are similar to
rian anomalies, myomas, previous cesareans) and those of singleton pregnancies.
therefore observations cannot be extrapolated to 8. In cases of VE before 9 weeks, no higher rate
the general population. Some authors find the of neurological sequelae, cerebral palsy, or
“take-home baby” rate would be higher than in developmental disorders was found.
initially twin pregnancies, although they express 9. In cases of VE before 9 weeks, it is not nec-
doubt about the ethical considerations that may essary to adjust the biochemical parameters
arise when performing an ER in these cases [60]. of trisomy 21 with nonconclusive results
between weeks 9 and 12.
10. ER in multiple pregnancies is a model of iat-
9.4 Conclusions rogenic VE that allows to determine the
influence of early placentation on the growth
9.4.1 Key Points of the other twins and on the abortion rate.
However, the most frequent model of sponta-
1. The VT syndrome is defined as the loss of neous VE, i.e., the reduction of two twins to
one of the twins during the first trimester. a singleton pregnancy, is rare in the applica-
Two thirds of the cases occur during the first tion of ER techniques and therefore the data
8–9 weeks. obtained are less reliable.
2. The incidence is variable, with a mean value Finally, the information given to a pregnant
of about 12% (9–20%). woman experiencing a VE during the first trimes-
3. It is important to perform an accurate ultra- ter, particularly before 9 weeks, should be posi-
sound diagnosis to avoid confusing the VE tive and management should be expectant, with
high possibilities of take-home baby and without 15. Bellver J, Ayllon Y, Ferrando M, Melo M, Goyri E,
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P, Melotte C, et al. Chromosome instability is com-
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Part II
Male Infertility
The Effect of Cancer Therapies
on Sperm: Current Guidelines 10
Akanksha Mehta and Mark Sigman
Abstract
Advances in treatments and improved survival for young men with cancer
have led to attempts to preserve or restore fertility in these patients.
Baseline fertility is often impaired at the time of cancer diagnosis and is
often worsened by subsequent cancer therapies. Fertility is most com-
monly impaired in testicular cancer followed by Hodgkin’s lymphoma,
non-Hodgkin’s lymphoma, and leukemia, and even in those with other
solid tumors. Sperm banking prior to therapy remains the most important
tool for fertility preservation. Recovery of spermatogenesis may take
months to years following chemotherapy or radiation therapy. Attempts to
protect spermatogenesis from the effects of these therapies remain experi-
mental and largely unsuccessful. Both chemotherapy and radiation therapy
cause damage to sperm DNA with temporary increased rates of aneu-
ploidy. Despite this, most studies show no increase in the rates of congeni-
tal anomalies in children born to men following cancer therapies. Future
efforts are being directed at protection of spermatogenic cells from dam-
age and germ cell transplantation and in vitro maturation.
Keywords
Leukemia • Hodgkin’s lymphoma • Chemotherapy • Radiation therapy •
Cancer • Gonadotoxic • Type Ad spermatogonia • Type Ap spermatogonia •
Type B spermatogonia • Spermatocytes • Spermatids • Testicular cancer •
Alkylating agents • Bone marrow transplantation • Retroperitoneal lymph
node dissection • Retrograde ejaculation • Sperm banking • Sperm cryo-
preservation • Testicular sperm retrieval • Germ cell transplantation •
Sperm aneuploidy • Congenital anomalies • Testicular allograft
M. Sigman (*)
Division of Urology, Rhode Island Hospital,
Warren Alpert Medical School at Brown University,
Providence, RI, USA

118 A. Mehta and M. Sigman
10.1 Introduction 10.2 Overview of Spermatogenesis
During the last two decades, the survival rates Spermatogenesis is an elaborate cell-differentiation
of young men suffering from various types of process that takes place in the seminiferous
cancers have improved dramatically, due to tubules of the testes, starting with the spermatogo-
advanced diagnostic techniques and better nial stem cell and terminating with a fully differ-
treatment modalities. Greater attention is there- entiated and highly specialized male gamete
fore being paid to patients’ quality of life. called the spermatozoa. In humans, spermatogen-
Testicular cancer, lymphoma, and leukemia esis requires approximately 64 days.
remain the most common malignancies diag- On histologic examination of the seminifer-
nosed in men of reproductive age, and infertility ous tubules, spermatogonia are located along
problems stemming from cancer diagnosis as the base of the seminiferous epithelium and
well cancer therapy are a growing concern for consist of two classes: Type A and Type B cells.
cancer survivors. The Type A spermatogonia, in turn, consist of
In many cancer patients, sperm quality is the pale type (Ap) and dark type (Ad) cells. The
already impaired before receiving any form of Ap spermatogonia are mitotically active cells,
treatment. Further deterioration in semen which divide into Ap spermatogonia and type B
parameters and endocrine parameters typically spermatogonia. The Type B spermatogonia
ensues due to the damaging effects of chemo- undergo mitotic division to produce primary
therapy or radiotherapy, and this deterioration spermatocytes, which then undergo two meiotic
may be temporary or permanent. Many of divisions to form spermatids. Thus, Ap sper-
these patients are young men who have either matogonia are primarily responsible for replen-
not started, or not completed their families. ishing their own populations, as well as the
While our understanding of the gonadotoxic entire spermatogenic process. This process is
effects of specific cancer therapy regimens is summarized in Fig. 10.1.
improving, it is impossible to predict with any Ad spermatogonia rarely divide and are
degree of certainty which patients will have believed to represent dormant reserve germ
impaired spermatogenesis and which patients cells. When the number of Ap spermatogonia is
will have improvement in their gonadal func- diminished, for example, after irradiation, Ad
tion to reestablish normal spermatogenesis. As spermatogonia become active, transform into Ap
such, preservation of fertility potential and the spermatogonia, and then start to proliferate [1].
use of assisted-reproductive techniques have The hormonal regulation of spermatogenesis
become increasingly important in the long- is via the hypothalamic-pituitary-gonadal axis,
term management of cancer patients of repro- which is based on hormonal feedback mecha-
ductive age. nisms. Pulsatile release of gonadotropin-releasing
This chapter discusses the effects of the can- hormone (GnRH) from the hypothalamus regu-
cer disease process as well as the associated ther- lates the pulsatile release of follicle-stimulating
apies, including surgery, chemotherapy, and hormone (FSH) and luteinizing hormone (LH)
radiation therapy on gonadal function and sperm from the anterior pituitary. LH acts on Leydig
quality in male cancer patients. The availability cells to regulate testosterone (T) secretion,
of assisted-reproductive techniques has undoubt- while FSH acts on Sertoli cells to initiate and
edly improved the chances of a successful preg- sustain spermatogenesis, in the presence of
nancy for these patients, but there is some testosterone.
controversy as to the safety of these techniques Elevated serum levels of LH and FSH, as
when using sperm from irradiated or postchemo- well as depressed levels of testosterone, have
therapeutic patients. been associated with impaired spermatogenesis.
10 The Effect of Cancer Therapies on Sperm 119
Fig. 10.1 Overview of spermatogenesis
In particular, elevated FSH has been used as a hormone action. The role of testosterone in
marker for impaired Sertoli-cell function. spermatogonial development is less obvious.
Recently, Inhibin B, secreted by Sertoli cells, has Normal spermatogenesis requires high intrates-
been proposed as a marker for Sertoli-cell func- ticular testosterone concentrations. However, the
tion, especially in prepubertal boys and male role of intratesticular testosterone in return of
adolescents [2, 3]. Bordallo et al. compared spermatogenesis in impaired testes is more com-
Inhibin B/FSH ratios in 21 males after chemo- plicated. Animal studies have shown that high
therapy for Hodgkin’s lymphoma with 20 healthy, intratesticular testosterone concentrations can be
matched controls. There was no significant dif- detrimental to spermatogonial differentiation,
ference in LH, T, Inhibin B concentrations, or and suppression of testosterone is required to
T/LH ratio between the two groups, but serum promote spermatogonial development in irradi-
Inhibin B was correlated with serum FSH levels ated rodents and mouse models of juvenile sper-
and Inhibin B/FSH ratio was positively correlated matogonial depletion [6]. During normal early
with sperm count [2]. The authors concluded that pubertal development, when spermatogonial
serum Inhibin B levels may be an important divisions are very active, testosterone levels are
marker of seminiferous tubule function in men very low [1]. Fine regulation of intratesticular
treated with chemotherapy and should be inter- hormone concentrations during the spermatogen-
preted in the context of serum FSH. esis cycle appears key to normal sperm produc-
Each Sertoli cell supports a defined number of tion and differentiation.
germ cells, and therefore, spermatogonia. It is
evident that reduction in the Sertoli cell popula-
tion directly impairs spermatogenesis. Hormonal 10.3 Cancer
cues are equally important. FSH plays a major
role in the regulation of spermatogenesis, The most common cancers in men of reproduc-
although the mechanism of action of FSH on the tive age are leukemia, lymphoma, and testicular
Sertoli cell is unknown. Several animal studies germ cell tumors. In past years, patients suffering
have indicated that Type A spermatogonia are from these cancers were most concerned about
affected by the withdrawal of FSH [4], or by the disease recurrence and treatment side effects. As
administration of GnRH antagonists [5], indicat- survival rates after treatment have improved,
ing that the first mitotic division is a site of there is growing concern about quality-of-life
issues, such as preserving fertility potential. potential is not restored at the conclusion of
A recent study on semen quality in 205 adolescent cancer therapy. Improvement in and increased
males with various solid and hematologic cancers availability of in vitro fertilization (IVF) and
found that semen parameters such as sperm intracystoplasmic sperm injection (ICSI) tech-
count, motility, and semen volume were signifi- niques have allowed for successful pregnancy
cantly lower in cancer patients compared to outcomes even in instances of severe male factor
healthy controls [7]. It is important to determine infertility.
patients’ pretreatment fertility potential, in order
to better understand how their cancer disease and
its treatment will affect their fertility potential in 10.3.1 Testicular Cancer
the future.
It is well accepted that malignant disease, in The incidence of testicular cancer has increased
and of itself, can lead to impaired gonadal func- worldwide over the past five decades [12]. There is
tion, through hormonal alterations and metabolic controversial evidence of a concurrent decline in
conditions [8]. Reproductive hormones may be male reproductive health, as manifest by decreas-
low in some patients as a result of physiologic ing semen parameters and increasing frequency of
stress associated with the cancer state, or down- other genital abnormalities such as hypospadias
regulation by endocrine substances produced by and cryptorchidism, and it has been suggested that
some tumors, or even direct tumor invasion of the development of testicular cancer is etiologi-
endocrine organs. Malignancy may also result in cally linked to these genital abnormalities.
malnutrition, with deficiencies in vitamins, min- Several studies have documented lower sperm
erals, and trace elements needed for optimal counts in patients with testicular germ cell carci-
gonadal function. Hematologic malignancies noma compared to controls. Based on sperm con-
such as Hodgkin’s lymphoma are often accompa- centrations, a striking 50–75% of patients with
nied by constitutional symptoms, especially unilateral testicular carcinoma are subfertile at
fevers, which negatively influence spermatogen- the time of diagnosis, with sperm concentrations
esis. Some authors have described the develop- below 10 million/mL [12]. It is well known that
ment of antisperm antibodies as a result of approximately 10% of patients with testicular
disruption of the blood–testis barrier by cancer, cancer will have a history of cryptorchidism, and
leading to poor semen analysis results [9]. Lastly, this may explain in part, but not entirely, the
tumor-released cytokines such as interleukins effect on spermatogenesis. Previous histologic
and tumor necrosis factor can affect spermato- studies have shown severe abnormalities in 24%
zoal function, resulting in low sperm motility. of the biopsies from contralateral testes in men
This effect may be local, as demonstrated by the who underwent orchiectomy for unilateral tes-
observation that the number of spermatogenesis ticular germ cell tumor. Eight percent had no
defects in testicular tissue is highest in the tissue sperm production, 16% showed varying degrees
closest to the tumor, while spermatogenesis is of spermatogenesis, and 5% had carcinoma in
uniform in orchiectomy samples that have benign situ (CIS) [13].
tumors [10, 11]. The question of whether Leydig cell dysfunc-
There is no concrete evidence to support the tion preexists in patients with testicular cancer
hypothesis that pretreatment spermatogenic func- remains unresolved, but evidence from current
tion predicts posttreatment spermatogenic function. literature does support this notion. Men who
Rather, the purpose of studying pretreatment undergo orchiectomy for testicular cancer have
fertility potential and sperm quality is to deter- lower testosterone and higher serum LH levels
mine whether semen cryopreservation prior than men who undergo orchiectomy for benign
to initiation of therapy can provide sperm of causes [14]. Petersen et al. [15] compared gonadal
adequate quality to allow for use with assisted- function between two groups of men who
reproductive techniques in the event that fertility underwent orchiectomy for unilateral testicular
carcinoma with or without CIS of the contralateral CIS is found in the contralateral testis of 5%
testicle. Significantly higher LH levels and lower of patients with testicular germ cell cancer [19].
testosterone levels were found in the group with The management of CIS is important because the
CIS, indicative of markedly altered Leydig cell majority – if not all – cases of CIS will eventually
function in this group. Additionally, patients with progress to invasive disease. Seminiferous tubules
CIS had significantly lower sperm concentration containing CIS are devoid of spermatogenesis,
and total sperm count. Similar results were and the neighboring tubules often demonstrate
reported by Jacobsen et al. who studied gonadal severely impaired spermatogenesis as well. This
function in 63 men with bilateral testicular can- explains the frequent finding of oligospermia or
cer after their first orchiectomy and before addi- azoospermia, in combination with elevated serum
tional treatment. Results were compared with LH and FSH levels in patients with CIS [15].
those from 174 patients with unilateral cancer
after orchiectomy [16]. Although testosterone
levels did not differ between the two groups, LH 10.3.2 Lymphoma
levels were significantly higher in the subset of
patients who developed a contralateral testicular Hodgkin’s lymphoma affects patients of all ages,
tumor. Taken together, the above results indicate but has a first incidence peak between 18 and 40
that abnormal serum testosterone and LH profiles years. Presently, 5-year survival from the disease
after orchiectomy are not simply a result of is around 90%. Infertility is one of the most sig-
removal of the testis, but rather may be attributed nificant side effects in long-term survivors of
to some inherent impairment of Leydig cell func- Hodgkin’s lymphoma, as treatment usually
tion in the contralateral testis. involves chemotherapy, radiation, or a combina-
Patients who develop testicular carcinoma in tion of both.
one testis are 500–1,000 times more likely to To this end, several authors have investigated
develop carcinoma in the contralateral testis, the pretreatment fertility status of Hodgkin’s
compared to the general population. Metachronous lymphoma patients. In a large observational
tumors are far more common than bilateral syn- study, Lass et al. [20] demonstrated no difference
chronous tumors [17]. While bilateral radical in semen parameters such as sperm count and
orchiectomy remains the standard of care for motility between patients with lymphoma vs.
these patients, there is growing enthusiasm for leukemia, but patients with Hodgkin’s lymphoma
testis-sparing surgery, which has been supported had significantly lower sperm quality compared
by the publication of specific guidelines from the with non-Hodgkin’s lymphoma. Rueffer et al.
German Testicular Cancer Study Group [18]. [21] investigated semen quality in 158 Hodgkin’s
In theory, testis-sparing surgery should be asso- lymphoma patients, classified as having either
ciated with improved fertility and endocrine early-, intermediate-, or advanced-stage disease
function. However, a major concern when sparing and found that 70% of patients had inade-
a tumor-bearing testis is the high prevalence of quate semen quality at baseline, with 21% of
adjacent foci of CIS. Given the exquisite radio- patients demonstrating severe damage such as
sensitivity of CIS, supporters of organ-preserving azoospermia or oligoasthenoteratozoospermia.
surgery for testicular carcinoma generally rec- Advanced stage of disease was associated with a
ommend that every patient undergoing partial greater likelihood of having semen abnormali-
orchiectomy be treated with adjuvant local ties, possibly because advanced Hodgkin’s lym-
radiation therapy to the testicular parenchyma, phoma is a biologically active disease, often
regardless of whether the tumor was seminoma presenting with systemic symptoms and elevated
or nonseminoma. Such treatment, intended to ESR, which may have a significant impact
minimize the risk of local recurrence, invariably on spermatogenesis. These results are in agree-
arrests spermatogenesis and culminates in infer- ment with those of the German Hodgkin Study
tility [16]. group trial, wherein only 20% of patients had
normozoopermia in pretreatment analysis, with 10.3.4 Other Solid Tumors

11% of patients having azoospermia and 69%
other dyspermia [22]. Although much less frequent than testicular germ
FSH and Inhibin B have both been proposed cell tumors and hematologic malignancies, fertil-
as markers of gonadal function in patients ity impairment is nevertheless present in solid
with Hodgkin’s lymphoma [22, 23]. A prospec tumors involving other organ systems, ranging
tively randomized trial of the German Hodgkin from carcinomas and sarcomas to CNS tumors.
Study group examined pre- and posttreatment Lass et al. [20] evaluated 231 men diagnosed
fertility parameters in groups of 202 and 112 with malignant disease, categorized as testicular
patients, respectively, and found a significant dif- tumors, hematologic malignancy, or other can-
ference in FSH levels between patients with cers. Men with testicular tumors had significantly
azoospermia and those with preserved spermato- lower sperm concentration and motility than the
genesis [22]. However, some authors have argued other two groups of patients. Men with other
that FSH is not a reliable marker for fertility in solid tumors had semen quality abnormalities
postchemotherapy patients, as the frequency of similar to patients with hematologic malignan-
elevated FSH levels after chemotherapy varies cies, but significantly better than testicular cancer
from 35 to 100% and depends on the cumulative patients.
doses of alkylating agents used [24]. Van Beek
et al. showed that male survivors of Hodgkin’s
lymphoma treated in childhood with alkylating 10.4 Cancer-Associated Therapies
chemotherapy agents have significantly lower
levels of inhibin B, when compared to men not Treatment options for cancer patients consist of
treated with these agents. Additionally, they found surgical intervention, chemotherapy, radiotherapy,
Inhibin B to be a stronger and more independent or multimodal therapy, depending on the type of
determinant of sperm concentration compared to malignancy. Both chemotherapy and radiation
FSH and recommended Inhibin B be used as a therapy act by interfering with rapidly dividing
screening tool to detect gonadal damage in men cells. Germ cells are also rapidly dividing, and
treated for Hodgkin’s lymphoma in childhood. thus become a target for these therapies. Recovery
for surviving germ cells can happen, but is unpre-
dictable and is often a lengthy process. Leydig
10.3.3 Leukemia cells, with their slower rate of turnover, are more
resistant to gonadotoxic therapy, resulting in
Like patients with testicular carcinoma and preservation of androgen production even when
Hodgkin’s lymphoma, patients with leukemia patients are infertile.
also appear to have significant pretreatment Because of their high mitotic activity, sper-
impairment of spermatogenesis. Hallak et al. [25] matogonia are more vulnerable to DNA damag-
examined semen characteristics in 25 men with ing agents such as radiation and chemotherapy,
acute or chronic leukemia and compared them compared to Sertoli and Leydig cells and sperma-
with 50 healthy controls and found that patients tids. Spermatogonia have also been shown to be
with leukemia had significantly lower prefreeze more vulnerable than spermatocytes, which carry
and postthaw sperm motility, motile sperm count, out meiotic divisions only [1].
and curvilinear velocity. In this analysis, the type As previously mentioned, spermatogenesis is
of leukemia did not have an effect on prefreeze or already impaired to some degree in men with
postthaw semen quality. Most importantly, the cancer, even prior to cancer therapy. Interestingly,
quality of the postthaw sperm was sufficient for some patients who are azoospermic prior to can-
use with ICSI, and therefore, cryopreservation of cer therapy do go on to have normal sperm
sperm was recommended for leukemia patients parameters after treatment. This observation has
despite the abnormalities noted in the analysis. led some authors to suggest that the mechanism
of infertility before treatment is reversible and due to interruption of the retroperitoneal sympathetic
distinct from the mechanism following treatment nerves. During the past two decades, the fre-
for cancer [26]. Therefore, even in patients with quency of these complications has been reduced
preexisting impairment of spermatogenic func- from approximately 75% to approximately 33%,
tion, it is still worthwhile to select the least with the introduction of a modified right- and
gonadotoxic treatments possible. left-sided template for RPLND, with no effect on
For patients with testicular cancer, treatment relapse rate [12]. The introduction of nerve-spar-
typically consists of radical orchiectomy, fol- ing techniques has allowed for even greater pres-
lowed by retroperitoneal lymph node dissection ervation of ejaculatory function. While surgical
(RPLND), chemotherapy, or radiation therapy, treatment for testicular germ cell tumors can cer-
based on the histopathology and stage of the germ tainly impact fertility, the impact is not as great as
cell tumor. Patients with lymphoma and leukemia that of chemotherapy and radiation therapy.
are primarily candidates for chemotherapy, with
or without radiation therapy, with a variety of
regimens. Patients who undergo bone marrow 10.4.2 Chemotherapy
transplantation (BMT) for hematological malig-
nancies are subject to a combination of chemo- Chemotherapy agents are divided into classes,
therapy and total body irradiation (TBI), each with specific mechanisms of action
significantly increasing their risk of gonadotoxic- (Table 10.1) [8, 26, 27]. Because these cytotoxic
ity compared to patients who do not undergo agents interfere with cell division cycle, rapidly
multimodal therapy. dividing cells are more susceptible to their effects.
During spermatogenesis, chemotherapeutic agents
most severely affect Type B spermatogonia [26].
Recovery of spermatogenic function therefore
10.4.1 Surgery depends on survival of Type A spermatogonia. If
cytotoxic therapy destroys both Type A sper-
Radical orchiectomy remains the gold standard matogonia in addition to Type B spermatogonia,
for surgical treatment of testicular germ cell car- permanent azoospermia results. The time to
cinoma, followed by RPLND or chemotherapy or impairment of spermatogenesis after initiation of
radiotherapy, as indicated by tumor histopathol- chemotherapy can be a few weeks, but recovery
ogy. Radical orchiectomy for testicular cancer is of spermatogenesis after cessation of therapy can
associated with a decrease in postoperative sperm take a few years [16].
concentrations. Petersen et al. [12] noted a Maymon et al. [28] have argued that Sertoli-
decrease from 20 million/mL before orchiectomy cell damage may be a contributory factor to che-
to 10 million/mL after orchiectomy in a group of motherapy-induced azoospermia, in addition to
29 patients. This observation is in agreement with germ cell damage. By staining testicular biopsy
other investigators who found azoospermia in specimens for specific cell-differentiation fac-
10–56% of men after orchiectomy [26]. Fertility tors, the authors found a mixed histologic pattern
is nevertheless possible in men after orchiectomy. consistent with dedifferentiation of Sertoli cells
Jacobsen et al. [16] compared fertility in in a patient who had undergone chemotherapy for
63 patients with bilateral and 174 patients with testicular carcinoma [28]. Because Sertoli cells
unilateral testicular cancer and found that 74% of are involved in maintaining the blood–testis bar-
men with unilateral disease and 40% patients rier, damage to even a fraction of Sertoli cells in
with bilateral disease were able to achieve pater- a given tubule can have a disproportionately
nity after their first orchiectomy, with or without larger impact on spermatogenesis.
the use of assisted-reproductive techniques. The testes are highly sensitive to alkylating
The most common complications of RPLND agents. Chemotherapy regimens that include
are loss of ejaculation and retrograde ejaculation, alkylating agents are more gonadotoxic than
Table 10.1 Classes of chemotherapeutic agents and risk vinblastine, and dacarbazine) cause transient
for impairment of spermatogenesis azoospermia in a third of patients, who usually
Group Drug names Risk go on to make a full recovery [29]. For this very
Alkylating agents reason, chemotherapy cocktails used in the treat-
Nitrogen mustards mechlorethamine High ment of non-Hodgkin’s lymphoma are much less
chlorambucil High gonadotoxic than those used for Hodgkin’s lym-
cyclyphosphamide High
phoma [30].
melphalan High
ifosfamide High A large study by the European Organization
Nitrosureas carmustine Medium for Research and Treatment of Cancer (EORTC)
lomustine Medium analyzed FSH levels as a measure of fertility in
Others busulphan High men treated with chemotherapy or nonpelvic
Antimetabolites radiotherapy [26]. FSH was elevated in 60% of
Folate antagonists methotrexate Low patients treated with alkylating chemotherapy,
Pyrimidine analogs cytarabine Medium compared to 8% of patients treated with nonal-
fluorouracil Low
kylating chemotherapy and 3% of patients treated
gemcitabine Low
with radiotherapy. Recovery of spermatogenesis
Purine analogs mercaptopurine Low
thioguanine Low is, however, often associated with a normaliza-
Cytotoxic antibiotics tion of FSH level. Patients treated with alkylat-
Anthracyclines doxorubicin Medium ing agents were less likely to return to normal
Others bleomycin Low FSH levels than those treated with nonalkylating
dactinomycin Low agents. In addition, recovery, if it did occur, took
Plant alkaloids considerably longer in patients treated with alky-
Vinca alkaloids vincristine Low lating vs. nonalkylating chemotherapy and was
vinblastine Low
dependent on the dose of alkylating agents
Topoisomerase etoposide Low
inhibitors received.
Others Standard treatment for testicular cancer con-
Platinum compounds cisplatin Medium sists of platinum-based combination chemother-
carboplatin Medium apy including bleomycin, etoposide, and cisplatin
Miscellaneous procarbazine High (BEP). The cytotoxic effects of cisplatin are
dacarbazine High mediated by induction of DNA cross-linking,
MOPP nitrogen mustard, High while etoposide causes toxicity due to its interac-
vincristine,
procarbazine, tion with Topoisomerase II. Shortly after initia-
and prednisone tion of chemotherapy, semen analysis demon-
BEP bleomycin, Medium strates oligospermia, decreased sperm motility,
etoposide, decreased chromatin integrity, and increased
and cisplatin abnormal morphology [31]. Not surprisingly, the
ABVD adriamycin, Medium
gonadotoxicity of the BEP regimen for testicular
bleomycin,
vinblastine, cancer is thought to be less than that of regimens
and dacarbazine that include alkylating agents. While individual
responses may be quite variable following che-
motherapy for testis cancer, spermatogenesis, if it
recovers, will do so in the majority of patients
those that do not contain alkylating agents. After within 4 years.
six cycles of MOPP (nitrogen-mustard, vincris- The risk of permanent azoospermia after che-
tine, procarbazine, and prednisone) or a compa- motherapy is drug-specific and dose-dependent.
rable regimen, 90–100% of patients experience Pont and Albrecht concluded in their review that
prolonged azoospermia. In sharp contrast, regi- cumulative doses of >400 mg/m2 of Cisplatin
mens like ABVD (adriamycin, bleomycin, given to testicular cancer patients led to irreversible
impairment of gonadal function [30]. Similarly, Several studies have confirmed that subdia-
gonadal dysfunction is well documented in over phragmatic irradiation can cause profound, dose-
80% of patients who receive >300 mg/m2 of dependent impairment of spermatogenesis due to
cyclophosphamide [30]. radiation reaching the testis. The effect is due to
scatter, more so than direct irradiation of the tes-
tes, and the use of a gonadal shield have been
10.4.3 Radiation Therapy shown to be beneficial even in cases of radiother-
apy focused on, and limited to, the para-aortic
The testis is one of the most radiosensitive tis- region [35]. In the absence of a testicular shield,
sues in the body. Radiotherapy produces lethal the median testicular dose received has been cal-
cell damage to most tissues, including the male culated to be as high as 1.7 Gy [36].
gonad, by inducing double-strand DNA breaks Radiotherapy has a much more deleterious
not amenable to repair. Like chemotherapy- effect on fertility than chemotherapy. In a large,
induced damage, radiotherapy-induced damage retrospective study of 451 patients with testicu-
is dose-dependent; the speed of onset of this lar germ cell tumors, patients were divided into
damage, chance of reversal, and time to recov- two groups, based on whether they were treated
ery of spermatogenesis are all related to the tes- with orchiectomy and chemotherapy, or orchiec-
ticular dose of irradiation [30]. Spermatogonia tomy and radiotherapy [37]. Only two thirds of
can be affected by doses as small as 0.15 Gy, the couples who attempted a pregnancy after
although spermatogenesis has been shown to treatment for testicular cancer were successful,
recover at doses of up to 1–3 Gy. Permanent echoing the data from other studies that have
azoospermia can be seen at doses exceeding shown a 30% decrease in fertility in testicular
4 Gy [32]. It is possible that men with germ cell cancer patients compared to the general popula-
neoplasia might be more vulnerable to the tion [37–39]. With respect to specific treatment
effects of irradiation because spermatogenic modalities, the authors found that cumulative
function in these individuals is already abnor- conception rates were much lower for patients
mal before irradiation. treated with radiotherapy compared to those
In contrast, Leydig cells are more resistant to treated with chemotherapy [37]. The recovery of
radiation, and doses exceeding 20 Gy are usually sperm production following radiotherapy can
needed to produce hypogonadism [8]. Graded take up to 60–80 weeks after a dose of 0.32 Gy
doses of radiation therapy (14–20 Gy) in patients [38], but has been reported to take up to 9 years
with testicular germ cell neoplasia have been in some cases [36]. However, most patients can
linked to a gradual and continued decline in tes- expect spermatogenic recovery within 2 years
tosterone levels at a rate of approximately 3.6% of radiation.
per year [33]. In one study, this decline lasted
over 5 years, irrespective of the treatment, and
40% of the patients required androgen supple- 10.4.4 Multimodal Therapy
mentation [33].
Men with testicular germ cell cancer are Over the last two decades, BMT has become an
treated with radiotherapy given in fractionated important treatment modality for hematological
doses, which is known to be more gonadotoxic malignancies, and a combination of high-dose
than the bioequivalent dose given in one single chemotherapy and TBI is frequently used in pre-
sitting. In men with CIS in the contralateral testis, parative regimens for BMT. The primary aims of
treatment with fractionated radiotherapy (20 Gy the preparative regimens are myeloablation in
given in ten fractions of 2 Gy) led to eradication order to enable grafting of donor bone marrow
not only of the CIS, but also of germ cells [34]. and the eradication of malignant cells that might
Sertoli and Leydig cells were preserved in this have survived previous therapy [40]. Bakker et al.
patient population. investigated pubertal development in 21 prepubertal
boys treated with TBI and BMT. The majority of to some extent can be seen months to years after
boys had elevated serum LH and FSH, with con- end of treatment. However, sperm quality may be
comitant low testicular volumes, suggestive reduced compared with pretreatment levels, as
of severe impairment of reproductive gonadal reported in the case of testicular cancer and
function [40]. A second group of authors has hematological malignancies [43, 44]. In a large
examined semen parameters in BMT patients French study of 451 testicular cancer survivors,
conditioned with a single alkylating agent, two the fertility rate was found to be 30% lower after
alkylating agents, or an alkylating agent plus irra- cancer treatment, but was nevertheless significant
diation. Recovery of spermatogenesis was best in at 67% [37]. Other authors have reported similar
patients receiving single or dual-agent chemo- fertility rates of 71–82%, with a small proportion
therapy (90 and 50%, respectively), followed by of these pregnancies achieved using assisted-
patients receiving chemotherapy and irradiation reproductive techniques [32]. Brydoy et al. have
(17%) [41]. Patients treated with a single alkylat- reported a 15-year actuarial posttreatment pater-
ing agent also had better sperm quality and faster nity rate of 71% among survivors of unilateral
recovery of spermatogenesis compared to the testicular cancer, without the use of cryopre-
other two groups of patients. In patients receiving served semen [44]. Not surprisingly, patients with
a combination of chemotherapy and radiation, the highest intensity of treatment had the longest
recovery of spermatogenesis was delayed as time to conception.
much as 9 years after cessation of therapy in Among survivors of lymphoma, posttreatment
some cases [41]. fertility rates of 49–71% have been reported,
Recently, nonmyeloablative stem cell trans- depending on the treatment modality and chemo-
plantation has been introduced, which employs therapy cocktail used [26]. For Hodgkin’s lym-
conditioning regimens of reduced intensity, in phoma patients, use of the ABVD regimen has
an effort to reduce gonadotoxicity. Kyriacou been associated with a lower incidence of gonad-
et al. have compared the impact of myeloabla- otoxicity compared to the MOPP regimen, with
tive and nonmyeloablative regimens on the germ 90% of patients recovering normal sperm counts
cell and Leydig cell compartments in 32 men. 12 months after cessation of therapy [8].
Study results showed that the nonmyeloablative Posttreatment fertility is lowest among leuke-
regimen was gonadotoxic and affected both the mia survivors, especially those who undergo che-
germ cell and Leydig cell compartments, but motherapy in combination with TBI. This
damage to the germ cell compartment was more regimen has been shown to result in permanent
severe with myeloablative regimens that azoospermia in 83% of patients, severe oli-
included TBI [42]. gospermia in the remainder, with spontaneous
Even when multimodal therapy is used, the conceptions rare [41].
irradiation component of therapy appears to have Although conception is unlikely while patients
impact of a greater magnitude and longer dura- are on cytotoxic therapy, contraception is recom-
tion on the spermatogenesis cycle. Limiting the mended for at least 6 months following therapy,
radiation dose in multimodal therapy may given the theoretical risk of teratogenic effects of
improve posttreatment gonadal function in these treatment. Tempest et al. used fluorescence in situ
patients. hybridization analysis to evaluate aneuploidy in
sperm DNA from testicular cancer and Hodgkin’s
lymphoma patients and found increased aneu-
10.5 Fertility Following Cancer ploidy frequency prior to, and up to 2 months
Treatment from the start of, chemotherapy [45]. They there-
fore recommended delayed attempts at concep-
Although cancer treatment can induce azoo- tion 2 years after the end of treatment. Most
spermia in many men, fortunately, this effect is authors recommend a waiting period between
usually temporary and recovery of spermatogenesis 6 and 24 months.
only means of ensuring semen availability for

10.6 Fertility Preservation future use, and studies have documented the use
of cryopreserved sperm with good result up to
Any discussion of fertility in cancer survivors
15 years after initial preservation [27].
must involve both biologic and psychosocial con-
In order to compensate as best as possible for
siderations. Survivors may be less likely to find a
tumor-induced impairment of spermatogenesis,
partner, or less inclined to want children because of
Schrader et al. have described the use of testicu-
the psychological effects of the disease or treat-
lar sperm extraction (TESE) prior to the onset of
ment, or because of fear of an increased risk of con-
cancer therapy [48]. The authors noted success-
genital malformation in the offspring of parents
ful sperm retrieval in 8 of 17 azoospermic
who have received cytotoxic therapy. Nevertheless,
patients (47%), with no delay in initiating can-
a recent report by Schover et al. revealed that 51%
cer treatment. Two patients chose to use the
of men with cancer expressed their wish to preserve
cryopreserved sperm; there was one successful
their capacity for procreation in the future, includ-
pregnancy.
ing 77% of men who were still childless when their
Down-regulation of spermatogenesis prior to
cancer was diagnosed [46]. Strategies of fertility
the initiation of cytotoxic therapy has been pro-
preservation can be broadly divided into two cate-
posed as a means of preventing the deleterious
gories: pre- and postcytotoxic therapy. Semen cry-
effects of cancer therapies on sperm production.
opreservation prior to initiation of cancer therapy
However, given the length of the spermatogen-
remains the cornerstone for fertility preservation in
esis cycle in humans, hormonal inhibition may
the majority of patients. Recent research has
require several weeks to completely inhibit
focused on methods of improving posttreatment
sperm production, thereby delaying cancer ther-
fertility. However, the alternatives to semen cryo-
apy for weeks as well. Therefore, such a strat-
preservation, such as testicular stem cell transplan-
egy, although logical, may not be technically
tation or testicular tissue transplantation, are
feasible [27].
experimental at present.
10.6.1 Pretreatment Options 10.6.2 Posttreatment Options
Patient counseling and access to sperm banking Men who remain azoospermic long after under-
should be a standard component of patient educa- going chemotherapy have traditionally been con-
tion and decision-making before the initiation of sidered sterile. However, sperm retrieval in these
cytotoxic cancer therapy. Yet, a significant per- patients is possible using microdissection TESE.
centage of patients do not bank sperm before Even in men previously treated for testicular
undergoing treatment. Based in part on the type germ cell tumors, despite the presence of a soli-
of malignancy, the treatment modality, the avail- tary testis, the sperm retrieval rate with TESE is
ability of resources, the urgency to treat, and the 67% [49]. Chan et al. reported on the success rate
knowledge of the oncologist regarding reproduc- of TESE followed by ICSI in a cohort of
tive technology, the options of sperm cryopreser- 17 patients. Sperm retrieval was accomplished in
vation are not always discussed with patients. 45% of the patients, with biochemical pregnancy
Furthermore, in patients who are prepubertal or in four couples, clinical pregnancy in three cou-
young adolescents, obtaining mature sperm for ples, and live deliveries in two couples [50]. In a
cryopreservation is not always possible or appro- separate study, Mesegue et al. showed that sperm
priate [47]. Even patients who chose cryopreser- recovery rates were higher in patients with tes-
vation are not always able to pursue it, if ticular cancer than with hematological malignan-
spermatogenesis is sufficiently impaired due to cies (67% vs. 20%), and that sperm were more
the underlying malignancy. Nevertheless, pre- often retrieved from patients treated with chemo-
treatment sperm cryopreservation remains the therapy alone or with RPLND, compared to those
receiving chemotherapy and radiation therapy Although considerable research effort is being
[51]. From a histopathological point of view, directed to evaluate new options for fertility
sperm were retrieved from patients with Sertoli restoration in cancer patients, the majority of
cell-only syndrome, severe hypospermatogene- these options are not currently tangible or repro-
sis, and maturation arrest [51]. Data from other ducible. Use of ART still remains the leading
studies report similar success rates [52]. For option for interested patients.
patients suffering from persistent azoospermia
after cytotoxic cancer therapy, the combination
of TESE and ICSI has certainly introduced new 10.6.3 Ethical Considerations
opportunities for fertility.
Spermatogenesis restoration by hormone While the availability of assisted-reproductive
treatment after cytotoxic therapy has been stud- techniques has enabled some cancer patients with
ied in animal models using GnRH agonists. The azoospermia to father children, concerns have
resulting suppression of FSH and testosterone been raised about the safety of IVF and ICSI in
secretion has been shown to remove the block patients who have received chemotherapy or
in spermatogenesis [53]. However, only one of radiation therapy. DNA damage induced by cyto-
seven trials using hormone suppression to pre- toxic cancer therapies may result in genetic errors
vent gonadotoxicity in humans showed a positive that cannot be repaired in late-stage spermatids
result. The value of application of this treatment and spermatocytes. These germ cell mutations
is currently uncertain. might increase the risk of congenital malforma-
Germ cell transplantation is being considered tions, growth disturbances, and even cancer in the
as a new tool for fertility preservation in cancer offspring of cancer patients.
patients [54] and has been applied to restore sper- DNA and chromosomal aberrations that can
matogenesis in mice. However, when the tech- be transmitted by sperm include aneuploidy,
nique was applied in primates, no recovery of structural aberrations, epigenetic modifications,
sperm counts was noted [55]. Although promis- nucleotide repeats, and gene mutations. Contro
ing in theory, at present, germ cell transplantation versy exists as to the degree and reversibility of
remains very much an experimental technique in DNA damage secondary to chemotherapy and
humans. radiation therapy. Unfortunately, most studies
In the same vein, testicular tissue allograft has addressing this topic are very limited in size.
been proposed as a treatment to restore fertility if In testicular cancer patients treated with BEP
no recovery of spermatogenesis can be estab- chemotherapy, an abnormally high percentage of
lished after cessation of cytotoxic therapy. The DNA-damaged sperm has been found by Spermon
technique has been successfully performed in et al. [57]. The same chemotherapy regimen has
animal models [54], but human studies to date are also been linked with altered chromatin quality in
lacking. One concern with this approach has been sperm from a mouse model [31]. Similarly,
the possibility, albeit small, of reintroducing increased rates of human sperm aneuploidy have
malignant cells via the transplanted testicular been reported for up to 18–24 months following
issue. For example, as few as five reintroduced initiation of chemotherapy [58]. On the contrary,
cells are sufficient for leukemia to recur [8]. Thomson et al. have shown that, despite a reduc-
In vitro culture and maturation of spermatogo- tion in sperm concentration, the sperm produced
nial cells in specific stages of development have by cancer patients carries as much healthy DNA
also been described. Tesarik et al. previously cul- as the sperm produced by healthy, age-matched
tured primary spermatocytes in premeiotic arrest controls [59]. Other authors have reported
to produce morphologically abnormal but devel- chromosomal aneuploidies after alternative
opmentally competent gametes, which were then chemotherapy regimens, but these anomalies
used for successful micromanipulation-assisted have been transient and self-limited [60]. Most
fertilization [56]. recently, Thomas et al. have reiterated the absence
of long-term effects of chemotherapy or radiation to the onset of cytotoxic therapy. Fertility rates in
therapy on sperm aneuploidy rates, based on a these patients have already improved with the use
cohort of 38 patients [61]. Based on current evi- of TESE and ICSI. Based on current evidence,
dence, recommending a delay from the cessation there appears to be no increased biologic or
of treatment to attempt pregnancy of 18–24 genetic risk to offspring of cancer survivors. With
months is prudent. ongoing research, a better understanding of fertility
To date, epidemiologic studies have not shown preservation in these men is expected.
an increased risk of congenital anomalies, genetic
diseases, or chromosomal abnormalities in the
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Environmental Insults
on Spermatogenesis 11
Stefan S. du Plessis and Ashok Agarwal
Abstract
The frequency of defective spermatogenesis and accompanying decreases
in sperm parameters such as sperm count and motility appears to be on the
increase. Arguably one of the most compelling reasons for this phenome-
non is the influence of environmental factors on male reproduction. Despite
spermatogenesis being a function of only the mature testis, environmental
insults during maternal, perinatal, and prepubertal phases can indirectly
influence eventual sperm production in the adult male. It is believed that
exposure during these phases of the developing testis leads to irreversible
effects on spermatogenesis, while the accompanying effects of adulthood
exposure are in all probability reversible.
This chapter explores the various environmental factors that can influ-
ence spermatogenesis, both directly and indirectly (i.e., exposure during
all the stages from the developing fetus all the way up to and in the adult
male). In this overview, not only are the effects of environmental chemi-
cals and toxins discussed, but the focus is also on several lifestyle factors
and occupational exposure that can impinge on the process of sperm pro-
duction. Furthermore, the role of epigenetic defects that can result in
defects in transgenerational inheritance due to environmental insults is
also investigated briefly.
Despite the lack of conclusive studies, it is evident from this overview
that there are enough compelling reasons to believe that the future of male
gamete production may be actively affected by the environment.
Keywords
Spermatogenesis • Environmental • Chemical • Lifestyle factors
• Epigenetics • Oxidative stress • Endocrine disrupting • Maternal expo-
sure • Testis
A. Agarwal ()
Center for Reproductive Medicine, Cleveland Clinic,
Cleveland, Ohio, USA

134 S.S. du Plessis and A. Agarwal
variety of forms [11]. Changes in male reproduc-

11.1 Introduction tive neuroendocrine function in response to envi-
ronmental challenges have also been implicated to
Production of gametes in the male occurs as a
affect reproductive function and spermatogenesis.
process known as spermatogenesis and occurs in
This phenomenon is more evolutionary by nature
the testes. This stepwise progression from diploid
and does not have acute effects on sperm produc-
spermatogonia to ultimately turn into haploid
tion [12].
spermatozoa is a truly remarkable process that
The disruption of spermatogenesis seems to
requires delicate control and takes approximately
involve two fundamentally different routes of
64 days to complete in humans. Spermatogenesis
exposure. As spermatogenesis only occurs in the
is a highly synchronized event that involves
mature testis, environmental insults can directly
stage- and testis-specific gene expression, mitotic
affect male germ cells throughout adulthood. The
and meiotic division, and histone-protamine sub-
second route by which environmental insults
stitution [1]. Sperm production starts at puberty
exert an influence on spermatogenesis is less
and continues uninterrupted until death, unlike
direct. It can act on events preceding gamete pro-
oogenesis in females. Due to the complexity of
duction through exposure of women during preg-
germ cell development, spermatogenesis is
nancy and subsequent disruption of testicular
dependent on optimal conditions. It is a critical
development in the male fetus as well as during
period during which many things can go wrong,
infancy [11, 13].
making spermatogenesis inherently more suscep-
The evidence for and against environmental
tible to disruption by external factors [2].
insults as key factors in decreased spermatogen-
In recent decades, couple infertility has been
esis and male infertility is not very clear-cut, but
on the increase with male factor infertility identi-
nonetheless evidence indicates that this is a seri-
fied as one of the most common causes thereof.
ous health issue requiring closer scrutiny [11].
It is estimated that paternal factors contribute
This chapter will therefore aim to highlight and
30–50% towards all infertility cases [1]. Male
address various environmental insults which can
reproductive health and semen quality seem to be
directly impede the process of spermatogenesis
on the decline with Toppari et al. [3] reporting that
in the mature testis, while it will also briefly refer
mean sperm counts in the general population has
to and discuss any preceding events influencing
decreased by 50% in the past 50 years [4–8]. The
spermatogenesis indirectly. For the purpose of
incidence of testicular cancer and cryptorchidism
this review, both lifestyle and occupational fac-
increased in parallel to defective spermatogenesis.
tors will be included under the umbrella term of
This decline in male fertility is proposed to be the
environmental factors.
result of a variety of environmental factors that
impact both directly and indirectly the process of
spermatogenesis [9, 10]. The key motivation for
this argument is that significant lifestyle and envi-
11.2 Maternal and Perinatal
ronmental changes have occurred during this same Exposure to Environmental
period of time. This is substantiated by the rapid Insults Affecting Future
expansion of the chemicals industry in both the Spermatogenesis
developed and developing worlds during the latter in the Offspring
part of the twentieth century which has resulted in
the release of a plethora of toxins and xenobiotics Spermatogenesis occurs in the male gonads only
into the environment [11]. These foreign mole- during adulthood. All the cells and structures
cules, including pesticides, herbicides, cosmetics, involved in this process differentiate during fetal
preservatives, cleaning materials, municipal and development. Initially, the embryo has bipotential
private waste, pharmaceuticals, and industrial by- structures that can develop into male or female
products have worked their way into our lives in a gonads. The medullary region of this sexually
11 Environmental Insults on Spermatogenesis 135
indifferent ridge starts to differentiate into the delimit the final number of these cells and impact
testes, with the Sertoli cells being the first recog- sperm count. The testicular dysgenesis syndrome
nizable testicular cell type. During this process, (TDS) hypothesis furthermore suggests that these
the Sertoli cells enfold the germ cells to form and other disorders such as cryptochidism, hypos-
seminiferous tubules, while the mesenchymal padias, and testicular germ cell cancer have a
cells differentiate into Leydig cells in the intersti- universal fetal precursor triggered by malforma-
tial spaces. tion of the testis [33].
Spermatogonia and Sertoli cells are crucial to There is very little concrete evidence to prove
allow normal spermatogenesis during adulthood. that a relationship exists between maternal expo-
It is important that germ cells multiply and dif- sure to environmental insults and impaired sper-
ferentiate normally during fetal development to matogenesis in humans. One of the problems
ensure a sufficient pool of spermatogonia [14]. faced with such studies is the long periods of time
This involves proliferation of the fetal germ cells, that may have elapsed between maternal expo-
before loss of their pluripotency. They subse- sure and the appearance of a condition such as
quently become quiescent for a period of time offspring infertility [11]. However, results from
before migrating to the basal lamina of the semi- many animal studies are reason enough to sup-
niferous tubules [15]. In humans, the latter phase port the theory that exposure to environmental
might only occur and be finalized just prior to the insults during development can determine sper-
onset of puberty [16]. matogenesis and fertility in adulthood [2].
Sertoli cell number and function play a major In utero exposure of the male fetus to maternal
role in regulation of spermatogenesis and altering lifestyle factors and environmental insults can be
rates of sperm production in the adult testis [17, 18]. an important determinant of its future fertility pro-
It is well recognized that the number of Sertoli file. The result of these insults on male testicular
cells is related to the level of spermatogenesis as development and adult spermatogenesis is likely
measured by daily sperm production per testis to be irreversible [2]. This is evidenced by a study
because each Sertoli cell can only support a finite of Mocarelli et al. [34] stating that dioxin expo-
number of developing spermatozoa [19, 20]. sure, from infancy through puberty, affects semen
Functions of Sertoli cells include providing nutri- quality while adult exposure showed no effect.
tion and structural support to developing germ From the aforementioned information, it is
cells, phagocytosis of degenerating germ cells, clear that hormones, specifically testosterone, are
production of protein hormones (e.g., inhibin), important role players in promoting testicular dif-
and finally release of the spermatids [17, 21–28]. ferentiation. Maternal lifestyle or exposure to any
According to Sharpe et al. [29], increase in Sertoli environmental chemicals with endocrine-disrupting
cell numbers occurs during fetal development, properties, especially antiandrogenic activity, can
immediately postnatal and again near puberty. therefore impinge on testicular development and
Testosterone secreted by the newly developed spermatogenesis in the adult testis. Maternal
Leydig cells appear to regulate the first two phases smoking and obesity have both been implicated
of proliferation [30], while follicle-stimulating in causing reduced sperm counts in developing
hormone (FSH) is more likely involved in the male offspring. A significant decrease in total
prepubertal phase [29, 31, 32]. sperm count was reported in male offspring
Therefore, any exposure of these developing whose mothers smoked substantially during preg-
structures to environmental insults, while the nancy [35–38]. In all probability it was the effect
basis for sperm production is set in place, can of polycyclic aromatic hydrocarbons (PAHs) or
impact indirectly spermatogenesis during adult- other components in the cigarette smoke which
hood (Fig. 11.1). Sharpe [2] advocates that expo- activate the aryl hydrocarbon (Ah) receptor and
sure during any of the three Sertoli cell antagonize the androgen receptor (AR)-mediated
proliferation periods, as well as during the first action. Ultimately, this manifests as reduced
and last phases of germ cell differentiation, could Sertoli cell numbers [2, 39, 40].
Fig. 11.1 Both maternal and adult exposure to environmental insults together with epigenetics can have an influence
on spermatogenesis in the mature testis
On the other hand, maternal obesity can theo- reduced spermatogenesis in the mature testis of
retically encroach on testicular development via their sons [42].
increased aromatization, thereby disrupting the Xenobiotics (environmental estrogens) can
testosterone: estrogen ratio in the developing have long-term impacts on male fertility as they
fetus. Various organochlorines, including many are associated with reduced sperm counts. One
herbicides/pesticides and polychlorinated biphe- explanation involves the capacity of these envi-
nyls (PCBs), are lipophilic and accumulate in fat ronmental estrogens to suppress production of
of obese expectant mothers. During pregnancy FSH by the fetal pituitary gland, consequently
and lactation, these accumulated compounds can leading to decreased Sertoli cells [43]. Alter
be delivered to the fetus and neonate [41]. natively, the xenobiotics impair Leydig cell
Anabolic steroids present in meat consumed by development or function, thereby affecting tes-
expectant mothers have also been linked to tosterone production and germ cell differentiation
Table 11.1 A list of the most common environmental in all boys wearing plastic-lined nappies. They
chemicals, including xenobiotics, which can possibly subsequently hypothesized that increased scrotal
impact on future spermatogenesis in the offspring due to
maternal exposure temperatures in boys, as a result of disposable
diaper use, could be an important contributor
Environmental chemicals References
towards a decline in sperm production observed in
Polychlorinated biphenyl (PCB) [64]
(used as coolants and dielectric fluids recent years [3].
in transformers)
Dioxin (by-product of combustion) [34]
Polycyclic aromatic hydrocarbon (PAH) [2]
11.3 Environmental
(constituent of exhaust fumes, smoke, Insults Impinging
and cooking processes) on Spermatogenesis
Diesel exhaust fumes (mixture of [238]
particulate and gas phase pollutants)
in the Mature Testis
Polybrominated compounds [239]
(used as flame retardants) Males have the capacity to reproduce continu-
Phthalates (present in cosmetics, [240] ously, unless inhibited by environmental factors.
toiletries, and medications) On average, men produce around 200 million
Organochlorine compounds (pesticides [241] sperm cells daily, which would have taken
such as dichlorodiphenyltrichloroethane
(DDT); 1,2-dibromo-3-Chloropropane)
between 9 and 10 weeks to pass through the vari-
Nonylphenol (industrial surfactant) [61, 234] ous stages of development. To allow for continual
Genistein (plant-derived phytoestrogens [61, 242] sperm production throughout adult life, a sub-
present in, e.g., soybeans) population of primordial germ cells remains as
Bisphenol A (BPA) (used to improve [52, 243] part of the stem cell pool at the outer edge of the
polycarbonate plastics) seminiferous tubule during mitotic division. One
Arsenic (used as pesticides, herbicides, [226] of the daughter spermatogonia moves towards
insecticides, and in various alloys)
the lumen to continue the process of spermato-
Vinclozolin (common fungicide used [237]
in vineyards) genesis. Spermatogonia undergo a finite number
of mitotic divisions to form primary spermato-
cytes which enter the first meiotic division. The
accordingly. The action of androgen receptors second meiotic division of secondary spermato-
may also be inhibited by these environmental cytes results in the production of haploid sperma-
estrogens within the fetal testes. Another possible tids. Following meiosis II is the developmental
mechanism is that some of these environmental phase known as spermiogenesis. This involves
estrogens are metabolized and converted to qui- the differentiation and transformation of round
nones. These molecules can cause cellular dam- spermatids into elongated spermatozoa. Sperma
age by either generating reactive oxygen species tids undergo restructuring of the chromatin archi-
(ROS) or by binding to DNA [43]. A list of envi- tecture, replacing the majority of histones first by
ronmental chemicals, including xenobiotics, transitional proteins and then protamines which
which can possibly impact spermatogenesis due are required for nuclear compaction. Thereafter,
to maternal exposure is given in Table 11.1. the spermatozoa are released into the lumen of
A marked change in the testicular thermal envi- the seminiferous tubules during spermiation and
ronment postnatally is an additional factor that can then enter the epididymis [46]. Transit
might influence the outcome of adult spermato- through the epididymis takes about 12 days as
genesis [44]. Partsch et al. [45] reported the effect spermatozoa complete their final developmental
of reusable cotton vs. plastic-lined disposable dia- stages. Spermatogenesis is under hypothalamic-
pers on scrotal temperature during infancy and pituitary-gonadal control and appears to require
early childhood. Significant lower recto-scrotal FSH, luteinizing hormone (LH), and testosterone,
temperature differences, as well as higher mean with FSH acting as the primary agent responsible
24-h scrotal skin temperatures, were observed for stimulating sperm maturation.
Evaluation of the results of environmental compounds affect LH-stimulated Leydig cell

insults on spermatogenesis in human sperm stud- function, which in turn influence androgen secre-
ies is hampered by inconsistencies in biological tion and interrupt the endocrine regulation of
analytical methods, in controlling for confound- spermatogenesis. Not only can endocrine disrup-
ing factors, and in weakness of study design [47]. tors affect the testosterone:estradiol ratio and
It is also recognized that documentation of cause their respective roles in the feedback and regula-
and effect in human studies is exceedingly more tion of the hypothalamus-pituitary-gonadal axis,
difficult than in laboratory animals because of but it can also disturb the prooxidant/antioxidant
ease of intervention and measurement in animal system of the cells. This can lead to the genera-
studies, as well as the shorter gestation and devel- tion of free radicals and ROS. These free radicals
opment in animal studies [2]. Furthermore, ani- are harmful to cells once they destabilize the elec-
mals vary less with regard to their spermatogenesis trolytic balance within the cells. As spermatozoa
profile than humans [48]. Sharpe also emphasizes contain a large amount of polyunsaturated fatty
that, in reality, males are constantly exposed to a acids (PUFA’s) in their membranes, they are more
mixture of environmental chemicals that may lead susceptible to ROS attack and lipid peroxidation.
to additive or interactive effects on spermatogen-
esis. This may confound interpretation of studies 11.3.1.1 Chemicals Produced
in which only one factor is evaluated [2]. Even so, in the Plastics Industry
there is reason to suggest that human spermato- Plasticizers are polyphenolic chemical additives
genesis in the mature testis is affected by environ- used to enhance the flexibility and toughness of
mental factors during adulthood as it is becoming plastic. They are found in all clear, heat-resistant,
increasingly apparent from analysis of the bio- and unbreakable plastics. A few of these com-
logical fallout from environmental pollution that a pounds are reportedly toxic to the male reproduc-
major target of this chemical barrage is the repro- tive system and in specific spermatogenesis.
ductive system, particularly in the male [11, 47]. Bisphenol A (BPA) is used to improve polycar-
Furthermore, dramatic changes in Western life- bonate plastics and can be found in most dispos-
style, diet, and exercise suggest that one or more able plasticware, especially in the coverings of
of these factors appear to be involved in the etiol- food containers (e.g., milk, water, and infant bot-
ogy of declining male fertility and the impairment tles), as well as in dental materials. Potential
of sperm production. In addition, spermatogenesis human exposure occurs due to migration of BPA
in normal men is badly organized and extremely from food containers into food or migration from
inefficient, making men highly susceptible to dental sealants and composites (fillings), ulti-
these environmental and lifestyle insults [2]. mately ending up in the systemic circulation
The remainder of this section will highlight [49, 50]. It is estimated that 90% of Americans
and deal with a few environmental chemicals, have BPA present in their blood. Due to it being so
lifestyle factors, and occupational exposures dur- common, BPA is one of the more harmful known
ing adulthood that can contribute to impaired chemicals that reduce sperm count, motility, and
spermatogenesis (Fig. 11.1). viability in a time- and dose-dependant manner.
Chitra et al. [51] reported that BPA generates ROS
in various rat tissues including the reproductive
11.3.1 Environmental Chemicals, organs. BPA was shown to increase the levels of
Toxins, and Endocrine Active H2O2 and TBARS (thiobarbituric acid reactive
Compounds substance) in rat testicular tissue. This subse-
quently leads to the depletion of the antioxidant
Many environmental chemicals contribute to defense system. Kabuto also reported that BPA
increased toxic load and have the potential to dis- administration induces overproduction of H2O2 in
rupt fertility. Some have estrogenic properties the kidneys, liver, and testes of rats [52, 53].
and are toxic because they affect the endocrine Phthalate esters are also used in the plastics
system (endocrine disruptors). Some of these industry to provide flexibility and resilience to
various plastic products and commonly occur in used as an additive in paint and petroleum, leading
many consumer products such as plastic bags, to widespread exposure via fumes. However, due
inflatable recreational toys, blood storage bags, to its nature it can accumulate in fish, providing
plastic clothing, soaps, and shampoo. One of the for an alternative source and form of exposure
most commonly used and best studied phthalate [70]. Lead has an inhibitory effect on the delta
esters in the plastics industry is Di(2-ethyhexyl) amino levulanic acid (ALA) synthase enzyme.
phthalate (DEHP) [54–57]. Animal studies pro- The subsequent accumulation of ALA causes
vided clear evidence of reproductive toxicity due ROS generation and peroxidation of PUFA’s in
to DEHP; however, human studies lack to provide plasma membranes. Spermatozoa are more sus-
similar effects. DEHP and its most toxic metabo- ceptible to lead-induced oxidative stress due to
lite mono (2-ethylhexyl) phthalate (MEHP) were the high content of PUFA’s in their plasma mem-
shown to induce testicular atrophy and impinge brane. Another target of lead is the enzymatic
on spermatogenesis in laboratory animals. Admin antioxidant systems of the cell [65]. Lead has
istration of phthalate esters to rats increased ROS also been shown to hinder the activity of superox-
generation within the testis with a simultaneous ide dismutase (SOD), catalase, and glutathione
decrease in antioxidant levels, culminating in peroxidase by inhibiting the functional sulfhydril
impaired spermatogenesis [58]. DEHP leads to group of these enzymes [65, 71].
the depletion of zinc in the testis that can cause the The toxic effect of Cadmium on the reproduc-
induction of oxidative stress and germ cell apop- tive system is well established. It has been shown
tosis. This is possibly the mechanism via which to be present at significantly higher levels in both
it can exert its negative effect on reproductive the seminal plasma and blood of infertile men
function [59, 60]. It is also speculated that germ when compared to that of the normal population
cell apoptosis can be the result of redox-mediated [72], while a negative correlation exists between
activation of nuclear factor-kB and up-regulation cadmium and sperm concentrations. First, it has
of Fas ligand on Sertoli cells by MEHP [59]. been shown to have antisteroidogenic effects.
Nonylphenol, another synthetic plastic addi- It lowers testosterone production due to reducing
tive, has estrogenic properties and can accumu- the expression of steroidogenic acute regulatory
late in tissues due to its lipophylic nature. It is protein in the adult rat testes [73]. Yang et al. [74]
commonly used in detergents, paints, personal reported that cadmium has direct toxic effects on
care products, food processing, and the packag- the Leydig cells and thereby on testosterone levels.
ing industry. Adult exposure to nonylphenol has Furthermore, cadmium is regarded as a prooxi-
been shown to decrease sperm count [61, 62]. dant and exposure causes alterations in trans-
Despite studies reporting a link between cription of genes and expression of L type
phthalate exposure and decreased sperm counts, voltage-dependent calcium channels [75]. These
it is suggested that more conclusive studies are channels regulate calcium as well as cadmium
needed [2, 63, 64]. entry into the cell. The negative effects of cad-
mium may be mediated by indirect generation of
11.3.1.2 Heavy Metal Toxicity hydroxyl radical, superoxide anion, H2O2 or NO,
Several studies reported impaired spermatogene- and reduction of the zinc content. Chronic zinc
sis and decreased sperm counts due to heavy deficiency is associated with increased sensitivity
metal (e.g., lead, cadmium, mercury) toxicity in to oxidative stress, mainly because it serves as an
men even after moderate exposure [65–68]. antioxidant and has a protective effect on sper-
Metals such as aluminum and vanadium are matogenesis [75]. In support of this theory, it was
believed to also have effects on male fertility. shown that chronic low-dose cadmium exposure
Occupational lead exposure negatively affected produced a time- and dose-dependent reduction in
fecundity of male workers [69]. Inorganic lead sperm motility. Finally, it was recently discovered
can disturb the prooxidant and antioxidant bal- that cadmium impinges on spermatogenesis by
ance and cause development of oxidative stress disruption of the inter-Sertoli cell tight junctions
[65]. Before being banned, lead was commonly and thus disrupting the blood-testis barrier [76].
Oxidative damage to sperm DNA due to inha- weight, low sperm count, reduced seminiferous
lation of metal fumes by battery and paint factory tubule diameters [96], decreased sperm motility,
workers/welders has been blamed for the increase and increased abnormal sperm morphology [97].
in infertility and miscarriage observed in their A study done in rats revealed that carbendazim’s
partners [77, 78]. An assessment of heavy metal deleterious effects on male reproduction are medi-
status may be necessary during infertility treat- ated via its ability to increase oxidative stress, by
ment depending on the lifestyle and occupational impairing steroidogenic enzymes and antioxidant
exposures of the couples affected [79–81]. cellular defenses. It also enhanced H2O2, hydroxyl
radicals, and lipid peroxidation in the Leydig cells
11.3.1.3 Agriculture Fertilizers, [98]. Chlorpyrifos, an organophosphate pesticide,
Pesticides, and Herbicides is another agricultural chemical that can lead
Due to the extensive use of chemical fertilizers in to ROS-induced DNA strand breaks and lipid
agricultural areas, nitrogen and ammonia soil peroxidation of spermatozoa [99].
saturation is occurring. Plenty of research has
been performed on the effect thereof on crops, 11.3.1.4 Solvents
grasslands, and surrounding areas or the impact Humans are primarily exposed to toluene, a
of nitrogen loading on aquatic ecosystems [82–84]. widely used organic solvent, via vapor inhala-
These fertilizers can stimulate NO production, tion. Toluene is found in paint, rubber, gasoline,
and when in excess, it negatively affects sper- adhesives, and various cleaning fluids [100].
matogenesis as seen by the decrease in motility, Recent investigations have shown that toluene
viability, acrosome reaction as well as ability to may induce reproductive dysfunctions. In a study
penetrate the oocytes [85]. done in male rats, toluene administration led to a
Results from a study by Jurewicz et al. [86] decrease in epididymal sperm count and serum
suggest that there are consistent indications that testosterone levels [101]. It mediates reproduc-
some pesticides like dichlorodiphenyltrichloro- tive toxicity via oxidative damage by either
ethane (DDT), ethylenedibromide, and organo- inducing excessive ROS generation [101] or
phosphates affect sperm count. Studies from decreasing the antioxidant status [100].
Wong et al. [87] and Oliva et al. [88] support Abnormally high concentrations of another sol-
these findings by reporting that men exposed to vent, xylene, have been found to be present in the
pesticides, e.g., farmers, have a higher incidence blood and semen of workers exposed to a working
of infertility. environment where the air concentration of xylene
Various pesticides and herbicides such as lin- exceeded the maximum allowable concentration.
dane, methoxychlor, and dioxin-TCDD have all These men experienced decreased sperm vitality,
been linked with testicular oxidative stress and motility, and acrosin activity [102]. Studies have
decreased sperm counts [89–92]. Lindane expo- shown that xylene is capable of inducing complete
sure can damage the morphology of the seminif- inhibition of mitochondrial respiration and is
erous tubule by affecting both gap and tight known to usually enhance mitochondrial ROS
junction proteins in Sertoli cells, resulting in generation [103, 104]. This propensity of xylene to
decreased spermatogenesis [93]. Not only does generate ROS is more than likely the basis of its
lindane lead to testicular oxidative stress, but it harmful effects on male reproduction.
also decreased activity of steroidogenic enzymes
and steroidal regulatory and transport proteins
(e.g., androgen-binding protein), resulting in 11.3.2 Lifestyle and Occupational
decreased circulating testosterone levels [94]. Exposure
Carbendazim (methyl-2-benzimidazole car-
bamate) is a systemic broad-spectrum fungicide, Exposure to certain lifestyle and occupational fac-
but is also used as a preservative for fruits, paint, tors can influence the adult testis directly and lead
textiles, and leather [95]. Its detrimental effects on to impaired spermatogenesis. A few of the most
male reproduction include decreased mean testes common issues will be subsequently discussed.
11.3.2.1 Smoking A recent study showed that motility is one of

It is well established that smoking has detrimen- the first sperm parameters affected and astheno-
tal effects on spermatogenesis as it has been cor- zoospermia may be an early indicator of reduced
related with significantly lower sperm counts semen quality in light smokers. This study also
(10–17%), decreased motility, and impaired found the incidence of teratozoospermia to be
morphology [105–108]. A meta-analysis per- significantly higher in heavy smokers when com-
formed by Vine et al. [109] substantiates this pared to nonsmokers [117].
association.
Smoking not only interferes with oxygen sup- 11.3.2.2 Alcohol Consumption and Drugs
ply, but also exposes smokers to thousands of There is a growing body of evidence suggesting
potentially harmful substances (e.g., alkaloids, that alcohol is a lifestyle factor that impacts sper-
nitrosamines, nicotine, hydroxycotine). Several matogenesis. Moderate alcohol consumption did
of these toxins can lead to the generation of ROS not show a significant impact on sperm count
and reactive nitrogen species, ultimately leading [118, 119]; however, chronic alcoholism appears
to oxidative stress when reaching pathophysio- to harm spermatogenesis and male fertility [120,
logical concentrations [110, 111]. 121]. Not only are impotence, testicular atrophy,
Saleh et al. [107] demonstrated that cigarette and loss of sexual interest associated with alco-
smoking leads to an increase in ROS levels and holism, but it is often accompanied by reduced
decreases in ROS-TAC scores in semen of smok- FSH, LH, and testosterone levels [122]. Goverde
ers showing a 100-fold increase in oxidative et al. furthermore reported that semen volume,
stress and up to 5× higher cadmium levels. sperm count, motility, and number of morpho-
Furthermore, it was reported that smokers have logically normal sperm were significantly
high levels of leukocytospermia and suggested decreased in alcoholics when compared to nonal-
that oxidative stress develops due to ROS gener- coholics [123]. Excessive alcohol consumption
ation by activated leukocytes [107]. Smoke has also been linked to oxidative stress in the tes-
metabolites and several compounds of cigarette tis as a significant reduction in testosterone,
smoke may act as chemotactic stimuli, thereby increase in lipid peroxidation by-products, and a
inducing an inflammatory response, recruitment drop in antioxidants were observed [124].
of leukocytes, and subsequent ROS generation Many processes and factors are involved in
[107, 112]. It was furthermore reported that alcohol-induced oxidative stress. One possible
smokers have decreased levels of seminal plasma mechanism is the generation of ROS molecules
antioxidants such as Vitamin C [113] and Vitamin in response to the metabolism of ethanol by the
E [114]. microsomal ethanol-oxidizing system (MEOS)
Approximately half of the arterial blood sup- [125, 126]. Alcohol metabolism results in NADH
ply to the testes is drained off via arterio-venous formation which enhances activity of the respi-
anastomoses in the spermatic cord. This, coupled ratory chain, including heightened oxygen use
with the evidence that the testes have high meta- and excessive ROS formation [127]. ROS are
bolic requirements due to the ongoing process of natural cellular renegades and wreak havoc in
spermatogenesis, translates to the testis being biological systems through tissue damage, alter-
regarded as physiologically on the verge of being ation of biochemical compounds, corrosion of
hypoxic [2, 115, 116]. Sharpe [2] speculated that cell membranes, and killing outrightly [128].
any factors which compromise oxygen supply to Alcohol also induces hypoxia that results in tis-
the testis, including smoking, would have detri- sue damage. In addition, many alcohol abusers
mental effects on spermatogenesis. follow diets deficient in protective antioxidants
An alternative route whereby components [129, 130].
of cigarette smoke might affect spermatogenesis The association between alcohol and infertility
is through the interaction of PAH with the Ah is of great concern because the use of alcohol is
receptor and the subsequent effects on the AR widespread. In many countries, alcohol usage is
[2, 39, 40]. extensive, especially in the teenager. The evidence
regarding moderate alcohol intake is less clear, changes in diets and eating habits. Not only are
but most experts agree it is best to avoid more people eating more highly refined carbohydrate-
than 1 or 2 drinks/day. rich food, but they are simultaneously consuming
Certain drugs, whether therapeutic, recre- less fresh fruit and vegetables [139].
ational, or performance enhancing, can have The importance of fresh fruits and vegetables
adverse effects on spermatogenesis. Several in the diet was suggested in a study where
prescription drugs used for therapeutic purposes, decreased intake of these nutritional substances
especially when used chronically, can impact on was correlated with subfertility [87]. Apart from
spermatogenesis. Antibiotics and cancer chemo- containing antioxidants, essential nutrients like
therapy usually damage the germinal epithelium micronutrients, vitamins, and folate are found
[131, 132]. Some drugs may cause spermatogenic in fruit and vegetables and these substances play
arrest (mechlorethamine) (U49), while many an important role in spermatogenesis as they
antimicrobials (e.g., tetracycline derivatives, sulfa are involved in DNA and RNA synthesis. It is
drugs) impair spermatogenesis and chronic use not clear if the mechanism of higher sperm
can lead to infertility [132–134]. In a very inter- production in patients with a fruit/vegetable
esting study by Hayashi et al. [135], it was shown diet is related to dietary factors or reflects an
that men who switched or stopped treatment of epiphenomen.
the most common medications (allergy relief, Nutritionally deficient diets, lacking antioxi-
antiepileptics, antibiotics) had a 93% improve- dant vitamins and synergistic minerals, do not
ment in semen quality. In general, the severity of enable the quenching of reactive oxygen mole-
testicular influence is related to the class of thera- cules. For example, Vitamin C and Vitamin E are
peutic agent used, as well as the dose and dura- essential antioxidants that protect the body’s cells
tion of therapy [136]. from damage due to oxidative stress and free
Concrete evidence for the effect and mecha- radicals. Vitamin C is the most abundant antioxi-
nism of recreational drug abuse, such as marijuana dant in the semen of fertile men, and it contrib-
and cocaine, on sperm production is still lacking. utes to the maintenance of healthy sperm by
Several articles report that it does have detrimen- protecting the sperm’s DNA from free radical
tal effects and that the mode of action is more than damage [140]. Vitamin E is a fat-soluble vitamin
likely via receptor binding and endocrine disrup- that helps protect the sperm’s cell membrane
tion or direct effect on the spermatozoa. from damage. Studies have shown that Vitamin E
The use of anabolic steroids, predominantly improves sperm motility and morphology.
used to enhance body image or improve perfor- Vitamin C functions to regenerate Vitamin E,
mance among athletes, is on the increase and thus these vitamins may work together to improve
reaching epidemic proportions [136]. This can sperm function [140–143]. Selenium is a mineral
result in oligozoospermia as spermatogenesis is that also functions as an antioxidant and selenium
reduced because steroids cause suppression of LH supplements have been shown to increase sperm
secretion and consequently suppress intratesticu- motility. A combination of selenium and Vitamin
lar testosterone levels. Hypogonadotropic hypog- E has been shown to decrease damage due to free
onadism is therefore the most common cause for radicals and improve sperm motility in infertile
impairment of sperm production in this group men [144]. Diets deficient in these substances
[137, 138]. These defects can be reversed in the will then be unable to provide the benefits men-
mature testis within a couple of months after dis- tioned above.
continuation of anabolic steroid use [138]. Obese, overweight, and high body mass index
(BMI) subjects may be at risk of infertility [145–
11.3.2.3 Diet and Obesity 147]. Men with BMI’s greater than 25 are up to
Diet and obesity are important lifestyle factors three times more at risk of infertility due to reduc-
that can influence spermatogenesis. Accom tion in sperm count and increased DNA fragmen-
panying modern Westernized lifestyles are tation. Many clarifications have been given to
explain the link between obesity and decreased periods of exams [160]. Eskiocak et al. [161]
spermatogenesis. Firstly, excess adipose tissue were also able to link periods of psychological
leads to the conversion of more testosterone to stress with a reduction in sperm quality, mediated
estrogen. This subsequently results in the devel- by an increase in seminal plasma ROS generation
opment of secondary hypogonadism through and a reduction in antioxidant protection [161,
hypothalamic-pituitary-gonadal axis inhibition 162]. It has also been established that stress can
[148], thereby decreasing the levels of circulating lead to increased levels of glucocorticoids and
testosterone and increasing the levels of estradiol decreased levels of testosterone [163].
[36, 37, 149–151]. This change in testosterone: Similarly, Ruffoli et al. [164] demonstrated
estradiol ratio is often accompanied by reduced that chronic noise stress can cause accumulation
levels of LH and FSH [145]. More than likely, of lipofuscin in the testis of mice. Moreover, it
the decreased testosterone levels in the testis are subsequently led to a decrease in testosterone
responsible for impaired spermatogenesis [152]. production in these animals. It was previously
Secondly, accumulation of suprapubic and established that corticosterone administration
inner thigh fat in severely obese men could lead caused free radical production in the mitochon-
to infertility via increased scrotal temperatures. dria of Leydig cells and that free radicals are
Deposition of fat around the scrotal blood vessels known to stimulate lipofuscin formation [165,
can also cause impaired blood cooling and ele- 166]. Ruffoli et al. [164] went on to conclude that
vate testicular temperature [153]. Obese men also exposure to above the normal levels of noise in
tend to be more sedentary which would exacer- the workplace or environment may be detrimen-
bate any temperature increases as discussed else- tal to testicular function.
where under the Sect. 11.3.2.5.
Finally, obesity and several of its accompany- 11.3.2.5 Scrotal Heat Stress
ing complications, namely, insulin resistance and The exteriorization of the male gonads into the
dyslipidemia, are associated with systemic proin- scrotum is a uniquely mammalian feature. The
flammatory states and increased levels of oxida- most plausible explanation for such evolution is
tive stress [154, 155]. Oxidative stress causes that optimal spermatogenesis requires a tempera-
sperm membrane lipid peroxidation, which ture (±34°C) markedly lower than core abdomi-
results in the impairment of sperm motility, DNA nal temperature (±37°C) [167, 168].
damage, and impaired sperm-oocyte interaction The concept that an elevation of testicular
[156, 157]. Conversely, adipose tissue releases temperature results in impairment of spermato-
proinflammatory adipokines that increase leuko- genesis is widely accepted. For instance, rats
cyte production of ROS [158]. whose testes were immersed into a luke warm
Both endocrine and exocrine (spermatogene- water bath (43–45°C for 15–30 min) showed
sis) functions of the testis are believed to be influ- deterioration in spermatogenesis [169]. It was
enced in direct proportion with the current trend found that apoptosis of the pachytene spermato-
of increased BMI and obesity in men throughout cytes and round spermatids were induced via the
the world. mitochondrial pathway [170–175].
Various mechanisms have evolved to allow
11.3.2.4 Psychological and Noise Stress for the testes to be kept 3–4°C below core body
Mental stress is associated with lower levels of temperature. First, the testes descend into the
antioxidants, such as glutathione (GSH) and very bottom of the scrotum – as far away from
SOD, as well as higher levels of prooxidants the body surface as possible. Second, the pre
[159]. This can create oxidative stress. Various sence of a countercurrent heat exchanger in the
studies have shown a correlation between stress form of the pampiniform plexus, as well as a
and semen quality. One such study in particular vascular-rich corrugated scrotal surface (third
reported that students have lower sperm counts mechanism) through which heat loss can occur,
and sperm quality during the highly stressful ensures further cooling of the testes.
The lower temperature leads to reduced rates these studies are in agreement that the type
of oxidative DNA damage and consequently of clothing can increase genital temperature
fewer mutations in resulting sperm cells [168, [190–194]. A few studies found no significant
176]. Spermatozoa are stored in the epididymis differences [195, 196], but in general most of
often for many days or weeks. Storage occurs these studies were not well controlled. Recently,
specifically in the cauda epididymis which is Jung et al. [197] analyzed the influence of the
found in the coolest area of the scrotum, thereby type of underpants under highly controlled con-
reducing metabolic rates and oxidative damage ditions and reported that scrotal temperatures
of these spermatozoa [177]. were significantly higher for tight fitting, less for
Scrotal pathologies such as varicocele and loose fitting, and the least for no underwear.
cryptorchidism can increase testicular tempera- Prolonged hot baths and saunas also increase
ture excessively; however, lifestyle and occupa- scrotal temperature and have been implicated to
tion can also lead to chronically elevated scrotal impair spermatogenesis [192, 198–200]. Further
temperatures that can contribute to the global more, obesity and the accompanying accumula-
trend in declining male reproductive parameters tion of adipose tissue within the groin region also
[167]. result in raised testicular temperature. This has
Occupational exposure to increased ambient been linked to the generation of ROS and devel-
temperatures (welders, bakers, stokers, foundry opment of oxidative stress in the testis resulting
workers, furnace operators) has been shown to in reduced sperm quality [201–203].
increase scrotal temperatures, decrease sperm Febrile illnesses (e.g., influenza, Malaria) are
density, motility and morphology, as well as sig- another factor that can lead to increased scrotal
nificantly prolong time to pregnancy [178–180]. temperatures. Several studies demonstrated a
A few studies, however, reported no differences decrease in total sperm counts and semen quality
in sperm concentration and semen quality in men even up to a few months after these episodes of
with similar occupations [181, 182]. Therefore, fever [183, 204, 205].
the influence of occupational heat exposure not Failure to cool the scrotum adequately there-
yet allows for definitive conclusions [169]. fore appears to be associated in the adult with
Extended periods of sitting have also been impaired spermatogenesis.
correlated with increased scrotal temperatures
[183, 184]. Hjollund reported a progressive 11.3.2.6 Cell Phone and Ionizing
increase in scrotal temperature in office workers Radiation
in relation to sedentary time with sperm density Stopczyk et al. [206] demonstrated that radiofre-
being significantly decreased in men with more quency electromagnetic waves (RF-EMW), pro-
than 75% of their daytime scrotal temperature duced by cell phones, significantly depleted
above 35°C [185, 186]. Various other studies SOD-1 activity while increasing the concentra-
support these findings. More generally speaking, tion of malonyldialdehyde (MDA) after 1, 5, and
sperm density is believed to decrease by 40% per 7 min of exposure in a suspension of human
1°C increment of median daytime scrotal tem- blood platelets. On the grounds of these results,
peratures [186, 187]. The difference in scrotal the authors conclude that oxidative stress after
temperatures between sitting in the office chair exposure to microwaves may be the reason for
and sitting in a car was shown to be statistically many adverse changes in cells and could cause a
insignificant [183]. Professional drivers such as number of systemic disturbances in the human
lorry and taxi drivers have also been shown to body. Several animal studies also demonstrated
experience reduced semen quality and increased that cell phone radiation can increase levels of
time to pregnancy [178, 188–190]. MDA and decrease the levels of antioxidant
Several studies have investigated the impact enzymes, while other studies consistently
of tight vs. loose fitting underwear. On the whole, reported that cell phone radiation leads to a
decrease in male fertility. A few studies, however,

showed conflicting results [207]. 11.4 Environmental Insults,
Various epidemiological studies proposed that Epigenetics, and
cell phone usage may cause decreases in sperm Spermatogenesis
count and other sperm parameters [208–212].
A study by Agarwal et al. [213] provided evidence The ability of environmental factors to promote a
that cell phone radiation can lead to generation of phenotype or disease state not only in the indi-
ROS. Results showed a significant increase in vidual exposed, but also in subsequent progeny
ROS production and a decrease in sperm motility, for successive generations is termed transgenera-
viability, and ROS-TAC score in exposed semen tional inheritance. The majority of environmental
samples. A plausible explanation for the ROS factors such as nutrition or toxins do not promote
production is stimulation of the spermatozoa’s genetic mutations or alterations in DNA sequence.
plasma membrane redox system (increase in the However, these factors do have the capacity to
activity of spermatozoal NADH oxidase) by alter the epigenome. Epimutations in the ger-
RF-EMW or the effect of EMW on leukocytes mline which become permanently programmed
present in the neat semen. Recently, Friedman can allow transmission of epigenetic transgenera-
et al. [214] also suggested that RF-EMW stimu- tional phenotypes [221]. Skinner et al. [221] fur-
late plasma membrane NADH oxidase in mam- thermore define epigenetics as molecular factors
malian cells and cause production of ROS. and processes around DNA that are mitotically
EMW-dependent decrease in melatonin, an anti- stable and regulate genome activity independent
oxidant, can also predispose sperm to oxidative of DNA sequence.
stress [215]. Epigenetic elements identified include DNA
It is furthermore speculated that RF-EMW methylation and histone modifications [222, 223].
emitted from cell phones might also influence A special category of genes, called imprinted
spermatogenesis via a thermal molecular effect genes, are subjected to epigenetic programming
[208]. Leydig cells are among the most suscepti- and can be influenced by environmental expo-
ble cells to EMW and injury to these cells sure[224]. Epigenetic influences have been obser
may affect spermatogenesis. A decrease in ved with environmental compounds (Fig. 11.1),
seminiferous tubular diameter and epithelium nutritional factors (X45 X46), inorganic contami-
thickness was also observed after RF-EMW nants such as arsenic [225, 226], PAHs [227],
exposure [216]. endocrine disruptors such as BPA [228–230], phy-
Although many animal as well as in vitro stud- toestrogens [231, 232], and chemicals used as fun-
ies have provided evidence that cell phone radia- gicides and pesticides [233, 234].
tion may lead to a decrease in sperm parameters, The crucial period for epigenetic regulation
considerable research is still required to confirm and modification of the germline is during the
that cell phone usage impact spermatogenesis. period of primordial germ cell migration and
Chronic exposure to ionizing radiation can gonadal sex determination. The permanent altera-
also cause decreased sperm quality coupled to tion in the epigenetic programming of the ger-
endocrine disruption [217–219]. Results are mline appears to be the mechanism involved in
mainly from animal studies, but prisoners volun- the transgenerational phenotype [233, 235]. The
teering for testicle X-ray irradiation as well as endocrine disruptor vinclozolin was used in one
men exposed to radiation after the Chernobyl of the initial studies to demonstrate the ability of
nuclear accident were also studied. In all cases, an environmental factor to modify epigenetic pro-
the doses of exposure seem to be the important gramming of the male germline. Embryonic expo-
determinant of the effect on spermatogenesis sure of rats through maternal administration led to
with higher dosages leading to even permanent adult onset of disease in the first through fourth
sterility [47, 218, 220]. subsequent generations [233]. This phenomenon
was found to be caused by male germline changes As spermatogenesis exclusively occurs in the
in DNA methylation, resulting in heritable changes mature testis, adulthood exposure impacts
in transcription in tissues such as the testis [236]. directly sperm number and quality. On the con-
It also correlated with reduced sperm number trary, perinatal exposure to environmental insults
in the adult due to increases in apoptotic germ can have lasting and more severe effects on gam-
cell numbers in the testis of pubertal and adult ete production, even transgenerationally. These
animals [237]. emphasize the importance of environmental
Transient early life exposures in the affected impacts throughout the life course.
individual, or transgenerational exposures if the With the onset of assisted reproductive tech-
germline is involved, are now included as causal nologies, specifically intracytoplasmic sperm
factors for adult onset disease. Endocrine dis- injection, a major proportion of infertile men is
ruptors are one of the most prevalent groups of now afforded the opportunity and ability to pro-
environmental compounds humans are exposed create. The dilemma of this situation is that the
to daily. Although these compounds disrupt the fundamental and root cause of male fertility is
endocrine system, it is the long-term response of not addressed, only the symptoms. Tragically,
molecular processes such as epigenetics that these advances in infertility treatments are also
will promote downstream developmental-events negating any advances in understanding the etiol-
and adult onset disease [1, 221]. Epigenetic ogy and treatment of male infertility. It is there-
events in the testis have just begun to be studied. fore paramount that research on the reasons for
New work on the function of specific histone male infertility, specifically with regard to the
modifications, chromatin modifiers, DNA meth- effects of environmental insults on spermatogen-
ylation, and impact of the environment on sper- esis, be performed in order to solve for preven-
motogenesis suggests that the setting of the tion and cure and not leaving it to future
epigenome is required for male reproductive generations to try and resolve when it might
health [1]. already be too late.
11.5 Conclusion
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Sperm DNA Damage: Causes
and Guidelines for Current Clinical 12
Practice
Aleksander Giwercman, Marcello Spanò,
and Mona Bungum
Abstract
Infertility is a common health problem affecting approximately 15% of all
couples. It is estimated that, in at least 50% of all cases, impairment of
semen quality is a factor contributing to the problem of the couple.
Furthermore, 10–15% of all infertility cases are “unexplained”. Traditional
sperm parameters – concentration, motility, morphology – were shown to
have a poor predictive value, both in relation to fertility in vivo and in vitro.
Thus, there is an urgent need of finding new sperm quality markers that
can be used for the evaluation of the chance of a couple to conceive in a
natural way, and also, if necessary, for the selection of the right assisted
reproductive technique (ART). In this context, testing of sperm DNA
integrity seems to be a potential candidate. The causes of sperm DNA
damage are multi-factorial, including testicular and post-testicular, genetic
and exposure-related mechanisms. For the assessment of sperm DNA
damage, a number of different techniques is available. The results obtained
by these methods are generally correlated, but, on the other hand, they
seem to measure slightly different elements of sperm DNA integrity and
they provide limited information on the nature of the lesions detected. So
far, assessment of sperm DNA has proven to be the most powerful in pre-
dicting in vivo infertility, which means cases where the chance of preg-
nancy by natural conception or intrauterine insemination is close to zero.
High percentage of sperms with DNA defects seems also to have a nega-
tive impact on the results of standard in vitro fertilisation (IVF), but not on
that of intracytoplasmatic sperm injection (ICSI), even if the issue is debat-
able and under active scrutiny. Clinically applicable threshold values
A. Giwercman (*)
Reproductive Medicine Center, Skåne University
Hospital, Malmö, Sweden
and
Department of Clinical Sciences, Lund University,
Malmö, Sweden

156 A. Giwercman et al.
have been defined for one of the methods for assessment of sperm DNA
integrity – the sperm chromatin structure assay (SCSA) – but not yet for
the other techniques. There is a need for standardisation of technology for
these assays in the same way as it has been achieved for SCSA. Thus,
well-designed studies should be carried out in order to define the clinical
role of these new measures of sperm quality. This is important from the
diagnostic as well as therapeutic point of view, since a more nuanced char-
acterisation of sperm DNA defects will not only lead to an improved
choice of the ART methods, but may also help in developing cause-related
therapy of male subfertility. From the biological point of view, it needs to
be investigated whether sperm DNA damage transmitted to the embryo by
use of IVF and ICSI can be hazardous to the offspring, taking into account
the wealth of animal data demonstrating the link between sperm DNA
damage and abnormalities in embryo development.
Keywords
Infertility • Sperm DNA • SCSA • TUNEL • Comet • Strand breaks •
Insemination • Assisted reproductive technology • IVF • ICSI •
Pregnancy
of IUI were unsuccessful. Adam delivered one

12.1 Case Story more semen sample and the examination revealed
again perfectly normal volume, concentration,
Adam, 28 years old, has been referred for andro-
motility and morphology. As additional test,
logical examination due to involuntary childless-
sperm chromatin structure assay (SCSA) was per-
ness. Together with Linda, 27 years, he has for
formed, showing a DNA fragmentation index
1 year tried to beget a child without succeeding.
(DFI) of 36%. The SCSA was repeated this time
Linda has never been pregnant and Adam has no
showing a DFI of 39%. The couple was wonder-
children and has never made any woman preg-
ing why they were unable to achieve pregnancy
nant. They have intercourse 2–3 times per week.
and how they should proceed.
Gynaecological examination of Linda, including
measurement of hormonal status and tubal pat-
ency, revealed no abnormalities. Adam’s andro-
logical history is unremarkable and the physical 12.2 The Need for New Tests
examination did not indicate any abnormality. for the Assessment of Semen
He delivered two semen samples for examina- Quality
tion both showing perfectly normal volume,
sperm concentration, motility and morphology. The case story presented above shows some of
Since the infertility problem of the couple the basic problems of today routine infertility
was characterised as “unexplained” and they work-up:
were relatively young, the advice was to con- (a) The predictive value of standard semen anal-
tinue the attempt to achieve spontaneous preg- ysis in relation to the chance of achieving
nancy. One year later, the couple was again spontaneous pregnancy is rather poor [1, 2].
referred to the infertility clinic since Linda has (b) The same is true in relation to choice of the
not got pregnant. They were thereafter referred method of assisted reproduction technique
for intrauterine insemination (IUI). Three attempts (ART), which means that couples may
12 Sperm DNA Damage: Causes and Guidelines for Current Clinical Practice 157
undergo treatment a priori having a very low nucleus is needed. In fact, a mature sperm nucleus
chance of success. containing the haploid genome must adopt a vol-
Traditional semen analysis is based on the ume 40 times less than that of a normal somatic
guidelines of the World Health Organization nucleus [8]. In order to achieve such architectural
[3], the main parameters to be assessed being compression, the nucleosomal organisation is
volume, sperm number, motility and morphology. dismantled in the last part of spermiogenesis, and
According to the most recent WHO guidelines [4], histones are replaced by a new set of nuclear pro-
the 15 × 106/mL is to be considered as the lower teins, the protamines, more basic and much
reference limit. However, a population-based study smaller than histones [9]. The fundamental pack-
has shown that optimal male fertility potential is aging unit of mammalian sperm chromatin is a
not achieved before the sperm concentration toroid containing 50–60 kb of DNA. Individual
reaches the level of 40–50 × 106/mL [1, 2]. On the toroids represent DNA loop-domains highly con-
other hand, spontaneous pregnancies are possible densed by protamines and fixed at the nuclear
with sperm concentration far below this lower matrix. The toroids are further compacted by the
reference limit [1]. The wide overlap between intra- and inter-molecular disulfide cross-links,
“fertile” and “non-fertile” is true not only for formed by oxidation of sulfhydryl groups of
sperm concentration, but also for the other param- cysteine present in the protamines [9, 10]. Thus,
eters, i.e. motility and morphology [2]. Thus, when each chromosome represents a garland of toroids,
evaluating the results of semen analysis, we quite and all 23 chromosomes are clustered by cen-
often are unable to predict the chance of the couple tromeres into a compact chromocenter positioned
to achieve a natural pregnancy. well inside the nucleus with telomere ends united
The other emerging problem related to the clas- into dimers exposed to the nuclear periphery [11,
sical semen parameters is the poor link between the 12]. This condensed, insoluble and highly
pathogenesis and the result of semen investigation. organised nature of sperm chromatin acts to pro-
Thus, oligo-astheno-teratozoospermia may be due tect genetic integrity during transport of the
to different types of testicular as well as post- paternal genome through the male and female
testicular pathologies [5]. This limitation highly reproductive tracts as the mature sperm is a
restricts our possibilities to dig into the causes of repair-deficient cell. It also ensures that the pater-
male-related subfertility, and thereby, for finding nal DNA is delivered in the form that sterically
and testing cause-related new treatment modalities. allows the proper fusion of two gametic genomes
Finally, even when the decision of performing and enables the developing embryo to correctly
ART has been made, standard semen parameters express the genetic information [11, 13].
are only of limited value to select the best option In many animal species, protamines comprise
among the different techniques available as IUI, 95% of the spermatozoal nucleoproteins. How
standard in vitro fertilisation (IVF) or intracyto- ever, in humans this percentage is usually only
plasmatic sperm injection (ICSI). 85%. This may be one of the reasons why human
For these reasons, during the recent years, a sperm chromatin is less compacted and more
lot of attention has been given to sperm DNA frequently contains DNA strand breaks [14].
damage as an additional and useful marker of In comparison with other species [15], human
sperm function and as an alternative indicator of sperm chromatin packaging is also exceptionally
the mechanisms behind male subfertility [6, 7]. variable, both within and between men. Moreover,
in contrast to the bull, cat, boar and ram, whose
spermatozoa contain only one type of protamine
12.3 Human Sperm Chromatin (P1), human and mouse spermatozoa contain a
second type of protamine (P2), which contains
Due to the limited size of the head of the sperma- fewer cysteine groups [16]. Consequently, the
tozoa, a significantly more compact packaging of diminished level of disulfide cross-linking could
the genetic material than in the case of a somatic be one of the responsible factors underlying the
looser human sperm chromatin packaging as 12.4.2 Abortive Apoptosis

compared to the sperm chromatin of other spe-
cies containing P1 alone [17]. Under the process of spermatogenesis, germ cells
are under the control of Sertoli cells resulting in
induction of apopotosis in 50–60% of the germ
12.4 Origin of Human Sperm DNA cells before they enter meiosis I [21]. For several
Damage reasons including (a) inability of spermatozoa to
undergo “programmed cell death” due to lack of
The origin of human sperm DNA damage is het- transcriptional and translational capacity; (b)
erogeneous involving, in a not mutually exclusive reduced nucleosome content because of extensive
ways, both testicular and post-testicular mecha- protamination and thereof following inability to
nisms. The most common types of DNA damage exhibit the characteristic DNA laddering seen in
include abasic sites, chemical modification of a somatic cells; and (c) endonucleases activated in
base, inter- and intra-strand cross-links, and sin- the cytoplasm or released from the mitochondria
gle or double DNA strand breaks. Both sperm being prevented from physically accessing the
nuclear and mitochondrial DNA can be damaged, DNA due to the physical architecture of the sper-
but since the latter makes no contribution to the matozoa, the apoptotic process in male germ cells
functionality of the male gamete, the attention cannot be identical to that in other cell types.
will be given to impairment of the integrity of the Therefore, ideally, the spermatozoa earmarked by
nuclear genetic material. Testicular mechanisms the apoptotic markers of Fas type should be elimi-
include (a) alterations in chromatin modelling nated by being phagoctosed by the Sertoli cells.
during the process of spermiogenesis and (b) However, this process seems not to be completely
abortive apoptosis, whereas post-testicular fac- efficient leading to some of the “abortive apoptosis”
tors are mostly related to the action of (c) reactive meaning that some of the cells escape the apoptosis
oxygen species (ROS) and (d) activation of cas- and may, thereafter, appear in the ejaculate [20].
pases and endonucleases [18, 19].
12.4.3 Reactive Oxygen Species

12.4.1 Chromatin Modelling During
the Process of Spermiogenesis Oxidative stress is supposed to be the major con-
tributory factor behind defective sperm function
The process of chromatin condensation and and is supposed to be due to an imbalance
packing during the spermiogenetic steps includes between the amount of ROS produced and the
the induction of DNA nicks to provide relief of ability of the antioxidants to scavenge these.
torsional stress to aid chromatin arrangement Spermatozoa seem to be susceptible to the attack
during the displacement of histones by protamines. of free radicals due to superabundance of polyun-
Type II DNA topoisomerase is the enzyme saturated fatty acids, necessary for a proper fer-
responsible of the induction and sealing of these tilisation process. The sources of the oxidative
nicks because of the endonuclease and ligase stress leading to DNA damage in the germ cells
activity of the enzyme [20]. The intermediate include factors as loss of antioxidant protection,
breaks allow the DNA to be untangled or infection and/or intrinsic (spermatozoa), and
unwound, and at the end of these processes, the extrinsic radical production [22].
DNA is reconnected again. Any impairment of Spermatozoa are relatively deficient of ROS-
this process, occurring in round spermatids, will scavenging enzymes, for that reason the antioxi-
lead to long-term consequences such as an dant protection being of a crucial importance for
increased number of sperm DNA strand breaks their DNA integrity. Epididymis secretes an array
and/or higher susceptibility to other assaults. of antioxidant factors including vitamin C, uric
acid, taurine and thioredoxin, superoxide dismutase There are a variety of etiologic factors that
and gluthatione peroxidise. Secretion of some of have been associated with sperm DNA fragmen-
those factors is under androgenic control. Even the tation and/or defective chromatin. Sperm DNA
seminal plasma may provide some antioxidant breaks have also been linked to exposure to can-
protection; however, unlike the epididymis, sperm cer treatment, radiotherapy and chemotherapy
spend very little time in seminal plasma and the [25], as well as to some environmental toxicants,
role of seminal antioxidants is not completely clar- e.g. PCBs, phthalates and air pollution [26, 27].
ified. However, it has been shown that the degree The exact mechanisms behind the effects of
of sperm DNA fragmentation in ejaculated sper- such exposures in relation to sperm DNA integ-
matozoa can be higher than in those obtained from rity are not known. However, these might include
the testis and caput or corpus epididymis [22, 23]. a direct effect on the DNA of the spermatozoa as
Leukocytes, mainly neutrophils and mac- well as indirect effect caused by changes in the
rophages, generate ROS and are important con- endocrine milieu of the testis and/or epididymis
tributors to the general seminal level of ROS. and, thereby, lowered activity of the testoster-
Infection is the major cause of leukocytic infiltra- one-dependent DNA enzyme topoisomerase as
tion into the male genital tract, and in a vast well as reduced epidymal production of
majority of cases, the spermatozoa get in contact antioxidants.
with the leukocytes at ejaculation [23]. Both single- (SSB) and double-stranded
However, the origin of ROS which may be (DSB) DNA breaks can occur. Generally, SSB is
deleterious to the integrity of sperm DNA may easier to repair and has a better prognosis than
not only be extrinsic but even intrinsic, the major the DSB. SSB are usually due to unrepaired DNA
source being sperm mitochondria [24]. The net nicks introduced during the spermiogenesis or as
result in terms of the degree of sperm DNA dam- a consequence of the action of ROS. However,
age depends on the balance between extrinsic and the latter may also lead to DSB, which also can
intrinsic ROS production and the protective abil- be a consequence of abortive apoptosis and/or
ity of the seminal fluid. the action of caspases and endonucleases. DSBs
are considered the nastiest DNA lesions that, if
left unrepaired, would lead to gross structural
12.4.4 Activation of Caspases alteration of the chromosome complement.
and Endonucleases Therefore, a sperm DSB is considered to be a
most serious DNA lesion which has potential
We should also mention that sperm DNA frag- deleterious impact of the healthy development of
mentation can even be induced by activation of the progeny [7].
sperm’s own caspases and endonucleases acti- Some recent review articles [18, 19] provide
vated by oxygen radicals and physicochemical more finely detailed reviews of the possible
factors, including heat exposure. This also means mechanisms entangled behind sperm DNA dam-
that ROS may induce DNA strand breaks in the age. From the clinical point of view, it is impor-
spermatozoa by exerting a direct effect, or indi- tant to stress that the cause of sperm DNA
rectly, by inducing a cascade of events involving fragmentation may be multi-factorial and is partly
the above-mentioned enzymes [19]. unknown. Most of the available techniques for
The proposed mechanisms to explain the very detection of sperm DNA damage provide limited
origin of sperm DNA damage are obviously not information on the nature of the DNA lesions
mutually exclusive and, recently, a two-step detected and none of them enables us to depict
hypothesis has been put forward where faulty the exact aetiology and pathogenesis of impair-
spermatogenesis can lead to defective chromatin ment of sperm DNA, this diagnostic problem
remodelling with the DNA more susceptible and reducing our possibilities of giving a cause-
vulnerable to a variety of stressors [22]. related efficient therapy.
easure of the amount of DNA damage in the

m
12.5 Methods for Assessment cell. The cells are stained with a fluorescent
of Sperm DNA Damage DNA-binding dye. The overall intensity of the
fluorescence for the whole nucleoid and the flu-
Several methods for assessment of sperm DNA orescence of the migrated DNA is measured by
damage are now available. Below some details of image analysis. The stronger the signal from the
the most commonly applied techniques will be migrated DNA, the more damage there is pres-
given. ent. The overall structure resembles a comet
(hence “Comet assay”) with a circular head cor-
responding to the undamaged DNA that remains
12.5.1 TdT and TUNNEL in the nucleus and a tail of damaged DNA. The
brighter and longer the tail, the higher the level
The terminal deoxynucleotidyl transferase of damage. Comet assay can be performed under
(TdT)-mediated 2¢-deoxyuridine 5¢-triphosphate- neutral as well as alkaline conditions. By choos-
nick end labelling (TUNEL) assay uses TdT, an ing different pH conditions for electrophoresis
enzyme able to catalyse, in the presence of DNA and the preceding incubation, different damage
nicks, the addition of dUTPs that are secondarily types and different levels of sensitivity can be
labelled with a marker, generally fluorescent. assessed. Under neutral (pH 8–9) conditions,
TdT preferentially labels the blunt 3¢-OH ends of mainly DSB are detected, although some SSB
double-stranded DNA breaks, but also works on due to the relaxation of supercoiled loops con-
the single-strand 3¢-OH [28]. The assay detects taining the breaks might also contribute to the
the DNA breaks directly, which means without observed comet [31]. Alternatively, under alka-
initial step of denaturation by introducing acid or line conditions, DSB and SSB (at pH 12.3) and
alkaline pH. However, the peculiar sperm chro- additionally alkali labile sites (at pH ³13) can
matin compaction may impede its effectiveness be visualised resulting in increased DNA migra-
and limit the access of the enzyme to all 3¢-OH tion in the electrophoretic field [32]. Comet
break ends [29]. TUNEL tests can be based on assay, requiring a much smaller number of cells
microscopic evaluation of 2–500 cells, and if the (typically, only 100) for analysis than other
labelling is fluorescent, flow cytometric analysis tests, has been considered as particularly useful
is also possible thus increasing statistical reli- and suitable for measures of testicular and oli-
ability (the number of scored cells can be gozoospermic sperm samples where cells are
5–10,000), precision, and sensitivity of the scarce [33].
measurements.
12.5.3 Sperm Chromatin Structure

12.5.2 Comet Assay
Comet assay [30] is a single cell gel electrophore- SCSA is a flow cytometric test where sperm DNA
sis of immobilised sperm, which involves their breaks can be evaluated indirectly through DNA
encapsulation in agarose, lysis (at different pH), denaturability. The assay measures the suscepti-
and electrophoresis. When the electric field is bility of sperm DNA to acid-induced DNA dena-
applied, the DNA, which has an overall negative turation in situ (the low-pH solution potentially
charge, is drawn towards the anode, which is pos- denatures only the DNA that is damaged), fol-
itively charged. Undamaged DNA strands are too lowed by staining with the fluorescence dye acri-
large and do not leave the nucleus, whereas the dine orange [34–36] and quantifying the
smaller the fragments, the farther they are free to metachromatic shift of acridine orange fluores-
move in a given period of time. Therefore, the cence from green (native DNA) to red (denatured
amount of DNA that leaves the nucleus is a DNA) under a blue light excitation. By using a
flow cytometer, 5–10,000 sperm can be analysed 12.5.5 Toluidine Blue Test
within a short time. Through a specific SCSA-
software (SCSA-Soft®, SCSA, Brookings, ND), Toluidine blue (TB) test is also based on micro-
the ratio of red to total (green plus red) fluores- scopic examination of smears stained with the
cence is calculated for each sperm and the fre- thiazine dye toluidine blue. Like in SCSA and
quency distribution, called DFI, of all the AOT, prior to staining, the DNA of the spermato-
accumulated measured sperm is generated, zoa is denaturated by lowering the pH. The test
together with scattergrams formed by a proper measures the availability of the sperm chromatin
combination of sperm fluorescent parameters. DNA phosphate residues for staining with TB,
From these data, the percentage of spermato- which is dependent on both the protein state and
zoa with an abnormally high DNA stainability DNA integrity. It has been reported [38, 39] that
and the level of DNA fragmentation are calcu- sperm nuclei with red fluorescence by AOT cor-
lated. The sperm fraction with the most inten- responded to those that stain dark-purple with
sive green colour is called high DNA stainable TB, whereas sperm with green fluorescence cor-
(HDS). It is still unclear precisely which mech- respond to those that stain light blue with TB.
anisms and types of DNA damage are lying The number of dark-purple-stained cells as per-
behind DFI and HDS; however, it is believed centage of all cells is assessed by microscopic
that DFI are related to the percentage of sperm examination. The results of the TB test correlate
with both SSB and DSB and/or impairment of well with the results of the SCSA and TUNEL
normal protamination. HDS is thought to rep- assay [40] and the test could be amenable of clin-
resent immature spermatozoa. One of the ben- ical application also because of its relative sim-
efits of SCSA is adhering to a standardised plicity and inexpensiveness.
protocol that minimises inter-laboratory varia-
tion [37].
These three methods are to be considered as 12.5.6 Sperm Chromatin Dispersion
major tests applied for clinical evaluation of
sperm DNA integrity. However, other potential Sperm chromatin dispersion (SCD) test is based
techniques can be mentioned. on the principle that sperm with fragmented DNA
fails to produce the characteristic halo of dis-
persed DNA loops that are observed on sperm
12.5.4 Acridine Orange Test with non-fragmented DNA, when mixed with
aqueous agarose following acid denaturation and
Acridine orange test (AOT) is based on exactly removal of chromatin nuclear proteins [41]. This
the same principle as the SCSA (see above). relatively simple procedure can be performed
However, the evaluation of the proportion of either with fluorescence or Diff-Quick staining,
cells with denaturated DNA is based on a micro- and consequently, the SCD can be assessed either
scopic examination of a smear. The main advan- by fluorescence microscopy or by bright field
tage is its simplicity and there is no need of microscopy. Approximately 500 cells are, after
expensive equipment. However, due to a low initial denaturation and staining procedure, eval-
number of cells which are scored, unpredictable uated under the microscope and the halo size of
artifactual of acridine orange on a glass surface each cell is evaluated by the relative parameter:
because of its critical equilibrium staining and area of the halo divided by that of the whole
the subjectivity in the assessment of the level of nucleoid. Since some sperm cells may have
intra- and inter-laboratory variation are signifi- different nuclear sizes, this relative parameter
cantly higher than the case is for the SCSA assay. avoids the distortion that would result if abso-
Such conditions could preclude use of AOT for lute sizes were considered. A complete kit
reliable clinical diagnosis and prognosis of a (Halosperm, INDAS Biotech, Madrid, Spain) has
semen sample. been developed.
12.5.7 Oxidised Deoxynucleoside 12.5.10 Evaluation of Anti- or

Pro-Apoptotic Proteins
Oxidised deoxynucleoside, 8-oxo-7,8-dihydro-
2¢deoxyguanosine (8-oxodG) level is considered Evaluation of anti- or pro-apoptotic proteins.
to be a biomarker of oxidative stress in sperm Antibodies against anti- (e.g. Bcl-xL) and pro-
which could also have negative effects on sperm apoptotic (e.g. caspase-3 and Fas) can be used for
function [42–44]. The levels of 8-oxodG and dG evaluation of the percentage of abortive apoptotic
in nuclear sperm DNA are chemically determined spermatozoa. Quantification can be done by use
by high pressure liquid chromatography (HPLC) of microscopy or flow cytometry [48].
after DNA extraction from nuclei isolated from Although these tests differ, both as we con-
the sample and digestion to the deoxynucleosides sider the technical principle but also what they
by means of nuclease P1 and alkaline phos- specifically are measuring, a number of investi-
phatase. The level of 8-oxodG is reported per 105 gations have shown a moderate association when
unoxidized dG. Recently, the formation of the same sample is assessed by use of two or more of
8-oxodG base lesion can also be measured using these tests [40] (Table 12.1). However, the corre-
fluorescently labelled anti-8-oxodG antibodies lations coefficients between the results of differ-
and their abundance can be assessed by measuring ent tests are usually of a magnitude not higher
the fluorescence level using flow cytometry [45]. than 0.4–0.6. To a certain extent, all these tests
may complement each other as each test could
address a particular facet of the complex pro-
12.5.8 Chromomycin A3 Staining
cesses involved in sperm nuclear packaging, and
consequently, in the formation and detection of
Chromomycin A3 (CMA3) staining is considered
sperm DNA breaks [49]. Finally, with the excep-
as a test assessing the packaging quality of the
tion of SCSA, each test requires a definitive pro-
chromatin in spermatozoa and may allow indirect
tocol propaedeutic to its standardisation.
visualisation of protamine deficiency [46]. It is
based on the ability of CMA3 to compete with
the protamines for binding to the minor groove of 12.6 Clinical Usefulness of Sperm
DNA. The fraction of CMA3 positive cells is
DNA Tests
commonly assessed by scoring 200 cells under a
fluorescence microscope.
Animal studies incontestably show that sperm
Furthermore, two other tests do not directly
DNA damage does impair normal embryo devel-
detect sperm DNA strand breaks, but detect ear-
opment and a healthy progress of pregnancy.
lier stages of the apoptotic cascade which ulti-
Whereas there is also no doubt that there is an
mately leads to DNA fragmentation.
association between the percentage of human
sperms with DNA damage and the fertility poten-
12.5.9 Annexin V-Binding Ability Assay tial of a couple, the question is what the clinical
usefulness of testing for sperm DNA integrity is?
Annexin V-binding ability assay is a method The literature does not give any unequivocal
based on the ability of Annexin V to bind to answer regarding this issue, the conclusions vary-
phosphatidylserine (PS), a well-established early ing from “very useful” to “not useful” or more
apoptosis marker [47]. Sperm suspension is often “more research is needed”. Some of the
washed and, thereafter, centrifuged. Annexin V confusion regarding the role of sperm DNA integ-
labelled with fluorescein isothiocyanate (FITC) rity testing in the clinical set-up is related to lack
is added to the pellet and subsequently a smear is of standardisation in methodology applied for
made and evaluated with a fluorescence microscope assessment of sperm DNA integrity (see above),
for assessment of the percentage of green- but also limited power of many studies due to a
fluorescing sperm (Annexin V positive) cells. low number of patients included. Furthermore,
Table 12.1 Correlation levels between results of different sperm DNA fragmentation assays from selected studies
(with >50 men)
General population Subfertile Correlation
Techniques or healthy donors, n men, n coefficient References
SCSA vs. Comet assay
SCSA vs. Comet, neutral pH 8 80 N.S. [116]
SCSA vs. Comet, alkaline pH 13 80 N.S. [116]
SCSA vs. Comet, alkaline pH 12.1 55 0.31 [117]
SCSA vs. TUNEL assay
SCSA vs. FlM-TUNEL 7 60 0.90 [118]
SCSA vs. FlM-TUNEL 25 55 0.50 [119]
SCSA vs. FCM-TUNEL 24 96 0.41 [112]
SCSA vs. FCM-TUNEL 666 0.56 Toft (personal
communication)
SCSA vs. FCM-TUNEL 58 0.27 [117]
SCSA vs. AOT
SCSA vs. AOT 185 N.S. [120]
SCSA vs. AOT 7 60 N.S. [118]
SCSA vs. SCD 7 60 0.87–0.98 [118]
SCSA vs. toluidine blue 63 79 0.47 [59]
FCM- vs. FlM-TUNEL assay
FCM-TUNEL vs. FlM-TUNEL 66 0.72 [121]
FCM-TUNEL vs. FlM-TUNEL 68 0.94 [122]
TUNEL assay vs. Comet assay
FCM-TUNEL vs. Comet 42 21 0.56 [123]
(alkaline pH 10)
FCM-TUNEL vs. Comet 58 N.S. [117]
(alkaline pH 12.1)
TUNEL assay vs. CMA3
FlM-TUNEL vs. CMA3 61 0.76 [62]
FlM-TUNEL vs. CMA3 132 0.53 [124]
FCM TUNEL vs. CMA3 39 28 0.83–0.96 [24]
TUNEL assay vs. SCD
M-TUNEL vs. SCD 30 60 0.6–0.9 [125]
FlM-TUNEL vs. SCD 7 60 0.9 [118, 126]
SCD vs. CMA3 78 r = 0.29 [126]
8-oxodG vs. TUNEL assay 94 0.25 (0.77)a [24]
Levels of correlation: £0.15 not correlated, 0.15–0.39 poorly, 0.4–0.45 moderately
AOT acridine orange test; CMA3 chromomycin A3; SCD sperm chromatin dispersion test; SCSA sperm chromatin
structure assay; TUNEL terminal deoxynucleotidyl transferase dUTP nick end labelling; M-TUNEL TUNEL assay,
bright field microscopy; FlM-TUNEL TUNEL assay, fluorescence microscopy; FCM-TUNEL TUNEL assay, flow
cytometry; N.S. not statistically significant
a
Calculated for Percoll high density separated semen fraction
the majority of reports focusing on ART do not 1. Is sperm DNA integrity testing a good predic-
discriminate between different methods (IUI; tor of the chance of achieving spontaneous
IVF; ICSI) applied for achieving pregnancy. pregnancy?
From a clinical point of view, the following ques- 2. Can it be used for selection of most appropri-
tions seem relevant to ask: ate method of ART?
3. How robust are the tests in terms of intra- These results based on couples from general
individual variation? population have been confirmed in a more recent
Below, we will try to give answer to these study of 127 men from infertile couples where
questions with focus on summarising of the exist- female factors contributing to the infertility prob-
ing knowledge rather than an extensive review of lem were excluded and 137 men with proven fer-
the literature. tility. Using SCSA as the method for assessing
sperm DNA and the group of men with DFI <10%
as reference group, the risk of being infertile was
12.6.1 Sperm DNA Integrity increased when DFI >20% (OR 5.1) in men with
and Chance of Spontaneous normal standard semen parameters, whereas if
Pregnancy one of the WHO parameters were abnormal, the
OR for infertility was increased already at DFI
Very few studies have addressed the issue of above 10% (OR 16) [54].
sperm DNA integrity in relation to fertility in the The above-mentioned study not only indicated
general population. The two major reports within that SCSA can be used in prediction of the chance
this area, one carried out in USA (the Georgetown of spontaneous pregnancy, independently of the
study, 165 couples) and the other carried out in standard sperm parameters (Table 12.2), but since
Europe (the Danish first pregnancy planners study, DFI above 20% was found in 40% of men with
215 couples), have been based on the use of otherwise normal standard parameters [54], it
SCSA. Both demonstrated that in couples from seems that in almost half of the cases of so-called
the general population, the chance of spontaneous “unexplained” infertility, sperm DNA defects are
pregnancy, measured by the time-to-pregnancy at least a contributing factor to the problem.
(TTP), decreases when DFI exceeded 20–30% Interestingly, a cut-off fraction around 20% of
[50, 51]. If the DFI was more than 30%, TTP defective sperm discriminating between normal
tended to become infinite and the chances of healthy donors and male factor infertile men has
spontaneous pregnancies were quite negligible. been found by two studies using the TUNEL
Stratifying the population into two groups, below assay evaluated by flow cytometry [28, 55].
and above a DFI threshold at 30%, the probability Also, other SCSA-based studies have shown
of pregnancy for the group with DFI <30% was higher DFI in men from infertile couples as com-
statistically significantly higher than that for the pared to fertile men or to donors [56, 57]. This is
group with DFI >30%. These two in vivo studies also true for reports based on use of other tech-
showed that the pregnancy rates are significantly niques as Comet assay [58], TUNEL [55], TB
higher for the group with DFI below the thresh- [59], 8-oxodG [60], AO [61], SCD [41] and CMA3
olds of 30% [52]. However, whereas DFI above [62]. However, although the vast majority of pub-
30% was highly predictive of low chance of spon- lished data of such difference was found, this is not
taneous pregnancy, in a significant proportion of true for all of them [63]. Furthermore, generally,
cases with DFI < 30% the couples were unable to perhaps due to a significantly less degree of stan-
conceive. Thus, these data indicated that sperm dardisation, the “threshold” values for infertility
DNA integrity testing, or at least SCSA, is a better differ significantly, from study to study, when
marker of “infertility” than of “fertility” in vivo. other techniques than SCSA have been applied for
In the above-mentioned population of Danish assessment of sperm DNA integrity [63].
first pregnancy planners [50], the likelihood of When evaluating infertile men (Table 12.3), it
pregnancy occurring in a single menstrual cycle has generally been noted that dyspermic men
was inversely associated with the level of 8-oxodG have also a higher fraction of chromatin defective
[53]. These data suggest that sperm DNA oxidative sperm, supporting the intuitive notion that, in the
damage influences fecundity, corroborating the presence of spermatogenic defects leading to
result of the previous SCSA analysis and reinforc- oligoasthenoteratozoospermia, the chance of an
ing the notion that oxidative DNA damage can play increased chromatin damage is also more proba-
a major role in the genesis of DNA breaks [18]. ble. This has been noted in a variety of studies
Table 12.2 Correlation levels between sperm DNA fragmentation and the WHO parameters of concentration, motility,
and morphology from selected studies (with >150 men)
General population Subfertile
Technique or healthy donors, n men, n Concentration Morphology Motility References
SCSA
SCSA 277 −0.31 −0.38 [100]
SCSA 215 −0.23 −0.25 [50]
SCSA 278 −0.12 [127]
SCSA 171 −0.26 −0.43 (−0.53a) [37]
SCSA 278 −0.12 −0.25 (−0.38a) [37]
SCSA 201 −0.22 −0.46 −0.56 [120]
SCSA 249 −0.36 −0.41 [70]
SCSA 707 N.S. −0.17 −0.23 [94]
SCSA 374 N.S. [69]
SCSA 209 N.S. −0.22 −0.19 [106]
SCSA 2,586 −0.22 −0.40 −0.61 [128]
SCSA 279 −0.41 −0.35 −0.50 [57]
SCSA 175 Negative Negative Negative [128]
M-TUNEL 332 −0.2 −0.2 [129]
Fl M-TUNEL 150 N.S. Negative Negative [91]
Fl M-TUNEL 262 N.S. −0.44 −0.60 [130]
Fl M-TUNEL 167 N.S. [131]
Fl M-TUNEL 167 0.12 [131]
FCM-TUNEL 298 −0.18 −0.34 −0.12 [92]
FCM-TUNEL 1,633 N.S. N.S. N.S.a [122]
Comet assay 257 −1.42 −0.05 −0.43 [132]
(neutral)
SCD 622 −0.13 −0.32 −0.28 [133]
AOT
AOT 187 −0.58 −0.48 −0.42 [134]
AOT 185 N.S. 0.18 N.S. [120]
AOT 234 0.21 [131]
AOT 234 0.20 [131]
Aniline blue 75 90 N.S. N.S. N.S. [90]
Levels of correlation: £0.15 not correlated, 0.15–0.39 poorly, 0.4–0.45 moderately
AOT acridine orange test; SCD sperm chromatin dispersion test; SCSA sperm chromatin structure assay; TUNEL termi-
nal deoxynucleotidyl transferase dUTP nick end labelling; M-TUNEL TUNEL assay, bright field microscopy; FlM-
TUNEL TUNEL assay, fluorescence microscopy; FCM-TUNEL TUNEL assay, flow cytometry; N.S. not statistically
significant
a
Evaluated by computer-assisted sperm analysis (CASA)
using the Comet assay [64], the TUNEL assay 12.6.2 Sperm DNA Integrity Testing
[65, 66], CMA3 and in situ nick translation [67], and ART
SCSA [56, 68–71], the SCD test [72], and toluid-
ine blue staining [40]. ART are widely used in cases when the chance
A higher fraction of sperm with DNA frag- of spontaneous pregnancy is considered as
mentation emerged also in men with abnormal non-existing or very low. If the infertility prob-
protamine 1/protamione 2 ratio, implying a lem of the couple is unexplained or the impair-
protective role of protamines against sperm DNA ment of semen quality is considered as “mild”,
damage [73]. the IUI may be the first choice treatment. If the
Table 12.3 Selected studies (with >100 individuals) reporting prevalence of DNA defective sperm in infertile men as
compared to normal controls
Technique Controls, n Subfertile men, n Notes References
SCSA 165 115 DFI significantly higher in infertility men (24.4%) [51]
than in controls (13.7 ± 7.2%)
SCSA 13 88 DFI significantly higher in infertile men [56]
(23.1 ± 1.7%) than in controls (10.8 ± 1.6%)
SCSA 16 92 DFI significantly higher in infertile men (28%) [71]
than in controls (15%)
SCSA 13 101 DFI significantly higher in infertile men [135]
(22.0 ± 1.5%) than in controls (10.8 ± 1.8%)
SCSA 100 200 DFI significantly higher in infertile men (20.2– [136]
23.8%) than in controls (5.2–6.6%)
SCSA 22 279 DFI significantly higher in infertile men (range [57]
3.8–90.8%) than in controls (range 6.43–25.74%)
SCSA 137 127 DFI significantly higher in infertile men (23%) than [54]
in controls (12%)
M-TUNEL 20 236 Fraction of TUNEL positive sperm significantly [82]
lower in controls (0.3 ± 0.4%) than in infertile men
FlM-TUNEL 23 87 Fraction of TUNEL positive sperm significantly [137]
(11.3 ± 4.9%)
FlM-TUNEL 49 61 Fraction of TUNEL positive sperm significantly [62]
(30.0 ± 7.1%)
FCM-TUNEL 47 66 Fraction of TUNEL positive sperm significantly [28]
(40.9 ± 14.3%)
Cut-off at 20%
FCM-TUNEL 25 194 Fraction of TUNEL positive sperm significantly [55]
(29.5 ± 18.7%)
Cut-off at 19.25%
CMA3 49 61 Fraction of CMA3 positive sperm significantly [62]
higher in infertile men (31.5 ± 7.1%) than in controls
(6.3 ± 1.8%)
Aniline blue 75 90 Fraction of condensed sperm significantly higher in [90]
control (78.0 ± 19.0%) than in infertile men
(55.0 ± 12.0%)
Toluidine blue 63 79 Controls have a significant higher fraction on normal [59]
toluidine blue light cells than infertile men (51 ± 21
vs. 32 ± 20%) and significantly less abnormal
toluidine blue dark cells (24 ± 13 vs. 47 ± 24%)
Cut-off for infertility 45% toluidine blue dark cells
8-oxodG 54 60 Levels of 8-oxodG significantly higher in infertile [60]
men than in controls (10.0 vs. 4.8/105
deoxyguanosines)
CMA3 chromomycin A3; DFI DNA fragmentation index, evaluated by SCSA; SCSA sperm chromatin structure assay;
TUNEL terminal deoxynucleotidyl transferase dUTP nick end labelling; M-TUNEL TUNEL assay, bright field micros-
copy; FlM-TUNEL TUNEL assay, fluorescence microscopy; FCM-TUNEL TUNEL assay, flow cytometry; 8-oxodG
8-hydroxydeoxyguanosine level evaluated by high-performance liquid chromatography
IUI treatment is unsuccessful or there is a serious within the range 0–20%, but started to decrease
female pathology, as, for example, occlusion of when the value exceeded the 20% level. This
the fallopian tubes, those couples may go directly result is well in accordance with the other SCSA
for IVF. In cases of poor semen quality and/or data based on in vivo conception, those based on
unsuccessful standard IVF, ICSI becomes the general population or couples with unexplained
method of choice. infertility (see above). In contrast, no correlation
Although the panorama of ART includes a num- was found between SCD results and pregnancy
ber of potentially powerful techniques, the results outcome in 100 Spanish IUI-patients [77].
are still somewhat disappointing and two third or
more of the treatment do not lead to a child birth. 12.6.2.2 IVF and ICSI
One of the reasons behind this problem is the low Earlier studies based on quite limited numbers of
predictive value of standard sperm parameters in couples indicated that DFI above 27% as mea-
relation to ART, which become quite meaningless sured by SCSA could be used as a cut-off value
in the case of ICSI. Therefore, better markers are for infertility, not only in vivo but even in vitro. It
needed in order to avoid ART treatment with an a was reported that in couples with a DFI >27%, no
priori low chance of success, but also to give the pregnancy could be obtained, regardless of the
most simple and least invasive type of therapy. type of ART applied [78, 79]. However, in 2004
It should be also kept in mind that the repro- three independent SCSA reports demonstrated
ductive end points in relation to ART may differ that a DFI level above 27% was indeed compati-
according to whether the fertilisation is achieved ble with pregnancy and delivery after both IVF
in vivo or in vitro. The latter scenario allows for and ICSI [70, 80, 81] meaning that use of ART
evaluation of not only pregnancy or birth rate, but compensates for poor sperm chromatin quality.
also of parameters as fertilisation, embryo quality Our own study [76] based on 388 IVF and 223
and early miscarriage. ICSI cycles has shown no statistically significant
difference between the outcomes of ICSI and ICSI
12.6.2.1 Intrauterine Insemination when DFI of 30% was used as a threshold. However,
Numerous studies have shown association in the DFI >30% group, the results of ICSI were
between level of indices of sperm DNA damage significantly better than those of IVF, no such dif-
and IUI pregnancy rate. The pioneering report by ference being seen for lower levels of sperm DNA
Duran et al. [74] has shown in 154 couples lack of damage. When comparing ICSI to IVF (reference),
pregnancy when DFI, as measured by the TUNEL the odds ratios (ORs) for biochemical pregnancy
assay on prepared semen, was above 12%. Similar (BP), clinical pregnancy (CP) and delivery (D)
findings have been reported by Saleh et al. [75] were 3.0 (95% CI: 1.4–6.2), 2.3 (5% CI: 1.1–4.6)
who performed a small study where 12 of 19 cou- and 2.2 (95% CI: 1.0–4.5), respectively.
ples had a DFI value as measured by SCSA above These data are in agreement with other previ-
28% and none of these couples achieved a preg- ous smaller reports using TUNEL or Comet
nancy. Boe-Hansen and colleagues used the assays, showing that sperm DNA damage is more
SCSA in a study on 48 IUI-couples. Only two of predictive in IVF and much less so in ICSI
the couples had a DFI value above 30%, and none [82, 83]. Also a very recent study of Simon et al.
of them obtained a pregnancy [69]. In our own [84], using Comet assay, has shown that the dif-
report based on 387 IUI cycles, DFI as assessed ference in percentage of sperms with impairment
by SCSA was shown to be a predictor of fertility of DNA integrity, when comparing successful
independent of other sperm parameters [76]. and unsuccessful treatment, is much more clear
While the proportion of children born per cycle for IVF than for ICSI. In contrast, one single
was 19.0% when the DFI value was below 30%, study reported that DFI threshold values were not
those with a DFI value above 30% only had a valid for prediction of IVF outcome [85].
take-home-baby rate of 1.5%. The chance of IUI However, this study did not discriminate between
pregnancy was unaffected by the variation of DFI standard IVF and ICSI.
Whereas several studies have shown some rates between high and low DFI groups as mea-
association between the ART outcome and DFI, sured by SCSA [79, 80], others have shown that
the other parameter measured by SCSA, HDS, has sperm DNA damage is negatively correlated with
not yet been shown of any clinical significance. embryo quality after IVF and ICSI [92, 93]. Two
The data regarding the relationship between studies have also reported that men with high lev-
sperm DNA fragmentation and fertilisation rates els of DNA fragmentation are at increased risk of
after IVF and ICSI are conflicting. Ahmadi and low blastocyst formation compared to men with a
Ng in a mouse model demonstrated that despite low DFI [94, 95].
DNA damage, a spermatozoa was able to fertilise
an oocyte [86]. Also, human studies have shown
that the fertilisation rate is the same for men with 12.6.3 Intra-Individual Variation
high number of sperm with damaged DNA as for in Sperm DNA Integrity
those with a low proportion of such cells, when
using SCSA [76, 79, 87] or other sperm DNA One of the limitations in use of standard semen
integrity assays [88–90]. On the other hand, the parameters in the daily clinical routine is the con-
presence of damaged sperm DNA was shown to siderable intra-individual variation with respect
have a significant inverse relationship with fertili- to sperm concentration, motility and morphology
sation in other studies [82, 91, 92]. [96–98]. Even DFI, as measured by use of SCSA,
Although fertilisation may be independent of seems to be a subject of such variation. Thus, for
sperm DNA integrity, it has been suggested that this parameter, CVs of 21–29% have been found
the post-fertilisation development of the pre- [99–101]. In this subgroup, a switch of DFI to a
embryo can be impaired by such incomplete or higher level may have implications for the selec-
aberrant sperm DNA repair by the oocyte leading tion of the ART treatment, since the DFI of 30%
to implantation failure, early miscarriages or in was found to represent a clinically relevant cut-
the worst cases diseases in the offspring [6, 22]. off level [76].
The human data regarding pre-embryo develop- Comparison between TUNEL and SCSA indi-
ment in relation to sperm DNA damage is also cated a less variation over the data collection
conflicting. While some authors have reported period for the latter method with a DFI within-
similar cleavage stage embryo developmental subject CVs of 47.4 and 22.3%, respectively [102].
Fig. 12.1 A two-step hypothesis of origin of human sperm DNA damage according to Aitken and De Iuliis [18]. The
figure does also visualise where in the male reproductive system the damage occurs
In another TUNEL study, the CV sperm DFI Interestingly, for PCBs, an ethnic difference in
ranged from 12.9 to 43.9%, whereas parallel susceptibility to the effect of the exposure has been
measurements on cell counts showed within- seen. Thus, high PCB levels in serum were associ-
donor CVs ranging from 16.7 up to 63.2% [103]. ated with increase in DFI in Caucasian, but not in
Although even DFI seems to be a subject of Inuit men [27]. Whether this difference is due to
some intra-individual variation, the question is genetic diversity or to other lifestyle factors, for
whether it is of any clinical significance? Our example, food intake, is not known. Knowledge to
own data (Oleszczuk, personal communication) the link between lifestyle/environment and sperm
have shown that among 616 men referred due to DNA integrity is interesting from a biological
infertility, 85% remained in the same category of point of view; the clinical implications are so far
DFI; £30% or >30% when comparing first and limited. These data are summarised in Table 12.4.
second SCSA test. This implies that only in 15% Furthermore, some studies evidenced that the
a repeated analysis implied a reclassification of association between sperm chromatin damage
the SCSA-based prediction of fertility potential and environmental exposure emerged if absent
of the patient. or resulted exacerbated if already present only
The biological background for the intra- in selected subpopulations characterised by a
individual variation in DFI is not completely particular gene polymorphism, thus implying that
known, although some lifestyle-related factors a suspected gene–environment interplay can be at
may be of an importance (Fig. 12.1). work [107–109].
12.7 The Effects of Lifestyle 12.8 Sperm DNA Integrity

and Environmental and Cancer
Factors
Males treated for cancer represent a potential
Several lifestyle- and environment-related factors source of worry in relation to sperm DNA integ-
have been linked to increase in percentage of rity. It could be expected that cancer therapy –
sperms with impairment of DNA integrity. both irradiation and cytotoxic drugs – might
A number of studies have shown positive associ- introduce sperm DNA defects, which could be
ation between the length of the abstinence period transmitted to the offspring, mainly if IVF or
and both SCSA DFI [100], although the correla- ICSI are used as methods of conception.
tions found were usually weak to moderate. The available data indicate that both adult and
Although it might be expected that cigarette childhood cancer per se can lead to a slight
smoking implies an impairment of sperm DNA increase in DFI [110, 111]. Cancer treatment may
integrity, the data in the literature are quite con- add to the impairment of sperm DNA integrity,
troversial with some of the largest studies not and this effect, even if transiently, seems to be
showing such effect [104, 105]. So far there is most pronounced for the irradiation treatment
lack of information regarding other lifestyle fac- [25], whereas it is limited or not seen at all in men
tors as consumption of alcohol, caffeine or recre- given chemotherapy [112]. Sperm cryopreserva-
ational drugs. tion has, in some studies, been shown to imply an
Among environmental factors, the most con- increase in percentage of sperms with abnormal
sistent association between exposure and DFI has DFI [113], although even these findings have
been found for polychlorinated biphenyls (PCBs) been questioned by others [110]. Epidemiological
and phthalates, although some reports have indi- data show that in vitro conceived children of men
cated impairment of sperm DNA integrity related treated for cancer have no increased risk of
to air pollution, styrene, lead, pesticide and congenital malformations as compared to those
dichlorodiphenyldichloroethylene (p,p¢-DDE – a IVF/ICSI children not having a father with a his-
metabolite of DDT) exposure [26, 27, 106]. tory of malignancy.
Table 12.4 Epidemiologic studies of ambient chemical exposures and sperm DNA damage
Noxia Subjects Indicator of exposure Technique Association References
Polycyclic aromatic hydrocarbons (PAH)
42 Taiwan coke-oven Urinary 1-hydroxypyrene SCSA Positive [138]
workers levels
273 Chinese fertile and Urinary 1-hydroxypyrene TUNEL Positive [139]
620 infertile men levels assay (FCM)
Air pollution
266 Young Czech Ambient air pollution SCSA Positive [140]
monitoring
36 Young Czech Ambient air pollution SCSA Positive [26, 108]
monitoring
Plastics
Styrene
21 Danish farmers Urinary mandelic acid SCSA Positive [141]
unexposed levels
23 Danish reinforced
plastic workers
Styrene (mandelic acid 46 Italian plastics Urinary mandelic acid Comet assay Positive [142]
urinary concentration) workers levels (alkaline)
27 Unexposed controls
Phthalates
168 US men from Phthalate metabolites Comet assay Positive [143]
infertile couples urinary concentration (neutral)
379 US men from Phthalate metabolites Comet assay Positive [105]
infertile couples urinary concentration (neutral)
234 Young Swedish Phthalate metabolites SCSA N.S. [144]
from general population urinary concentration
300 Indian men (200 SCSA Positive [136]
infertile, 100 fertile)
from infertility clinics
Acrylonitrile 30 Chinese acrylonitrile Ambient air monitoring Comet assay Positive [145]
exposed workers of acrylonitrile in the (alkaline)
30 Non-exposed controls workplace
Persistant organic pollutants (POPs)
DDT
212 US men from Serum p,p¢-DDE levels Comet assay N.S. [146]
infertile couples (neutral)
176 Swedish fishermen Serum p,p¢-DDE levels SCSA N.S. [104]
514 European and 193 Serum p,p¢-DDE levels SCSA N.S. [27]
Inuit men from general
population
452 European and 200 Serum p,p¢-DDE levels TUNEL N.S. [48]
Inuit men from general assay (FCM)
population
116 Mexican residents in Serum p,p¢-DDE levels Aniline blue Positive [147]
malaria areas
209 South Africans Serum p,p¢-DDE levels SCSA Positive [106]
living in malaria areas
PCBs
212 US men from Serum concentration of Comet assay N.S. [146]
infertile couples congeners PCB 118, PCB (neutral)
138, PCB 153
176 Swedish fishermen Serum concentration of SCSA Positive [104]
PCB congener 153
(continued)
Table 12.4 (continued)
Noxia Subjects Indicator of exposure Technique Association References
514 European and 193 Serum concentration of SCSA Positive (in [27]
Inuit men from general PCB congener 153 Europeans
population only)
452 European and 200 Serum concentration of TUNEL Positive (in [48]
Inuit men from general PCB congener 153 assay (FCM) Europeans
population only)
Hexachlorobenzene 212 US men from Serum concentration of Comet assay N.S. [146]
(HCB) infertile couples HCB (neutral)
Pesticides
169 Danish pesticide Interview SCSA N.S. [148]
spraying farmers
82 Danish not spraying
farmers
33 Mexican agricultural Urinary concentration of SCSA Positive [149]
workers diethylthiophosphate
(DETP)
21 Chinese pesticide Ambient air monitoring Comet assay Positive [123]
factory workers of fenvalerate in the (alkaline)
23 Internal controls workplace and TUNEL
19 External controls assay (FCM)
260 US men from Urinary metabolite Comet assay Positive [150]
infertile couples concentrations of (neutral)
chlorpyrifos
(3,5,6-trichloro-
2-pyridinol) and
carbaryl (1-naphthol)
207 US men from Urinary concentration of Comet assay Positive [151]
infertile couples pyrethroid metabolites (neutral)
3-phenoxybenzoic acid
and cis- and trans-3-(2,
2-dichlorovinyl)-2,2-
dimethylcyclopropane
carboxylic acid
54 Mexican agricultural Interview ISNT assay Positive [107]
workers
16 Chinese pesticide Ambient air monitoring TUNEL Positive [152]
factory workers 30 of carbaryl in the assay (FCM)
internal and external workplace
controls
Metals – lead
362 European Blood Pb concentration SCSA Positive [153]
Pb-exposed workers
141 Non-exposed men
68 Mexican fertile men Pb concentration in NCD Positive [154]
seminal fluid, spermato-
zoa, and blood
80 Taiwan battery plant Blood Pb concentration SCSA Positive [155]
workers
DDE p,p¢ dichlorodiphenyldichloroethylene; DDT 1,1,1 trichloro-2,2-bis(chlorodiphenyl)ethane; FCM flow cytometry;
ISNT in situ nick translation assay; NCD nuclear chromatin decondensation test; PCBs polychlorinated biphenyls;
SCSA sperm chromatin structure assay; TUNEL terminal deoxynucleotidyl transferase-mediated dUTP nick end label-
ling; N.S. not statistically significant
related to the outcome of IVF than that of

12.9 Clinical Recommendations ICSI.
(d) In several “meta analyses” and review papers,
Whereas the testing of sperm DNA integrity has
no discrimination is made between the results
proved to be an important tool both in reproduc-
obtained by different methods for sperm DNA
tive and in environmental research, its role in the testing, SCSA, Comet assay and TUNEL.
daily clinical practice is still widely debated. So far, clinical threshold values have only
Thus, the Practice Committee of the American been established for the SCSA, perhaps because
Society for Reproductive Medicine [114] con- the methodology has been highly standardised,
cluded: “Sperm DNA damage is more common which is witnessed by very low intra- and
in infertile men and may contribute to poor repro- inter-laboratory variation [99]. When evaluating
ductive performance. However, current methods the clinical applicability of this method, the poor
for evaluating sperm DNA integrity do not predictive value of standard semen parameters
reliable predict treatment outcomes, and no treat- should also be taken into consideration.
ment for abnormal DNA integrity has proven Furthermore, although the SCSA is based on use
clinical value”. Furthermore, “At present, the of expensive equipment – flow cytometer – the
results of sperm DNA integrity testing alone do analysis can be centralised. We, therefore,
not predict pregnancy rates achieved with inter- consider the use of SCSA testing as clinically
course, IUI, IVF and ICSI”. valuable in following cases:
On the other hand, in an Editorial in Molecular 1. Unexplained infertility and normal standard
Human Reproduction, Barratt [115] claims that semen parameters combined with mild or
“… there is an urgent need to develop new assess- treatable female subfertility.
ments of male reproductive potential and testing (a) DFI above 20% can, at least partly, explain
of DNA and its packaging in the human sperma- the infertility problem of the couple.
tozoa is likely to be a very important tool in the (b) If the DFI is above 30%, the couple can
armamentarium. Importantly, there are already go directly to IVF/ICSI.
considerable data to support the clinical use of (c) If the DFI is below 30% (at least two sam-
DNA damage assays”. ples), treatment of the female in order to
There are several reasons for the confusion improve the chance of the couple to achieve
regarding the clinical usefulness of sperm DNA spontaneous pregnancy can be tried.
integrity testing: 2. Mild impairment of semen quality (e.g. only
(a) The vast majority of studies are based on one of the standard parameters: concentration,
small patient cohorts and are, therefore, motility or morphology below the reference
underpowered. range) and relatively short (<2 years) history
(b) Furthermore, most available reports focus on of infertility.
difference between fertile and infertile sub- (a) DFI below 10% and the female partner
jects in percentage of sperms with DNA without any pathology and below the age
damage rather than on predictive value of of 35 years: achieving spontaneous preg-
these tests in a clinical set-up. nancy can be tried (for 1/2–1 year).
(c) Uniform criteria for selection of patients for (b) DFI below 10% and the female partner
these studies are lacking. Thus, in many with treatable subfertility: treatment of
studies, no consideration is taken to the pos- the female in order to improve the chance
sible contribution of the female partner. of the couple to achieve spontaneous
Furthermore, in order to reach higher number pregnancy can be tried.
of patients, couples receiving different types 3. A couple referred for IUI and fulfilling
of ART are pooled together. As indicated criteria – including standard semen analysis –
above, sperm DNA testing seems to be the for this treatment.
most powerful predictor for in vivo fertility, (a) DFI above 25–30% (in two samples): the
and among the two in vitro methods, more couple should go directly for IVF/ICSI.
Fig. 12.2 Schematic flow chart illustrating clinical guidelines for use of SCSA DNA fragmentation index (DFI) analysis
4. A couple referred for standard IVF and fulfill- d ifferent clinical situations and treatment
ing criteria – including standard semen analy- modalities. Since the techniques for measuring
sis – for this treatment. sperm DNA damage are not completely overlap-
(a) DFI above 30% (in two samples): ICSI ping, it cannot be excluded that highest predictive
should be considered. value can be obtained by use of a panel of well-
5. These recommendations are summarised in characterised and standardised methods.
Fig. 12.2. From the biological point of view, we still
6. So far, it seems that the SCSA analysis of need more information about the mechanisms
sperm DNA integrity, to be clinically useful, behind sperm DNA damage and an improved
should be performed on raw and not on pre- methodology to discriminate between the underlying
pared semen. pathologies. This will enable us to develop tar-
geted, cause-related therapies, so far an almost
non-existing option within the field of male
12.10 Future Perspectives subfertility.
Furthermore, we need to learn more about the
The clinical role of different methods for testing implications of sperm DNA damage, not only in
sperm DNA integrity needs to be established. relation to the fertility, but also as considers pos-
Standardisation of the methodology, mainly for sible transmission of wrong genetic information
techniques other than SCSA, is needed and so are to the offspring. This knowledge seems of par-
clinical studies based on sufficiently large and ticular interest in the current era of ICSI, which is
well-defined patient cohorts. This will, hopefully, bypassing the natural biological mechanisms pre-
lead to defining relevant threshold values for venting a defective sperm in fertilising an egg.
18. Aitken RJ, De Iuliis GN. On the possible origins of

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The Emerging Role of the Sperm
Epigenome and its Potential 13
Role in Development
Sue Hammoud and Douglas T. Carrell
Abstract
The sperm genome has traditionally been thought to lack chromatin
structure significant to affect embryonic development, since during sper-
matogenesis nucleosomes are widely replaced by protamines, which are
believed to silence the genome, and the sperm DNA was known to be
hypermethylated in comparison to the egg. The notion of an irrelevant
sperm epigenome has been widely challenged due to many recent reports
that suggest that sperm chromatin is actually poised similar to an embry-
onic stem cell, a finding that has been reported in the germline of many
organisms. The significance of the mature sperm cellular epigenome is
unknown; however, one may foresee two potential roles: either a role in
developing embryo or a reminiscent memory of the spermatogonial stem
cell with no significance beyond ensuring proper sperm differentiation and
maturation. If these marks do help guide the embryo, then perturbations to
the epigenome may have implications on embryo quality, likelihood of
maintaining a pregnancy, or disease onset later in life.
Keywords
Epigenetics • Histone • Protamine • Imprinting • Methylation • Embryo
quality
13.1 Introduction:
The Significance
of the Cellular Epigenome
Epigenetic modifications on the DNA sequence

(DNA methylation) or on chromatin-associated
D.T. Carrell (*) proteins (i.e., histones) play an important role in
Andrology and IVF Laboratories, Departments of the regulation of gene expression and are essential
Surgery (Urology), Obstetrics and Gynecology and
Physiology, University of Utah School of Medicine,
for normal mammalian development. DNA meth-
Salt Lake City, UT, USA ylation usually occurs at CpG dinucleotides and is
e-mail: [email protected] commonly associated with gene silencing [1, 2].

182 S. Hammoud and D.T. Carrell
Non-CpG methylation at gene bodies, repetitive data in humans suggest that the gametes of
DNA, and pericentromeric regions was initially subfertile patients are epigenetically altered
described in plants [3, 4], but has recently been [15–19]. These findings bolstered the concerns of
reported in mammalian embryonic stem cells [5] the health outcome of offspring conceived by IVF
and unicellular/multicellular organisms [6, 7]. and raise the question whether epigenetic abnor-
However, its significance is unknown. On the malities can be inherited.
other hand, posttranslational modifications such The notion of transgenerational epigenetic
as acetylation, methylation, and phosphorylation inheritance in mammals has been raised on
on the core nucleosomes, H2A, H2B, H3, and H4, numerous occasions; however, the two waves
promote gene activation or repression. The com- of genome-wide epigenetic reprogramming
bination of DNA methylation and histone modifi- occurring during embryogenesis and gametogen-
cations refers to the cellular epigenome. esis [26–28] may limit its likelihood. Although
The cellular epigenome enables a dynamic embryonic reprogramming may potentially
and plastic cellular environment that facilitates explain the rare penetrance of epigenetic diseases,
the adaptation to cellular stress and environmen- some reports in mice have shown that the IAP
tal cues by modulating chromatin structure [8, 9], elements (retrotransposan) upstream of the Agouti
without altering the genomic content, to induce locus escape reprogramming [29, 30]. The data
changes in gene expression. Altering the cellular from mice suggest that a few gene classes may
transcriptome is a multifaceted process that escape reprogramming and the epigenome may
requires first the recruitment of specific transcrip- not be completely erased and reestablished from
tion factors and chromatin-modifying enzymes to a clean slate. Without a clear understanding of the
amend the histone code and/or DNA methylation extent of reprogramming, the safety and the risk
profile to allow gene promoters to become more assessment for transgenerational epigenetic
or less accessible for transcriptional machinery. inheritance for IVF patients is uncertain.
The role of the cellular epigenome has been
increasingly highlighted and has been implicated
in many cellular and developmental processes 13.2 Chromatin Remodeling
such as reprogramming (preimplantation embryos During Spermatogenesis
and primordial germ cell (PGC)), cellular differ-
entiation, imprinting, X-chromosome inactiva- During postmeiotic maturation of male haploid
tion, and genomic stability. germ cells, chromatin undergoes dramatic reorga-
Alterations in epigenetic profiles have been nization, including the exchange of canonical his-
seen in many complex diseases such as cancer tones for protamines (Fig. 13.1) [31]. This change
[10–12], obesity [13], and infertility [14–19]. in chromatin composition facilitates the compac-
Considerable attention in recent years has been tion of the paternal genome and protects the
devoted towards understanding the nature and genomic content from DNA damaging agents
the extent of the relationship between infertility during transit [32]. Several recent studies have
and epigenetic alterations [20–22]. These investi- shown that along with histone acetylation,
gations were prompted by the emerging data from histone ubiquination (upstream of H4) plays an
animal in-vitro fertilization (IVF) studies that essential role in the global histone replacement in
showed an increased incidence of the large off- spermatids. H2A/H2B ubiquination induces
spring syndrome (LOS) [14, 23]. LOS in animals H4K16 acetylation (H4k16ac) [33, 34], which is
is reminiscent to Beckwith–Wiedmann syndrome known to be a key step in the remodeling of chro-
(BWS) in humans, both resulting from imprint- matin to more open conformation [35, 36]. In
ing abnormalities [20, 24, 25]. Many have sug- addition to an elevated H4k16ac signal at the time
gested that the increased frequency of imprinting of histone to protamine transition, there was a
abnormalities in IVF patients in animals may be general wave of H3 and H4 hyperacetylation [37].
due to oocyte stimulation, gamete manipulation, Since acetylation adds negative charge to the
and/or embryo culture. However, more recent nucleosome, it has been postulated that H3/H4
13 The Emerging Role of the Sperm Epigenome and its Potential Role in Development 183
Fig. 13.1 Diagram highlighting the key and very well- replacement of the testis-specific histones by the transition
described histone modification events that facilitate the tran- proteins. Transition proteins are subsequently replaced by
sition of somatic histones to protamines. Somatic histones protamines 1 and 2, processed from a pool of RNP particles,
undergo site-specific methylation, phosphorylation, and undergo maturation before and during binding to the DNA
ubiquitination. H2A ubiquination is set by ring finger and replacement of the transition proteins. RING8 ring finger
protine 8, which later promotes H4 hyperacetylation. 8 protein; HAT histone acetyltransferase; Pygo2 pygopus
Hyperacetylation of H3/H4 relaxes the DNA coil to facilitate homolog 2 (HAT) (figure modified from Carrell et al. [63])
hyperacetylation loosens histone-DNA interaction many waves of histone modifications that appear
and enables the displacement of histone and to ensure proper chromatin remodeling and
replacement with transition proteins [38–40]. packaging.
These studies suggest that changes in chromatin Changes in histone ubiquination and acetyla-
composition are intricately governed by the tion patterns have significant ramifications on
spermatogenesis and reproductive potential. H2A s ubsequently replaced by protamines, which

in sperm is carried out by ring finger 8 (RNF8) signal the end of sperm chromatin reorganization.
and has been shown to have an important role in Protamines are highly basic sperm-specific nuclear
sex chromosome inactivation during meiotic pro- proteins that permit higher order of DNA
phase and in postmeiotic phases during the global packaging [48, 49]. Humans and mice express
histone replacement. However, RNF8−/− mice are two protamine proteins, protamine 1 (P1) and
proficient in meiotic sex chromosome inactiva- protamine 2 (P2), both of which are expressed in
tion and fail to replace histones with protamines, relatively equal quantities [50]. The P1–P2 ratio in
and the majority of the sperm cells undergo apop- human sperm donors of known fertility lies close
tosis [34]. To test the fertilization ability of the to 1 [51], ranging from 0.8 to 1.2 [32, 52], and
very few sperm cells that escaped apoptosis, IVF deviations from this ratio is inversely correlated
or intracytoplasmic sperm injection (ICSI) was with the amount of histones retained [53, 54].
used and showed that all microdrop samples Patients with either a low or high P1/P2 ratio had
resulted in no fertilization [34]. However, 5/28 significantly reduced semen parameter (lower
fertilized oocytes reached the blastocyt stage with sperm concentration, motility, and morphology),
ICSI [34]. These data suggest that histone replace- as compared to patients with normal P1/P2 ratios
ment is critical for normal sperm maturation, [52]. These patients also have increased sperm
escaping testicular and epididymal apoptosis, and DNA damage and reduced fertilization ability.
reproductive efficiency. The reduced fertilization resulting from the sperm
In addition to H2A ubiquination, histone abnormalities can be overcome by the use of ICSI;
acetylation (H3 and H4) is essential for the his- however, implantation and pregnancy rates were
tone to protamine transition. Enzymes involved significantly reduced [52, 55–58]. In mice, P1–P2
in H4 hyperacetylation in the round spermatid are knockout or happloinsufficency resulted in similar
unknown; nonetheless, two potential candidates findings to humans [59], which strongly suggests
expressed in the elongating spermatid at the time that protamine displacement may be an important
have shown to exhibit potent H4 acetylase activ- trigger for ensuring proper embryonic develop-
ity may be: testis-specific chromodomain protein ment, most likely through a secondary mechanism,
(CDY) and HAT (monocytic leukemia) such as epigenetic mechanisms.
4 (MYST4) [41, 42]. Similar to the RNF8−/− Alterations in chromatin remodeling at any
phenotype, mice treated with general histone point during the histone to protamine transition
deacetylase (HDAC) inhibitors resulted in results in severe male subfertility and poor
significantly reduced sperm counts and male embryo outcome following IVF [59–61]. The
infertility [43, 44]. On the other hand, H3 acety- reduction in reproductive efficiency resulting
lation in the elongating spermatid was shown to from improper sperm chromatin packaging
be Pygopus 2 (Pygo 2)-dependent and loss of prompts the question what is the true significance
Pygo 2 function leads to spermatogenesis arrest of chromatin reorganization: (a) protect the pater-
and infertility. The mechanism through which nal genome and prevent apoptosis, (b) complete
H3/H4ac elicits this chromatin exchange is spermatogenesis, as has traditionally been
unknown; however, a few studies have suggested thought, or (c) other possible reasons for the
the involvement of BRDT, a testis-specific pro- remodeling [32, 62, 63]. It has been proposed
tein. BRDT associates with acetylated H4 that the presence of protamines in the mature
nucleosomes [45–47] and mutating one of bro- sperm and their replacement at the time of fertil-
modomains (BD1) results in male sterility. ization may be important for reprogramming the
Furthermore, ectopic expression of BRDT in paternal pronucleus. Two potential mechanisms
somatic cells after treating cells with trichostatin through which this may be mediated are: first, the
A (a hyperacetylating agent) triggers a reorgani- DNA breaks resulting from protamine displace-
zation of chromatin [45–47]. ment may elicit the recruitment of the DNA
Histone hyperacetylation is thought to promote repair-mediated DNA demethylases (GADD45,
the incorporation of transition proteins, but is AID, MBD4) recently described in plants,
mouse, and zebrafish (described in detail modifications, can be enriched at particular genes/
below). Second, the short stretches of naked DNA loci of significance to the developing embryo.
(after protamine ejection and prior to histone Prior to the availability of genome-wide array
incorporation) may enable the recruitment of and modern sequencing technologies, two groups
demethylases to the paternal genome and enhance examined by qPCR the sperm chromatin compo-
reprogramming efficiency. Both mechanisms are sition at a few key loci required in embryogenesis
speculative and a better understanding of the sig- and both reported significant enrichment of his-
nificance of the protamine transition and its role tones at the developmental loci [66, 68]. This
in development is essential. suggested that histone retention maybe program-
Although sperm undergoes dramatic chromatin matically retained; however, these preliminary
changes, the exchange process is incomplete, observations were not confirmed genome-wide
retaining approximately 5–15% of the genome until recently by three independent labs [69–71].
packaged in nucleosomes [64–66]. The retained All three groups showed that histones were
nucleosomes are comprised of canonical histone retained at developmental transcription factors,
H2A, H3, and H4, but bear a testes-specific his- imprinted genes, cell cycle, and spermatogenesis
tone H2B with an unknown specialized function, gene promoters [69–71]. The spermatogenesis
along with very low levels of canonical H2B and cell cycle gene promoters retained high
[65, 67]. Histone retention raises an intriguing H3k4me, a mark of gene activation (Fig. 13.2)
question of whether the retained nucleosomes in [72], whereas developmental transcription factor
the sperm nucleus may simply be due to ineffi- gene promoters were bivalently marked as seen in
cient protamine replacement, leading to a low embryonic stem cells meaning that they retained
random genome-wide distribution with no func- both marks of activation and silencing (H3K4me
tion in the embryo, or whether the retained and H3k27me), respectively (Fig. 13.2) [69–72].
nucleosomes, along with their attendant epigenetic Furthermore, histone retention at imprinted loci
Fig. 13.2 Chromatin modifications determine gene state. associated with both active and inactive marks (bivalent
Histone modifications promote either gene activation (i.e., domains at developmental transcription factors and
spermatogenesis and cell cycle genes) or repression signaling factors). ac acetylation; me methylation; ub
(i.e., organogenesis gene promoters); however, in embry- ubiquitination (figure obtained from Carrell and
onic stem cells and sperm, a subset of genes is commonly Hammoud [72])
was intriguing and showed differential profiles family of methyl-binding domain proteins
depending on whether the gene was maternally (MBDs) that associate with chromatin-modifying
(lacked H3k4me) or paternally expressed (high enzymes, including HDACs and histone methyl-
H3k4me) [70]. Paternally expressed imprinted transferases (HMTs), that silence Pol II transcrip-
genes retained high levels of H3k4me, whereas tion [74–76]. Although the vast majority of DNA
maternally imprinted genes lacked H3k4me and a methylation resides on repetitive elements, the
few loci tested by qPCR enriched for H3k9me. methylation of CpG sites in promoters can lead to
This differential poising at spermatogenesis gene silencing [77].
and cell cycle versus developmental transcription Genome-wide methylation studies in sperm
factors were not only unique to humans, but have have shown that the sperm epigenome differs
been recently seen in mouse which retain less his- markedly from that of somatic cells, but is very
tone (~1%) and zebrafish sperm (complete his- similar to ES cells and embryonic germ (EG)
tone genome) [69, 73]. This conservation in cells [78–81]. An examination of human gene
histone marking raises the question of whether promoters by several labs has revealed several
the retained nucleosomal regions are important important conclusions. First, promoters with
for early embryonic development or are only “weak” CpG islands appear to be differentially
residual marks from the spermatogonial stem cell. methylated in sperm and somatic tissue or
Although a definitive answer for mammals is acquire methylation upon differentiation [70,
unknown, but in early zebrafish embryos H3k4me 78–81]. Second, the target gene promoters of
and H3k27me marks were erased and later rees- pluripotentcy network (e.g., OCT4, SOX2,
tablished after fertilization [73]. This suggests NANOG, KLF4, and FOXD3 proteins) [82, 83]
two possibilities either the histone marking in the are hypomethylated in sperm, but acquire meth-
paternal genome is not instructive to the early ylation upon differentiation to ensure cellular
embryo or the levels of H3k4me and H3k27me commitment, whereas several key members of
were very low beyond antibody detection limits. the pluripotency/self-renewal network, OCT4,
In contrast, Brykczynska et al. reported in his dis- NANOG, and FOXD3 themselves, acquire
cussion that paternal histones in mouse and human methylation throughout spermatogenesis, con-
embryos are retained in the embryo [69]. Whether sistent with recent studies in mice [78, 84]. To
these differences in findings are attributed to spe- summarize, developmental and signaling gene
cies variation or due to the difference in the onset promoters in mature sperm retain modified
of zygotic transcription, 2–8 cell stage (mouse nucleosomes and are DNA hypomethylated,
and human) vs. 500 cell stage (zebrafish) is which suggests that the sperm chromatin are
unclear. For humans, if an epigenetic role is poised for activation, and this poising may enable
ascribed to the retained nucleosomes, there are transcriptional competence/activation of devel-
obvious and profound implications for sperm with opmental regulators if needed in early embry-
abnormal histone retention and protamine levels onic development.
in humans, and for the use of such sperm, or any
immature sperm, to achieve a pregnancy with the
use of assisted reproductive technologies. 13.4 Imprinting and Alterations
at Imprinted Genes Have
Been Associated with
13.3 DNA Methylation and Infertility
its Role in the Germline
Genomic imprinting causes some genes to be
DNA methylation in mammals primarily involves monoallelically expressed and contributes to
the modification of cytosine in a CpG context by parental genome asymmetry. Approximately 100
a family of related DNA methyltransferases imprinted genes have been identified, many of
(DNMTs). This covalent mark is “read” by a which are important for prenatal growth, placental
development, and function and have been impli- o ffspring as compared to natural mating [99].
cated in certain diseases [85]. Imprints are erased These findings are concerning and require a bet-
and reestablished in the germ line and maintained ter understanding of the effects of existent or
into embryogenesis [26, 86, 87]. One of the inter- acquired epimutations on embryo quality, IVF
esting features of the organization of imprinted success rate, or the true risk for transmission to
genes is that most of them occur in clusters with counsel patients accordingly. However, more
long range cis-acting imprinting centers (ICs) [86]. recently, it has been shown that IVF culture media
These ICs carry allele-specific methylation marks, may not be solely responsible for these changes,
which subsequently influence epigenetic modifica- but also preexistent methylation alterations pat-
tions of additional cis-acting regulators important terns are common in the gametes of infertile men
for allele-specific, tissue-specific, or temporal- [14–19]. These observations beg the question of
specific regulation of imprinted genes [88, 89]. whether methylation defects as well as other epi-
Imprinted genes are classified as primary genetic defects (such as histone localization or
imprinted genes (such as H19/KCNQ1OT, etc.), modifications in the mature sperm) may affect
which acquire their imprinted status in the germ embryo development, growth of ART offspring,
line, whereas secondary imprinted genes are only and may last for future generations.
imprinted for a short period of time during devel- Evidence for germline epigenetic inheritance
opment (such as GTL2 IGDMR and CDKN1C) in humans has come almost exclusively from epi-
and then regain biallelic expression. Imprinting demiological studies. The strongest evidence for
in the female germ line is primarily achieved germline epigenetic inheritance in humans comes
using DNA methylation, whereas imprinting in from the work of Horsthemke and colleagues
males is primarily mediated through noncoding [100]. They have shown that the presence of
RNAs; however, two genes are known to be DNA epimutations and not genetic mutations, at the
methylated H19/Rasgraf. SNRPN–SNURF, was inherited from the pater-
A relationship between genomic imprinting nal grandmother [100]. More recently, a few
and infertility first stemmed from early work of reports have shown that a gain in methylation on
IVF in animal models. Offspring conceived by the paternal alleles of LIT1 or MEST in sperm is
assisted reproductive technologies in animals had maintained in the baby and associated with tran-
an increased incidence of LOS [23]. In humans, a sient neonatal diabetes [101] or Silver–Russell
meta-analysis showed that children born from syndrome [102]. Inheritance of epimutations
assisted reproductive technology (ART) have a from imprinted genes is intriguing since imprinted
fourfold increased incidence of BWS compared genes have tandem repeats in their gene promot-
with children conceived naturally [90–93]. ers and repeats may be incompletely erased.
Many speculations have been made suggest- Second, in mice the best known examples of
ing that hormone stimulation, gamete manipula- transgenerational inheritance were described at
tion, or embryo culture may alter oocyte/embryo two metastable epialleles known as the agouti
imprinting. The effects of culture on embryo viable yellow (Avy) allele (coat color ranges from
quality, embryo transcriptome and epigenetics, agouti to yellow) and the axin-fused (AxinFu)
and long-term behavioral effects have been previ- allele (normal to severe tail kinks). In both cases,
ously described [20, 94–96]. However, recently isogenic animals displayed variable expressivity
these findings have been confirmed by a very and phenotype variegation which correlated with
elegantly designed study using the agouti viable the differential level of methylation acquired at
yellow (Avy) allele in mice, which is one of the the IAP retrotransposon inserted at each of the
best-characterized alleles that display a labile loci [101–105]. To date, epigenetic inheritance in
epigenetic state [97–99]. This study shows that mice and humans has been limited to a certain
embryos cultured in commercially available gene classes; genes that contain repeat elements
embryo culture induced persistent epigenetic in their promoter regions. One hypothesis can be
changes that altered coat color in mouse IVF that epigenetic inheritance is limited to repeat
elements as they may escape reprogramming. Mammals, on the other hand, lack orthologues
However, epigenetic inheritance at a nonim- for any of the characterized demethylases in
printed gene was suggested by Suter et al. and plants or bacteria, but many of the recent findings
Chang et al., which showed that epimutations at suggest that the active DNA demethylation in
the promoters of DNA repair genes, MLH1 and mammals is achieved (embryo and PGCs), at
MSH2, were seen in some cases of familial col- least in part, by the base excision repair machin-
orectal cancer in successive generations [106, 107]. ery [123–134]. First, 5-me C is deaminated by
This study raises an interesting possibility that AID or Apobec, then followed by the T:G base
epigenetic marks may be maintained in families excision repair by glycosylases such as TDG/
and are not efficiently cleared in the genome- MBD4 creating an abasic site, which is then filled
wide reprogramming that takes place between by DNA polymerase and DNA ligase [134–136].
generations. Future studies are needed to deter- This multistep process results in a hypomethy-
mine the perdurance of these marks. lated genome. The use of AID and other factors
as a potential mechanism for active DNA dem-
ethylation was shown to be important for repro-
13.5 Genome-Wide gramming mouse PGC and enhancing the
Reprogramming in efficiency of somatic cell reprogramming. Recent
Preimplantation Embryos reports suggest that active DNA demethylation
and Primordial Germ Cells may be a more commonly used phenomena in
mammalian cells than previously thought; it is
Epigenetic reprogramming via demethylation of seen at gene specific promoters (interleukin genes
DNA occurs at two points in mammalian devel- and estrogen responsive gene promoters)
opment. First, in early preimplantation embryos undergoing transcriptional cycling [128, 133, 137].
where the vast majority of the genome is dem- Demethylation at these gene promoters correlated
ethylated, although some genes, including with the recruitment of Dnmt3a, Dnmt3b, TDG,
imprinted genes, escape this wave of DNA dem- and other BER enzymes [128, 133, 137]. Evidence
ethylation [29]. The second and more complete for such interaction between DNMTs (as
reprogramming happens in PGCs during which deaminases), glycosylases, and BER was previ-
imprinted genes are erased and reestablished. ously reported [138–140]. This raises the ques-
The two waves reprogramming, first in the early tion whether there may be two active DNA
embryo and a second in PGCs, may be needed to demethylase complexes, one specified for a robust
restore totipotency and limit the potential for genome-wide demethtylation which includes
transgenerational inheritance of epimutations AID (AID a mutagen – restricted to developmen-
[26, 108–115]. tal time-points) and the other being a gene/class-
Genome-wide reprogramming can be medi- specific reprogramming (involves DNMTs since
ated via two potential mechanisms: active dem- they have weak deaminase activity, therefore
ethylation, first described in plants, or passive their effect can be contained).
demethylation. In plants, the active DNA dem- In contrast to the active demethylation pro-
ethylation process is important for plant gene cess, the passive DNA demethylation occurs
imprinting and for general housekeeping func- when maintenance methyltransferases are inac-
tions to counteract the spreading of methylation tive during cell cycle following DNA replication
from repetitive sequences to genes [116–121]. and division, as seen in the female pronucleus
Loss of plant demethylases didn’t result in sig- shortly after fertilization or in early stages of
nificant changes in methylation on a genome- germ cell migration. Recent data examining
wide scale, but many genomic sites were allele-specific methylation status of H19 and
hypermethylated [3, 119]. Surprisingly, the accu- SNRPN in both migratory and postmigratory
mulation of methylation at many of the gene pro- PGC (e9.5–e11.5) suggest that erasure process
moters did not alter gene expression [119, 122]. may begin before the arrival to the gonad (e9.5)
[141]. During migration, the majority of the cells If epigenetic information can be inherited
analyzed were hypermethylated at the paternal from the sperm and the oocyte to regulate gene
H19 locus, but a very small subset of the alleles expression in an appropriate manner, then this
(~19%) get demethylated (<50% methylated) raises concern for the growing body of evidence
and the methylation continued to gradually that suggests that the ART procedure and/or
decrease until e11.5 [141]. Consistent with the infertility may provide an additional layer of
report above, Sato et al. 2003 showed that IGF2r complexity to the pregnancy and may elicit epi-
DMR2 starts demethylation at e9.5 in some genetic changes in IVF offspring. Underlying
migrating PGC, before the cells colonize the gen- support for these speculations comes from the
ital ridge, but the progression of demethylation is work published from the CDC in 2002, which
augmented after entering the genital ridge [142]. suggested that children conceived by IVF had an
These findings suggest that PGC reprogramming increased risk for preterm birth, low birth weight,
may begin as a passive demethylation process congenital anomalies, preeclampsia, and perina-
(because of cell doubling) followed by an active tal mortality even in singleton pregnancies
demethylation mechanism [143–145]. [146–148]. Low birth weight in naturally con-
A better understanding of the mechanism ceived babies has been connected to heart disease
responsible for erasing cellular memory and epi- and metabolic syndrome later in life; whether
genetic information provides a better understand- these risks apply to low birth weight IVF babies
ing for how epigenetic errors arise and potential too is unknown. Several groups have postulated
reversal. Most importantly, reprogramming is that these abnormalities may be epigenetic in
essential for the success and efficiency regenera- nature. Unfortunately, morphological assessment
tive medicine-based therapies such as stem cell of gametes at the time of fertilization and grading
transplantation, transdifferentiation, and gamete embryos at the time of transfer is incapable of
regeneration for azoospermic men, premature detecting epigenetic errors. Therefore, future
ovarian failure, or for fertility preservation for studies are needed to determine whether the
cancer patients who have undergone radiation or underlying epigenetic alterations in the gametes
chemotherapy. of infertile couple rather than the IVF technique
or both are responsible for these complications
seen in IVF babies.
13.6 Conclusions and Future
Directions
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Part III
Assisted Reproduction Techniques
ART and Epigenetic Disorders:
Should We Be Concerned? 14
Christopher N. Herndon and Paolo F. Rinaudo
Abstract
An ART treatment cycle – comprising a basic sequence of ovarian
stimulation, oocyte retrieval, embryo culture, and transfer – is, by conven-
tional measures, a routine and comparatively safe medical intervention.
The greatest risks from ART derive from the complications associated with
multiple pregnancies. Yet, studies indicate that even a singleton pregnancy
from ART confers greater health risks than that of natural conceptions. In
this section, we review the established associations of ART to rare epige-
netic disorders of imprinting. The biologic mechanisms through which
epigenetic programming might be altered within the process of ART are
examined. The still unknown long-term health associations of ART past 30
years are considered in the framework of emerging and increasing compel-
ling evidence that environmental conditions during the early development
are an important determinant of health during later adult years.
Keywords
Epigenetics • ART • ICSI • Imprinting • Morbidity
ART has emerged from a largely inefficient,

14.1 Introduction experimental protocol to a highly efficacious
therapeutic modality that is the standard of care
Since the first live birth from in vitro fertilization
treatment for many forms of infertility in devel-
over 30 years ago, greater than three million chil-
oped countries. Conceptions from ART in this
dren have been conceived through assisted repro-
decade comprise an estimated 1-3% of live births
ductive technologies (ART) [1]. Over this interval,
in many developed countries [1]. Although estab-
lished as an efficacious therapy, the safety and
P.F. Rinaudo () long-term health implications of in vitro fertiliza-
Division of Reproductive Endocrinology tion and culture on children conceived by ART
and Infertility, Department of Obstetrics, Gynecology are not well known. Importantly, longitudinal
and Reproductive Sciences, University of California,
San Francisco, CA, USA follow-up outcomes for any offspring resulting
e-mail: [email protected] from in vitro fertilization and culture do not

198 C.N. Herndon and P.F. Rinaudo
extend past puberty, and the impact past

adolescence remains entirely unknown [2–5].
14.2 Complications of ART
In perspective of its wide and increasing utiliza-
An ART treatment cycle – comprising a basic
tion, the long-term health implications of ART
sequence of ovarian stimulation, oocyte retrieval,
are an issue of paramount relevance and concern
to both the individual and society as a whole. embryo culture, and transfer – is, by conventional
The emergence of ART as a therapeutic measures, a routine and safe medical intervention.
modality has been characterized by an extraordi- Complications at that time of treatment cycle are
nary rapidity in its adoption, from proof of con- infrequent and include ovarian hyper stimulation
cept to clinical utilization in developed countries (OHSS), bleeding or infection from oocyte
worldwide. Intracytoplasmic sperm injection retrieval, and torsion of the stimulated ovaries.
(ICSI), the gamete micromanipulation technique The majority of these complications, although
that revolutionized the treatment of male factor serious, are rarely associated with significant long-
infertility, was first reported in 1992 [6]. Within a term morbidity and mortality. Significant adverse
few years, ICSI became widely adopted into clin- reactions from medications are exceedingly rare.
ical practice globally and currently it is utilized The greatest health risks associated with ART
in >60% of cycles in the United States [7]. are unquestionably attributable to multiple gesta-
Similarly, preimplantation genetic diagnosis tions that result from the transfer practice of more
(PGD), the technique that allows genetic testing than one embryo to optimize per cycle pregnancy
of an embryo following extraction of one or more rates. In 2008, twinning and higher order multiple
cells, is now widely offered by clinical centers gestations accounted for approximately one third
despite comparatively limited reporting of fol- of live births from ART in the United States [7].
low-up outcomes of live births [8]. Driving this The cost of medical care associated with multiple
progressive development and application of tech- pregnancies thus exceeds by far the cost of ART
nology in the clinical are the hopes and despera- treatment itself [10, 11]. Beyond the significant
tion of infertile couples [9]. The social and maternal, fetal, and neonatal risks of multiple
biological drives to conceive are powerful and gestations, studies have convincingly demon-
compelling and infertile couples are often willing strated that perinatal risk of singleton pregnan-
to accept new therapies, however unproven, that cies from ART is higher than natural conceptions
may give them a chance to have a biological [12–14]. ART singleton pregnancies have greater
offspring. risks of preterm delivery and low birth weight,
In this section, we summarize the known risks after adjustment to maternal age, parity, ethnicity,
and complications of ART, focusing on the or other cofounding variables.
reporting of increased risk of two rare epigenetic Significantly, there is a 30% increased risk of
disorders of imprinting. We cite evidence using congenital anomalies among children conceived
animal models to provide insights on the biologic by ART (3–4%), in comparison to the background
mechanisms by which epigenetic programming rate (2–3%) in pregnancies conceived in vivo
might be altered through the process of ART. In [12, 13, 15]. Despite initial concern, the incidence
this background, we introduce the concept that of congenital anomalies does not appear to be
the environment of early development has the higher among ART cycles in which ICSI is per-
potential to profoundly affect the phenotype in formed [16], although an slightly increased risk
later life. The still unknown long-term health of hypospadias has been reported [17]. It remains
associations of ART are considered within emerg- unclear if the increased incidence of congenital
ing and increasing unavoidable evidence that anomalies following ART is attributable to the
environmental conditions during the early devel- intervention itself or the intrinsic predisposition
opment are a significant determinant of health in of the infertile state [18]. Indeed, subfertile cou-
later adult life. ples who conceive without medical interventions
14 ART and Epigenetic Disorders: Should We Be Concerned? 199
are predisposed to increased incidence of a series The first identified and by far the best studied
of pregnancy complications, including preec- of these three modifications is DNA methylation,
lampsia, abruptio placentae, preterm birth, and in which a methyl group is added to a cytosine
perinatal mortality [19]. base located 5¢ to a guanosine, commonly referred
The first definite evidence that ART alters preg- to as “CpG” (p indicates the phosphate group
nancy outcomes came through the early reporting linking the two bases). Importantly, not all the
of an association of monozygotic twinning (MZT) CpG dinucleotides are methylated. An area of the
with ART. Studies have reported the incidence of genome (usually at least 200 bp long) with high
MZT following ART as 1.9–2.5% [20, 21], a rate incidence of CpGs (greater than 50%) is referred
consistently over a background incidence for to as “CpG island.” Approximately half of the
spontaneous conceptions, generally reported to be CpG islands are located near the transcription
around 0.4%. The mechanisms conferring this start site of genes, particularly for housekeeping
increased risk are not understood. Extension to genes; these CpGs are generally not methylated
blastocyst culture is the most consistent risk factor or have low levels of methylation. The remaining
identified, although use of ICSI and micromanip- 50% of CpG islands are intragenic or intergenic
ulation has also been reported [22]. and are believed to represent the transcription
start site of noncoding RNAs [27]; these CpG
islands are usually methylated [25].
14.3 Epigenetic Regulation Generally speaking, methylation silences gene
transcription through structural blocking of tran-
While the role played by Darwinian selective scriptional factor binding to DNA by the presence
pressure in determining trait inheritance is uncon- of a methyl group [28]. DNA methylation is
tested, modern biology suggests that the environ- responsible for X chromosome inactivation, silenc-
ment and life experience of an individual can ing of imprinted genes, and silencing of repetitive
modify future progeny, ironically somewhat reha- and transposable elements. A few genes, such as
bilitating the early and now widely debunked IGF2 and IGF2R, are activated by methylation
Lamarckan theory of inheritance. The field of epi- [29]. Methylation is catalyzed by DNA methyl-
genetics (epi- Greek: epί- over, above -genetics) transferases (DNMT), acting in conjunction with
studies the changes in gene expression, gene interacting corepressors, transcription factors, and
activity, and phenotype caused by mechanisms methyl-CpG-binding proteins. DNMT1 is a
not involving base pair changes in the underlying sequence independent methyltransferase and acts
DNA sequence [23–25]. Epigenetics can be to conserve the patterns of methylation status in
viewed as the link between environment and the process of DNA replication and cell division in
genes. This seminal concept was proposed by somatic cells [30]. Two isoforms, DNMT3A and
Waddington in the 1950s, far in advance of the DNMT3B, are responsible for de novo methyla-
current interest in the field [26]. The concept of tion during the process of gametogenesis and early
canalization was used to account for phenotypic life. Recently, the existence of active DNA dem-
changes that occur too rapidly to be explained by ethylation has been documented [31–33].
the natural progression of evolutionary changes Methylation is only one epigenetic marker;
in DNA sequence. epigenetic mechanisms include postranslational
The mechanisms and control of the epigenetic modifications of histone tails through addition of
alternations of DNA are still being unraveled. an acetyl, methyl, phosphate group, or more
Three mechanisms are thought to be involved: rarely, by ubiquitination, sumoylation, ADP-
1. DNA methylation. ribosylation, deimination, and noncovalent pro-
2. Posttranslational modification of histone pro- line isomerization. Histone modifications work
teins and remodeling of chromatin. often in conjunction or, occasionally, indepen-
3. RNA-based mechanisms. dently of DNA methylation [25]. The histone tail
modifications work through changing the affinity mothers were found to have diminished expression
of the basic histone proteins to the acidic DNA. of corticotropin-releasing hormone and a blunted
Acetylation, for example, neutralizes the positive response to stress [38]. These differences in their
charge of lysine, promoting disassociation of the adrenocortical axis emerged during the first week
histone protein from the negatively charged of life and persisted through the remainder of
DNA, resulting in a more open chromatin confor- their adult lives. Offspring who received less
mation. Generally speaking, DNA is least tran- grooming and reinforcement behaviors differed
scriptionally active when it is methylated and from the offspring who received more grooming
bound with unacetylated histones. in the methylation of just a single CpG dinucle-
The “histone code” is extremely complex and otide in the promoter of the glucocorticoid recep-
just being deciphered. The challenge of its study is tor [38]. Remarkably, these differences were
exemplified by the complexity of multiple levels reversed through cross-fostering with rat mothers
of modifications that can coexist. For example, who exhibited high levels of grooming and nurs-
arginine residues can be mono or demethylated ing behaviors. Furthermore, the stress-response
while lysine residues can accept one, two, or three phenotype was also modified at the molecular
methyl residues [23, 34, 35]. An additional epige- level through central infusion of a histone
netic control at the chromatin level is performed by deacetylase inhibitor.
two family of chromatin remodeling complexes.
The Brahma/SWI/SNF complex alters the position
of nucleosomes along the DNA, whereas the 14.4 Imprinting Disorders
SNF2H/ISWI complex mobilizes nucleosomes.
A third model of epigenetic control involves The reporting of an association of ART to two
noncoding RNAs (ncRNA, reviewed by Mattick rare disorders of imprinting opened a new per-
et al. [36]). It has become apparent that the spective into the effect of ART on early develop-
eukaryotic genome can be envisioned as an “RNA ment [39, 40]. Imprinting refers to the differential
machine,” in which the vast nonprotein-coding expression of genes dependent on parental inheri-
segments of DNA are important for generating tance. As a diploid species, humans contain two
abundant ncRNAs which play a central role in sets of autosomal genes, a copy inherited from
genetic and epigenetic processes. In fact, tran- each parental gamete. In classical Mendelian
scription factors and proteins involved in epige- inheritance, the genes from each parent are
netic modification (e.g., DNMTs and methyl expressed in the offspring in equal measure, inde-
DNA-binding domain proteins) can bind RNA. pendent of parental origin. In a small handful of
RNA can also contribute to chromatin structural known genetic disorders, the disease phenotype is
organization [36]. dependent on uniparental expression. The concept
of genomic imprinting was elegantly illustrated in
A unique feature of the epigenome is its plas- experiments in which diploid uniparental murine
ticity. While epigenetic marks are maintained embryos (either two male or two female pronu-
through cellular mitosis and are considered sta- clei) failed to undergo normal embryogenesis in
ble, they can be reversibly modified by the envi- comparison to control embryos (with one male
ronment. For example, it is clear that there are and one transplanted female pronucleus) [41].
important epigenetic changes associated with The first link of ART to an imprinting disor-
aging. Overall, aging is associated with global der was a report in 2002 of two cases of
DNA hypomethylation and isolated hypermethy- Angelmann syndrome (AS, OMIM 105830), a
lation of specific loci [37]. In addition, epigenetic rare disorder with an overall prevalence in the
programming can change during specific win- general population of approximately 1 in 15,000
dows of sensitivity. [42]. The syndrome is characterized by severe
In a seminal study, rat pups which received developmental delays, learning disabilities, pro-
higher level of grooming (pup licking) and nurs- found speech impairment, ataxia, microcephaly,
ing behaviors (arched-back nursing) from their and seizures. Affected individuals typically
exhibit a pathogenomic demeanor that has been The affected imprinted region is located on chro-
characterized as the “happy puppet syndrome,” mosome 11 (11p15) and includes several genes
manifested by frequent laughter, excitable per- and two differentially methylated regions (DMR1,
sonality, and affectionate nature. AS results from DMR2) that are either paternally expressed (IGF2,
loss of maternal expression of UBE3A gene KCNQ10T1) or maternally expressed (H19,
within 15q11-q13 [43]. This loss of maternal CDKN1C, KCNQ1). The overall prevalence of
expression most commonly results from a dele- BWS following ART has been estimated to be
tion within 15q11-q13 (~70%), but can also approximately 1 in 4,000, a four to fivefold higher
result in mutations in the UBE3A gene (~11%) level than in the background population [51]; the
or paternal disomy (7%). Epimutations, or abnor- relative risk is increased to 14-fold if only epimu-
malities of epigenetic marking rather than tation-associated cases are considered. While
directly in the sequence of genomic DNA, these numbers are worrisome, it should be noted
account for 3% of AS cases or an overall inci- that an association of BWS is not conclusive, as a
dence of approximately 1/300,000 cases in the few cohort studies have not found a correlation
general population [44]. between BWS and ART [52, 53]. The rare inci-
To date, seven cases of AS have been reported dence and sporadic occurrence of imprinting dis-
in children conceived by ICSI or IVF [39]. orders make it challenging to establish a clear
Importantly, five of these seven AS cases were association. In addition, the closer scrutiny and
secondary to epigenetic mutation suggesting a surveillance of offspring from ART may lead to a
strong correlation between AS and ART. The greater detection of the disorders in comparison
likelihood of these epimutations occuring by to natural conceptions.
chance is estimated to be less than 1 in 20 million Although not an imprinted disorder, the inci-
[40]. It is unknown whether the increased risk is dence of retinoblastoma (RB) in ART children is
attributable to embryo culture, gamete microma- noteworthy. One study found a nearly fivefold
nipulation, and/or ovarian stimulation. It is addi- increase in RB in ART children compared to the
tionally plausible that subfertile couples may be general population [54]. The occurrence of this
predisposed to AS. Cases of AS have been tumor of the retina requires two separate muta-
observed among subfertile couples who con- tions: the first mutation (“first hit”) is inherited,
ceived spontaneously or after ovarian stimulation and the second is acquired somatically [55]. It is
alone [45–47]. possible that methylation changes following
The second imprinting disorder found to be in vitro culture could alter the single functional
associated with ART is Beckwith–Wiedemann copy of the RB gene. While the presence of epi-
syndrome (BWS, OMIM 130650), a rare condi- genetic abnormalities was not evaluated in RB
tion with an incidence of 1 in 13,700 live births. cases among ART offspring, hypermethylation of
BWS is a heterogeneous disorder of overgrowth RB gene has been associated with the develop-
and characterized by macroglossia, anterior ment of RB [56, 57]. Of note, a large Danish
abdominal wall defects, visceromegaly, omphalo- cohort study detected no cases of RB among
cele, microcephaly, and embryonal tumors 6,052 IVF singletons [52].
(Wilms’ tumor, rhabdomyosarcoma, hepatoblas-
toma). Afflicted individuals in utero may present
with polyhydraminos, enlarged placenta, and 14.5 Are There Epigenetic
macrosomia. In 2003, three series were published Disturbances Following ART?
reporting an association between BWS and ART
[48–50]. To date, over 60 cases of BWS conceived Compelling evidence indicates that stressful envi-
through ART have been reported [39, 40]. A high ronmental conditions during sensitive periods of
percentage (>90%) of these cases have been asso- early development may predispose to chronic dis-
ciated with hypomethylation of the maternal ease later in life [58]. The premise of this postu-
allele, while 60% of BWS cases in the general late, known as the developmental origin of health
population derive as a result of epimutations [39]. and disease (DOHAD) hypothesis, is that the
plasticity of the conceptus to its environment is this mouse the transcriptional activity of agouti
an adaptive response, conferring a survival advan- protein, which decreases melanin production by
tage in low resource environments. In a different, melanocytes. As such, the coat color of the
high resource environment later in life, these agouti (= yellow) mouse is a highly useful tool
adaptations may predispose to metabolic and for assessing whether phenotypic variability is
cardiovascular dysfunction [59, 60]. In particular, attributable to epigenetic modifications. Mice
the increased incidence of low birth weight among without methylation at the intracisternal type-A
ART children is a reason of concern [61]. Low particle element (IAP) of the agouti gene have
birth weight is an indicator of in utero stress. increased agouti protein and therefore have a yel-
Barker’s hypothesis was formulated from obser- low coat color. In contrast, mice with increased
vations that regions of Britain with the greatest IAP methylation do not have agouti protein pro-
incidence of low birth weight correlated tightly to duction and have a brown coat color (a.k.a.
areas with the highest rates of cardiovascular dis- pseudoagouti). The phenotype from epigenetic
ease [58, 62]. Subsequent epidemiologic studies modifications is not restricted strictly to coat
in other countries have widely supported this color. Pseudoagouti mice are lean and have nor-
association [63]. Fetuses exposed to famine in mal glucose homeostasis, whereas yellow mice
early gestation during wartime Holland had a are obese, insulin-resistant, glucose-intolerant,
threefold higher risk of heart disease in fetuses and show a predisposition to diabetes [73].
exposed (3.0 OR, 95% CI 1.2–8.8) [64]. Importantly, stresses of a different nature (pres-
Low birth weight has additionally been ence of methyl supplement in the diet or in vitro
associated with other vascular morbidity includ- preimplantation culture) result in change in DNA
ing cerebrovascular disease and hypertension methylation [72, 74–77]. Specifically, in vitro
[65, 66]. In Scotland, incidence of stroke was culture of zygotes results in decrease in IAP
found to be inversely proportional to birth weight methylation (and therefore in increase of yellow
[67]. An association of low birth weight and mice) compared to natural mating [72]. No dif-
hypertension appears to be present, but the effect ference in IAP methylation (and coat color) was
appears modest. In one study, a 1 kg decrease in observed in pups resulting from embryo transfer
birth weight was correlated with a 2–4 mmHg alone, indicating that the culture conditions
increase in systolic blood pressure [68]. caused hypomethylation of the agouti locus.
Epidemiologic studies are compelling, but Epigenetic modifications are increasingly rec-
are limited by potential confounding variables. ognized to underlie a number of disease states
Controlled animal studies have confirmed the beyond imprinting disorders. Human malignan-
associations reported in epidemiological studies. cies have been found to have global genomic
Maternal undernourishment in rats and sheep hypomethylation (with activation of endogenous
during the period of conception and implantation, retroviral elements, oncogenes, and loss of imprint-
for example, leads to hypertension and cardiovas- ing) as well as hypermethylation of tumor sup-
cular dysfunction in offspring [69–71]. While the pressor genes [78]. Exposure to chemicals that
exact mechanisms responsible to alter the devel- function as reproductive endocrine disruptors (e.g.,
opmental pattern of the organism are unknown, diethylstilbestrol, vinclozolin, and bisphenol A) is
epigenetic alterations are believed to play a fun- associated with specific epigenetic changes [79].
damental role. This concept has particular rele- Even syndromes characterized by birth defects
vance to ART, as gametes and preimplantation including ICF syndrome (immunodeficiency, cen-
embryos are exquisitely sensitive to the environ- tromere instability, and facial anomalies syn-
ment and susceptible to epigenetic alterations. drome), ATRX syndrome (X-linked a-thalassemia
The agouti viable (Avy) mouse is an important – mental retardation syndrome [ATRX]), and
animal model because it displays a labile and Rubinstein–Taybi syndrome have characteristics
easily observable epigenetic state [72]. DNA of birth defect and have been attributed to epige-
methylation has been established to modulate in netic anomalies [80]. These associations raise the
question is the increase in birth defects observed in enable a sex-appropriate imprinted gene pattern.
offspring from ART have an underlying epigenetic Following fertilization, embryos undergo a
contribution. genome-wide demethylation; the male pronucleus
Limited studies of early adulthood are available is actively demethylated before zygotic genome
in context of young age of the majority of ART activation, while the female pronucleus is pas-
children. Developmental outcomes and physical sively demethylated in a process that requires
health in early childhood and during adolescent DNA replication. At the blastocyst stage, there is a
appear, however, to be comparable to naturally rapid remethylation process. Imprinted genes
conceived children [81–83]. However, recent evi- markings are normally unaffected by this process.
dence suggests that pubertal children (mean age The second wave of epigenetic reorganization
12.6 years old) conceived by ART have increased occurs only in the primordial germ cells (PGC),
blood pressure, are 2.5 times more likely to have starting on day 13 of embryonic life in the mouse
fasting glucose levels in the highest quartile [30]. During this phase, the entire PGC genome,
(³5.2 mmol/L, 95% CI 1.2–5.2) [3], and have dis- including imprinted genes, is demethylated and
turbed body fat composition [2, 4]. Although the then remethylated in a sex-specific manner, so
alterations are mild, the findings are concerning that eggs and sperm will have the appropriate
because they may represent sentinel observations, respective imprinted marks. The time course for
hinting towards the possibility of clinically signifi- remethylation of gametes is different for the two
cant increased risks of metabolic abnormalities sexes. Maternal imprints are established during
and chronic disease later in life. the development of the oocyte, from primordial
Recent studies have begun to explore differ- to maturation of antral follicles. While the imprint-
ences in epigenetic programming among human ing process progresses over time with increasing
embryos conceived in vitro. Katari et al. examined oocyte diameter, different imprinted genes com-
global methylation patterns at CpG sites using a plete the process at different times and, for sev-
microarray approach on samples of placenta and eral imprinted genes, the process is not completed
cord blood from ten children conceived in vitro vs. until ovulation [85, 86]. In contrast, the imprinted
13 children in vivo [84]. Although significant vari- gene pattern of spermatozoa is completed much
ability and overlaps in methylation patterns were earlier, at the prospermatogonial stage [87].
observed between groups, offspring from in vitro Disruption at different points during this pro-
conceptions had globally lower mean levels of cess results in varying degrees of epigenetic aber-
methylation at CpG sites in placental DNA and rations. Incomplete erasure of the imprints can
higher levels of methylation in DNA from cord result in epigenetic inheritance to the next genera-
blood samples in comparison to natural conception. More specifically, an epigenetic modification
tions. Of note, several of the differentially methy- could be transmitted to the offspring if one epige-
lated genes reported by Katari et al. have been netic mark is not erased in the parental germ cells.
implicated in chronic metabolic disorders, includ- Alternatively, a novel epigenetic error could occur
ing Type II diabetes mellitus and obesity [84]. after fertilization and before specification of PGC
[88]. Notably, ART interventions encompass both
critical periods of epigenetic remodeling [80, 89].
14.6 How ART Might Predispose Potential mechanisms by which ART could lead
to Epigenetic Problems to epigenetic errors include (Table 14.1):
Our understanding of the time course of epige- Hormonal superovulation. Differences in DNA
netic regulation in humans is almost entirely methylation of imprinted genes have been identi-
extrapolated from analysis of imprinted loci in fied among oocytes from stimulated ovaries,
murine models. For a review, see ref. [30]. In sum- both in mice and humans [90]. Importantly, a
mary, the epigenome is first reorganized after fer- dose–response effect was noted when mice
tilization and again during gametogenesis to oocytes were stimulated with different doses
Table 14.1 Potential mechanisms that can lead to individuals to epigenetic and imprinting errors
increased incidence of epigenetic disorders following ART [45, 80]. Sperm from oligospermic men has been
Mechanism demonstrated to contain a higher percentage of
Use of hormones to downregulate pituitary function imprinting alterations [96].
and to stimulate ovary for supernumerary oocyte
production
In vitro growth and/or in vitro maturation of oocytes
Use of immature sperm 14.6.3 Extraction and Injection
Use of sperm from infertile man carrying a mutation of Immature Sperm
Asynchrony between uterus and embryo
Use of ICSI While common for severely oligospermic patients,
In vitro culture extraction of sperm from the epididymis or testis
Cryopreservation of either gametes or embryos is occasionally performed in patients with prior
vasectomy or obstructive azoospermia. While it
of gonadotropins; higher gonadotropins were appears that imprinted gene marks have already
associated with increased alteration of imprinting been established at the spermatogonial stage [97],
gene methylation [85]. These experimental stud- genes involved in spermatogenesis are methy-
ies provide why patients undergoing ovarian lated only at the level of the epididymis [87] and
stimulation alone have an higher incidence of AS the spermatid is transcriptionally active [88]. In
[45]. Furthermore, the findings point to the ben- this way, the final epigenetic programming may
efit of the adoption of less aggressive ovarian not be present in nonejaculated spermatozoa. For
stimulation protocols. these reasons, and because one study found an
increased incidence of congenital malformation
[98], injection of round spermatid injection
14.6.1 In Vitro Growth and In Vitro (ROSI) is considered experimental.
Maturation of Oocytes
The processes of in vitro growth (IVG) and

14.6.4 Asynchrony Between Uterus
in vitro maturation (IVM) are complex and with
and Embryo
different efficiency in different species – see for
review [91]. Live offspring have been obtained
Human blastocysts reach the uterus 5 days postfer-
following IVG/IVM of mice primordial follicles
tilization. Blastocyst transfer was introduced to
[92]. On the contrary, only isolated preantral fol-
facilitate embryo selection and to better synchro-
licles have been grown in nonrodent species [91].
nize the embryo and the uterine receptivity.
In humans, the process of IVM involves the matu-
However, human embryos are routinely transferred
ration of germinal vesicle (GV) oocytes to meta-
into the uterus at the cleavage stage with good suc-
phase II (MII), with primary clinical indications
cess. Importantly, asynchronous embryo transfer
for women with PCOS or fertility preservation in
in sheep is associated with decreased implantation
patients preparing to undergo cytotoxic cancer
and altered growth trajectory of the implanted
therapy [93]. Although a small study detected no
embryos [99]. In particular, transfer of embryos to
increase in congenital malformation in children
an advanced uterine environment results in change
conceived by IVM [94], a mouse study found sig-
in fetal muscle development [100], a phenomenon
nificant histone acetylation changes in MII oocytes
that may be epigenetically regulated [101].
and early cleavage embryos, after IVM [95].
14.6.2 Sperm from Infertile Men 14.6.5 Intracytoplasmic Sperm

Injection
An increased prevalence of epigenetic defects
could be present in the gametes of infertile ICSI differs in multiple obvious aspects com-
couples, and thus predispose offspring of those pared to in vivo fertilization. Beside the lack of
the physiologic sperm egg interaction, ICSI entitles 3 months of age (corresponding to the postpuber-
the introduction of the sperm tail into the ooplasm tal age in humans) [110]. Similarly, one-cell
which may effect oocyte cellular pathways and mouse embryos cultured for 4 days in KSOM
result in suboptimal sperm nuclear decondensa- medium in the presence of 10% fetal calf serum
tion [102]. In addition, ICSI zygotes exhibit or 1 g/L bovine serum albumin showed specific
shorter calcium oscillations with altered pattern behavioral alterations in anxiety and deficiencies
[103]. The cleavage of the ICSI embryos occurs in implicit memories [111]. Disturbingly, fetal
at a slower rate; the resulting blastocysts have cord serum was used in several human IVF labo-
reduced cell numbers and reduced hatching rate. ratories until recently [112].
While several reports suggested that fertilization Extension of embryo culture until the preim-
by ICSI was associated with increase in imprint- plantation stage is associated with epigenetic
ing errors [42, 104], a recent study did not find an alterations. In the rodent model, Doherty et al.
increased incidence of chromatin or methylation [113] showed that the H19 gene is imprinted and
errors in ICSI embryos compared to embryos fer- exclusively expressed from the maternal allele
tilized through conventional IVF [105]. It is still in vivo. However, mouse blastocysts that develop
possible that ICSI plays an indirect role, by allow- in vitro show biallelic expression. After culture of
ing the creation of embryos with immature or two-cell embryos to the blastocyst stage in WM,
suboptimal male gametes, a fact that could not the normally silent paternal H19 allele is aber-
occur in vivo. rantly expressed; in contrast, as in the in vivo situ-
ation, little paternal expression is observed after
culture in KSOMAA medium. IVF and embryo
14.6.6 In Vitro Culture of culture can generate abnormal epigenetic marks
Preimplantation Embryos at levels of histones as well as modify global
DNA methylation patterns [114]. In mice, changes
Compelling evidence from animal models indi- in global DNA methylation from embryo culture
cates that in vitro culture is in fact a stressful and are evident as early as the two-cell stage [115].
unfavorable state for embryos. Rodent studies Studies in sheep and cattle conceived through
underline that the development rate and global ART show an increased incidence of elevated birth
patterns of gene expression are abnormal in mice weight and increased perinatal morbidity, a condi-
embryos cultured in vitro [106, 107]. A lower tion known as the “large offspring syndrome”
number of trophectodermal cells are found in (LOS), reviewed by Sinclair et al. [116]. Although
mouse blastocysts conceived in vitro in compari- the differences in size levels off at 1 year of age,
son to in vivo controls [107]. Suboptimal oxygen examination of in vitro conceived animals revealed
concentrations and culture medium increase abnormal organ and skeletal development [117].
aberrant gene expression [108]. Mouse fetuses LOS is associated with epigenetic changes includ-
generated by IVF display delayed fetal developing alterations of global methylation levels
ment in comparison to controls. In particular, cul- [118, 119] and specifically a lack of methylation at
ture in suboptimal conditions (using Whitten’s IGF2 receptor, an imprinted gene in mice [120].
medium, WM) resulted in a more severe pheno- Hiendleder et al. analyzed the effects of two in vitro
type as opposed to culture in optimized condi- fertilization protocols (IVF1 and IVF2) on the
tions (K simple optimized medium with amino phenotype and tissue methylation levels in bovine
acids, KSOMAA) [109]. Intriguing are the behav- fetuses [119]. The two protocols differed only in
ioral abnormalities observed in mice offspring the amount of follicle-stimulating hormone,
in vitro culture. Offspring of mice cultured for luteinizing hormone, and cow serum in the culture
2 days in vitro (from the two-cell stage to the medium. IVF1 fetuses were similar to controls fer-
blastocyst stage) in different media (KSOMAA or tilized in vivo, but IVF2 fetuses were significantly
WM) showed decreased anxiety and decreased heavier and longer, with increased heart and liver
memorization of spatial information. These weights, displaying an overgrowth phenotype.
behavioral changes were not observed before Quantification of genomic 5-methylcytosine by
capillary electrophoresis indicated that both IVF1 Are there reasons to be concerned? Animal
and IVF2 fetuses differed from in vivo controls studies strongly suggest that stress in specific
[118, 119]. Lastly, there were significant differ- windows of development can be associated with
ences in DNA hypomethylation in liver and muscle long-term health complications. The low birth
of IVF1 fetuses and significant hypermethylation weight outcomes associated with ART live births
in liver of IVF2 fetuses. may predispose to a greater extent of vascular
and metabolic disorders. The late stages of game-
togenesis and early embryo development are a
14.6.7 Cryopreservation of Gametes/ highly sensitive window for potential epigenetic
Embryos programming influenced by environmental states.
Studies in animal models and initial studies in
Mouse oocytes subjected to vitrification show an humans suggest that multiple aspects of the pro-
increase in H3K9 methylation and H4K5 acetyla- cess of ART can result in epigenetic dysregula-
tion [121]. This epigenetic alteration could be tion. The current evidence endorses the important
secondary to the freezing and thawing process or need for longitudinal studies for surveillance of
to the use of cryoprotectants. One study found the health outcome of ART conceptions as well
that the methyltransfer activity of DNMT3a is as basic research geared to understanding the
stimulated by the addition of dimethyl sulfoxide mechanisms that can lead to epigenetic errors.
(DMSO), a cryprotectant commonly used in vit- Unfortunately, despite million of people suffer-
rification or cryopreservation process [122]. It ing from fertility-related problems, the NIH in
should, however, be noted that epidemiological 2009 spent only 75 million dollars out of a 31.2
evidence suggests reduced perinatal morbidity in billion budget (0.002%) to fund research related
children born after cryopreservation of embryos to infertility [124]. In addition, given the pro-
or oocytes [123]. Reasons to explain the better found effects of culture media on embryo gene
outcome include the lack of adverse effect of hor- expression, it is worrisome that the composition
mone stimulation and the fact that embryos sur- of commercial proprietary media used for human
viving freezing and thawing might be of better IVF is not disclosed. Until the risks and safety of
quality than fresh embryos. ART are better defined, its use should be reserved,
as with any medical treatment, to patients with
clear indication after exhausting other more con-
14.7 Conclusions servative therapeutic interventions. Research and
interventions to optimize in vitro culture and
At our current state of knowledge, the vast major- other aspects of ART may have potential to trans-
ity of children conceived by ART are healthy and late to lower risk of disease of later life with
the established epigenetic risks from IVF are attendant lower morbidity, mortality, and eco-
confined to two rare and sporadic disorders. nomic cost.
Although both conditions are associated with
profound disability, the absolute risks of AS and Acknowledgment This work is supported by NICHD
grant R01 HD 062803 - 01 A1 to PFR.
BWS are remote. In this context, these disorders
are unlikely to defer prospective infertile couples
from pursuing treatment. For many patients, ART
represent the only therapeutic option to have a References
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Novel Approaches of Sperm
Selection for ART: The Role 15
of Objective Biochemical Markers
of Nuclear and Cytoplasmic Integrity
and Sperm Function
Gabor Huszar and Denny Sakkas
Abstract
With the technological advancements in assisted reproduction treatment, it
is now feasible to cause fertilization and pregnancy by injection of a single
spermatozoon into the oocyte via the ICSI method. The ultimate goal in
this respect is the selection of a spermatozoon that has genetic and cellular
attributes comparable to those sperm that interact with the zona pellucida
under physiological or conventional IVF fertilization conditions. In light
of this aim, this review focuses largely on objective biochemical markers
within the nuclear and cytoplasmic compartment of spermatozoa, which
define and reflect sperm developmental and genetic characteristics that are
optimal for fertilization as well as for paternal contribution to the develop-
ing embryo. The ICSI sperm selection methods, including those based on
hyaluronic acid binding and high-magnification visualization, are dis-
cussed along with the research evidence that supports the interrelated
developmental and genetic integrity of the selected sperm.
Furthermore, the review examines the use of other sperm diagnostics
which may guide us in the treatment of male factor infertility patients.
The diagnostic techniques examined include those focusing on the sperm
nuclear integrity, in particular the Sperm Chromatin Structure Assay
(SCSA).
The fundamental information being generated from more molecular
and biochemical-based analysis of sperm will create a basis for identifying
key deficiencies in spermatozoa that might be implicated in the defective
sperm function observed in a significant proportion of infertile males and
may aid in both diagnosis and treatment.
D. Sakkas ()
IVF Laboratories, Department of Obstetrics, Gynecology
and Reproductive Sciences, Yale University School of
Medicine, New Haven, CT, USA

212 G. Huszar and D. Sakkas
Keywords
Sperm nucleus • Spermatogenesis • Spermiogenesis • Intracytoplasmic
sperm injection • Male infertility • Protamine • Hyaluronic acid • Apoptosis
15.1 Introduction 15.2 Beyond Routine Sperm

Analysis
Infertility has been commonly defined as failure
to conceive after 12 months of regular inter- 15.2.1 Sperm Biochemical Markers
course. Based on this definition, the prevalence
of infertility has been calculated to be 10–15% A major approach in the discovery of sperm bio-
[1]. A multicenter study conducted by the World chemical markers, independently from sperm
Health Organization (WHO) concluded that in concentration and motility in the semen, is based
20% of infertile couples, the predominant cause on the recognition that there are objective mark-
of infertility is the male factor, while in a further ers of sperm function that focus on elements of
27% of couples both partners contribute [1]. In spermatogenesis and spermiogenesis that are
the 2008 national US statistics, 35% of the abnormal in the process of sperm development
IVF-ET cycles reported in the United States were from spermatogonium to spermatozoa. One of
diagnosed with male factor infertility either as a the first recognized morphological markers was
single (17%) or combined (18%) diagnosis [2]. cytoplasmic retention, which was measured by
While these statistics underlie the importance of the excess cytoplasm in sperm; biochemically,
male factor in reproduction, the clinical and ana- this observation translated to an increased sperm
lytical methodologies used to diagnose male creatine phosphokinase (CK) activity [8]. The
infertility are still evolving [3]. variation in CK activity in the various semen
Semen analysis is routinely used to evaluate samples has been well documented, as well as the
the male partner of the infertile couple. Although fact that sperm recovered from pellets of density
widely used parameters for normal semen mea- gradient centrifugation had substantially lower
surements have been published by the WHO, the CK content [9]. A logistic regression analysis of
current normal values for sperm concentration, 180 couples with oligozoospermic husbands
motility, and morphology fail to meet rigorous when treated with intrauterine insemination indi-
clinical, technical, and statistical standards. With cated that the increased levels of CK activity in
the advent of ICSI [4], the onus to understand the these oligospermic men predicted a lower likeli-
biology of which sperm fertilized the egg and the hood for pregnancy [8].
ongoing research focusing on biochemical mark- The connection between arrested cytoplasmic
ers of sperm function [5] have taken higher prom- extrusion and diminished sperm fertilizing func-
inence, as the pathology in male factor infertility tion was also established by experiments using
patients, who require ICSI treatment, is likely to CK-immunocytochemistry of sperm-hemizona
be of higher complexity. complexes; only the sperm with clear heads with-
There are, however, valid concerns about the out cytoplasmic retention were bound to the
use of ICSI in these low fertility patients [6], even hemizona [10]. This important finding led to a
though large follow-up studies do not show any concept, described in the latter part of this chap-
major differences between ICSI, IVF, and normal ter, relating to one of the new methods available
conceptions [7]. In this chapter, we examine the for sperm selection.
current and new methods available to identify Other aspects of the spermatogenetic and
and select the optimal sperm to be selected to fer- spermiogenetic processes, including (a) specific
tilize the oocyte. protein expression in human sperm (for example
15 Novel Approaches of Sperm Selection for ART 213
the HspA2 chaperone); (b) persistent histones fragmentation values in aliquots of the same
which indicate abnormal histone-transition pro- sperm fraction used for IVF, measured by
tein-protamine replacement process; (c) apop- TUNEL, were significantly correlated with preg-
totic processes; (d) and reactive oxygen species nancy outcome. This is in sharp contrast to the
(ROS) production within the sperm or in the sur- results reported by Bungum et al. [15] who found
rounding seminal fluid [5, 11–13] will all be no correlation between sperm DNA fragmenta-
assessed in relation to novel methods of sperm tion values in the samples used for IVF, as mea-
selection. sured by the SCSA test, and pregnancy outcome.
Another misconception is that TUNEL is unique
to apoptosis, which is studied in many sperm
15.2.2 Sperm Nuclear DNA Integrity DNA assessment papers; this correlation to apop-
tosis in sperm is an overestimation of this test’s
Over the past two decades, a number of tests have capability [30].
been introduced for the analysis of sperm nuclear
DNA fragmentation [14, 15]. These tests include
TdT-mediated-dUTP Nick-End Labeling (TUNEL) 15.2.3 SCSA Issues and Usefulness
[16], COMET [17], chromomycin A3 (CMA3)
[18], in situ nick translation [19, 20], DBD-FISH In 1980, Evenson et al. [31] published a pioneer-
(DNA breakage detection fluorescence in situ ing paper in Science entitled “Relation of mam-
hybridization) [21], the sperm chromatin disper- malian sperm chromatin heterogeneity to fertility”
sion test (SCD) [22], and the Sperm Chromatin in which they used flow cytometry measurements
Structure Assay (SCSA) [23]. of heated sperm nuclei to reveal a significant
One of the important questions related to sperm decrease in resistance to in situ denaturation of
DNA fragmentation analysis is the type of breaks spermatozoal DNA in samples from bulls, mice,
occurring in the DNA strands. First, whether the and humans of low or questionable fertility when
breaks are single or double-stranded and what is compared with others of high fertility. They then
needed to detect them. The different tests therefore went on to suggest that flow cytometry of heated
sometimes require an initial step of denaturation in sperm nuclei and testing of DNA chain integrity
order to detect DNA breaks: such as the SCSA [24, could provide a new and independent determi-
25], SCD [22], or COMET at acid or alkaline pH. nant of male fertilizing potential and the ability
Other tests such as TUNEL [16] (in situ nick trans- of sperm to provide paternal contribution.
lation [19]) and COMET at neutral pH [26] do not The SCSA test has been extensively studied
require an initial denaturation step and, therefore, to date from the clinical point of view [31–36].
measure single-stranded or double-stranded DNA The SCSA test measures sperm susceptibility to
breaks directly. In general, single-stranded DNA DNA denaturation after exposure to a mild acid.
damage provides a better prognosis and is easier to DNA of sperm with normal chromatin structure
repair than double-stranded DNA damage. Single- do not denature, while if the DNA is somewhat
stranded DNA fragmentation may be caused by damaged and contains breaks, DNA strands can
unrepaired DNA nicks generated during the pro- reach different degrees of denaturation, which
cess of meiosis chromatin remodeling. It might be is detected after mild acid treatment and with
also caused by oxygen radical-induced damage. acridine orange staining [35]. The degree of
Double-stranded DNA damage is more likely to DNA damage is measured by flow cytometry
lead to failure of an individual sperm to pass on its and is expressed as a DNA Fragmentation Index
paternal DNA [27]. or DFI. Previous studies indicate that a DFI
Regarding tests that measure DNA damage value >27% is associated with pregnancy fail-
directly, some controversy still exists regarding ure in ART [37, 38]. However, recent reports
which test is best [28]. A recent study reported challenge the predictive value of the SCSA test
by Borini et al. [29] showed that sperm DNA [15, 39, 40].
One possible reason for the discrepancy between In 1988, a paper by Aitken and Clarkson [45]
studies is the variability of sperm concentrations demonstrated that centrifugal pelleting of unse-
within the samples studied. In other words, for fer- lected sperm fractions from human ejaculates
tilization to occur, there is a minimal threshold caused the production of ROS (superoxide and
number of normal fertile sperm that are necessary. hydroxyl radicals) within the pellet. ROS produc-
If this amount of fertile sperm is present (the thresh- tion has induced irreversible damage to the sper-
old level depends on the conception model), the matozoa and impairment of their fertilizing
additional presence of nonfertile sperm, as assessed ability. Superoxide radicals cause peroxidation of
by SCSA or any other equivalent test, whether 20, sperm plasma membrane phospholipids [46] and
40, or 70% of the total sperm content of the semen, elevated superoxide production, through a mem-
is irrelevant. The importance of examining nuclear brane-bound NADPH oxidase system, which has
DNA integrity is clearly highlighted as numerous been implicated in defective sperm function at
studies have emphasized its relationship to human the cellular level [47].
fertility success [28, 41, 42]. As an alternative concept, addressing the
In animal studies, an inverse correlation was question whether all sperm are equally affected
shown between increasing levels of DNA dam- by ROS propagation, using the sperm biochemi-
age inducers, such as heat and radiation, and cal markers, three related points were addressed
good reproductive outcome. For example, the in the Huszar laboratory [9]. First, a relationship
studies by Ahmadi and Ng [43, 44] showed that was found between cytoplasmic retention as
fertilization rates, blastocyst development, and measured by CK activity and ROS production.
the rates of live births are very much related to Thus, retention of the excess cytoplasm was pro-
the degree of DNA damage secondary to con- portional with the ROS production. The second
trolled amounts of radiation. point with respect to the CK and ROS production
In relation to natural conception and intrauter- was the fact that sperm fractions purified by
ine insemination rather than fertility treatments Percoll gradient centrifugation showed lower
by conventional IVF or IVF-ICSI, if an advanced ROS production and CK content. Third, in order
sperm selection method is used, the ICSI results to study whether the iatrogenic cause of increased
are not related to the results of DNA integrity ROS production indeed propagates from sperma-
within the whole sperm sample. The single final tozoa to spermatozoa, the following experiments
determinant of pregnancy success is the qual- were performed. Semen pairs [A and B] were
ity of sperm DNA in the actual fertilizing studied by first assessing the sperm destruction
spermatozoa. by ROS following measurement of malonyldial-
dehyde (MDA), an end product of sperm lipid
peroxidation. The two semen samples were
15.2.4 Sperm Preparation by Gradient assayed independently, and the mixture of the
Centrifugation two samples was also subjected to MDA assess-
ment. All three semen fractions (A, B, and A + B)
The two-phase gradient centrifugation and frac- were then centrifuged for 10 min, and the sperm
tionations of sperm is based on the specific pellets were incubated at 37°C for 30 min.
gravity differences between normally developed Further, the MDA has been remeasured in all
spermatozoa (ie. sperm with completed cytoplas- three samples. The data indicated that the aggre-
mic retention and a head containing only DNA gate MDA levels were the same as in the original
plus plasma membrane), as opposed to sperm A and B semen samples before the mixing, cen-
affected by dysmaturation (excess cytoplasm in trifugation, and incubation of the two semen
the sperm head). Normal sperm will sediment in the samples, whether the initial MDA measurements
pellet, whereas the lighter, dysmature sperm with were high (ROS present) or low [9].
cytoplasmic retention will remain at the interface Thus, we could deduce that in the case of iat-
or in the top portion of the gradient media. rogenic generation, the propagation of the ROS
production does not apparently apply to the nor- from oligozoospermic samples, even with high
mally developed sperm without cytoplasmic amounts of nonmotile gamete and/or nongamete
retention. Further, based on the data, Huszar and cell contamination.
colleagues concluded that the increased sperm
MDA levels and ROS production are apparently
not “acquired,” but arise from an “inborn” error 15.2.5 Considerations Related to Men
of spermatogenesis and spermiogenesis [9]. This with Excessive Semen ROS
experiment was carried out in 1993–1994, yet the Production
data, with respect to “inborn errors” and the role
of objective biochemical markers in the detection Sperm preparation for assisted reproduction in
of sperm function and fertility, have remained men with high seminal ROS may improve sperm
valid and confirmed in the ensuing years with quality if media containing antioxidants such as
more studies [5, 31, 48]. reduced glutathione and catalase/EDTA is used
Using the various sperm biochemical attri- [55–57]. Such an approach may improve the
butes, it has been confirmed that sperm quality is quality of gametes used by protecting the sper-
improved by density gradient centrifugation matozoa from high oxidative stress.
techniques. In addition to the decline of ROS and In case of oxidative stress-related infertility,
CK levels in sperm following gradient centrifu- the appropriate treatment strategy is the elimina-
gation [5, 45, 49], Gandini and coworkers mea- tion of the underlying cause, whether lifestyle
sured DFI and HDS on both the raw and gradient and environmental factors that enhance oxidative
semen purified aliquots [39, 50]. They found stress, infections and antibiotic treatment, or use
that enriched cell suspensions contained sperm of sperm for IVF-ICSI arising from the testicular
with better motility, morphology, SCSA, and DFI. sperm extraction (TESE sperm). It is also advis-
The Sakkas laboratory had also shown that when able to avoid using cryopreserved spermatozoa
sperm samples from different men were prepared which are sensitive to DNA damage [58].
using density gradient techniques and stained Regarding the utilization of TESE sperm, it is
using the CMA3 fluorochrome, which indirectly established that sperm originating in the adlumi-
demonstrates a decreased presence of protamine, nal area are protected from oxidative attack,
or with in situ DNA nick translation which exam- whereas most ROS-initiated DNA fragmentation
ines for the presence of endogenous DNA nicks, occurs during epididymal storage [59]. In studies
a significant (P < 0.001) decrease in both CMA3 of sperm DNA integrity within the same individ-
positivity and DNA strand breakage was observed uals in ejaculated sperm or epididymal sperm,
[9, 20, 48, 51, 52]. there were significant improvements in sperm
Another technology that could aid in the prep- DNA quality in TESE samples [60, 61]. Thus, the
aration of sperm populations is microfluidics use of testicular sperm in men with poor DNA
[53]. It has been reported that the use of a micro- quality is recommended if more conservative
fluidic device, designed with two parallel laminar treatments such as antioxidant administration had
flow channels that could preferentially separate failed.
motile spermatozoa into a separate outlet, Another approach is offered by the hyaluronic
increased sperm motility in a sample from 44 to acid-mediated sperm selection, as it will be
98% and morphology from 10 to 22% following described later, at selection for single sperm for
processing [54]. This microfluidic device pro- IVF/ICSI. The rationale for this approach is as
vided a novel method for isolating motile, mor- follows: (a) A close correlation has been shown
phologically normal spermatozoa from semen between ROS production and cytoplasmic reten-
samples without centrifugation. This may limit tion which represent gamete dysmaturity [9].
some of the iatrogenic preparation problems Thus, it is expected that dysmature sperm, which
described earlier. This technology may also produces and/or have been affected by ROS,
prove useful in isolating motile spermatozoa would have a high amount of DNA degradation,
and due to the arrested spermiogenesis, would 8-OHdG type, followed by double-stranded
not bind to hyaluronic acid, and (b) Sperm selec- DNA fragmentation that may be mediated by
tion by HA-binding is also helpful as hyaluronic caspases and endonucleases. The implications of
acid-bound sperm is also devoid of excess persis- combined nucleotide damage and DNA strand
tent histones, DNA fragmentation, and the apop- fragmentation, in addition to its diagnostic value
totic process. Thus, sperm selection by deselection (oxygen radical-induced damage may be treated
of dysmature spermatozoa and positive selection with antioxidants), are the fact that this type of
of normally developed spermatozoa with low or DNA fragmentation causes a diminished sperm
no ROS production is an appropriate and practi- functional integrity because the great majority of
cal solution [5, 10, 83]. the sperm cells will be affected [27].
In line with the above discussion regarding
ROS-damaged sperm, most negative effects may
15.2.6 Impact of Sperm Chromatin be avoided by using testicular sperm. A number
Maturation on Sperm Function of reports indicate that sperm DNA damage is
significantly lower in the seminiferous tubules
Protamine replacement of histones and transi- compared to the cauda epididymis or ejaculated
tion proteins may also facilitate the modulation sperm [59, 60]. The use of testicular sperm in
of the expression pattern of the genome, as well couples with repeated pregnancy failure in ART,
as the imprinting pattern of the gamete [62, 63]. and high sperm DNA fragmentation in semen,
This methylation lends variability to the pater- resulted in a significant increase in pregnancy
nal genome during zygotic gene expression rates [61, 64]. Moreover, pregnancy rates in first
events with individual consequences in devel- cycles of TESA-ICSI are relatively high [27].
opmental effects. There are several studies on These results are consistent with the notion that
the impact of sperm chromatin methylation levin couples with long-standing infertility, or
els in IVF success regarding both fertilization repeated pregnancy failure in ART without an
and pregnancy rates. Some support the idea that apparent cause, sperm DNA fragmentation could
children conceived by ART do not show a be an underlying factor. Thus, use of testicular
higher degree of imprinting variability. The sperm with very low levels of sperm DNA frag-
present understanding with respect to chroma- mentation reduces the burden of sperm DNA
tin methylation during the remodeling process repair by the oocyte. In the majority of cases,
is not complete, but it is known that the DNA sperm DNA damage occurs or is increased in the
methylation and chromatin recycling, during epididymis and, therefore, DNA fragmentation
the histone-to-protamine exchange, are impor- levels in testicular sperm originating in the semi-
tant [63]. niferrous tubuli could be relatively lower [61].
With respect to sperm in the seminiferous tubuli
and in the epididymis, it is relevant to consider
15.3 Source of Sperm for Assisted that both in horses and in humans, sperm devel-
Reproduction opment with respect to cytoplasmic extrusion
and HspA2 expression occurs within the adlumi-
It could be argued that whatever sperm prepara- nal area [63]. Thus, regarding TESE spermato-
tion technique is used, the protection of ejacu- zoa, the usefulness of this approach is limited to
lated spermatozoa may be late due to the the location of the testicular origin, i.e., whether
testicular and epididymal exposure to ROS. sperm extracted from the testes would originate
Posttesticular sperm DNA damage induced by in the seminiferous tubuli, in which the sperma-
hydroxyl radicals or after exposure to ionizing tozoa was already released from the adluminal
radiation is associated with nucleotide damage. compartment after all aspects of the sperm cellular
In the first stage, the damage produced is of the development have been completed [63].
h istones [71–75]. Accordingly, based on the

15.4 Deselection of Spermatozoa variations in sperm maturity, staining intensity
with Apoptotic Marker was light, intermediate, and dark tone and repre-
Proteins sent sperm with mature, moderately immature,
and severely immature developmental status,
The presence of apoptotic sperm in the ejaculate respectively [30].
was shown by the Sakkas laboratory and has It is clear that sperm chromatin is essential for
since been verified by a number of other studies, sperm function and subsequent embryonic devel-
for example, Fas, phosphatidylserine, Bcl-XL, opment because defects in sperm chromatin are
p53, etc. [51, 65–67]. The expression of these linked to natural reproductive malfunctions like
apoptotic marker proteins on the surface mem- spontaneous abortion as well as assisted repro-
brane of ejaculated spermatozoa indicates that ductive failure [15, 73]. DNA damage is the main
this phenomenon could be utilized to select non- cause of implantation failure in embryos derived
apoptotic spermatozoa from semen samples [68]. from healthy eggs fertilized by protamine com-
A novel and promising technique of annexin promised sperm [74, 76].This requirement for
V-conjugated microbeads (ANMB) – magnetic- histone-to-protamine exchange in order to main-
activated cell sorting (MACS) – has been shown tain chromatin integrity facilitates the correct
to remove spermatozoa with phosphatidylserine folding of DNA and preserves DNA chain integ-
externalization (marker of apoptosis) and pro- rity. The relationship between persistent histones
duce a higher quality nonapoptotic sperm frac- and DNA degradation was highlighted in a recent
tion [68]. Furthermore, these nonapoptotic cells report from the Huszar laboratory. In sperm with
display higher fertilization rates when used in dark aniline blue staining, representing extensive
animal models for IVF and ICSI. levels of persistent histones, there was a lack of
fluorescence in situ hybridization (FISH) chro-
mosome probe binding [77]. Thus, deficiency in
15.5 Sperm Chromatin sperm chromatin development, improper DNA
Maturation and its folding, and consequentially diminished DNA
Importance chain integrity caused diminished FISH probe
binding, as there were limited number of long
The formation of mature spermatozoa is a unique DNA sequences that would facilitate the binding
process involving a series of developmental steps of FISH probes. In addition, environmental stress
in both the nuclear and cytoplasmic compartments also can disturb chromatin maturation and ulti-
including histone-transition protein-protamine mately lead to an abnormal chromatin structure
replacement. In this process, first somatic histones that is incompatible with fertility [12, 75].
are replaced by testis-specific histone variants,
which are then replaced by transition proteins
(TP1 and TP2) in a process that involves exten- 15.6 Sperm Binding
sive DNA rearrangement and remodeling [69]. to Hyaluronic Acid
A greater than tenfold compaction of sperm
chromatin is achieved during the final phases of Various biochemical sperm markers indicate that
spermatogenesis when the histone that is bound to human sperm bound to HA exhibit attributes sim-
DNA is almost completely replaced by protamine ilar to that of zona pellucida-bound sperm, includ-
1 and protamine [70]. ing minimal DNA fragmentation, normal shape,
Earlier studies showed an association between no persistent histones, and low frequency of
diminished histone-transition protein-protamine chromosomal aneuploidies [5]. Jakab et al. [78]
exchanges that may be detected by aniline blue reported that the frequency of chromosomal
staining of the excess persistent lysine-rich aneuploidies in HA-bound spermatozoa was
s ignificantly decreased after the HA-mediated Another aspect of the markers of sperm
sperm selection, regardless of the frequency of d evelopment is the enhancement of sperm with
chromosomal aberrations in the initial semen. It normal morphology in the HA-bound sperm frac-
was suggested that clinical use of HA-mediated tion. Studies in the Huszar laboratory indicated
sperm selection could ultimately solve the perti- that there was a 2–3-fold enrichment of Tygerberg
nent problem of ICSI with increased frequency of normal sperm compared to the respective semen
aneuploidies in the offspring when the ICSI is sperm fraction, which, interestingly, agreed with
performed with embryologist-selected sperm the finding of the Tygerberg group with respect
from samples with high proportion of dysmature to the enrichment of normal morphology sperm
spermatozoa. in the zona pellucida-bound vs. semen sperm
Subsequently, a number of studies have now fraction [81].
indicated that HA-bound sperm used in the ICSI
procedure may lead to increased implantation
rates. In one such study, Parmegiani et al. [79] 15.6.1 Selection of Single Sperm
showed that in 293 couples treated with HA-ICSI for ICSI by Hyaluronic Acid (HA)
vs. 86 couples treated with conventional ICSI Binding: Normally Developed
(historical control group), all outcome measures Spermatozoa Selectively Bind
of fertilization, embryo quality, implantation, and to Solid State HA
pregnancy were the same or improved in the
HA-bound sperm group. The implantation rate The data on biochemical markers have also
was increased from 10.3% in conventional ICSI revealed that the sperm membrane remodeling
to 17.1% in the HA group. The authors concluded process is inherently related to upstream sper-
that if multicenter randomized studies confirm the matogenetic and spermiogenetic events. First, the
beneficial effects on ICSI outcome, HA could be Huszar group looked at the sperm markers of
considered as a routine choice for “physiologic” LDH-C, cytoplasmic retention, DNA integrity,
sperm selection prior to ICSI. A smaller clinical sperm shape, chromatin abnormalities, apoptotic
trial assessing the same technology by Worrilow markers, and enzymes [5]. Sperm with cytoplas-
et al. [80] has also shown that clinical pregnancy mic retention (arrested cellular maturation)
rates are improved when using HA-selected sperm showed a positive signal with these probes; how-
compared to conventional ICSI. Furthermore, the ever, sperm aliquots that were bound to a
sperm HA-binding score (the proportion of sperm hyaluronic acid-coated slide contained no sperm
that underwent plasma membrane remodeling in with the positive signals for any of the dysmatu-
spermatogenesis and binds to hyaluronic acid) is rity and DNA degradation markers.
an important diagnostic indicator. Men with Furthermore, in a more refined experiment,
<55% binding score would particularly benefit, as semen aliquots were smeared on glass slides and
their ICSI success rates were improved by (20– the sperm were fixed for the various markers.
30% higher pregnancy rates) by using the Another aliquot of the same semen was incubated
HA-mediated sperm selection [80]. Thus, it is on hyaluronic acid-coated slides. After the 15 min
important that in IVF programs, the Andrology binding process, the unbound sperm were gently
Laboratory performs the sperm-HA-binding test rinsed off, and the HA-bound fraction was fixed.
for the husbands of IVF-ICSI couples and that the Finally, both the whole semen fraction on glass
IVF team triages the couples according to their slides and the HA-bound sperm fraction on the
HA-binding score. This score provides the infor- HA-coated slides were stained for the biochemical
mation on the proportion of sperm with attributes markers and were evaluated. In the semen sperm
of dysmaturity, such as DNA fragmentation, chro- fraction, there were sperm with cytoplasmic reten-
mosomal aneuploidies, persistent histones, cyto- tion, DNA degradation (detected in individual
plasmic retention, and lack of HA-binding ability, spermatozoa with DNA-nick translation), aber-
consequential to dysmaturity. rant shape, and persistent histones with aniline
blue staining, whereas in the HA-bound sperm 15.6.3 Does Sperm HA-Binding Test
fraction, there was no presence of sperm with any Predict High DNA Integrity
of the cytoplasmic or nuclear defects [5, 9]. in the HA-Bound Sperm?
A recent example of DNA integrity in the

15.6.2 Sperm Dysmaturity HA-selected sperm was developed by acridine
and Persistent Histones orange [AO] staining of HA-bound spermatozoa, a
reagent which provides green fluorescence for
Regarding the interrelationship between persis- DNA with high chain integrity and orange fluo-
tent histones and arrested sperm development, a rescence for damaged DNA. Baker et al. reported
recent report by the Huszar laboratory indicates previously that the zone pellucida-bound sperm
that diminished sperm fertility and reduced pater- has mostly green fluorescence [82]. In the Huszar
nal contribution of sperm to the embryo may not laboratory, the AO assay was performed with
originate only in the arrested histone – protamine sperm bound to the HA-spot of the PICSI dish,
remodeling, but along with the persistent his- which is used for ICSI sperm selection in IVF
tones, there are other associated factors of dys- laboratories. The data indicated, using the very fine
maturity in the same spermatozoa [30]. In these Polyscience Inc. acridine orange, that literally
double staining studies, a method was developed: 100% of the hundreds of HA-bound sperm were of
First, sperm are stained with aniline blue for green fluorescence. Thus, whether we probe DNA
probing persistent histones. Second, after record- integrity with nick translation or acridine orange
ing the aniline blue-stained sperm fields, and a reagent, the DNA of HA-bound sperm had high
subsequent destaining step, the same sperm are integrity and no cellular dysmaturity defects [5, 83].
probed for sperm shape, for cytoplasmic reten- Thus, one more experiment supports the concept
tion with creatine kinase immuno-staining, for that HA-mediated sperm selection by the PICSI
the apoptotic process with caspase 3 immuno- dish is an optimal tool for ICSI sperm selection.
staining, and for DNA chain fragmentation with Regarding the HA-mediated ICSI sperm selec-
in situ DNA-nick translation. tion, in addition to the DNA integrity, there is now
The evaluation of the same sperm after the first focus on the increase in chromosomal aneuploi-
and second probes convincingly showed an dies with the embryologist-selected sperm, as the
approximately 75% agreement between the ani- aneuploidy frequencies are 3–4 times higher in
line blue staining patterns whether light (no probe the ICSI offspring. Pointing out the relationship
presence), intermediate (some probe detection), between meiotic and late spermiogenetic events,
and dark staining (heavy probe presence) and of it was shown that sperm with cytoplasmic reten-
the various other biochemical probes and sperm tion defects and sperm dysmaturity also have
morphology. This indicated that indeed there is a increased frequencies of chromosomal aneuploi-
relationship between the attributes of arrested dies with a significant correlation at the level of
development or dysmaturity within the same r > 0.75, P < 0.001 [84]. Thus, these data, along
sperm. Also, the experiment demonstrated that the with the experiments by Jakab et al. with FISH
nuclear and cytoplasmic as well as the early and studies of >20,000 spermatozoa [78], suggest
late events of sperm development are related. It that the filtering effect of the zona pellucida has
also suggests that persistent histones and arrested been reconstructed and tested by hyaluronic acid
chromatin modulation fail to initiate protamine binding. No matter how high the aneuploidy fre-
transport and proper DNA folding in the nucleus quency was in the initial semen sperm fraction, in
and are associated with the events contributing to the sperm bound, and removed from HA, there
diminished sperm function. Thus, the data support was a 4–6× decline in disomy and diploidy frequen-
the idea that the later manifestations of arrested cies within the 0.1–0.2% normal range, which
sperm development may originate in common is customary in babies conceived with natural
upstream events of early spermatogenesis [30]. conception or with conventional IVF [5, 78].
15.6.4 Relationship Between the that the HA-bound sperm lacked any of the attri-
Various Biochemical Sperm butes of arrested sperm development, including
Markers of Dysmaturity and cytoplasmic retention, no persistent histones, and
Diminished Sperm Function no DNA fragmentation (detected by two meth-
ods: in situ DNA-nick translation, and acridine
Along with sperm dysmaturity, that is detectable orange fluorescence), and had improved
by defects of the histone-transition protein- Tygerberg strict morphology (approximately 3×
protamine replacement, and by the consequential enrichment of normal sperm vs. the semen sperm
strong aniline blue staining due to excess histone population), as well as normal frequency of chro-
retention (which also has consequences in DNA mosomal aneuploidies, regardless how elevated
folding and DNA chain vulnerability/integrity), the aneuploidy and diploidy levels were in the
there were several other sperm attributes that are semen sperm fraction [5, 9, 77, 78, 89].
associated with arrested cellular development Finally, regarding the validation of the sperm
contributing to diminished sperm fertilizing characteristics discussed above, there are two
potential. points of interest. First, in a study of comparing
The key elements, confirmed with objective sperm binding to hemizonae and HA, there
biochemical markers, include (a) Cytoplasmic was a high correlation and a significant rela
retention. Indeed, high sperm creatine kinase tionship (r = 0.76, P < 0.001), which validates the
activity of oligozoospermic men treated with HA-binding assay and reinforces the idea that the
intrauterine insemination has predicted the lack formation of the zona pellucida and HA receptors
of pregnancies, independently from sperm con- are related during the spermiogenetic plasma
centration and motility [85]; (b) The diminished membrane remodeling [5]. The location of these
expression of the HspA2 chaperone protein. Men receptors is also common in the acrosomal region,
with low sperm HspA2 levels failed to achieve as sperm binding to the zona pellucida and HA
pregnancy in couples treated with conventional follows identical patterns [5, 9]. Second, it was
IVF in two independent studies, one in a Yale- recently published that sperm with dark aniline
Norfolk collaboration and one of a Yale IVF blue staining show no DNA staining with probes
study [86, 87]; (c) Men with sperm cytoplasmic for in situ fluorescence hybridization, or with a
retention and low HspA2 expression had a higher DNA probe [77]. The data suggest that sperm
incidence of sperm with aniline blue staining, with high levels of persistent histones and dimin-
indicating elevated content of histones; (d) In ished protamine content suffer major DNA chain
semen samples with increased frequency of sper- fragmentations, thus the FISH probes are unable
matozoa with cytoplasmic retention, there were to bind and the fragmented DNA dissipates from
increased levels of sperm creatine kinase, and sperm during the multiple steps of the FISH
aniline blue staining; (e) Sperm with cytoplasmic process.
retention have a higher rate of aneuploidy with a
statistically significant relationship (i.e., sperm
with cytoplasmic retention vs. Y disomy: R = 0.78, 15.7 Other Sperm Selection
P = <0.001) [84]; (f) Further, with the establish- Techniques
ment that sperm, after the decondensation step
necessary for FISH or DNA integrity studies, 15.7.1 Sperm Selection by Sperm
maintain their initial shape as it was in semen Charge Properties
[88], an association was demonstrated between
sperm shape and aneuploidies within the same The Aitken group recently reported a novel elec-
spermatozoa. trophoretic sperm isolation device utilizing a
Conversely, study of hyaluronic acid (HA)- separation strategy based on sperm size and
bound spermatozoa that underwent spermioge- electronegative charge. The suspensions gener-
netic plasma membrane remodeling has indicated ated by the electrophoretic separation technique
contained motile, viable, and morphologically Hazout et al. [94] also reported a positive asso-
normal spermatozoa, while exhibiting low levels ciation between high-magnification selection of
of DNA damage. Reportedly, the electropho- sperm cells with normal nuclear shape and preg-
retic sperm isolation procedure is both time- and nancy outcome in patients with repeated conven-
cost-effective [90] and the first pregnancy using tional ICSI failures. In a subgroup of patients
this method for a couple suffering from exten- (n = 72) involved in the study, a noticeable
sive sperm DNA damage was reported [91]. improvement in clinical outcomes (implantation
and birth rates) was observed both in patients
with an elevated (>40%) and moderate (30–40%)
15.7.2 Intracytoplasmic degree of sperm DNA fragmentation and in those
Morphologically Selected with normal sperm DNA status (<30%) using the
Sperm Injection (IMSI) TUNEL assay.
The use of IMSI appears promising [95].
In 2001, Bartoov et al. [92] reported the selection Some drawbacks are, however, present; in par-
of spermatozoa with normal nuclei to improve ticular, the belief that it is a complicated tech-
the pregnancy rate with intracytoplasmic sperm nique that cannot be routinely performed [96].
injection. They went on to verify this technique Future simplification of the selection procedure
by performing ICSI using morphologically nor- by automated ICSI may, however, be possible.
mal sperm, strictly defined by high-power light As the high-magnification approach is increas-
microscopy (x > 6,000). Sixty-two couples, with ingly used, more studies examining high magni-
at least two previous consequent pregnancy fail- fication selected sperm and their respective levels
ures after ICSI, underwent a single ICSI trial pre- of cytoplasmic retention, persistent histones,
ceded by morphological selection of spermatozoa DNA chain integrity, aneuploidy rates, tyrosine
with normal nuclei. Fifty of these couples were phosphorylation, or apoptotic markers should be
matched with couples who underwent a routine performed.
ICSI procedure at the same IVF center and exhib- Another microscopic technology that may
ited the same number of previous ICSI failures. show promise is the birefringence analysis of the
The matching study revealed that the preg- sperm head. Gianaroli et al. [97] previously pos-
nancy rate after modified ICSI was significantly tulated that this could represent both a diagnostic
higher than that of the routine ICSI procedure tool and a novel method for sperm selection.
(66.0 vs. 30.0%). Recently, they performed a prospective random-
More recently, Antinori et al. [93] conducted a ization including 71 couples with severe male fac-
prospective randomized study to assess the tor infertility and performed ICSI using polarized
advantages of intracytoplasmic morphologically light for sperm selection which permitted analysis
selected sperm injection (IMSI) over the conven- of the birefringence sperm head [98]. Twenty-
tional ICSI procedure. A total of 446 couples three patients had their oocytes injected with
with 3 years of primary infertility, the woman aged acrosome-reacted spermatozoa, 26 patient’s
35 years or younger, without workup for female oocytes were injected with nonacrosome-reacted
factor and husbands of severe oligoasthenoterato- spermatozoa, and in 22 patients with both reacted
zoospermia were randomized to eye selected ICSI and nonreacted spermatozoa. They found no
(n = 219; group 1) and IMSI (n = 227; group 2) effect on the fertilizing capacity and embryo
treatment groups. The data showed that IMSI development of either type of sperm, whereas the
resulted in a higher clinical pregnancy rate (39.2 implantation rate was higher in oocytes injected
vs. 26.5%; P = 0.004) than ICSI. In spite of their with reacted spermatozoa (39.0%) vs. those
initial poor reproductive prognosis, patients with injected with nonreacted spermatozoa (8.6%).
two or more previous failed attempts benefited The implantation rate was 24.4% in the group
from IMSI in terms of pregnancy (29.8 vs. 12.9%; injected with both reacted and nonreacted
P = 0.017) and miscarriage rates (17.4 vs. 37.5%). spermatozoa.
6. Devroey P, Van Steirteghem A. A review of ten years

15.8 Future Technologies experience of ICSI. Hum Reprod Update. 2004;10(1):
19–28.
A number of novel new technologies are being 7. Ponjaert-Kristoffersen I, Bonduelle M, Barnes J, et al.
International collaborative study of intracytoplasmic
developed in sperm selection for classical IVF or sperm injection-conceived, in vitro fertilization-
IVF-ICSI. An example is Raman Spectroscopy conceived, and naturally conceived 5-year-old child
which noninvasively distinguish the DNA pack- outcomes: cognitive and motor assessments.
aging and protamine content between normal and Pediatrics. 2005;115(3):e283–9.
8. Huszar G, Vigue L. Spermatogenesis-related change
abnormal cells [99]. Differences identified include in the synthesis of the creatine kinase B-type and
the vibrational marker modes that Raman spectra M-type isoforms in human spermatozoa. Mol Reprod
of individual sperm cells can distinguish and this Dev. 1990;25(3):258–62.
may be used to assess the efficiency of DNA 9. Huszar G, Vigue L. Correlation between the rate of
lipid peroxidation and cellular maturity as measured
packaging for each cell. The attributes followed by creatine kinase activity in human spermatozoa.
are the relative protein content per sperm cell and J Androl. 1994;15(1):71–7.
DNA packaging efficiencies throughout wide 10. Huszar G, Vigue L, Oehninger S. Creatine kinase
ranges for sperm cells with both normal and immunocytochemistry of human sperm-hemizona
complexes: selective binding of sperm with mature
abnormal shapes. Data indicate that single-cell creatine kinase-staining pattern. Fertil Steril. 1994;
Raman spectroscopy could be a valuable tool in 61(1):136–42.
assessing the quality of sperm cells. 11. Huszar G, Stone K, Dix D, Vigue L. Putative creatine
Other molecular techniques may allow the kinase M-isoform in human sperm is identified as the
70-kilodalton heat shock protein HspA2. Biol Reprod.
future analysis of sperm populations. For exam- 2000;63(3):925–32.
ple, DNA methylation patterns of key develop- 12. Aoki VW, Emery BR, Liu L, Carrell DT. Protamine
mental genes have been shown to differ in levels vary between individual sperm cells of infertile
spermatozoa and this may impact embryo devel- human males and correlate with viability and DNA
integrity. J Androl. 2006;27(6):890–8.
opment. In addition, the fundamental information 13. Seli E, Sakkas D. Spermatozoal nuclear determinants
being generated from Proteomic [100] and RNA of reproductive outcome: implications for ART. Hum
[101] analysis of sperm will also create a basis for Reprod Update. 2005;11(4):337–49.
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new tests of sperm genetic integrity into semen analy-
be implicated in the defective sperm function sis: breakout group discussion. Adv Exp Med Biol.
observed in a significant proportion of infertile 2003;518:253–68.
males and aid in both diagnosis and treatment. 15. Bungum M, Humaidan P, Axmon A, et al. Sperm
DNA integrity assessment in prediction of assisted
reproduction technology outcome. Hum Reprod.
2007;22(1):174–9.
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The Role of the Oocyte
in Remodeling of Male Chromatin 16
and DNA Repair: Are Events During
the Zygotic Cell Cycle of Relevance
to ART?
Liliana Ramos and Peter de Boer
Abstract
Transmission of male genetic material through highly differentiated and
specialized sperm cells is a highly dynamic process, in which the zygote
plays a major role. Chromatin remodeling and DNA repair are involved,
both during spermatid nuclear elongation and after gamete fusion at chro-
matin remodeling from a protamine dominated towards a nucleosomal
chromatin state. Roles for DNA repair are further envisaged for zygotic
G1 and S-phases, with an active role of the maternal complement toward
the male PN. In this chapter, findings from mainly the mouse, extending
into paternal DNA demethylation in the zygote, have been integrated.
Although biological insight into the roles of the oocyte toward chromatin
of the sperm cell is developing, there is a gap between the current knowl-
edge for mouse and the observations made on human sperm DNA and
chromatin, fertilization efficiency (after IVF and ICSI), and the subse-
quent zygote and cleavage stage development of human embryos. In part,
this is due to the use of normal mouse sperm for research, whereas in male
factor infertility, the fraction of normal gametes can be sharply reduced.
On the other hand, human oocytes will not be homogeneous in chromatin
remodeling and DNA repair capacity. Nevertheless, there are enough
experimental data in transmission biology to make a plea for more careful
monitoring of DNA and chromatin in ART children and adults.
L. Ramos ()
Department of Obstetrics and Gynecology, Radboud
University Nijmegen Medical Center, Nijmegen,
The Netherlands

228 L. Ramos and P. de Boer
Keywords
ART • Base excision repair • Chromosomes • Nucleus decondensation
• DNA demethylation • DNA damage • DNA repair • Double-strand breaks
• Embryo, epigenetics • Gametes • GammaH2AX • Fertilization • Histones
• Homologous recombination repair • ICSI • Immunofluorescence
• Imprinting • Matrix-associated regions • DNA methylation • Mutagenesis
• Nonhomologous end joining • Nucleosomes • Nucleotide excision repair
• Oocytes • Oxydative stress • Pronucleus • Protamines • Polycomb pro-
teins • Spermatozoa • Spermiogenesis • S-phase • Synchrony • Zygote
insight that is required is not readily available as

16.1 Introduction experimental mouse models use normal sperm
cells [2–4]. When in the mouse an experimental
In recent years, exciting new insights have been condition such as unprotected sperm freezing is
made regarding mouse/human gametes: (a) non- applied, detrimental phenotypic effects arise [5].
random distribution of nucleosomes in sperm, The problem in ART is exacerbated because of
(b) different DNA repair requirements between the fundamental differences between human sper-
the parental chromosome sets, (c) DNA demeth- matogenesis and that of experimental animals
ylation involves DNA repair, and (d) the presence (rodents and farm animals) that usually have very
of small RNA molecules in sperm. homogeneous sperm populations. On the con-
Seemingly unrelated, these findings all touch trary, human sperm, especially in cases of male
upon one underlying aspect: the transmission of factor sub- and infertility, is among the most vari-
genetic and nongenetic (epigenetic) information to able cell populations to be found in the body [6].
the next generation. Although already a topic fas- This chapter will focus mainly on mouse data
cinating for biologists, it gains in medical rele- concerning the remodeling of sperm chromatin
vance due to the widespread and increasing use of by the zygote, particularly during sperm pronu-
artificial reproductive techniques (ART). As a con- cleus formation. Thereafter, repair of DNA
sequence, ART babies are often conceived from damage and removal of DNA methylation will be
gametes that in normal reproduction would never covered, ending with differences in DNA replica-
have functioned. What are the de novo genetic and tion between male and female pronuclei as asso-
epigenetic effects of this type of reproduction? ciated with DNA repair. At the end, we will
The answer to this question is currently largely introduce information from human sperm and
unknown, but the new studies yield strong hand- embryo culture that can be interpreted against the
holds that can guide and focus future research. presented background of mouse data. Although
Although in ART, the term fertilization is used much uncertainty remains, the conclusion is
synonymous to pronucleus formation (2PN), it is probably justified that the first 26 h of our exis-
actually a more lengthy process that encompasses tence will hallmark our life more than at any
both cytoplasmic and nuclear fusion, hence other short period of time.
includes first cleavage. Fusion between male and
female pronuclei is never obtained, only apposi-
tion, in the human earlier after gamete fusion
than in the mouse. The two chromosome sets do 16.2 Male Chromatin Remodeling,
not intermingle at the spindle and only gradually Preparation in the Male
mix in next rounds of cell division [1]. Germline
The premise of this chapter is that, in ART, the
borders of the normal interaction between gam- Chromatin remodeling during the nuclear elon-
etes are stretched due to the use of gametes that gation phase of spermiogenesis and chromatin
in vivo would never play a role. The biological remodeling after sperm entry in a number of
16 The Role of the Oocyte in Remodeling of Male Chromatin and DNA Repair 229
aspects are mirror images. Loss of nucleosomes, exposed and chromosome abnormalities were
hence histones, and acquiring protamines to sta- observed in the zygote at the first cleavage divi-
bilize the DNA lead to chromatin condensation sion. In agreement with the standing concept,
needed for sperm formation. Male chromatin DNA repair decreases from the onset of chroma-
decondensation in the zygote involves the reverse tin remodeling in elongating spermatid nuclei on
order, though at a much higher rate. No until completion of spermiogenesis. Our under-
protamines, as observed at the level of immune standing of DNA metabolism in this important
fluorescence (IF), are retained in zygotic male phase of male reproduction is rudimentary. This
chromatin [7]. Nucleosomes consist of an octamer is illustrated by the reported decline in dsDNA
of four histone molecules, of which each is pres- breaks upon inhibition of TopoII beta by etopo-
ent twice (two times a H2A-H2B dimer and twice side [29]. The anticancer cell drug etoposide
a H3-H4 dimer) and around which 146 bp of inhibits the ligation of the TopoII-induced dsDNA
DNA is wrapped [8]. break. In the zygote, and in agreement with
Both stages involve Topoisomerase II (TopoII) expectation, etoposide treatment induced persis-
inflicted DNA-double-strand (dsDNA) breakage tent dsDNA breaks [10].
and ligation (zygote: [9–11]; elongating sperma- There is no proof for stabilization of free
tids: [12, 13]). During spermiogenesis, a dsDNA ends during the later stages of spermio-
nucleosomal chromatin state is only selectively genesis, the current opinion being that religation
lost [14–17]. Where protamines dominate, high is complete [29]. Techniques to make this an
DNA content doughnut-like toroids are formed absolute statement are currently lacking. As
[18]. These are believed to encompass only one already mentioned in this section, from human
loop domain of about 50 kb that is attached to a and mouse sperm mutagenesis studies [30–33], it
nuclear matrix [19]. The current insight is that as has been deduced that DNA breaks induced at
TopoII religates its break [20], most TopoII- these later remodeling stages and in sperm are not
induced cuts, needed to alleviate supercoiling of repaired in situ upon induction, but are processed
nucleosomal DNA, will not require backing up by the oocyte after sperm penetration [34]. The
by other DNA repair systems. The extent of non- statement that dsDNA break repair is principally
ligated TopoII-induced breaks present in elongat- impossible in sperm chromatin can, however, not
ing spermatids is currently hardly known [21]. be made, although it is unlikely yet alone for the
PARP-1 and PARG are involved in the regulation adaptation of chromatin structure that accompa-
of polyribosylation of chromatin proteins in nies DNA repair [35, but see 25]. Methods to
mouse elongating spermatids [22]. This is an measure DNA breaks directly (by neutral and
argument for active DNA break repair at this alkaline comet assay, TUNEL assay) and indi-
stage. In man, PARP activity was established by rectly (by Sperm Chromatin Structure Assay
situ histochemistry on testis sections [23, 24]; the [SCSA] and sperm chromatin dispersion [SCD]
presence of PARP-1, PARP-2, and PARP-9 was tests) are by far not accurate enough to test human
determined on sperm by western blotting [25]. sperm for active DNA repair [36, 37].
Also, transition proteins (that form an intermedi- We do know for a long time that during sper-
ary chromatin state after histone shedding [26]) matid nucleus elongation, replacement of
and protamines are supposed to act as DNA- nucleosomes by protamines is not complete, less
stabilizing factors. Experimental proof for this so for the human than for mouse. Before the
has been obtained for transition protein 1 [27]. In advent of chromatin immunoprecipitation fol-
a classical mouse mutagenesis study, Marchetti lowed by DNA identification, histones have been
and Wyrobek analyzed the effect of diepoxybu- determined by gel electrophoresis [14, 38], by IF
tane (DEB), a bifuctional alkylating agent that on artificially expanded sperm nuclei [39, 40], and
induces inter- and intrastrand DNA crosslinks as by IF on the expanding mouse sperm nucleus
the basis of its mutagenicity. DEB is the metabo- before and after gamete fusion [41]. The most
lite of 1,3 butadiene, a tobacco smoke mutagen plausible explanations for their presence are the
[28]. Postmeiotic male germ cell stages were restoration of the embryonic somatic chromosome
architecture [41] and determining gene function remodeling was obtained when the corresponding
towards embryo pattern formation [16, 17]. mRNA probe with HA tag was injected in the
zygote, and specific staining of the male PN was
obtained [51]; for a review on the role of H3.3 in
16.3 Male Chromatin Remodeling, reproduction and development see [52].
Paternal vs. Maternal Histones As histones are among the most conserved
proteins in biology, the mouse-derived H3.1 anti-
Expansion of sperm chromatin in the oocyte ini- body used above also detects the human protein.
tially involves reduction of the protamine cysteine The fact that histones are more abundant in
S-S bridges and acceptance of the shed protamines human sperm compared to mouse sperm was
by an unknown maternal mechanism [42]. For used to probe for this protein by IF after heterolo-
mammalian oocytes, reduced glutathion (GSH) gous ICSI between human sperm and mouse
acts as the reductor of protamines, and without oocytes [15]. H3.1 could be followed in male
this step, no shedding of protamines is possible chromatin up to the start of DNA replication.
[43, 44]. In the toad Xenopus, the chromatin Thereafter, massive de novo nucleosome forma-
chaperone nucleoplasmin 2 (NPM2) is impli- tion, using H3.1/H3.2-H4 dimers, obscures the
cated in the removal of protamines [45, 46]. sperm-specific nucleosome signal. These experi-
When testing this gene by maternal depletion in ments were repeated in human tripronuclear
the mouse [47], no effect on sperm decondensa- (3PN) zygotes, collected in the early PN stage
tion was shown. Instead, heterochromatin organi- which is before S-phase. Male and female PNs
zation around the primary oocyte nucleolus and could easily be recognized on the basis of the
zygote nucleolus precursor bodies and fusion density of H3.1 signal, heavier staining being
product (that was absent) failed. In the mouse, no observed in the female PN. Hence, transfer of
maternal mutation for sperm nuclear deconden- sperm histones to the male PN was shown again
sation has yet been picked up. As a parallel to the [15, 41]. The meaning of paternal histones for
in vitro situation, where heparan and heparan sul- embryo development has received support from
fate can decondense the sperm nucleus, Romanato the analysis of the epigenome of the human sperm
et al. showed the presence of heparan sulfate in nucleus [16, 17].
the mouse oocyte, suggesting a role in nuclear From earlier sperm chromatin research, it was
expansion, as it does in vitro on sperm [48, 49]. already known that nucleosome density could
When nucleosomes are built on free DNA, his- vary within one gene [53]. The advent of chroma-
tone dimer assembly complexes (also called chap- tin immuno precipitation (CHIP) on chip tech-
erones) are involved. Some are specific for one of nology, connecting histone modification with
the two major histone 3 (H3) variants contained in DNA sequence technology, was thereafter used
the H3–H4 dimer that, as a tetramer, forms the to decipher the whole sperm epigenome, i.e.,
core of the nucleosome [50]. Dimers between histone occupancy/density in combination with
H3.1/3.2 and H4 are used during DNA replication; histone modification [16, 17]. One conclusion is
dimers between H3.3 and H4 are used in all other shared: genes that play a role in embryonic cell
circumstances involving de novo nucleosome differentiation and pattern formation and are non-
assembly such as transcription. HIRA, a replica- transcribed during the cleavage divisions are
tion-independent H3.3–H4 specific chaperone, under suppression of the Polycomb complex, as
was found in the mouse remodeling sperm nucleus deduced by promoters that are more heavily
after gamete fusion (using IF [7]). In line with the occupied with nucleosomes. The Polycomb
absence of DNA replication at this early stage, the signature is picked up because of their H3
male PN did not stain with an antibody specific N-tail posttranslational modification (PTM). The
for H3.1/3.2, which, as expected, was detected at H3 N-tail mark trimethylation of lysine 27
the onset of paternal DNA synthesis [7]. Proof for (H3K27me3) stands for methyltransferase activ-
the exclusive use of H3.3 in male chromatin ity of Polycomb repressive complex 2 (PRC2),
hence gene suppression by Polycomb repressive equalized between male- and female-derived
complex 1 (PRC1). Contrary to Hammoud and chromosomes [60]. Theoretically, this agrees
coworkers, Bryszinska and colleges found that with the notion, like for DNA methylation
nucleosomes were present everywhere in the [61, 62], that under the influence of oocyte
genome, though at a low density. A regular distri- cytoplasm, male and female chromatin becomes
bution of histones (i.e., nucleosomes) over the more equal to each other at the onset of cell dif-
genome would be compatible with the 50 kb ferentiation from the blastocyst inner cell mass.
spaced sensitivity for endogenous endonucleases Epigenetic asymmetry has also been described in
in mouse sperm nuclei [19]. Digestion of more human 3 PN zygotes [63].
open chromatin at matrix-associated regions
(MARs), which leads to the indicated regular
loop domain representing DNA fragments, is 16.4 Male Chromatin Remodeling,
then made possible because of the association DNA Repair is Involved
between matrix association and nucleosome
occupancy. Taking the evidence together, is it The evidence for DNA breaks during paternal
highly likely that histones do play a role in the chromatin remodeling in the mouse oocyte is
transmission of nuclear information via the male based on inhibition of TopoII [9–11]. TopoII beta
germline, other than by DNA code. The histone has been found in the late stage of sperm nucleus
landscape of the sperm nucleus also gives testi- remodeling and early female PN. TopoII alpha
mony to past functioning by showing increased was not found there, but was abundant on the sec-
density of nucleosomes at promoter sites of genes ondary oocyte metaphase II chromosomes, where
involved in spermatogenesis. These nucleosomes TopoII beta was absent [64]. Nucleosome deposi-
were picked up because of the di and trimethy- tion and protamine shedding most likely occur
lated states of H3 lysine 4 (H3K4me2 and 3), a simultaneously and commence within 30 min
mark of gene activity [16, 17]. after gamete fusion [7]. Remodeling starts at the
As introduced by the above examples, PTMs, posterior (caudal) side of the sperm nucleus,
both by general nuclear characterization as which first unfolds after fusion with IVF [7] and
obtained by IF and by analysis at the level of the after ICSI in the Rhesus monkey [65]. As soon as
gene, can be used to read the functional status of nucleosomes appear, the H2A variant H2AX
both the gene and chromatin domains [54–56]. apparently is incorporated (next to H2A), as gam-
So, different chromatin states, constitutively maH2AX focal staining (the common expression
inactive (heterochromatic) vs. facultatively for phosphorylation of H2AX at serine 139) is
inactive or active, can be illustrated by PTMs at routinely observed at this stage in the mouse [10].
lysine (K) residues of particularly the N-tails of After an initial stage of chromatin expansion,
H3 and 4. When staining by IF for these N-tail condensation takes place, a process that can be
modifications, a superficial picture of the overall followed by the H3S10Ph marker [10, 41]. The
state of chromatin is obtained, annexed to the most likely explanation for this phenomenon is
development of constitutive heterochromatin that that as male chromatin remodeling occurs, female
usually is found around the centromeres. chromosomes are in anaphase/telophase of the
When these methods were applied to mouse second meiotic division [42]. Male chromatin
zygotes, it was soon apparent that male and apparently does not entirely escape the influence
female pronuclei greatly differ for H3, H4 lysine of a lowering level of MPF (maturation promot-
methyl PTMs: these are strikingly underrepre- ing factor, alias for CDC2/cyclin B). If oocyte
sented in the male PN [7, 57–59]. This observa- activation is incomplete and a metaphase II spin-
tion is known as the epigenetic asymmetry of the dle is maintained, this process ends with prema-
zygote. What happens during the first cleavage ture chromosome condensation (PCC) [66]. The
divisions, as nicely illustrated by Puschendorf male chromatin contraction phase that normally
et al., is that the overall histone PTM pattern is occurs [42] allows a sharp delineation of large
gammaH2AX foci, which are induced by sperm to the very low levels in somatic cell nuclei [79],
irradiation in a dose-dependent manner [10]. are testimony to the fact that dsDNA repair is a
These large foci are taken to represent dsDNA normal event during male chromatin remodeling
breaks for which gammaH2AX is a chromatin after gamete fusion.
indicator [67, 68]. Large foci are also induced by This high level of foci is an underestimate of
the inhibition of TopoII religation by etoposide the real number of dsDNA breaks dependant on
[10]. Also, many small gammaH2A foci can be active DNA repair (as contrasted to just TopoII
seen; these are not induced by ionizing irradia- religation). Using the heterologous ICSI model
tion of sperm before entry [10]. The meaning of to count gammaH2AX foci of human sperm in
small gammaHAX foci is uncertain, maybe indi- mouse oocytes [78], sperm selected by ICSI cri-
cating other DNA irregularities such as bulky teria from normospermic and oligospermic
DNA lesions that challenge nucleotide excision donors was compared. Average levels of foci
repair (NER) [69]. An indication for DNA repair were about the same. Only the fraction of sperm
of this male stage was found long before, using without foci was smaller in the oligospermic
UV-irradiated mouse sperm, by spotting the donors. Strikingly abnormal male patterns of
uptake of radioactive (3H) Thymidine in the male gammaH2AX were found when nonmotile sperm
PN 3–4 h after setting up in vitro fertilization was selected. Also, an intense H2AX phosphory-
[70]. DNA synthesis is typical for NER that lation was often imposed upon female chromatin.
repairs UV-induced DNA helix distortions. Also, This result is one of the warnings against the use
very much unlike the situation in somatic nuclei, of ejaculated nonmotile sperm in human ICSI.
UV irradiation of sperm before fertilization Using heterologous in vitro fertilization and
results into structural chromosome-type abnor- cytogenetic techniques at first cleavage, Tateno
malities (the abnormality occurs before S-phase, et al. [80] showed hamster oocytes to faithfully
is doubled during S-phase, and is scored by mor- repair (and misrepair) radiation-induced DNA
phology at first mitosis) [71]. damage to human sperm, enabling dose–response
The use of oocyte genotypes that are deficient curves to be made for misrepaired dsDNA breaks
for specific DNA repair routes can yield unex- that lead to structural chromosomes abnormali-
pected information into the matter of DNA repair ties. Human sperm was more irradiation sensitive
in male chromatin remodeling after gamete than were hamster and mouse sperm [30]. More
fusion. Classical DNA.PKcs-dependent nonho- experiments are necessary to monitor chromo-
mologous end joining (NHEJ), one of the major somal stability in human fertilization [81].
routes to repair dsDNA breaks [72, 73], usually is Unfortunately, the only human zygotes ethically
tested in hypomorphs of the DNA.PKcs holoen- suitable for experimental use, such as monopro-
zyme, such as spontaneously found in the BALB/c nuclear and tripronuclear ones, occur at a reduced
and C.B17 mouse genetic backgrounds (with rate. Most instructive would be studies on zygotes
10% activity) [74]. In the C.B17 scid mouse, only evolving from severe male factor infertility.
residual NHEJ activity remains [75]. These It seems that oocyte activation can relate to
oocyte genotypes show more male gammaH2AX the fidelity of break repair management during
large foci when using the same source of mouse male chromatin remodeling. Using an oligo-
sperm [76]. This effect is exacerbated when astheno-teratozoospermia (OAT) mouse model,
sperm is irradiated before IVF. Hence, NHEJ is Baart et al. [66] observed that after ICSI with
involved in sperm chromatin remodeling in the cauda epididymal sperm, absence of activation
mouse. This defect also leads to high levels of with resulting PCC was relatively common.
chromosome abnormalities at the first cleavage Often, abnormal male chromosomes were pro-
division [76, 77]. The high levels of gamma- duced with many acentric (without centromere)
H2AX foci (around one per nucleus for both con- fragments and composite multicentrics in the
trol mouse and human sperm [10, 78]), compared same male complement. Both lack of dsDNA
break repair and illegitimate religation of breaks chromatin. In the absence of aphidicolin, DNA
had occurred, a cytogenetic picture typical for synthesis was shown (by an in situ nick translation
NHEJ malfunctioning. This result could suggest assay) both at the sperm chromatin remodeling
that oocyte activation by the sperm affects male stage (in keeping with the earlier statement that
chromatin remodeling, maybe with implications DNA repair is involved with sperm chromatin
for DNA repair. remodeling) and in the G1 PN [88].
Another interesting observation by Wossidlo
and coworkers is colocalization at G1 of the
16.5 Paternal DNA Demethylation universal DNA repair enzyme PARP-1 with
and DNA Repair at Zygotic gammaH2AX, in a focal pattern indicative for
G1, Is There a Link? dsDNA repair. It was subsequently found that a
chromatin bound form of the repair enzyme
From mouse mutagenesis studies, we know for XRCC1, which as PARP-1 can both function in
long that zygotes at the early PN stage are extremely base excision repair (BER) and in DNA.PKcs-
sensitive to DNA insult [82]. Sensitivity for irra- independent NHEJ (hence dsDNA repair [92]), is
diation increases when the pronuclei have been present in the male PN before S-phase, at the time
formed [83] and decreases towards S-phase [84]. of active demethylation of 5 methyl cytosine [93].
Recent research into active paternal demethylation The puzzle of male DNA metabolism at zygote G1
of DNA is shedding some light on this matter, is further enhanced by the BER-inducing mutagen
although as expected major uncertainties remain. methyl methane sulfonate (MMS), which is capa-
By using an antibody against 5 methyl cyto- ble of strongly enhancing the number of PARP-1
sine, in the mouse, the phenomenon of active gammaH2AX colocalizations [88]. The mutagenic
paternal DNA demethylation after gamate fusion effect of MMS on mouse sperm is long known
has been discovered [85, 86]. This observation [71], leading to chromosome-type abnormalities in
was subsequently proven by bisulfite sequencing the mouse zygote, a result that cannot be explained
[87] and shown to be sequence-specific such that on the basis of classical somatic cell cytogenetics.
already differences were noted between two types Similar to the experiments involving UV-irradiated
of retrotransposons [88]. Active demethylation mouse sperm, in the zygote, repair upon mutagenic
also occurs in the human PN [89] and is not treatments that lead to transitory single-strand
genome wide. In mouse and man, methylation of breaks (which is the case with BER and NER)
paternal (and also maternal) imprinting control leads to chromosome-type abnormalities that in
regions (ICRs) is preserved. The demethylation somatic nuclei are mainly caused by dsDNA
reaction extends over most of the first cell cycle, breaks. A further riddle is that after a low dose of
as shown by an in situ reporter construct [90]. For 0.5 Gy X-rays, the female G1 PN is more sensitive
a period of time now, demethylation has been to the induction of chromosome abnormalities
linked to DNA repair activity [91]. At zygotic than the male one, a result not expected as sponta-
Gap1 (G1, pre S-phase) in the mouse, spontane- neous gammaH2AX foci are absent [10, 83, 88].
ous gammaH2AX foci only occur in the male PN Apparently, repair is more directed towards the
and never in the female one [10, 76, 88]. At this male PN. Final proof for the involvement of DNA
stage, these foci were found to sharply increase in repair with paternal CpG demethylation will await
the male PN in the presence of aphidicolin, an the use of gene knockout mouse models.
inhibitor of many DNA polymerases [88]. As Recently, an alternative approach, based on
S-phase has not yet commenced and DNA repair siRNA gene knockout technology for candidate
often involves DNA synthesis, this may suggest a proteins and a reporter construct to stain for unm-
DNA repair event associated with the removal ethylated CpG residues, was developed [90]. By
of the so-called fifth base (5 methyl cytosine) this procedure, proteins involved with RNA poly-
in order to demethylate CpG dimers in paternal merase II elongation (i.e., transcription), such as
Elp1, 3 and 4, were found to affect active paternal ICRs were affected, but as for Stella, not system-
demethylation. atically. Zpf57 already affects the methylation
status of a maternally imprinted ICR in the
oocyte (such as the Snrpn gene, linked to the
16.6 A Role for the Zygote human Prader-Willi and Angelman imprinting
in Maintenance of Imprinting syndromes). The gene-specific aspect in protec-
tion for demethylation is also illustrated by
After many years of molecular research into the the fact that for the H19 DMR, the 5 methyl
control mechanisms that ensure monoallelic cytosine-binding protein MBD3 is needed to
inheritance from both the mother and the father, maintain imprinting during preimplantation
the CpG methylation status of the differentially development [99].
methylated region (DMR) of the imprint control As expected, matters are more complicated, as
regions (ICRs) has proven to be a reliable indica- has nicely been demonstrated by Terranova and
tor of the epigenetic status of the germline. By coworkers. Another reflection of the imprinting sta-
axioma, the methylation status of the ICR must tus is chromatin organization. Around the Beckwith
be resistant to the paternally active and mater- Wiedemann syndrome (Kcnq1)-imprinted gene
nally passive demethylation that results into cluster, paternal silencing is already found at the late
largely demethylated genomes at the blastocyst zygote stage, due to the involvement of Polycomb
stage [62]. Hirasawa et al. confirmed this expec- proteins and histone-repressive PTMs colocalizing
tation at the blastocyst stage, combining methy- with the inactivating RNA transcript [100]. These
lation sensitive (bisulfite) sequencing with allele authors did not determine the CpG methylation
differentiation enabled by an intraspecies mouse status of the ICR (the promoter region of the func-
cross, using two paternally imprinted ICRs (of tional-inactivating Kcnq1ot1 RNA). Summarizing,
the H19 and Rasgrf1 gene clusters) and two the message must be that the oocyte is mandatory
maternally imprinted ones [94]. When the main- for fine regulation of imprinting maintenance. In the
tenance DNA methylase DNMT1 was not pres- mouse, the stability of both male and female
ent in both the oocyte and in the embryo (using a imprinting maintenance was in a gonadotrophin
null-allele from the father), the CpG methylation dose-dependent mode affected by the induction of
imprint was eradicated [94]. Apart from mecha- superovulation [101]. In this light, the increase in
nistic copying of the methylation status during imprinting disorders associated with ART is of
DNA replication, there is active though selective significance [102].
protection of the methylated status at the zygote
stage. This has been illustrated in zygotes from
oocytes that lack the germ cell-specific protein
Stella [95]. Maternal absence of Stella, as 16.7 Is Zygotic S-Phase a Backup
observed in late zygotes, affected both maternal System to Further Repair Male
and paternal imprints though not systematically. DNA?
Such embryos develop poorly beyond the mater-
nal to zygote transition (the activation of the The classical cytogenetic outcomes of dsDNA
embryonic genome) that in the mouse is at the breaks during S-phase and the G2 phase of the
late 2-cell stage and between the 4 and 8-cell cell cycle are chromatid-type chromosome abnor-
stage in the human [96, 97]. Another DNA- malities in which the chromatid is the unit of
interacting protein has recently been discovered breakage. When the break occurs before S-phase,
(Zfp57, member of a family of transcription fac- the replication machinery simply copies the
tors) that clearly shows a maternal effect as to abnormality leading to a chromosome-type
maintenance of the mCpG mark at the DMR exchange. When misrepair involves the union of
[98]. Both paternally and maternally imprinted two ends of different chromatids, a so-called
chromatid exchange occurs by which four chro- polymerase inhibitor aphidicolin, indicating
matids now adhere (sister segment by sister seg- stalled replication forks, is much stronger in the
ment) in a so-called quadriradial. These changes male PN than in the female one [88].
always lead to mosaicsm in the preimplantation Paternal replication problems likely underlie
embryo. The chromosomal make up of the two impaired first cleavage in two OAT mouse models
daughter blastomeres now depends on the orien- that were used for ICSI to study testicular vs.
tation of centromeres in the cleavage spindle, cauda epididymal sperm cells. In the T(1;13)70H/
always leading to blastomere inequality for the T(1;13)1Wa double translocation heterozygous
genetic content of the two involved chromo- mouse, a full autosomal meiotic synapsis often is
somes. Derijck et al. [76] observed that extremely not achieved. The unsynapsed autosomal chro-
low doses of the free radicals generating mutagen matin then joins the sex body. When using cauda
4-nitroquinoline 1-oxide (4NQO) in an oocyte epididymal sperm, the majority (around 70%) of
impaired for homologous recombination dsDNA ICSI zygotes blocks in S-phase, the female PN
break repair (HRR, by ablation of Rad54/Rad54B) adjusting to the male one that seems more
resulted into a high incidence of quadriradials, severely affected. This problem is alleviated
especially in the male PN. This is another indica- when testicular sperm is used [66]. The epididy-
tion for the genetic vulnerability of male DNA. mal environment, by for instance ROS (reactive
This finding nicely combines with the long- oxygen species), likely causes damage to the
standing fact that de novo human reciprocal sperm nucleus that is fatal at DNA replication.
translocations in great majority are from the male This finding was subsequently confirmed in a
germline [103]. At this moment and incorporat- mouse model, deficient for transition protein 1
ing the available information from spermatogen- and haploinsufficient for transition protein 2
esis, the zygote is the most likely stage at which (Tnp1,Tnp2; −/−, +/−) in which the error occurs
these occur. Compared with other mammals, the during chromatin remodeling at spermatid nuclear
human has a high frequency of de novo occur- elongation [105]. In the reduced amount of meta-
rence of reciprocal translocations (1:2,000 by phase zygotes from cauda epididymal sperm, an
classical cytogenetics analysis on amniocentesis elevated rate of chromosome abnormalities was
cases) [104]. found.
Because the presence of gammaH2AX sym- An element that is much overlooked and is
bolizes ongoing repair activity, foci on zygote predicted to relate to the difference in S-phase
metaphase chromosomes (involving one or two behavior between male and female PN is
chromatids) means transportation of the repair explained in a recent review on male transgener-
event to the next cell generation. Using two muta- ational epimutation and genetic mutation [106].
gens on S-phase zygotes, 4NQO and X-rays and Attention is given to the loop domain structure of
maternally defective DNA repair genotypes, it sperm nuclei that can be interpreted as a prereq-
was found that especially targeting HRR yields uisite for zygotic DNA replication as is explained
transmission a majority of male chromatin repair by Lemaitre et al. for Xenopus zygotes [107].
foci to the two-cell stage [76]. Ablation of Rad54/ A dogma in cell biology is that per loop domain,
Rad54b in combination with these mutagenic one origin of replication is active. Hence, the pro-
treatments leads to a sharp increase of stalled rep- cess of DNA replication takes longer when loop
lication forks in especially the male PN, as if the domains are big, and shorter when loop domains
zygote expects problems in male DNA replica- are small. When the cell experiences difficulty in
tion mainly [76]. A likewise indication for the finishing S-phase, at metaphase an adjustment of
sensitivity of male S-phase for obstruction in loop domains towards shorter ones is made by
DNA metabolism was reported by Wossidlo and imposing activity on existing MARs that then
coworkers in the mouse. At this stage (as in G1), also become attached to the scaffold/nuclear
the gammaH2AX focal response to the DNA matrix [107, 108]. These attachments now are in
the memory system of the cell and the corre- patients [114]. Generally, the standard spermio-
sponding origins of replication will be used in gram values, concentration, motility, and mor-
forthcoming S-phases. In this way, differentia- phology are affected when P1/P2 ratios are
tion problems of the male gamete could lead to deviant [115, 116]. A low level of P1 has more
S-phase obstacles in the zygote [106]. obvious clinical consequences [113, 116], affect-
ing fertilization rates and implantation rates [112,
117]. Also, the presence of preP2 in spermatozoa
16.8 Indications from Human seems to influence the chance of pregnancy,
higher ratios of preP2 to P2 being favorable [112]
ART for Early Roles of Oocyte
despite the earlier finding that the level of preP2
and Sperm in Determining positively correlates with the level of sperm DNA
Ongoing Embryonic damage [118]. Principally, these observations
Development show that the chromatin composition of sperm
influences fertilization and implantation (caus-
Direct observations on human chromatin remod- ally or not). DNA damage, found in sperm with
eling and DNA damage responses in elongating deviant P1/P2 ratios, could be one detrimental
spermatids, remodeling sperm nuclei after gam- factor [115, 119]. Of notice, the P1/P2 ratio of
ete fusion, and in pronuclei before, during, and prepared sperm of human donors with known
after zygotic S-phase are largely lacking. For fertility (90% PureSperm layer, PS) tends to be
zygotes, ethical and legal implications prevent lower than 1.0 [120]. This tendency was con-
these experiments. However, data on fertilization, firmed by de Mateo et al. [109]. Interestingly,
embryo development, and pregnancy from ART sperm from the 90% PS layer of OAT samples
practice can sometimes be interpreted in the light presented with a higher P1/P2 ratio than in fertile
of mouse experimental data on DNA repair and controls (1.02 and 0.93, respectively).
OAT sperm as presented here. However, seem- In ART literature, early paternal effects are
ingly contradictory results and alternative inter- separated from later paternal effects, the later
pretations will remain in this complicated field. ones suggested to be linked to DNA damage
[121]. Mouse data on mutagenized normal sperm
cells do agree with this subdivision. In an over-
16.8.1 Maternal vs. Paternal Factors view of several mutagens applied to sperm,
Marchetti and coworkers calculate that when
Sperm in situations of male factor infertility is misrepair in the zygote yields more than four
more often not fully protaminated [39, 40, 109]. chromosome abnormalities at first cleavage, the
Earlier, it had been found that in a RNA FISH for result is preimplantation loss, which in the mouse
protamines 1 and 2, the frequency of “positive” will be apparent as delayed development from
round spermatids was lower for infertile patients the morula stage on [122, 123]. Zygotes with
[110]. More recently, protamine 1 and 2 RNA lev- fewer chromosome abnormalities are able to
els in mature sperm were found to correlate posi- implant and lead to later embryonic death [123].
tively with fertilization success in conventional Irradiation of bull sperm that was subsequently
IVF [111]. The etiology of these observations will researched in an IVF setup yielded a comparable
have aspects in common with the earlier finding result. The lowest dose of 1.25 Gy already
that the two human protamine species P1 and P2 resulted into embryonic death showing apoptotic
that normally are present in a ratio of between 0.8 blastomeres and leading to a shortage of day 7
and 1.3 (P1/P2, [112–114]) in infertile patients blastocysts [124]. In the mouse, the kinetics of
often show deviations in both directions. the first cleavage division have been reported not
Larger ratios outnumber smaller ones [113]. to be altered by radiation-induced sperm and
A shortage of P2 relative to P1 has been reported early zygote DNA breakage (5 Gy [83, 125]).
in 40% and a shortage of P1 in 13.6% of infertile However, using a dose of 6 Gy and 3H Thymidine
uptake to monitor S-phase progression, an effect early predictors of normal development. The
on DNA replication hence the length of the first value of zygote PN observation has been touched
cleavage division was found in another mouse upon above, but also synchrony of the division
strain and shown to be under control of p53 [122, from 2 to 4 blastomeres has been used to assess
126]. Mouse data on irradiated sperm can be of embryo development [133].
relevance for human ART because of the impli- One rationale is that when S-phase encounters
cated oxidative stress. difficulties in the sphere of DNA management,
Oxydative stress is a prime suspect for causing sperm nuclear structure, this may well lead to
DNA damage in human sperm [127, 128]. Zini delayed first cleavage as in human ART pronu-
et al. have, in a review and meta analysis, evalu- clear blocks are rare. In many laboratories, indi-
ated the existing literature on a possible relation cations for the value of early first cleavage
between the level of (directly and indirectly) for predicting pregnancy have been obtained
measured DNA damage in spermatozoa and [134–140]. Direct demonstration of a longer
reproductive outcome in ART. They found the duration of DNA replication would involve a dif-
patient sperm DNA damage level to be associated ference in the frequency of stalled replication
with an increased risk for pregnancy loss [129]. forks between early and late cleavage human
As an argument for a negative role for the epididy- embryos. Of note here is that corrected for the lag
mal environment including length of storage, tes- phase of sperm entry in IVF, the first cell cycle in
ticular sperm has been shown to improve fertility IVF is 1 h longer than an ICSI cycle (27 vs. 26 h)
when DNA damage was dominant in ejaculated [141]. Also, differences between IVF and ICSI
sperm [130]. have been observed around the second cleavage
Eight-oxoguanine (8-oxoG) is the accepted division that is less synchronous after ICSI [141].
genotoxic indicator for oxidative DNA stress. In Nevertheless, and despite the value of synchrony
an oocyte donation program to which younger of cleavage for reproductive success [132, 133]
women contributed, the effect of the level of and the expected larger load of detrimental pater-
8-oxoG in the sperm nucleus on embryo develop- nal factors with ICSI, no differences in efficiency
ment in ART could be determined, not being between IVF vs. ICSI have been found.
hindered by variation in oocyte quality. Marked This leads to the paradox in ART. On a casuis-
levels of oxidative stress in sperm were found to tic basis, there is almost no limit to the source of
affect zygotes using PN grading by nucleolus the sperm cell (testis, epididymis, ejaculate), and
precursor body numbers and positions and were within certain margins with respect to head mor-
visible in day 3 blastomere symmetry [131]. phology and motility, to achieve reproductive
However, stalled pronuclear development as was success [140, 142–145]. On the other hand, male
found when cauda epididymal sperm from OAT inheritance assumes a more differentiated land-
mouse models was used for ICSI [66, 105] is scape, with the likely involvement of histone
rarely encountered in human ART practice. occupancy, the presence of microRNA species
Conclusively, sperm nuclear effects, both on [146], and DNA damage to affect heredity. These
the level of DNA and of chromatin protein distri- aspects, for instance DNA damage, may well be
bution, can lead to early and late embryonic more serious at older age [23, 147] as is also indi-
effects. Early paternal effects of course are also cated by an oocyte donation program that avoids
related to centrosome and tubuline aster behav- bias for the maternal factor [148, 149].
ior, which together with oocyte activation could
be summed up as the non-nuclear paternal contri-
bution [121, 132], which is of a lesser concern in 16.9 Conclusion
the context of this review.
In an IVF setting, early zygote/embryo events This review serves to show to what extent DNA
can be recorded by time lapse microscopy. This and chromatin remodeling is occurring in late
technology has been stimulated by the search for spermiogenesis and in the zygote. Roles for male
and female gametes are strikingly divided. There 3. Caperton L, Murphey P, Yamazaki Y, et al. Assisted
are many indications that more male information reproductive technologies do not alter mutation fre-
quency or spectrum. Proc Natl Acad Sci USA. 2007;
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5. Fernandez-Gonzalez R, Moreira PN, Perez-Crespo
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as small RNA species and nucleus loop domain health and behavior of adult offspring. Biol Reprod.
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male nucleus changes that are involved, for mouse: presence of DNase I hypersensitive sites
instance, the adaptation of loop domains for DNA in situ and a putative role for topoisomerase II. Zygote
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Proteomic/Metabolomic Analysis
of Embryos: Current Status 17
for Use in ART
Mandy G. Katz-Jaffe and Susanna
McReynolds
Abstract
The selection of embryos for transfer is currently based on morphological
indices; though successful, the field of assisted reproductive technologies
(ART) would benefit from a noninvasive quantitative method of embryo
assessment. Omics technologies, including proteomics and metabolomics,
have already begun providing evidence that viable embryos possess unique
molecular profiles with potential biomarkers that could be utilized for selec-
tion purposes. Of particular interest in ART is the secretome (extracellular
proteins and metabolites) that are present in the surrounding environment of
the embryo, the microdrop of culture media. Defining the human embryonic
secretome has the potential to expand our knowledge of embryonic cellular
processes and may also assist in identifying those embryos with the highest
implantation potential. Advances in proteomic and metabolomic technolo-
gies have allowed for the noninvasive profiling of the human embryonic
secretome with ongoing research focused on correlation with outcome that
may result in improved IVF outcomes and routine single embryo transfers.
Keywords
Omics • Embryo • ART • Proteomics • Metabolomics • Secretome
• Embryo quality • Implantation
based on detailed embryo morphology with

17.1 Introduction the highest implantation rates observed follow-
ing detailed morphological assessment [1, 2].
The assessment of embryo competence is a
Though this information contributes to the pre-
crucial component of assisted reproductive tech-
diction of implantation potential and is relatively
nologies (ART). Current selection methods are
successful in improving pregnancy rates and
reducing multiple gestations, morphology has
M.G. Katz-Jaffe ()
limitations, with more than 70% of in vitro fertil-
Colorado Center for Reproductive Medicine,
Lone Tree, CO, USA ization (IVF) embryos failing to implant. This
and failure is likely due to both the absence of devel-
National Foundation for Fertility Research, opmentally competent embryos in an IVF cohort
Lone Tree, CO, USA
as well as our inability to select the competent

246 M.G. Katz-Jaffe and S. McReynolds
embryo(s) present. The field of human ART embryos and secreted at any given time into the
would benefit from more quantitative and accu- surrounding culture medium. Indeed, proteomic
rate methods of embryo viability assessment. In assessment of secreted human embryonic proteins
turn, the ability to select the most viable embryo may assist in identifying noninvasive biomark-
in a cohort will allow for routine single embryo ers that reflect developmental competence and
transfer, while maintaining or improving preg- viability [7]. To date, the investigation of the
nancy rates [3]. human embryonic secretome and its correlation
Recent developments in omics technologies to embryo viability and outcome has been chal-
(genomics, transcriptomics, proteomics, and lenging, but holds promise with recent develop-
metabolomics), including improvements in plat- ments and increased sensitivity of both targeted
form sensitivity, are highlighting the potential of and profiling proteomic approaches.
these technologies to investigate diverse biologi-
cal samples. In particular, proteomic and metabo-
lomic analysis provides a snapshot of cellular 17.2.1 Noninvasive Targeted
physiology and function, as well as a direct link Single Protein and Molecular
with phenotype. Both these omics technologies Analysis
allow for analysis of IVF spent culture media that
would represent a noninvasive minimal risk The initial studies of the human embryonic secre-
approach to assessing embryo viability [4]. In this tome involved targeted analysis of individual
chapter, we discuss the role of proteomics and proteins and molecules. The soluble factor,
metabolomics in examining the mammalian 1-o-alkyl-2-acetyl-sn-glycero-3-phosphocholine
embryonic secretome and their applicability to the (PAF), was one of the first targeted molecules to
assessment of embryo viability in ART. Defining be identified in the human embryonic secretome
and characterizing the mammalian embryonic and was shown to be produced and secreted by
secretome will also expand our knowledge of mammalian embryos during preimplantation
early embryogenesis and advance our understand- development [8]. PAF has been shown to act in
ing of the embryonic role during implantation. an autocrine fashion as a survival factor, as well
as influencing a range of maternal physiology
alterations including platelet activation and
17.2 Noninvasive Proteomic immune function [8].
Analysis of Embryos for ART Leptin, a 16 kDa small pleotrophic peptide,
has been observed in blastocyst-conditioned
Knowledge of the human embryo proteome is medium while studying the interaction between
very limited despite recent advances in pro- the embryo and endometrial epithelial cells
teomic technologies. The combined effect of (EEC) [9]. The authors showed that competent
limited template, low protein concentration, human blastocysts secreted higher leptin concen-
deficient platform sensitivity, and limited pro- trations into the surrounding medium than
tein database information has contributed as the arrested embryos. Leptin has been hypothesized
main hurdles. The human proteome is diverse to initiate and establish a molecular dialog with
and dynamic, with over one million proteins leptin receptors in the maternal endometrium
constantly changing through both internal and during the window of implantation [10]. Another
external interactions and stimuli. The secretome reciprocal embryo–endometrial interaction that
is of particular interest to researchers investigat- could transform the local uterine environment
ing the dynamics of a specific physiological impacting both embryo development and the
condition or disease state and is defined as those implantation process is HOXA10 expression by
proteins that are produced by cells and secreted epithelial endometrial cells and the unknown
at any given time [5, 6]. In ART, the secretome soluble molecule secreted by human blastocysts
incorporates those proteins that are produced by that modulates its regulation [11].
17 Proteomic/Metabolomic Analysis of Embryos: Current Status for Use in ART 247
Human leukocyte antigen G (HLA-G) is these studies sHLA-G was most likely to be
thought to play a role at the maternal embryonic maternally derived either from the oocyte or fol-
interface during implantation. HLA-G is pro- licular environment. Perhaps the analysis of
duced by human oocytes and embryos at both the embryonic sHLA-G on day 5 of development
mRNA and protein level and has also been will be more indicative of embryo developmental
detected extracellular in embryo spent culture competence. Another explanation for the lack of
media [12–14]. The presence of soluble HLA-G reproducibility and association observed to date
(sHLA-G) in embryo spent culture media has could be the lack of sensitivity of the current
been linked by several studies to successful preg- sandwich ELISA assays used for most sHLA-G
nancy outcome and suggested as a noninvasive analysis. Thus, it would appear that in order to
marker to predict embryo quality and implanta- determine the true importance of sHLA-G in
tion success, especially when used in conjunction regards to embryo development and implantation
with current morphological embryo assessment outcome, a more sensitive (picogram level),
methods [15]. These results, however, have not reproducible, and quantitative detection method
been absolute with pregnancies established from for analysis is required to be tested on individual
sHLA-G negative embryos and data revealing spent culture media samples throughout all stages
undetectable levels of sHLA-G in embryo spent of embryo development including the blastocyst,
culture media [15–17]. postembryonic genome activation [16].
In a multicenter study, a wide range of sHLA-
G concentrations were observed across the differ-
ent ART clinics, as well as differences between 17.2.2 Noninvasive Protein Profiling
sHLA-G in relation to implantation. Indeed, a of Embryos
significant association between sHLA-G-positive
embryo spent culture media and successful The targeted secretome studies described above
implantation was only established in one of the have been focused on only a single protein or
three clinics involved in the study [18]. Another factor; however, it would be reasonable to assume
recent study was unable to detect any association that more than one molecule would be required
between sHLA-G expression and implantation to predict developmental competence and/or
rates, but concluded that miscarriage rates were implantation potential considering the multifac-
significantly lower when embryos were selected torial nature of embryonic development. With the
based on a graduated embryo morphology recent advances in proteomic technologies
score and sHLA-G levels vs. the morphol- including increased sensitivity, it has become
ogy score alone [19]. In addition, no correlation possible for more extended investigations of the
has been observed between the concentration of proteins and peptides produced and secreted by
sHLA-G in embryo spent culture media and either the human embryos.
embryology morphology [20] or chromosome Mass Spectrometry (MS) has rapidly become
aneuploidy by FISH for up to 11 chromosomes an important technology in proteomics research.
(8, 9, 13, 15, 16, 17, 18, 21, 22, X and Y) [21]. Searching for consistent and significant alteration
There are numerous factors that could influ- in protein expression between specific groups of
ence the presence of sHLA-G in embryo spent samples has revealed underlying mechanisms of
culture media including the culture system itself, physiological processes and disease states [22].
the extent of cumulus removal, single vs. group MS typically involves an ion source for produc-
embryo culture, media composition, microdrop tion of a charged species in the gas phase, and an
volume, and the day of media collection [15, 16]. analyzer, which can separate ions by their mass-
Most of the studies to date have investigated the to-charge (m/z) ratio. Several commonly used
expression of sHLA-G on day 3 of embryo devel- ionization methods include electrospray ioniza-
opment at the time of embryonic genome activation (ESI), matrix attenuated laser desorption/
tion. It would be reasonable to conclude that in ionization (MALDI), and surface-enhanced laser
desorption/ionization (SELDI). These are coupled protein turnover [26, 27]. Interestingly, ubiquitin
to either time-of-flight (TOF), ion trap, or quadru- has been implicated to play a crucial role dur-
pole analyzers, which can occur in tandem for ing mammalian implantation by controlling
peptide sequencing. For reliable and reproducible the activities and turnover of key signaling
proteomic data, a consistent protocol needs to be molecules [28].
followed during sample collection, storage, and In a retrospective study by Dominguez et al.
handling due to the dynamic and sensitive nature [29], protein microarrays that contained 120 tar-
of the human proteome. Data prejudice during gets were used to compare implanted vs. nonim-
processing can be controlled for by running sam- planted blastocyst-conditioned media. With a
ples in replicates, routinely performing internal lower detection limit of only 10 pg/mL for this
and external calibrations, and including suitable system, samples were pooled following single
control samples with every run [23]. embryo transfer according to pregnancy outcome.
SELDI-TOF MS, with specific surface affinity An increased expression of the soluble TNF
protein chips, has allowed for fast, cost-effective, receptor 1 and Interleukin-10 (IL-10) and the
high-throughput application of small sample vol- decreased expression of MSP-a, SCF, CXCL13,
umes (mL range) and enables sensitivity to be in TRAILR3 and MIP-1b were observed in the con-
the picomole to femtomole range. The technol- ditioned media compared to control media [29].
ogy has been applied to a variety of biological Interestingly, there were no proteins significantly
tissues and fluids with specific focus on oncopro- increased in the conditioned media of implanted
teomics [24]. Using SELDI, Katz-Jaffe et al. [25] blastocysts compared to nonimplanted blasto-
were the first to successfully analyze the protein cysts. The presence of two significantly decreased
profile of individual human embryos. The authors proteins, CXCL13 and GM-CSF, were observed
observed distinctive protein secretome signatures in the pooled implanted blastocyst-conditioned
at each 24 h embryonic developmental stage from media indicating consumption of these proteins
the time of fertilization to the blastocyst stage. by the human blastocyst. These results are cor-
Unique proteins were observed in the human roborated by studies showing that GM-CSF pro-
embryonic secretome after the activation of motes embryo development and implantation
the embryonic genome. A clear association was when present in both human and mice blastocyst
observed between protein expression profiles and culture media [30].
morphology, with degenerating embryos exhibit- A follow-up study comparing protein secre-
ing significant up-regulation of several potential tome profiles between the EEC coculture system
biomarkers that might be involved in apoptotic and sequential microdrop culture media revealed
and growth-inhibiting pathways. differential protein secretome profiles [31].
In addition, the secretome of developing blas- Several molecules were increased in the EEC
tocysts revealed a significantly higher expression coculture profile including IL-6, PLGF, and BCL
of an 8.5 kDa protein in comparison to the secre- (CXCL13), while other proteins were decreased
tome of degenerating embryos, potentially indi- (consumed) such as FGF-4, IL-12p40, VEGF,
cating an association between this protein and and uPAR. IL-6 was the most secreted protein by
developmental competence. Tandem MS and the EEC coculture system. Using an IL-6 ELISA
database peptide sequence identification indi- assay, the sequential culture media secretome of
cated that the best candidate for this 8.5 kDa viable blastocysts displayed an increased uptake
protein was ubiquitin, a component of the compared to blastocysts that failed to result in a
ubiquitin-dependant proteosome system that is pregnancy, suggesting a potential role for IL-6 in
involved in a number of physiological processes blastocyst development and implantation [31].
including proliferation and apoptosis. Secreted Current methods used to screen for chromo-
ubiquitin has been shown to be upregulated some aneuploidy in embryos involve biopsy pro-
in body fluids in certain disease states and this cedures which are invasive and could compromise
accumulation provides evidence for an increased further embryo development. A noninvasive
method for aneuploidy screening of human substrates, as well as contributing to protein

embryos would be a significant advantage to synthesis and pH regulation [34]. The end prod-
embryo selection in ART. Using SELDI-TOF ucts of embryo metabolism and cellular processes
MS, protein profiles of embryo spent culture are low molecular weight molecules called
media from individual blastocysts relative to the metabolites (<1,000 Da) that reflect the efficiency
chromosomal constitution of all 23 pairs of chro- of the processes in response to a variety of nutri-
mosomes were investigated [4]. The secretome ent or environmental changes; these could repre-
of individual blastocysts identified protein sent potential biomarkers of embryo viability
expression that allowed for discrimination [35]. An estimated 8,500 metabolites, represent-
between euploid (correct number of chromo- ing a wide dynamic and diverse range, have to
somes) and aneuploid chromosomal constitutions date been identified in the human metabolome
[4]. A novel set of nine differentially expressed which is considerably fewer than the >1 million
biomarkers was identified with statistical signifi- proteins in the human proteome. Noninvasive
cance and was reproducible in all spent culture quantitative techniques assessing the metabolic
media samples analyzed, classifying a euploid parameters of the developing embryos have been
blastocyst from an aneuploid blastocyst. Ongoing under investigation for over a decade with prom-
research by this group is focused on further vali- ising advances for predicting embryo viability
dation to discriminate between euploid and aneu- and pregnancy outcome. The “quiet embryo
ploid blastocysts and on a future blinded hypothesis” has been proposed in which nonvia-
prospective study. The ability to noninvasively ble embryos are more metabolically active than
assay for the combination of developmental com- developmentally competent embryos who are
petence and chromosomal constitution could rep- more metabolically quiescent [36].
resent a powerful viability selection tool in ART,
but to date the lack of sensitivity and the com-
plexity of the human embryonic proteome have 17.3.1 Pyruvate and Glucose
been limiting factors. The challenge is to discover
the proteins of interest; however, once identified, Pyruvate is one of the main sources of energy dur-
immunodetection using ELISA or radioimmuno- ing the early stages of embryogenesis through to
assay will allow for sensitivity, high throughput, compaction on day 4, when carboxylic acid-based
and should be cost-effective for application in a metabolism is predominant [32, 33]. The uptake
clinical setting. of pyruvate by embryos in culture has been inves-
tigated as a possible marker of embryo develop-
ment and viability, with inconclusive results to
17.3 Noninvasive Metabolomic date. Initial studies showed a higher uptake of
Analysis of Embryos for ART pyruvate by embryos that develop to the blasto-
cyst stage [37, 38]. This was later supported by
Complementing proteomic analysis, information Gardner et al. [39] who reported a significantly
regarding human embryo metabolism and metab- higher pyruvate uptake on day 4 in embryos that
olomic profiling may also assist in the search for developed to the blastocyst stage compared to
noninvasive methods to distinguish competent embryos that arrested and failed to develop into
embryos in culture. During the preimplantation blastocysts. In contrast, there have been other
stage, the nutrient requirement of the human findings suggesting a lower pyruvate uptake by
embryo changes as it travels through the repro- the early cleavage stage embryo (two to eight
ductive tract, from relatively high levels of pyru- cells) in association with outcome [40]. It has also
vate and low levels of glucose in the oviduct to been observed that embryos display a wide varia-
lower pyruvate and higher glucose concentration tion in pyruvate uptake, with the lowest uptake
in the uterus [32, 33]. In addition, amino acids representing embryos that successfully implant
are essential for development acting as energy [41]. Inconsistent data regarding pyruvate uptake
would indicate that it would not be a reliable turnover than those which arrest in culture, con-
candidate for embryo viability assessment. sistent with the “quiet embryo hypothesis” [44].
In comparison to pyruvate, glucose uptake, In a follow-up study, HPLC was used to exam-
which is low during the cleavage stages, increases ine the concentration of amino acids on day 2
significantly at the transition from morula to blas- from individual-cultured human embryos [45].
tocyst stage and indeed appears to correlate with An association was observed between the turn-
the developmental competence and viability of over of three amino acids (decreased glycine and
human blastocysts. Glucose uptake by human leucine levels, and increased asparagine levels) in
embryos on day 4 has been reported to be signifi- the embryo spent culture media with clinical
cantly higher in embryos that developed into blas- pregnancy and live birth. Proton nuclear mag-
tocysts compared to those embryos that arrested netic resonance (H1-NMR) has also been used to
prior the blastocyst stage [39]. Furthermore, this identify amino acid biomarkers. Analysis of day
study also determined that the greatest glucose 3 embryo spent culture media showed that higher
uptake belonged to higher morphological grade glutamate levels correlated with clinical preg-
blastocysts. While there may be a relationship nancy and live birth [46].
between glucose uptake and embryo quality, the A more recent publication has indicated an
technology currently used for glucose analysis on association between amino acid turnover and
embryo spent culture media are microfluoromet- chromosome constitution. Following the screen-
ric enzymatic assays. These assays are technically ing of six chromosomes by FISH (13, 18, 19, 21,
difficult, low throughput and require technical X and Y), asparagine, glycine, and valine turn-
expertise rendering them unsuitable for clinical over were significantly different between euploid
application. Advances in microfluidics allow for and aneuploid day 3 embryos, while serine, leu-
high throughput and potentially more user- cine, and lysine displayed differences in their
friendly analysis and could move glucose uptake profiles between these two groups on day 4 of
analysis towards clinical applicability [42]. embryo development [47].
There are several potential explanations for
the amino acid differences observed in the stud-
17.3.2 Amino Acids ies outlined above including the culture system
itself, type of culture media, day of analysis, and
Great attention has been devoted to the profiling platform used for measurement. Overall, these
of amino acids during human embryo develop- studies suggest an association between amino
ment. Amino acids play key roles during embryo acid turnover and embryos of different reproduc-
metabolism and several studies have assessed tive potential with promise for the development
their relationship to embryo viability. High- of noninvasive clinical assays. However, there is
performance liquid chromatography (HPLC) has a need for further exploration and validation in
been used to examine the amino acid turnover relation to the prediction of embryo reproductive
(depletion and/or appearance) during preimplan- potential as well as a more user-friendly screen-
tation embryo development. Initial studies ing method that can rapidly and routinely be used
observed a correlation between amino acid turn- in the IVF clinic.
over and embryos that arrested in culture com-
pared to competent embryos that developed to
the blastocyst stage [43]. Leucine was the most 17.3.3 Noninvasive Metabolomic
consistently depleted amino acid and Alanine the Profiling of Embryos
highest secreted amino acid throughout develop-
ment by embryos that formed blastocysts. Metabolites are compounds with diverse chemi-
Furthermore, the data also indicated that compe- cal and physical properties, found in a variety of
tent embryos displayed a lower amino acid concentrations. Since the metabolome is defined
as a spectrum of these small metabolites, there is In summary, metabolomic profiling appears to

a shift away from the analysis of single or defined show promise for selecting viable human embryos
metabolites towards more comprehensive analy- and has also been shown to be an independent
sis and metabolomic profiling. Metabolomic pro- parameter to morphology [51]. However, in each
filing systematically analyses this wide range of study described above, there was considerable
diverse metabolites that represent a functional overlap between the viability index for embryos
phenotype [48]. that implanted and those that failed to result in a
Raman, measuring the vibrations of bonds, and pregnancy, with both inter-patient and inter-
Near Infrared (NIR) Spectroscopy, which primar- embryo variation. Moreover, to date, no prospec-
ily measures overtones and combination vibra- tive trial has proven that such a metabolomic
tions, are two optical spectroscopy methods that algorithm has selective capabilities superior to
have successfully been used to profile human morphological evaluation alone. Therefore, the
embryo spent culture media in relation to preg- question still remains unanswered as to whether
nancy outcome [49]. The advantages of optical metabolomic profiling will prove to be a reliable
spectroscopies include no sample processing, high and reproducible clinical assay for human embryo
throughput, and rapid turnaround (<1 min/sam- viability assessment.
ple). However, metabolomic profiling does not
identify or quantitate specific metabolites. Instead,
it relies on identifying metabolites displaying an 17.4 Conclusion
association with a specific biological phenotype.
Under this circumstance, reproducibility of pro- Proteomics and metabolomics are two comple-
files is even more crucial for validity. It appears mentary omics platforms under investigation that
that the spectral regions of the metabolomic pro- show promise for the development of noninvasive
files most predictive of outcome corresponded to methods for embryo selection in the field of ART.
markers of oxidative stress, including –CH, –NH, With the ability to assay both proteins and metab-
and –OH groups. These findings could indicate an olites in embryo spent culture media, generating
association between embryo developmental com- complimentary but different molecular profiles, a
petence and oxidative modification. combined omics contribution seems reasonable
Using a mathematical model, Seli et al. [49] to fully characterize the human embryonic secre-
generated an algorithm based on metabolomic tome. Further, taking into account the complexity
profiling of day 3 human embryo spent culture and diversity of the human embryo, it is feasible
media with known outcome to calculate viability to propose that embryo viability assessment may
indexes for positive vs. negative pregnancy. The include a combination of both these omics
mean viability score for embryos that resulted in platforms used alone or in conjunction with
pregnancy was significantly higher than those morphology.
that failed to implant. Follow-up studies analyz- Defining the embryonic secretome could result
ing embryo spent culture media from single in a rapid, user-friendly, cost-effective, noninva-
embryo transfers on day 2, 3, and 5 of develop- sive, reliable, and reproducible method for quanti-
ment using NIR spectroscopy and bioinformatics tative embryo assessment. Recent proteomics and
consistently showed higher mean viability metabolomics research is encouraging; however,
scores for embryos that resulted in clinical to date no technique has proven true clinical pre-
pregnancy [3]. In addition, a blinded pilot study dictive value or been examined in prospective
on retrospectively collected data, using the randomized control trials. However, once true effi-
Raman spectroscopy platform, revealed an over- cacy is proven, a noninvasive assay for the selec-
all diagnostic accuracy for predicting embryonic tion of viable human embryos could lead to routine
reproductive potential, either delivery or a failed single embryo transfers, a reduction in early preg-
implantation, at 80.5% [50]. nancy losses, and increased singleton deliveries.
16. Vercammen M, Verloes A, Haentjens P, Van de Velde

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Ultrasound-Guided Embryo Transfer
Robert L. Gustofson and William B. Schoolcraft
18
Abstract
The complexity of in vitro fertilization is culminated in the embryo
transfer. Failure to achieve a successful transfer can negate the effort of the
entire process. Any method to facilitate an easier transfer with higher
pregnancy rates is considered a necessary step. Ultrasound-guided embryo
transfer has become integrated in most IVF centers because of its ease of
use, reassurance of proper embryo placement, and improved pregnancy
rates.
Keywords
Ultrasound guidance • Embryo transfer • Pregnancy rate • ART • Catheter
• Embryo
control is required to maintain consistent embryo

18.1 Introduction quality week in and week out. The final steps in
the IVF process include adequate endometrial
In vitro fertilization consists of a series of
receptivity, proper luteal support, and finally, the
mutually dependent and intricate steps. The pro-
quality of the embryo transfer itself. The embryo
cess begins with patient selection and is followed
transfer is the final step in this complex chain of
by controlled ovarian hyperstimulation and
events. Failure during embryo transfer will negate
oocyte retrieval, which leads the process into the
the months of careful work and sophisticated
laboratory. Within the lab, fertilization and
technology predating this final stage.
embryo culture is accomplished. The quality of
The variables affecting embryo transfer suc-
the culture media, the maintenance of proper
cess have been enumerated in the literature. These
temperature and pH regulation, and proper air
include the performance of a trial of transfer [1],
quality are all critical for the production of viable
contamination of the catheter tip with blood [2, 3],
embryos. Constant quality assurance and quality
mucus, or endometrial tissue, the presence of
retained embryos, the type of catheter [4–8], the
W.B. Schoolcraft ()
volume and type of transfer media, the presence of
Colorado Center for Reproductive Medicine,
Lone Tree, CO, USA bacteria in the cervix or on the catheter tip [9, 10],
e-mail: [email protected] and the utilization of ultrasound guidance during

256 R.L. Gustofson and W.B. Schoolcraft
the transfer. It is this last variable of ultrasound downwards toward the cervix. Indeed, Woolcott
guidance that will be the focus of this chapter. and Stanger [25] demonstrated that in 17% of
Since 1985 when ultrasound guidance was first blind transfers, the catheter actually abutted the
described by Strickler et al. [11], technological fundal endometrium. In addition, the outer guid-
use of ultrasound has become widely debated but ing cannula indented the endometrium in 25% of
now routinely incorporated by most IVF centers. transfers. The catheter actually was embedded
within the endometrium in 33% of cases, and in
7% of cases, ultrasound detected a catheter that
18.2 Benefits of Ultrasound was actually cannulating the fallopian tube,
Guidance which would almost have guaranteed tubal place-
ment of the embryos. A study involving hystero-
There are many potential benefits to utilizing scopic examination of the uterine cavity following
ultrasound guidance during transfer. First, it can embryo transfer resulted in visualization of sig-
help the operator avoid a difficult transfer. There nificant endometrial trauma with difficult trans-
are several studies demonstrating that technically fers as opposed to easy transfers [17].
difficult transfers are associated with reduced As mentioned earlier, uterine contractions are
pregnancy rates [12–16]. One potential link clearly stimulated by difficult transfers, and ultra-
between difficult transfers and a lower implanta- sound guidance is a means to minimize such con-
tion rate is trauma to the endometrium [17]. This tractions. Increased subendometrial contractions
can lead to uterine contractions, which negatively are associated with a traumatic embryo transfer.
influence pregnancy outcome [18]. In addition, If seen prior to embryo transfer, pregnancy rates
the contamination of the catheter with blood is are substantially decreased [18].
also more common with difficult transfers and
blood itself is associated with lowering of preg-
nancy outcome [2]. Indeed, a meta-analysis by 18.3 Optimization of Ultrasound
Sallam and Sadek [19] demonstrated a lower Guidance
incidence of difficult embryo transfer with the
use of ultrasound guidance vs. clinical touch. With abdominal ultrasound guidance, a full blad-
The other benefit to ultrasound guidance is der is necessary for optimal visualization. A full
that it can facilitate proper placement of the cath- bladder itself is a benefit as demonstrated by
eter tip. Several studies have shown that place- Lewin who showed that the presence of a full
ment of the embryos approximately 1.5–2 cm bladder without ultrasound guidance was associ-
from the fundus results in optimal implantation ated with improved pregnancy rates as compared
rates [20, 21]. In addition, placement of the cath- to an empty bladder [26]. In addition, Abou-Setta
eter close to the fundus increases the rate of ecto- [27] and Sundstrom et al. [28] demonstrated not
pic pregnancy [22, 23]. Ultrasound guidance only a higher pregnancy rate with full bladder but
avoids these complications associated with high a significantly increased incidence of easy embryo
transfers, i.e., less than 1 cm from the fundus, as transfers with such full bladders, suggesting a
well as inadvertent low transfers that fail to clear passive straightening of the cervicouterine angle.
the internal cervical os. This optimal placement Various ultrasound techniques have been eval-
of the embryos minimizes the risk of endometrial uated to determine best application. Ultrasound
trauma, minimizes the incidence of uterine con- location, transabdominal vs. transvaginal, has
tractions, and lowers the incidence of retained been evaluated as well and appears to be equal
embryos within the catheter [24]. efficacy [29]. Transabdominal ultrasound, how-
Ultrasound guidance also avoids placing the ever, may be advantageous due to ease of use and
catheter in a subendometrial location and patient comfort compared to the transvaginal
detects a catheter that potentially might loop approach. Further, more advanced ultrasonogra-
upon itself, thereby directing the embryos back phy with 3D/4D approach may further enhance
18 Ultrasound-Guided Embryo Transfer 257
embryo transfer to an exact location. Current analysis an odds ratio (OR) of 1.5 in favor of
studies suggest 3D/4D ultrasound-guided embryo ultrasound guidance. When the study by Drakeley
transfer is at least equal to 2D imaging, however, was incorporated into the Cochrane Database
may be limited by availability of 3D technology again, Brown found there still remained an
[30, 31]. increased ongoing pregnancy rate (OR 1.38);
however, there was no difference in live birth
rate [35]. Several other studies [36–38] have
18.4 Controversy: Randomized come to similar conclusions in favor of ultra-
Trials vs. Meta-Analysis sound guidance.
Controversy still remains regarding the use of

ultrasound-guided embryo transfers and its ben- 18.5 Colorado Center
efits to pregnancy rates. One large randomized for Reproductive
trial involving ultrasound guidance did not show Medicine Experience
a benefit [32]. In this study of 373 patients, there
was no significant difference in pregnancy or At the Colorado Center for Reproductive
implantation rates. It should be noted, however, Medicine, our protocol for embryo transfer is as
that there was a trend toward fewer patients with follows. We prepare the patient with oral diaze-
prior ART failure in the control group as com- pam and ask her to fill her bladder about
pared to the study group. In addition, there were 30–40 min prior to the anticipated transfer.
more male factor cases and less unexplained Ultrasound guidance is accomplished abdomi-
infertility in the controls, characteristics likely to nally by a licensed ultrasonographer with exper-
favor a better success in this group. Further, the tise in gynecologic evaluations. Upon inserting a
ultrasound technologists “received training” speculum, the cervix is washed with culture
before the study as opposed to having years of media, gently lavaged, and all excess mucus is
experience with such techniques. Patients had no aspirated with an 18-gauge angiocatheter outer
requirement for a full bladder, suggesting that sheath [39, 40]. A trial of transfer is then per-
visualization would not be optimal in all cases. formed just to the internal os. At this point,
One clinician did all transfers in both groups and embryos maintained in a pediatric isolette are
was very meticulous with his technique, which loaded into a Wallace™ SureView Ultrasound
might minimize the difference between ultra- embryo replacement catheter (Smiths-Medical
sound and clinical touch. Finally, the transfer International) in a 30 mL volume. The fluid used
depth was greater in the ultrasound group, a vari- for embryo transfer is EmbryoGlue® (Vitrolife).
able known to negatively influence outcome. The catheter is placed as gently as possible with
In 2008, Drakeley et al. [33] performed a large, manipulation of the cervix using the speculum or
randomized trial with 2,295 embryo transfers a ring forceps, if necessary, to negotiate the inter-
comparing ultrasound guidance and clinical nal os. The embryo transfer catheter is loaded so
touch demonstrating no difference in pregnancy that there is initially a small column of air, fol-
rates. In the clinical touch arm of the study, the lowed by a 30 mL column of media, and then the
physician could select the catheter to utilize for embryos, followed by another small column of
transfer, adding a confounding variable. media just before the catheter tip. By utilizing
Despite these two large randomized controlled this consistent, meticulous, standardized tech-
trials, several meta-analyses demonstrated that nique, we believe it contributes to the overall suc-
the cumulative effects of ultrasound-guided cess of our program.
embryo transfer lead to a better IVF outcome In cases of difficult embryo transfers, often
with respect to implantation and pregnancy associated with cervical stenosis, pre-cycle cervi-
rates. In 2007, prior to the large study by Drakeley, cal dilation can be valuable. We have found that
Abou-Setta et al. [34] demonstrated in their meta- cervical laminaria is the most effective method to
258 R.L. Gustofson and W.B. Schoolcraft
achieve useful dilation along with other authors in vitro fertilization-embryo transfer. Fertil Steril.
using laminaria [41], hygroscopic rods [42], or 1998;70(5):878–82.
3. Sallam HN, Agameya AF, Rahman AF, Ezzeldin F,
malecot catheters [43]. Approximately 1 month Sallam AN. Impact of technical difficulties, choice of
prior to a scheduled embryo transfer, a patient has catheter, and the presence of blood on the success
a medium cervical laminaria placed through the of embryo transfer–experience from a single provider.
internal os. This is maintained in position over- J Assist Reprod Genet. 2003;20(4):135–42.
4. Abou-Setta AM, Al-Inany HG, Mansour RT, Serour
night and removed the next day. This prolonged GI, Aboulghar MA. Soft versus firm embryo transfer
dilatation seems to retain its benefits up to and catheters for assisted reproduction: a systematic
through the time of the transfer the following review and meta-analysis. Hum Reprod. 2005;20(11):
month. In contrast, cervical dilatation at the time of 3114–21.
5. Buckett WM. A review and meta-analysis of prospec-
retrieval or thereafter lowers pregnancy rates [44]. tive trials comparing different catheters used for
embryo transfer. Fertil Steril. 2006;85(3):728–34.
6. Ghazzawi IM, Al-Hasani S, Karaki R, Souso S.
18.6 Conclusion Transfer technique and catheter choice influence the
incidence of transcervical embryo expulsion and the
outcome of IVF. Hum Reprod. 1999;14(3):677–82.
Ultrasound guidance provides reassurance to 7. Urman B, Aksoy S, Alatas C, et al. Comparing two
both the physician and the patient that the cathe- embryo transfer catheters. Use of a trial transfer to
ter is in an optimal location for embryo deposi- determine the catheter applied. J Reprod Med.
2000;45(2):135–8.
tion. While not every case of embryo transfer 8. Saldeen P, Abou-Setta AM, Bergh T, Sundstrom P,
would necessarily benefit from ultrasound guid- Holte J. A prospective randomized controlled trial
ance, it is impossible to predict such cases in comparing two embryo transfer catheters in an ART
advance despite trial transfers, physician experi- program. Fertil Steril. 2008;90(3):599–603.
9. Egbase PE, al-Sharhan M, al-Othman S, al-Mutawa
ence, or prior patient transfer. Therefore, the rou- M, Udo EE, Grudzinskas JG. Incidence of microbial
tine use of ultrasound seems appropriate to growth from the tip of the embryo transfer catheter
minimize difficulty with unexpected problematic after embryo transfer in relation to clinical pregnancy
transfers. Ultrasound may also be helpful for rate following in-vitro fertilization and embryo trans-
fer. Hum Reprod. 1996;11(8):1687–9.
embryo transfer training. Residents and fellows 10. Moore DE, Soules MR, Klein NA, Fujimoto VY,
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Part IV
Evolving Controversies in Contemporary
Reproductive Medicine
IMSI as a Valuable Tool for Sperm
Selection During ART 19
Monica Antinori, Pierre Vanderzwalmen,
and Yona Barak
Abstract
Conventional semen parameters, routinely used by clinicians to assess male
reproductive potential, provide little information on the real fertility status.
Nevertheless, sperm morphology analyzed by strict criteria has demon-
strated its prognostic value regarding the outcome of both in vivo and
in vitro reproduction. However, ICSI introduction seemed to have decreased
the importance of sperm morphology in assisted reproduction, since fertil-
ization, embryo development, pregnancies, and healthy deliveries can be
achieved even in case of severe morphological impairments. This visual
assessment of sperm morphology, due to the limitations attributable to its
low magnification (200–400×) and concomitant low resolution, overlooks
minor morphologic defects potentially related to increased risk of chromo-
somal abnormalities in infants conceived with ICSI and decreased success
rates. Thus, based on the theory that subtle sperm morphological malfor-
mations might remain unnoticed during the routine sperm selection per-
formed prior to microinjection, negatively affecting its outcome, a new
technique, termed motile sperm organellar morphology examination
(MSOME), has been recently developed to perform a real-time detailed
morphological evaluation of motile spermatozoa under 6,600× high mag-
nification. When applied immediately prior the conventional ICSI tech-
nique, this new method of sperm morphological selection is defined:
intracytoplasmic morphologically selected sperm injection (IMSI).
Even though a rising number of studies have reported IMSI as having
remarkable clinical advantages in terms of fertilization, embryo quality,
pregnancy occurrence, and prosecution until delivery, this new method
continues to be debated regarding its routine application in the ART labo-
ratory. Although IMSI has not yet been standardized and requires further
validation, it can be considered one of the most promising issues in the
ART field. IMSI has been introduced to increase ART success in overcoming
severe male infertility.
M. Antinori (*)
Infertility Unit, RAPRUI Day Hospital, Rome, Italy

264 M. Antinori et al.
Keywords
Sperm morphology • High magnification • Selection • MSOME • IMSI
• Embryo quality
19.1 Introduction assessment of sperm morphology, limited by its

low magnification (200–400×) and concomitant
Clinicians routinely use conventional semen low resolution [21], overlooks minor morpho-
parameters attempt to obtain a reliable overview logic defects potentially related to sperm func-
on the male reproductive potential. However, in tional impairment. At the same time, since men
most cases little information on the fertility status with oligoasthenoteratozoospermia were shown
is provided unless semen parameters are grossly to have significantly elevated levels of sperm
abnormal [1, 2]. Among them the best fertilization numerical chromosomal aberrations, and DNA
predictor is morphology as assessed according to chain fragmentation [25–31], and since incidence
strict criteria [3]. Since its introduction [4], sev- of chromosome aneuploidy in spermatozoa might
eral advantages to the outcomes of conventional be related with the severity of sperm defects [32],
in vitro fertilization [3, 5], intrauterine insemina- there is great concern regarding the increased risk
tion [6], and in vivo reproduction have been shown of chromosomal abnormalities in infants con-
[7]. Therefore, it is clear how a spermatozoon’s ceived with ICSI [33–35].
morphological normality reflects its function, in Moreover, although ICSI has provided treat-
relation to its ability to reach, recognize, bind, ment for new groups of couples with male infer-
penetrate, and deliver its genome to the oocyte. tility who were previously untreatable by IVF, at
Differently, once the sperm is mechanically present the resulting pregnancy rates are only
injected into the oocyte by an ICSI method, and between 30 and 45% [36–38] while the European
hence has crossed both barriers of the zona pellu- average “take-home baby” rates remain similar
cida and oolemma, its abnormal morphology does [76] to those of a decade ago [39, 40]. The rising
not seem to interfere with its fertilizing capacity. demand for improvement of success rates compels
In this respect, most authors do not notice any cor- us to reassess male fertility potential through the
relation between ICSI outcomes and the strict development of new tests with clinical relevance
morphology of the sperm used for microinjection for each ART procedure.
[8–11]. In patients with a poor prognosis (4% nor- Conventional means of light microscopy can-
mal sperm morphology) [12], and even in extreme, not identify the entire variety of sperm morpho-
specific cases of total teratozoospermia [13], logical defects, especially in the head structure
globozoospermia [14, 15], and megalozoospermia [41–43]. In 1999, electron microscopy (scanning
[16] fertilization can be achieved with ICSI. electron microscopy, SEM and transmission elec-
Several points of negative impact, both genetic tron microscopy, TEM) enabled Bartoov et al.
and epigenetic, have been identified in embryos [44] to correctly identify the ultra-morphological
following the ICSI procedure [4, 17–19]. state of seven sperm subcellular organelles
Nonetheless, major morphological anomalies (acrosome, postacrosomal lamina, nucleus, neck,
lead to decreased rates of fertilization, pregnancy, axoneme, mitochondrial sheath, and outer dense
implantation [16, 20], embryo quality [21–23], fibers). These subcellular organelles were found
and blastocyst formation [21, 24]. Hence, during to be highly predictive factors for male fertility
an ICSI procedure the embryologist selects a potential. Due to the fact that the applied technol-
motile, normal-looking spermatozoon and ogy was expensive, and often not available in
discards the most distorted forms. This visual conventional laboratories, its application was
19 IMSI as a Valuable Tool for Sperm Selection During ART 265
limited only to those cases in which the male 19.2 IMSI in Practice
infertility factor could not be clearly identified by
routine tests or following repeated ART failures. Due to the usage of the optics of Nomarski, and in
As for conventional sperm morphology, this new accordance with the current literature, carrying out
evaluation turned out to be useful only in the con- of IMSI requires a (sterile) glass-bottomed dish
text of a reproductive prognosis assessment, con- with the following characteristics (Fig. 19.2):
sidering that the single sperm used for fertilization – On the left side, 4 mL observation droplets of
might not reflect the peculiarity of the analyzed sperm culture medium containing between
sample. Moreover, since the evaluation was based 0 and 10% polyvinyl pyrrolidone (PVP) solu-
on the examination of fixed and stained sperm tion. Small bays extruding from the rim of the
cells, it did not provide the patient with any infor- droplets are created in order to capture
mation about the single sperm used for ICSI.
Taking all the aforementioned into consideration,
a few years later a new approach was developed
for a real-time detailed morphological evaluation
of motile spermatozoa. The sperm analysis,
termed motile sperm organellar morphology
examination (MSOME), was performed using an
interference phase contrast inverted, microscope,
with the optics of Nomarski, that combines maxi-
mal optical magnification (100×), magnification
selector (1.5×), and a video-coupled magnifica-
tion to reach a final video magnification of
6,600×. This application was developed to allow
the visualization of subtle sperm morphological
malformations that might remain unnoticed dur-
ing the routine sperm selection performed prior
to microinjection. This method was introduced to Fig. 19.1 A multivacuolated sperm clearly showing a
improve the success of intracytoplasmic sperm large nuclear vacuole next to small vacuoles
injection. Of the six sperm subcellular organelles
examined (acrosome, postacrosomal lamina,
neck, mitochondria, tail, and nucleus), the sperm’s
nucleus was demonstrated to be the most impor-
tant parameter affecting ICSI outcome [45]
particularly in the form of large nuclear vacuoles
that were proposed to reflect damages in the
nuclear DNA content and organization [46, 47]
(Fig. 19.1).
Presently, only a few studies have been carried
out to investigate the real meaning of nuclear
vacuolization and its relationship to DNA status.
The opportunity to carry on additional research
justifies the application of this new technique
which requires specially trained personnel and
can be considered extremely time-consuming
and expensive, to be routinely inserted in the
ART laboratory. Fig. 19.2 The IMSI dish
the heads of motile spermatozoa. The temper- nuclear chromatin mass containing no more
ature of the sperm sample and the PVP con- than one vacuole, which occupies less than
centration are coordinated with the intensity 4% of the nuclear area (0.78 ± 0.18 mm).
of the sperm motility. Spermatozoa with abnormal head size are
– In the middle, 4 mL selection droplets of sperm excluded by superimposing a transparent cellu-
culture, where selected sperm cells are located loid form on the motile examined gametes, rep-
after MSOME evaluation. Three distinct drops resenting the correct sperm size, which is
are made to host spermatozoa with different calculated by the ratio of expected normal
morphological features. sperm size to the actual size visualized on the
– On the right side, 4 mL droplets of sperm cul- monitor screen. Spermatozoa with severe mal-
ture medium that will host the oocytes to be formations, such as a pin, amorphous, tapered,
injected in the following ICSI procedure round, or multinucleated head, which can be
(injection droplets), one for each oocyte identified clearly even by low magnification
available for microinjection. (200–400×), are not assessed by MSOME.
All microdroplets are placed under sterile liquid Spermatozoa with a doubtful determination are
paraffin. excluded from selection. In order to perform a
correct sperm evaluation, the embryologist fol-
lows each motile single sperm cell with appar-
19.2.1 MSOME Criteria and Evaluation ent suitability by moving the microscopic stage
Procedure in the x, y, and z directions until also the small-
est details are visualized. Actually some mor-
Based on data collected by scanning and trans- phological defects, such as large vacuoles, can
mission electron microscopy [44], the MSOME be revealed only during sperm movement, and
criteria for normally shaped nuclei were defined therefore motility can be advantageous to the
as size (average length and width to be morphological observation. Furthermore, it is
4.75 ± 0.28 and 3.28 ± 0.20 mm, respectively) relevant to emphasize how a single sperm eval-
smoothness, symmetry, oval configuration (an uation is reliable only when it is carried out on
extrusion or invagination of the nuclear mass a motile sperm cell; on the other hand, static
was defined as a regional nuclear shape malfor- sperm images only allow evaluation of the visi-
mation, Fig. 19.3), and homogeneity of the ble part, leaving some morphological altera-
tions undiscovered.
Additionally, in order to minimize the subjec-
tive nature of sperm evaluation, a cooperation of
two embryologists working together at the same
time on the analysis of the same sample is
recommended.
Finding normal-looking spermatozoa is variable
according to the quality of the semen sample.
19.2.2 IMSI Step-by-Step
Freshly ejaculated semen is subjected to routine

morphological selection of motile spermatozoa
on the basis of a two-layer density gradient sys-
tem: 1 mL of post-ejaculated liquefied semen is
Fig. 19.3 An example of nuclear disorder, visible as a placed onto the gradient and centrifuged at 375 × g
blebs protruding from the sperm head surface for 15 min at 25°C. The sperm cell pellet is
s uspended by adding 3 mL of sperm culture

medium and then recentrifuged for 10 min. The
19.3 IMSI Reproductive Outcomes
supernatant is removed and replaced by sperm
A new approach for real-time motile sperm mor-
culture medium to bring the final concentration
phological evaluation (MSOME) was introduced
of motile sperm cells to about 4 × 106 spermato-
[45]. The MSOME enabled the visualization of
zoa per milliliter. In severe oligozoospermic cases
some morphological anomalies which conven-
with sperm density below 1 × 106 spermatozoa
tional light microscopy cannot detect at 200–
per ejaculate, liquefied semen is placed onto
400×. Assuming that spermatozoa with severely
1 mL of the low density layer only, centrifuged as
impaired morphology show reduced fertilization,
previously described, and the final sperm cell
pregnancy, and implantation rates [16, 20], the
pellet is resuspended in 0.1–0.2 mL of sperm cul-
study aimed to determine whether subtle mor-
ture medium.
phological anomalies affect ICSI outcome and
The sperm cell suspension obtained after
identify those that are most relevant. Having ana-
semen preparation is used for real-time high-
lyzed a total of 10,000 spermatozoa (100 sperm
magnification motile sperm organelle morphol-
samples, 100 spermatozoa each), it was demon-
ogy examination (MSOME) [48] that is performed
strated that in routine IVF–ICSI cycles patients
on the observation droplets by means of an
who exhibited less than 20% spermatozoa with
inverted microscope (Olympus IX81, Tokyo,
normal nucleus, defined by MSOME, did not
Japan) equipped with Nomarski differential inter-
achieve any pregnancy. With respect to the ICSI
ference contrast optics, an Uplan Apo ×100
fertilization rate, the morphological normalcy of
oil/1.50 objective lens previously covered by a
the entire sperm cell, according to MSOME crite-
droplet of immersion oil, and a 0.55 NA con-
ria, showed a positive and significant correlation
denser lens. The images are captured by a DXC-
(r = 0.52, P £ 0.01) and a very high predictive
990P video camera having 1/2-in., 3-chip power
value (area under the ROC curve, 88%), whereas
HAD CCD and visualized on a color monitor
no association with pregnancy outcome was
screen with diagonal dimension of 355.6 mm.
found. The normalcy of the sperm nucleus
Calculation of the total magnification is based on
(shape + chromatin content), defined by MSOME,
four parameters: (1) objective magnification
was significantly and positively correlated with
100×; (2) magnification selector 1.5×; (3) video
both: fertilization rate (r = 0.42, P £ 0.01) and
coupler magnification 0.99 (UPMTV X 0.3, PE
pregnancy occurrence (r = 0.38, P £ 0.01). Else the
X 3.3); and (4) (a) CCD chip diagonal dimension
predictive value of normalcy of the nucleus turned
8 mm and (b) television monitor diagonal dimen-
out to be significantly higher (areas under the
sion for a calculated video magnification (b/a) of
ROC curve, 72 and 74%, respectively). Hence,
44.45. Thus, total magnification = microscope
the authors could conclude that sperm nucleus is
magnification (150×) X video coupler
the most important sperm parameter affecting
magnification (0.99×) X video magnification
ICSI outcome [45].
(44.45×) = 6,600×.
A further utilization of high power magnifica-
Only motile spermatozoa with morphologi-
tion in real-time of ICSI was applied. Using
cally normal nuclei are retrieved from the obser-
MSOME evaluation for the injected sperm in
vation droplets and aspirated into a sterilized
real-time was termed as intracytoplasmic
glass non-angulated pipette with a 9 mm inner
morphologically selected sperm injection (IMSI)
diameter tip. Sperm cells are then placed into
[48]. Fifty IMSI couples were compared to 50
the selection droplet and finally used to be
ICSI couples in a matched control manner (e.g.,
injected into the oocytes for the classical ICSI
couples with the similar number of previous ICSI
procedure [49].
failures). Implantation and pregnancy rates after
This procedure is performed using a motorized
IMSI were significantly higher, and the abortion
micromanipulator system (TransferMan NK2,
rate was significantly lower, compared to the
Eppendorf, Germany).
c urrent ICSI trial (F = 18.0, P £ 0.01; x2 = 4.4, where only sperm cells with minimal impairment
P £ 0.01; and x2 = 4.4, P £ 0.05). In addition, the were used for microinjection, since no “best”
IMSI attempt produced a significantly higher sperm cells were available in those cases, demon-
value of top embryo percentage, compared to the strated that fertilization rate, percentage of top
current ICSI treatment (F = 6.5, P £ 0.01). embryos, implantation, pregnancy, and delivery
Moreover, the 12 cases of the unmatched IMSI rates per cycle were significantly higher, and the
group with an average of 9.1 ± 1.2 previously abortion rate was significantly lower in the “best”
failed ICSI attempts achieved a 50% pregnancy group than in the “second best one” (F = 10.5,
rate following their first IMSI trial. The obtained P £ 0.01; F = 4.6, P £ 0.03; F = 23.4, P £ 0.01;
results demonstrated that IMSI improves signifi- x2 = 15.5, P £ 0.05; x2 = 19.6, P £ 0.01; and x2 = 5.5,
cantly the success rate in couples with at least P £ 0.02, respectively) [50].
two previous ICSI failures. These authors restricted the application of this
With the intention of eliminating the proba- new procedure to cases with over two previous
bility that the increased pregnancy outcome was implantation failures. Actually, according to
linked to the sperm preparation technique recent publications, those couples seem to have
adopted for IMSI and not to the nuclear mor- the worst reproductive prognosis with a dramatic
phology of the selected spermatozoa a compara- reduction in pregnancy and implantation rates as
tive study was conducted. Thirty-eight transfers compared with couples who underwent 0–1 pre-
of embryos derived from “second best” morpho- vious failed IVF attempts [51, 52]. Hence,
logically evaluated sperm cells (negative group) Antinori et al. [53] designed a prospective ran-
following IMSI cycles took place, vs. 38 trans- domized controlled protocol to assess the poten-
fers derived from morphologically normal nuclei tial advantages of the IMSI procedure in the
(positive group). Comparison between the groups treatment of patients with severe oligoasthenot-
revealed that fertilization rate, percentage of top eratozoospermia regardless of their previous
embryos, and implantation rates were signifi- failed ICSI attempts, followed by a subgroup
cantly higher in the positive group than in the splitting according to the number of previous
negative group [46]. failed attempts (subgroup A: no previous
Out of six pregnancies achieved in the nega- attempts; subgroup B: 1 previous failed attempt;
tive group, four turned into a first trimester abor- subgroup C: ³2 previous failed attempts). The
tion. Interestingly, three abortion cases occurred comparisons between the two different tech-
when microinjection was conducted with sper- niques were made in terms of pregnancy,
matozoa exhibiting large nuclear vacuoles, abortion, and implantation rates.
whereas in the other abortion case the sperm cells Pregnancy and implantation rates resulted sta-
exhibited combined malformations: large vacu- tistically better in IMSI than ICSI cycles (PR:
oles associated with narrow formed head shape. 39.2 vs.26.5%; P = 0.004) (IR: 17.3 vs. 11.3%;
A subsequent enlarged study by the same P = 0.007).
group [47] confirmed all the previous findings as However, cases with two or more failed
follows: pregnancy rate was significantly higher attempts benefited most from IMSI, with a statis-
and the abortion rate significantly lower following tically significant doubling of pregnancy rate
IMSI, compared with ICSI attempts (x2 = 20.1, (12.9 vs. 29.8%; P = 0.017) and a remarkable
P £ 0.01; and x2 = 5.1, P £ 0.03, respectively). 50% reduction in the abortion rate (17 vs. 35%).
Furthermore IMSI provided a higher percentage Comparisons did not show any statistical differ-
of top quality embryos with a better implantation ence in terms of abortions but the clinical trend
rate than ICSI. was clearly in favor of the IMSI method in cases
A comparison between a “best group,” in with two or more previous failed attempts.
which embryos were obtained from microinjec- Based on the above results, it is likely that in
tion exclusively performed using spermatozoa those couples, male factor could be featured by
with intact nuclei, and a “second best group” semen impairment, undetected by conventional
diagnostic tools, thus reducing the effectiveness chromosomal or DNA defect, sperm cells with
of previous ICSI treatments. and without large vacuoles were selected from
the same ejaculate and examined by different
biochemical methods from an external analytic
19.3.1 IMSI and Vacuolated system [47].
Spermatozoa A first step in this direction was taken by
Hazout et al. [59], who compared the outcomes
Implantation and pregnancy achieved by IMSI of 125 couples with at least two previous ICSI
seem associated with morphological nuclear nor- failures and an undetected female infertility
malcy of the sperm. Nonetheless, spermatozoa factor that underwent conventional and high-
with a morphologically abnormal nucleus show magnification ICSI in two sequential attempts.
low fertility potential, even if some with certain Following sperm injection into the oocytes with-
severe nuclear abnormalities may still be able to out nuclear alterations, a double pregnancy rate
produce pregnancy following ICSI [13–16]. and a 50% decrease in the abortion rate were
Some reports found that head malformations recorded as against similar cases treated by con-
negatively correlate with DNA integrity [54–56]. ventional ICSI. In 72 out of 125 patients involved
Else, a clear negative association between the in the study, the degree of sperm DNA fragmen-
existence of sperm nuclear vacuoles and natural tation was determined by TUNEL and the out-
male fertility potential was reported by others comes of high magnification ICSI were compared
[57, 58], it can be assumed that within the cate- in cases with different sperm DNA fragmenta-
gory of specific morphological malformations, tion degrees. However, this test was not per-
existence of large vacuoles in the sperm nuclei formed directly with the sperm samples used for
indicates more damage to the nuclear DNA con- ICSI. A marked rise in clinical implantation and
tent and organization than nuclear shape or size birth rates was observed in patients with both
impairment. normal (<30%), moderately (30–40%) and highly
The impact of sperm with normal nuclear (>40%) increased percentage of DNA-fragmented
shape but large nuclear vacuoles on pregnancy spermatozoa in the ejaculate.
outcome compared to those with strictly defined The extent of DNA fragmentation (TUNEL
morphologically normal nuclei, including shape assay) and the presence of denatured single-
and content, was investigated by Berkovitz et al. stranded or normal double-stranded DNA (acri-
in 2006 [47]. A lower pregnancy rate per cycle, dine orange fluorescence method, AOT) in
and a higher early spontaneous abortion rate per spermatozoa with large nuclear vacuoles (LNV)
pregnancy were observed in the study group in selected by high magnification compared to those
comparison with the same parameters in the with normal nucleus were evaluated by Franco
control group (18 vs. 50%, Pearson’s x2 = 6.4, et al. [60]. The percentage of positive DNA frag-
and 80 vs.7%, Pearson’s x2 = 10.9, respectively, mentation was significantly higher (P < 0.0001)
P = 0.01). in LNV spermatozoa (29.1%) than in NN sper-
MSOME revealed that the ejaculates of males matozoa (15.9%). Similarly, the percentage of
routinely referred for ICSI exhibit on average denatured-stranded DNA was significantly higher
30–40% spermatozoa with a vacuolated nucleus. (P < 0.0001) in the former (67.9%) than in the lat-
This sperm malformation, identifiable as a preg- ter (33.1%).
nancy risk factor on the basis of the previous A direct correlation in DNA quality has not
findings, can easily be missed by the standard been tested in single selected spermatozoa. Thus,
selection prior to ICSI, and have, therefore, a the chromatin structure (sperm DNA integrity by
chance to be chosen for microinjection of at least acridine orange; DNA fragmentation by TUNEL
30%. assay) was analyzed, in correlation to sperm ane-
In order to verify that the large nuclear vacu- uploidies (FISH test). The study was conducted
oles in the sperm cell reflect some underlying by Garolla et al. [61] in ten patients with severe
testicular damage (severe oligozoospermia) on when seen upfront that looks like a vacuole in
single immotile sperm cells morphologically the following image in motile acrosome-reacted
selected by high-magnification microscopy spermatozoa that were analyzed by MSOME.
(13,161×). From the sample of each patient, ten The authors concluded that the vacuole-free
spermatozoa with normal morphology and no spermatozoa microinjected during IMSI are
vacuoles (group A) and ten spermatozoa with mostly acrosome-reacted spermatozoa.
normal morphology and at least one large head-
vacuole (group B) were selected. Single cells
from group A showed a more physiological sta- 19.3.2 Classification of Sperm
tus of DNA integrity and DNA fragmentation
than cells from group B. Furthermore, FISH As was already mentioned, one of the main con-
analysis showed that no chromosomal alteration cerns of ICSI, for the embryologist is the aspira-
was present in cells from group A. Moreover, the tion of the good-quality spermatozoa for
authors reported that taking into consideration microinjection into an oocyte. It is likely that
spermatozoa with normal morphology and both some of the injected spermatozoa do have a good
presence or absence of large head-vacuoles, the developmental potential.
mean results (data not shown) from all tests were Else, as previously mentioned, it is accepted
significantly better with respect to those of unse- that the morphologic characteristics may not
lected cells performed in the first part of the study necessarily describe the quality of the specific
(all P < 0.001). single spermatozoon (in the processed sperm
Based on technical limits of the differential fraction) that was injected into the oocyte [66,
interference contrast (DIC), which doesn’t allow 67]. However, yet, morphology, is definitely the
intracellular evaluation [62–64], since in order to only tool to evaluate quality of the sperm selected
detect chromatin vacuoles the evaluation has to for oocyte injection. De Vos et al. [20] performed
be performed at 20,000× magnification by elec- such a study of the individual injected sperm and
tron microscopy, and because of their main the formation and development of the resultant
localization, in the anterior part of the sperm embryo. A reduced fertilization rate following
head, the acrosomal origin of these vacuoles was the injection of sperm cells with abnormal mor-
theorized [65]. The first experiment of this study phology, was observed. Nevertheless, reduced
consisted of MSOME evaluation on immotile implantation rates were noted, when only
spermatozoa followed by acrosomal status “abnormal-sperm-embryos” were transferred.
assessment of the same spermatozoon using These investigators admitted that the low magni-
Pisum sativum agglutinin fluorescein isothiocya- fication (i.e., routine magnification of 400× dur-
nate. The complete acrosome reaction corre- ing ICSI), and concomitant low resolution of the
sponded in most of the cases (70.9%) to sperm morphology assessment on motile sper-
spermatozoa of regular shape with absent or matozoa before ICSI, were a limitation of their
slight vacuolization, whereas those sperms with study.
incomplete or missing acrosome reaction showed IMSI definitely overcomes these limitations;
60.7% of vacuole presence. A second study com- by using a high power 1,500×, inverted, light
prised of ten patients whose immotile spermato- microscope with a zoom up to 6,100×, and
zoa were analyzed according to MSOME before higher, it was demonstrated that a normal sper-
and after the acrosome had been induced by ion- matozoon, and more precisely, a strictly defined
ophore A23587. Vacuole-free spermatozoa normal sperm nucleus, affects the fertilization
increased from 41.2 to 63.8% (P > 0.005) with a rate and the occurrence of pregnancy [45–48].
concomitant rise of the acrosome-reacted gam- Thus, the authors deemed it worthwhile to
etes from 17.4 + 7.8 to 36.1 + 12.7 (P < 0.001). develop a tool for determining which is the pref-
Moreover, it was possible to visualize large pro- erable spermatozoon that should be injected into
truding blebs as well as a sort of invagination an oocyte.
19.3.3 Establishment of Classification r eal-time, might increase embryo quality in terms

Scoring Scales of implantation [47]. These investigators reported
that, during the IMSI procedure, spermatozoa
In order to establish a detailed classification scor- were preselected under the magnification of
ing scale to choose the individual spermatozoon 6,000× and accumulated in a droplet. Classifi
with the highest predictive fertilizing and devel- cations of selected spermatozoa morphology
opmental potentials, in real-time, it is important to have been determined as “normal” and “second”
understand the correlation between the normalcy choice (one droplet for all the spermatozoa of a
of the sperm, its fertilizability, and early embryo specific “choice”). Spermatozoa were aspirated
development and to analyze which sperm defects from those accumulated motile sperm cell droplets
negatively affect the development of the embryo. and injected by the embryologist. One might only
One important point is that the authors assume speculate that during IMSI when no normal head
that the “experienced eyes” of the embryologists, spermatozoa can be found, the only alternative
omit, even under magnification of 200–400×, then is to select those motile sperm cells that
spermatozoa with rough morphological major are morphologically second best. However, the
abnormalities that are detectable under routine parameters used by those authors to define a
conditions. It is probable that the majority of normal sperm nucleus, normal nuclear shape, or
abnormalities observed under magnification of normal sperm, were not clearly portrayed [46, 48].
6,100×, are not visible using the routine condi-
tions. Thus, it is likely that the main benefit of a Vacuoles: The negative effect of vacuoles on
scoring scale of sperm classification in IMSI is a fertilization and embryo development and the
kind of “fine tuning” and is actually more benefi- possible correlation with DNA integrity has pre-
cial for those motile spermatozoa with normal viously been described. Vanderzwalmen et al.
morphological appearance, under magnification [68] classified the spermatozoa into four groups
of 200–400×. This was recently confirmed when according to the presence and size of vacuoles:
spermatozoon was first chosen for ICSI as rou- Grade I, normal shape and absence of vacuoles,
tinely performed under conventional conditions Grade II, maximum of two small vacuoles; Grade
using magnification of 400×. Thereafter, prior to III, more than two small vacuoles or at least one
injection, the spermatozoon was evaluated, inside large vacuole; and Grade IV, large vacuoles in
the ICSI pipette under high magnification [67]. conjunction with abnormal head shapes or other
abnormalities at the level of the base of the sperm
head. As previously explained, it has been postu-
19.3.4 Parameters for Motile Sperm lated that vacuoles of various sizes are most likely
Evaluation and Classification correlated to the integrity of sperm DNA and,
in Real-Time thus, may affect the fertilization potential of the
spermatozoon [53, 59, 60, 67].
In general, it is obvious that classification param- However, it is not clear yet, when vacuoles are
eters should be clear and will enable the embryol- formed and why they are such important indica-
ogist to score the aspirated motile spermatozoon tors of sperm quality. Nevertheless, the question
as fast as possible. Normalcy of the sperm nucleus: whether is there a correlation with DNA damage
was first detected by Bartoov et al. [45, 47] accord- in the sperm head is still debatable.
ing to the shape of sperm head. As already men-
tioned, normalcy of the nucleus was checked
(6,000×) in the leftover sperm fraction, in 100 19.3.5 Combination of Sperm
couples after using it for ICSI in three major IVF Characteristics in Real-Time
centers [45]. It was demonstrated that a normal
spermatozoon, with a normal nuclear shape, Cassuto and Barak scoring system was recently
observed under magnification of 6,000× in established to define more precisely the preferable
spermatozoon for ICSI [67]. In the latest, each Moreover, normalcy of the base appears to be
spermatozoon was initially aspirated using the a major factor, which affected embryo quality; a
routine ICSI method, i.e., magnification of 400×. higher rate of “top” and “good” day 2 and 3
Aspiration was then followed by immediate embryos were detected when the base of the
observation and scoring under magnification of sperm head was scored as normal. As the base of
6,100×, prior to ICSI. Utilization of this method the sperm head actually contains the centrosome,
made it possible to detect each single spermato- and is correlated to the sperm nucleus, it does
zoon in correlation to its contribution with a spe- contribute to embryo quality, as has been con-
cific oocyte/early embryo, and to examine the firmed by others [58, 69–71]. A friendly formula
effect of the morphology of an individual sperm of a model for the classification following cal
cell. Based on the knowledge accumulated in the culations of coefficients with: area = 0.618 of
field of fertility [4, 14, 19, 20, 26, 45], each aspi- the ROC curve, was designed: Score = Head × 2 +
rated spermatozoon was classified into one of Vacuole × 3 + Base.
three classes I, II, or III taking into consideration Values of the major sperm criteria were scored
the following characteristics: normalcy of head as normal = 1 and abnormal = 0. When calculated,
shape, acrosome, absence of vacuoles, normalcy the range of scoring varied between 0 and 6. The
of the base of sperm head, normalcy of tail, and recommended calculated Cassuto and Barak
clarity of head cytoplasm The investigators score for the injected spermatozoa should be 4–6
endeavour to comprehend which defect could (Class I).
effect the correlation between the normalcy of
the sperm, fertilization, and early embryo devel-
opment up to the blastocyst stage. A comparison 19.3.6 Scoring and Women’s Age
of the fertilization rates (FR) following the injec-
tion of spermatozoa of the three various classes Due to the data published by these investiga-
revealed a significant difference (P < 0.04; tors it seems that IMSI is more beneficial in
x2 = 6.31) when a pair wise comparison showed a “older oocytes” harvested from patients aged
significant difference between groups I and III 30 years and older. Statistical analysis for the
(P < 0.01; x2 = 6.3). Moreover, a significance was combination of the woman’s age and the scored
noted when comparison was made between the injected spermatozoa in correlation to embryo
rate of expanded blastocysts in the three groups quality, distinguished between the quality of
(P < 0.03; x2 = 6.71). No expanded blastocyst was embryos resulting from oocytes of patients
formed in embryos resulted following the injec- younger than 30 years and those which devel-
tion of class III spermatozoa. Statistical analysis oped from oocytes originating from patients 30
of the outcome and area under the best ROC years and older. A reduced rate of top D2 and
curve revealed that: head shape, absence of vacu- D3 embryos was observed when class II and
oles, and normalcy of head–base were the major class III spermatozoa were injected to oocytes
characteristics for sperm classification. In accor- of patients between 30 and 36 years old in
dance with the findings, the three classes of sperm comparison to the injection of class II and III
morphology impart new information that contrib- spermatozoa to oocytes harvested from patients
utes to the outcome of ICSI, in regard to fertiliza- <30 years (44.4 vs. 96.3%; P < 0.001; x2 = 19.52,
tion and early embryo development up to the respectively) [67].
blastocyst stage. Confirmed by others (and as The age-related decline in the quality of
previously mentioned), it has clearly been shown the oocytes, is a known phenomenon [72–74].
that vacuoles in the sperm head interfere already The outcome explains the importance of
from the first step of fertilization; a higher FR sperm scoring mainly in “older oocytes” har-
was achieved in oocytes injected with spermato- vested from patients aged 30 years and older.
zoa in which no vacuoles were observed in their This outcome is not surprising, as the young
heads. oocyte is capable of “repairing” the DNA of
the injected spermatozoon; ICSI may rescue References

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Blazar AS. Pregnancy outcome in in vitro fertilization 67. Cassuto NG, Bouret D, Plouchart JM, Jellad S,
decreases to a plateau with repeated cycles. Fertil Vanderzwalmen P, Balet R, et al. A new real-time
Steril. 2005;84:1043–5. morphology classification for human spermatozoa: a
53. Antinori M, Licata E, Dani G, Cerusico C, Versaci C, link for fertilization and improved embryo quality.
D’Angelo D, et al. Intracytoplasmic morphologically Fertil Steril. 2009;92(5):1616–25.
selected sperm injection: a prospective randomized 68. Vanderzwalmen P, Hiemer A, Rubner P, Bach M,
trial. Reprod Biomed Online. 2008;16:835–41. Neyer A, Stecher A, et al. Blastocyst development
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Thoughts on IMSI
Gianpiero D. Palermo, Jennifer C.Y. Hu,
20
Laura Rienzi, Roberta Maggiulli, Takumi
Takeuchi, Atsumi Yoshida, Atsushi
Tanaka, Hiroshi Kusunoki, Seiji Watanabe,
Queenie V. Neri, and Zev Rosenwaks
Abstract
ICSI achieves consistently high fertilization and pregnancy rates
regardless of sperm characteristics and even azoospermic patients have
fathered children by ICSI where spermatozoa were surgically retrieved. In
this review, we report multicenter study results after the use of IMSI in
patients with compromised semen parameters or in patients with previous
ART failure using sibling oocytes. These studies, based on higher magni-
fication screening for sperm surface irregularities, did not seem to benefit
the patients’ clinical outcome in independent investigations. We conclude
from these collaborative studies, IMSI does not bring any advantage in the
quest to find spermatozoa devoid of surface irregularities and that it sig-
nificantly lengthens the search time required.
Keywords
ICSI • IMSI • MSOME • Nuclear vacuoles • Spermiogenesis • Embryo
quality
f ertilization and pregnancy outcomes [4]. ICSI

20.1 Introduction achieves consistently high fertilization and
pregnancy rates regardless of sperm characteris-
Classically, sperm morphology has been a
tics and even azoospermic patients have fathered
valuable diagnostic tool [1] and has played an
children by ICSI where spermatozoa were
important role in predicting the fertility poten-
surgically retrieved [5–7].
tial of infertile men attempting IVF [2]. The
Notwithstanding these outcomes, several stud-
introduction of ICSI [3] rendered these basic
ies have shown that suboptimal sperm morphol-
semen parameters obsolete where this technique
ogy is often associated with aneuploidy, nuclear
was used because of their inability to predict
DNA damage, and at times an impaired ICSI out-
come [8–12] .
Not surprisingly, abnormalities of the male
G.D. Palermo () gametes may be a harbinger of developmental
The Ronald O. Perelman and Claudia Cohen Center
problems in embryos [13, 14]. Such effects, can
for Reproductive Medicine, Weill Cornell
Medical College, New York, NY, USA be separated into early and late events. Among
e-mail: [email protected] the early effects, failed fertilization, abnormal

278 G.D. Palermo et al.
zygote morphology, impaired cleavage arising called “intracytoplasmic morphologically selected

from centrosome dysfunction or lack of oocyte sperm injection” (IMSI), and its use is claimed to
activating factors are most obvious. Late paternal result in a higher pregnancy rate than with con-
effects include sperm aneuploidy, DNA damage, ventional ICSI [29, 30]. The beneficial effect of
or abnormal chromatin condensation and are IMSI has been demonstrated in a series of studies
considered responsible for repeated implantation in which the clinical outcome of patients treated
failures [13]. Of those factors, sperm DNA dam- by this procedure was compared with that of
age has been the most extensively investigated patients treated by conventional ICSI [31–34].
utilizing various methods considered to predict The early ultrastructural studies of human
the ART outcome [15–19]. sperm in the 1950s and 1960s, revealed that vac-
Sperm DNA integrity is currently assessed by uoles in the sperm nucleus have been seen in the
destructive methods such as TUNEL, COMET, large majority of human spermatozoa regardless
sperm chromatin dispersion (SCD) test, or by a of the fertility of the donors. Therefore, vacuoles
sperm chromatin structural assay (SCSA). in human sperm have been considered as a physi-
However, all of these require fixation and so ologic finding devoid of consequence on fertility
destruction of the sperm being assessed [15]. potential [35]. From this there is the need to
Therefore, we have explored the methods that revisit whether the presence of nuclear vacuoles
may be able to select sperm with a competent portend to sperm DNA defects with consequent
genome in a noninvasive manner to improve ART impaired developmental competence of the
outcome. spermatozoon.
One particular issue of interest in this context In this chapter, we revisit the actual definition,
are vacuoles in the sperm nucleus. In contrast to origin, and possible significance of human sperm
those of other mammals, nuclear vacuoles are a nuclear vacuoles. We report multicenter results
very common feature of human sperm, and are after the use of IMSI in patients with compro-
considered by some to reflect the presence of mised semen parameters or in patients with
DNA damage [20, 21] that may compromise the previous ART failure using sibling oocytes.
development of zygotes [16, 22, 23]. It has been A detailed relative quantification of sperm sur-
speculated that the presence of nuclear vacuoles face defects in ejaculated or surgically retrieved
is associated also with other aspects of sperm sperm was made by high magnification light
quality, such as a lower mitochondrial membrane microscopy, scanning, and transmission electron
potential, a higher incidence of chromo- microscopy (TEM) as well confocal microscopy.
somal abnormalities, and greater DNA damage In addition, the developmental competence of the
[20, 21, 24]. It has also been suggested that the embryo was assessed according to the presence
absence of nuclear vacuoles is indicative of or absence of vacuoles in their paternal sperm.
completion of the acrosome reaction [25]. This Finally, an assessment was made on the relation-
conclusion posits that spermatozoa devoid of ship between sperm surface irregularities and
vacuoles have likely undergone the acrosome genomic integrity in terms of the incidence of
reaction and are able to induce oocyte activation sperm chromatin fragmentation and karyotyping.
more effectively [26, 27]. In addition, deformities
of the midpiece section of the spermatozoon
assessed under high magnification microscopy 20.2 What Are Sperm Vacuoles?
has been linked to centrosomal dysfunction [28].
A technique called “motile sperm organellar The interest towards the male gamete began in
morphology examination” (MSOME) has been the seventeenth century and together with
proposed to assess living sperm morphology the concurrent refinement of new microscopic
under high magnification [29]. In this case, techniques have sparked the continued interest of
screening is used to select a spermatozoon for scientists wordwide towards this fascinating
ICSI with an optimal shape. The procedure is highly specialized cell. Structural analysis of the
20 Thoughts on IMSI 279
Fig. 20.1 Examples of nuclear vacuoles under transmission electron microscopy in normospermic (a, b) and globo-
zoospermic specimens (c)
mammalian spermatozoon has been studied fur- thus changing the configuration of the sperm
ther than most of the any other differentiated cell head to reach a more compact and hydrodynamic
type, yet the molecular mechanisms of motility, shape favorable for cell motility and penetration
sperm activation, zona penetration, syngamy, and through the egg vestments [38, 39].
chromatin decondensation still elude us. Vacuoles occur commonly in human sperm
In the majority of species, the uniformity of nuclei [35] but are generally observable only
the product of spermatogenesis is remarkable; with TEM. Nuclear vacuoles are irregular entities
hundreds of millions of sperm are produced each in the condensed chromatin and are not limited
day with very little variation in head shape and by a membrane (Fig. 20.1a-c). These vacuoles
with a surprisingly small percentage of nuclear are due to variably localized aberrations of
anomalies. However, human spermatozoa display nuclear decondensation during the histones-to-
considerable structural heterogeneity even after protamine exchange [37]. In an extreme example,
their maturation and transit through the epididymis it appears that the large majority of the round-
[36]. headed (globozoospermic) sperm nuclei pro-
Shaping of the nucleus takes place in late duced by some men have vacuoles verisimilarly
spermiogenesis as its chromatin is undergoing a related to incomplete chromatin condensation
remarkable condensation that renders the sperm (Fig. 20.1c) and their nuclei are characterized by
transcriptionally inert and highly resistant to DNA packing anomalies related to the reduced
digestion. Following the morphological transfor- protamine content.
mation of the nucleus in the testis, as sperm pass Another way to assess sperm nuclear vacuoles
through the epididymis there occurs a stabiliza- without the use of TEM would be to perform a
tion of the chromatin through establishment of decondensation assay [36] in which sperm are
disulfide bonds between the thiol-rich protamines exposed to sodium dodecyl sulfate (SDS) + dithio-
[37]. The human sperm nucleus is composed also threitol (DTT) that cleave weak and covalent
of the DNA condensing core and linker histones –S-S– bonds, respectively. In other mammals
that have been partially replaced by protamines, except man, sperm display a uniform rate of
the plication/vacuolization of the rostral plasma-

lemma during capacitation. As noted later in the
chapter, these vacuole-like structures or craters
appear in over 90% of spermatozoa from fertile
donors with normal semen parameters [43, 44].
20.3 Application of MSOME

in Men with Compromised
Semen Parameters
In a prospective randomized study in a single

center, patients with compromised semen param-
eters (<5 × 106/mL concentration and normal
Fig. 20.2 Selected spermatozoa devoid of surface irregu-
larities following exposure to sodium dodecyl sulfate + sperm morphology of <4% according to Kruger’s
dithiothreitol. Spermatozoa displayed several vacuole-like strict criteria) were allocated 1:1 to either the
structures visible at 200, 400, and 1,000× ICSI and the IMSI approach. In both groups,
three MII oocytes were inseminated per cycle
decondensation. In the case of the human sperm, in accordance with the Italian Law on Assisted
however, some decondense rapidly after treatment Reproductive Technology. Accurate sperm selec-
while others hardly react at all. Motile spermato- tion for ICSI was performed in both groups
zoa with regularly shaped heads following expo- (at 400×; Fig. 20.3a) and the morphology of
sure to a nuclear decondensing agent, displayed the selected spermatozoa in the IMSI group
multiple vacuole-like structures becoming visible (Fig. 20.3b) was recorded in detail. For IMSI,
under light microscopy (Fig. 20.2). The heteroge- spermatozoa were placed in a glass bottom dish
neity of human sperm nucleus decondensation and observed at 1,000× under mineral oil with
may reflect variation in the degree of –S-S– cross- Nomarski’s differential contrast (DIC) optics.
linking during nuclear compaction. It is also pos- The magnification was further increased by a
sible that this may be the result of an unusually digital imaging system in order to obtain a final
variable rate of epididymal transit by individual magnification of 6,600× [29]. In both groups,
spermatozoa that is characteristic of man. For in spermatozoa were first evaluated and when
humans, the rate of sperm passage from the testis selected were immobilized, and then placed in a
to the ejaculate, averages 12 days and ranges clean PVP drop before ICSI was performed. The
from 1–2 to 21 days. On the basis of their quasi- mean time employed to collect normal appearing
universal presence in human sperm heads, the spermatozoa (according to MSOME criteria) was
intranuclear vacuoles cannot be considered as 7.7 ± 3.1 min for ICSI and 108.3 ± 29.9 min for
degenerative structures [35, 40]. IMSI (P < 0.001). Oocytes were selected as pre-
A different type of sperm head irregularity is a viously described [45] and all viable embryos
surface “vacuole” or indentations, craters, dents, obtained were transferred on day 2, again as
or hollows on the surface. In such cases, during required by Italian law [46]. The primary aim
sperm morphogenesis, the outer acrosomal mem- was to evaluate the delivery rate per transfer pro-
brane misforms and generates what appears to be cedure and live births per transferred embryos,
a vacuole [41]. These vacuole-like structures dis- while secondary outcomes were related to fertil-
appear as the spermatozoon matures in the ization rates and day 2 embryo development.
epididymis or at the time of the acrosome reac- For this study, 33 couples were recruited – 16
tion [25]. In other circumstances, however, they assigned to ICSI and 17 to the IMSI approach
seem to increase with temperature (37°C) and (Table 20.1). The maternal age was comparable
incubation time (>2 h) [42], most probably due to in both groups as were the fertilization rates and
Fig. 20.3 Visualization
of a spermatozoon at 400×
(a) and at 6,600× (b)
Table 20.1 Benefits of No. of (%) ICSI IMSI

MSOME in oligoasthenot-
Cycles 16 17
eratozoospermic couples
Maternal age (M years ± SD) 34.9 ± 3 35.2 ± 3
2PN 40/48 (83.3) 42/49 (85.7)
Top quality day 2 embryos 27/40 (67.7) 29/42 (68.2)
Embryos transferred (M ± SD) 2.5 ± 0.8 2.5 ± 0.5
Delivery 6 (37.5) 5 (29.4)
Live birth 8/40 (20.0) 7/42 (16.7)
the number of embryos transferred. There was a basis of the rationale that the ART failure was
trend towards higher pregnancy (37.5 vs. 29.4%) due to an incompetent male genome regardless of
and implantation (20.0 vs. 17.6%) following ICSI the adequate semen parameters. In order to com-
indicating that IMSI did not yield any benefit pare the rates of implantation, clinical pregnancy,
over ICSI in regards to fertilization, pregnancy, and delivery in relation to the sperm selection
and implantation rates. This analysis indicated method, only embryos deriving from one proce-
that higher magnification microscopy did not dure or other were transferred to any one patient.
enhance the ability to select “good looking” sper- Briefly, oocyte-cumulus-complexes were retrieved
matozoa for injection while lengthening the from ovaries stimulated with exogenous gonado-
procedure. tropins after pituitary suppression with GnRH
agonists or antagonists. Embryo transfers were
performed under abdominal ultrasound guidance
20.4 ICSI Versus IMSI in Sibling on day 5 following oocyte retrieval.
Oocytes For IMSI, sperm selection was carried out
under an inverted microscope equipped with
In order to limit confounding factors, only women Nomarski’s DIC enhanced by digital imaging
less than 40 years of age were selected for this [24, 33]. Motile spermatozoa with the best avail-
study. Ejaculated spermatozoa were used in able morphology, devoid of or displaying only a
patients that had at least five metaphase II (MII) small vacuole occupying <4% of the nuclear area,
oocytes, half of them being injected using IMSI were selected for IMSI. Conventional ICSI was
and the other half by ICSI. Patients were grouped performed as described by Palermo et al. [47].
according to whether this was their first ART A total of 276 consenting couples with male
cycle or was a repeat following prior failed infertility were included in this study using ejacu-
attempts. Repeat attempts were undertaken on the lated spermatozoa. In the group for which this was
Table 20.2 High No. of (%) ICSI IMSI

magnification ICSI in ART
Women 214
couples at their first
attempt Injected MII oocytes 1110 1119
2PN 718 (64.7) 694 (62.0)
Day 3 embryos 409 (56.5) 400 (57.6)
Blastocyst development 235 (32.7) 233/610 (33.6)
Embryo replacements 107 107
Transferred embryos (M ± SD) 122(1.1 ± 0.3) 120(1.1 ± 0.3)
Clinical pregnancy (+FHB) 45 (42.1) 39 (36.4)
Implantation 51 (41.8) 45 (37.5)
Deliveries or ongoing 44 (41.1) 39 (36.4)
Table 20.3 High No. of (%) ICSI IMSI

magnification ICSI in
Women 62
couples with previous ART
failure Injected MII oocytes 376 359
2PN 235 (62.5) 216 (60.2)
Day 3 embryos 119 (50.6) 120 (55.6)
Blastocyst development 46 (19.6)a 61 (28.2)a
Embryo replacements 26 36
Transferred embryos (M ± SD) 37 (1.5 ± 0.5) 58 (1.6 ± 0.5)
Clinical pregnancy (+FHB) 8 (30.8) 17 (47.2)
Implantation 9 (24.3) 22 (37.9)
Delivered or ongoing 8 (30.8) 17 (47.2)
a
P = 0.04
the first ART attempt (n = 214), the mean maternal this did not translate to a superior pregnancy out-
age was 33.4 ± 3 years and the paternal age was come in comparison to ICSI. This indicates that
37.0 ± 6 years. The men had an average concentra- utilization of sperm void of surface irregularities
tion of 56.7 ± 57 × 106/mL with 36.4 ± 19% motil- does not grant a male genome with higher devel-
ity, and Kruger’s morphology of 2.9 ± 2%. In this opmental competence.
cohort, the rate of fertilization and embryo devel- In order to establish whether repeat treatment
opment were comparable between the two insem- with ICSI would have any impact on clinical out-
ination methods (Table 20.2). After embryo come, we assessed patients at Cornell of compa-
transfer, clinical pregnancies after ICSI (42.1%) rable age (£40 years) that failed to achieve a
did not differ from IMSI (36.4%). clinical pregnancy with ejaculated spermatozoa
In cases with previous failed ART attempts, at a first ICSI attempt (n = 3,018). Upon their
the average age of the women and men was return and subsequent treatment with ICSI, a
34.9 ± 3 years and 37.2 ± 4 years, respectively. clinical pregnancy rate of 44.2% (1,166/2,640)
The semen parameters were 48.8 ± 42 × 106/mL, was achieved.
34.0 ± 18% motility, with a Kruger’s morphology This finding suggested that an ART failures
of 3.2 ± 3%. In this multiple ART failure group, are multifactorial and cannot be simply blamed
fertilization rates again were comparable between on the spermatozoon. Even if the male genome
ICSI and IMSI, however, the frequency of full would be at fault, these findings confirmed that
blastocyst development was somewhat higher the genotype of the gamete cannot be simply pre-
after IMSI (P = 0.04) (Table 20.3). Nonetheless, dicted by its phenotype.
at 1,000× in ejaculated (Fig. 20.4a–c), epididy-

20.5 Qualitative Assessment mal (Fig. 20.4d), and testicular (Fig. 20.4e) sper-
of Sperm Nuclear Defects matozoa as well as in spermatids (Fig. 20.4f).
and Embryo Development Samples obtained from consenting men included
either testicular (from non-obstructive), epididy-
To determine whether “vacuoles” or “craters” on mal (obstructive azoospermic), or ejaculated
a human sperm head have any physiological sig- spermatozoa. In addition, spermatids of varying
nificance, their incidence and size were recorded stages were also assessed.
Fig. 20.4 Sperm craters under optic microscroscopy (a, d–f) and scanning electron microscopy (b, c) evidencing craters
in ejaculated (a–c), epididymal (d), testicular (e) spermatozoa, and spermatids (d). SEM images (b, c) confirmed the
presence of hollows in spermatozoa with and without acrosome (b)
Table 20.4 Full embryo Crater characterization for IMSI

development according to
No. of (%) Large Small None ICSI
crater size
Oocytes injected 23 63 20 256
Fertilization 14 (60.9)a 54 (85.7)a 16 (80.0)a 167 (70.8)a
Blastocyst development 7 (50.0) 28 (54.9) 4 (25.0) 85 (51.0)
a
P = 0.008
Regardless of the semen source, the large Morphologically, normal spermatozoa were
majority of spermatozoa had one or more craters assessed for nuclear vacuoles with DIC optics at
of various sizes. Ejaculated samples had the high- 1,000× (Fig. 20.5a). The sperm samples were
est proportion, the frequency ranging from 87.5 classified according to the presence, the size, and
to 97.7%, while early spermatids displayed the the localization of vacuoles as visualized by con-
largest craters. In the shaping of the nucleus dur- focal microscopy (Fig. 20.5b). Nuclear vacuoles
ing spermiogenesis, the number of craters were observed in over 90% of normally shaped
increased while their size diminished as cells spermatozoa from infertile and donor males alike.
moved through the epididymis. In fact, regularly shaped sperm without vacuole-
Fertilization of MII oocytes following IMSI like structures were extremely rare (2.4%). When
was compared to ICSI cases (Table 20.4). IMSI confocal microscopy was employed, the vacuole-
using small cratered spermatozoa yielded compa- like structures appeared more as surface craters.
rable fertilization rates to sperm without such Sperm chromosomal analysis was performed
craters, whereas large craters appeared to com- by injecting such classified spermatozoa into
promise fertilization. When compared to ICSI, mouse oocytes, while DNA fragmentation was
oocytes inseminated by IMSI with sperm having evaluated by TUNEL assay. In terms of chromo-
small vacuoles resulted in a higher fertilization somal abnormalities, there was no difference
(85.7 vs. 70.8%; P = 0.003). Interestingly, sper- between the spermatozoa with large nuclear vacu-
matozoa selected by MSOME was judged as oles and those without. In addition, we found no
being devoid of craters and generated the lowest correlation between the incidence of chromosomal
blastocyst rate (4/16). In addition, when DNA aberrations and the size or the location of “nuclear
integrity was assessed by TUNEL on large cra- vacuoles.” Moreover, the incidence of spermato-
tered sperm isolated by MSOME, there was no zoa with DNA fragmentation detected by TUNEL
evidence of chromatin damage (0/21). This was consistently low (3.1–3.5%) regardless of the
implies that craters do not reflect abnormalities presence of large vacuoles (Fig. 20.5c). Therefore,
of the sperm head genome, but may as well be it appears that nuclear vacuoles are not an indica-
common physiological variations occurring dur- tor of sperm nuclear chromosomal aberrations or
ing human spermiogenesis which unlike that in of sperm nuclear damage such as nicks and
animals may be partially affected by the higher breaks.
temperature brought by clothing [48].
20.7 Relationship Between

20.6 Frequency and Possible Phenotypic and Genomic
Significance of Sperm Sperm Anomalies
Nuclear Abnormality of the Sperm Nucleus
A further study investigated the possibility of an MSOME, often requested by patients, entails the
association between sperm nuclear vacuoles, live scanning of highly magnified spermatozoa
DNA fragmentation, and chromosomal abnor- for irregularities that may compromise the ability
malities. For this, sperm samples were obtained to fertilize and support embryo development. As
from 17 infertile patients (15 IVF and 2 ICSI) aforementioned, it has been claimed that genomic
and three fertile donors. integrity is impaired in spermatozoa displaying
Fig. 20.5 Sperm surface irregularities under high magnification DIC (a) and confocal microscopy (b). TUNEL assay
performed on spermatozoa with and without vacuole-like structures (yellow arrow) (c)
vacuoles or surface irregularities. However, the status, fixed spermatozoa were decondensed and
bearing of chromatin fragmentation on the health hybridized with locus specific probes for chro-
of the conceptus and its ability to implant has not mosomes X, Y, 13, 15, 16, 17, 18, 21, 22
been confirmed, nor has any link been demon- (MultiVysion™ PB probe mixture, Abbott;
strated between sperm head surface defects and OligoFISH™ probe kit, One Cell System).
genomic competence. Therefore, we aimed to The study cohort consisted of 35 men with an
assess the genomic and epigenetic implications average age of 38.4 ± 4 years. A total of 7,350
for male gametes exhibiting surface irregularities spermatozoa were evaluated for DNA fragmenta-
and “vacuoles.” tion and ploidy status, 3,890 of which had visible
At least 100 motile spermatozoa showing head surface defects and 3,460 were without. The
dysmorphism (with “vacuole”) were selected at assessment of sperm ploidy status revealed a com-
1,000× by micromanipulation, retrieved individ- parable proportion of chromosomal abnormalities
ually, and placed on pre-coated slides (Fig. 20.6). in the two groups (Fig. 20.7). The total fragmenta-
Motile sperm of normal morphology selected tion rate detected by SCD assay was 2.1% for
individually served as controls. Sperm DNA frag- vacuolated spermatozoa and 2.4% for the control
mentation was assessed by the Sperm Chromatin (Fig. 20.8). When the fragmentation was assessed
Dispersion Test (Halosperm® kit, INDAS labora- by TUNEL assay, 5.5% of spermatozoa with head
tories) and TUNEL assay (APO-BrdU™ TUNEL defects displayed DNA fragmentation versus
assay kit, Invitrogen). To determine their ploidy 5.6% observed in the controls (Fig. 20.9).
Fig. 20.6 Study design
Fig. 20.7 Semen characteristics and ploidy status assessed by FISH
In conclusion, the presence of sperm nuclear such as nuclear vacuoles and other head abnor-
defects assessed by high magnification micros- malities do not appear to directly reflect chro-
copy did not appear to correlate with the ploidy mosomal imbalances or presence of DNA
or status of the sperm chromatin. Thus, defects breakage.
Fig. 20.8 Semen characteristics and DNA fragmentation assessed by SCD test
Fig. 20.9 Semen characteristics and DNA fragmentation as assessed by TUNEL assay
Higher magnification screening for sperm sur-

20.8 Conclusions face irregularities did not seem to benefit the
patients’ clinical outcome in independent investi-
Since the development of ICSI, it has been sug- gations. This was true for patients with compro-
gested that more attention be paid to the selection mised semen parameters and for those either
of sperm while they are swimming in slow motion undergoing first or repeated ART attempts. More
in a viscous medium in order to select the “best detailed morphological observations indicated
looking” spermatozoon. By selecting individual that in human sperm heads visible irregularities
sperm cells, at the highest magnification possible, or vacuoles are almost ubiquitous, and appear to
ICSI has enabled even couples with severe male be a paraphysiologic finding. Analyses of sper-
factor to achieve conception. matozoa from different sources, ejaculated or
surgically retrieved, also revealed the varying 4. Nagy ZP, Liu J, Joris H, et al. The result of intracyto-
presence and size of vacuoles that develop during plasmic sperm injection is not related to any of
the three basic sperm parameters. Hum Reprod. 1995;
the dynamic processes of spermiogenesis and 10(5):1123–9.
maturation. This surface irregularity did not relate 5. Palermo GD, Schlegel PN, Hariprashad JJ, et al.
to the incidence of DNA fragmentation or aneu- Fertilization and pregnancy outcome with intracyto-
ploidy, nor to the ability of vacuolated spermato- plasmic sperm injection for azoospermic men. Hum
Reprod. 1999;14(3):741–8.
zoa to generate zygotes capable of developing to 6. Tournaye H, Devroey P, Liu J, Nagy Z, Lissens W,
blastocysts. Moreover, the safety of IMSI on how Van Steirteghem A. Microsurgical epididymal sperm
it is currently performed need to be confirmed. aspiration and intracytoplasmic sperm injection: a
In fact, it has been reported that infants born after new effective approach to infertility as a result of con-
genital bilateral absence of the vas deferens. Fertil
IMSI have a higher risk of low birth weight Steril. 1994;61(6):1045–51.
(<2500 g) [49]. 7. Schoysman R, Vanderzwalmen P, Nijs M, et al.
Even in light of the ability of the ooplasm to Pregnancy after fertilisation with human testicular
overcome certain sperm genomic anomalies, it spermatozoa. Lancet. 1993;342(8881):1237.
8. Tang SS, Gao H, Zhao Y, Ma S. Aneuploidy and DNA
should be kept in mind that the factors contribut- fragmentation in morphologically abnormal sperm.
ing to impaired embryo developmental compe- Int J Androl. 2010;33(1):e163–79.
tence cannot conveniently be identified in the 9. De Vos A, Van De Velde H, Joris H, Verheyen G,
simple analysis of the surface irregularities of the Devroey P, Van Steirteghem A. Influence of individual
sperm morphology on fertilization, embryo morphol-
sperm, particularly when we refer to chromo- ogy, and pregnancy outcome of intracytoplasmic
somal status or centrosomal function. sperm injection. Fertil Steril. 2003;79(1):42–8.
We conclude from these collaborative studies, 10. Templado C, Hoang T, Greene C, Rademaker A,
that a 16× factor magnification does not bring Chernos J, Martin R. Aneuploid spermatozoa in infer-
tile men: teratozoospermia. Mol Reprod Dev.
any advantage in the quest to find spermatozoa 2002;61(2):200–4.
devoid of surface irregularities, and significantly 11. Harkonen K, Suominen J, Lahdetie J. Aneuploidy in
lengthens the search time required. The presence spermatozoa of infertile men with teratozoospermia.
of vacuoles does not flag spermatozoa with frag- Int J Androl. 2001;24(4):197–205.
12. Tasdemir I, Tasdemir M, Tavukcuoglu S, Kahraman S,
mented DNA or aneuploidy. Finally, the putative Biberoglu K. Effect of abnormal sperm head morphol-
correlation between vacuolar size and genomic ogy on the outcome of intracytoplasmic sperm injec-
integrity was not confirmed. tion in humans. Hum Reprod. 1997;12(6):1214–7.
13. Tesarik J, Greco E, Mendoza C. Late, but not early,
Acknowledgement We are grateful to all the contribu- paternal effect on human embryo development is
tors for their efforts in allowing this endeavor to be related to sperm DNA fragmentation. Hum Reprod.
accomplished. We are thankful to Dr. J. Michael Bedford 2004;19(3):611–5.
for his critical review and Justin Kocent for his technical 14. Tesarik J, Mendoza C, Greco E. Paternal effects act-
assistance. Queenie V. Neri was funded by the grant ULI ing during the first cell cycle of human preimplanta-
RR024996 of the Clinical and Translational Science tion development after ICSI. Hum Reprod.
Center at Weill Cornell Medical College. 2002;17(1):184–9.
15. Zini A, Sigman M. Are tests of sperm DNA damage
clinically useful? Pros cons J Androl. 2009;30(3):
219–29.
16. Bungum M, Humaidan P, Spano M, Jepson K,
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Simultaneous removal of sperm plasma membrane 42. Peer S, Eltes F, Berkovitz A, Yehuda R, Itsykson P,
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31. Antinori M, Licata E, Dani G, et al. Intracytoplasmic ment for all forms of male factor infertility. Fertil
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Bacrie P, Tesarik J. High-magnification ICSI over- 49. Junca AM, Dumont M, Cornet D, Douard S, De
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33. Berkovitz A, Eltes F, Yaari S, et al. The morphologi- tal for pregnancy outcome? Fertil Steril. 2010;94:
cal normalcy of the sperm nucleus and pregnancy rate S31.
Index
A gonadotropins, 204
Acridine orange test (AOT), 161 hormonal superovulation, 203–204
Agouti viable yellow (Avy) allele, 187 ICSI, 198, 204–205
Amino levulanic acid (ALA) synthase enzyme, 139 immature sperm extraction and injection, 204
Angelmann syndrome (AS), 200–201 imprinting disorder, 200–201
Anti-Müllerian hormone (AMH) in vitro culture, preimplantation embryos,
aromatase, 22, 23 205–206
ART (see Assisted reproduction technologies) in vitro fertilization, 197
clinical applications, 24 IUI, 165, 167
corpus luteum, 23 IVF and ICSI, 167–168
follicle-stimulating hormone, 22, 23 male and female pronucleus, 203
gene and protein, 21 monozygotic twinning and perinatal outcomes, 92
intra-follicular concentrations, 22 oocytes, 203–204
menstrual cycle, 23 oocytes IVG and IVM, 204
OHSS, 17 ovarian hyperstimulation syndrome, 27–28
ovarian granulosa cells, 21 ovarian-stimulated cycles, 44
ovarian physiology, 20 ovulation induction, 26
ovarian response, COH, 66, 67 PGC, 203
PCOS (see Polycystic ovary syndrome) PGD, 198
sensitivity and specificity, 67, 68 sperm from infertile men, 204
sertoli cell maturation, 21 therapeutic modality, 197–198
serum concentration, 21, 22 uterus and embryo, asynchrony, 204
steroidogenesis, 23 Asthenozoospermia, 141
testosterone, 21, 22 ATRX syndrome, 202
Antinuclear antibodies, 6 Autoimmunity and female infertility
Antiovarian antibodies, 6 blastocyst, 4
Antiphospholipid syndrome, 4 implantation, 4
Antisperm antibodies, 6–7 pregnancy failure
Antithyroid antibodies, 5–6 antinuclear and antiovarian antibodies, 6
Antral follicle count (AFC), 27, 66 antiphospholipid antibodies, 4–5
Aromatase inhibitors, 15 antisperm antibodies, 6–7
Assisted reproductive technologies (ART) antithyroid antibodies, 5–6
complications, 198–199
controlled ovarian hyperstimulation, 26–27
embryo assessment (see Embryo assessment) B
epigenetic disturbances Beckwith–Wiedemann syndrome (BWS), 182, 201
agouti viable mouse, 202 Bisphenol A (BPA), 138
cardiovascular and metabolic dysfunction, 202 Body mass index (BMI), 36, 142
chronic metabolic disorders, 203 Bone morphogenetic proteins (BMP), 21
DOHAD hypothesis, 201
global genomic hypomethylation and
hypermethylation, 202 C
IAP methylation, 202 Cadmium, 139
epigenetic regulation, 199–200 Carbendazim, 140
gametes/embryos cryopreservation, 206 Chlorpyrifos, 140
gametogenesis, 203 Chromatin immuno precipitation (CHIP), 230

DOI 10.1007/978-1-4419-8456-2, © Springer Science+Business Media, LLC 2011
292 Index
Chromomycin A3 (CMA3) diet and obesity, 142–143

fluorochrome, 215 epididymis, 137
staining, 162 haploid spermatids, 137
Chronic zinc deficiency, 139 heavy metal toxicity, 139–140
Clomiphene citrate, 26 hypothalamus-pituitary-gonadal axis, 138
Colorado Center for Reproductive Medicine plastic industry chemicals, 138–139
(CCRM), 257–258 psychological and noise stress, 143
Creatine phosphokinase (CK), 212, 214 PUFA, 138
Cryopreservation, 120 scrotal heat stress, 143–144
smoking, 141
solvents, 140
D Western lifestyle, diet and exercise, 138
Developmental origin of health and disease testicular cancer and cryptorchidism, 134
(DOHAD), 201 toxins and xenobiotics, 134
Diamniotic pregnancies, 104
Dichorionic, diamniotic (DC/DA) twins, 94–95
Dichorionic twin pregnancies, 103–105 F
Diepoxybutane (DEB), 229 Follicle stimulating hormone (FSH)
Differential interference contrast (DIC), 270 environmental insults, spermatogenesis, 135
Di (2-ethyhexyl) phthalate (DEHP), 139 follicular activation and growth, 23
DNA and chromosomal aberrations, 128 follicular sensitivity, 59
DNA fragmentation index (DFI), 156, 172 gonadotropins, 26
Donor egg/recipient cycles, 86–87 menstrual cycle or cycle disturbances, 63
ovarian activity, 61
ovarian hyperstimulation, 26–27
E ovarian response, 66
Eight-oxoguanine (8-oxoG), 237 serum levels, 64
Embryo assessment sperm, cancer therapies, 118–119
implantation potential, 245 steroidogenesis, 60
mammalian embryonic secretome, 246 Follicular flushing, 16
non-invasive metabolomic analysis
amino acids, 250
non-invasive metabolomic profiling, 250–251 G
protein synthesis and pH regulation, 249 Gene ontology (GO), 51
pyruvate and glucose, 249–250 Gonadotrophin-releasing hormone (GnRH), 47
quiet embryo hypothesis, 249
non-invasive proteomic analysis
human embryonic proteins, 246 H
non-invasive protein profiling, 247–249 Homologous recombination repair (HRR), 235
single protein and molecular analysis, 246–247 Human chorionic gonadotropin (hCG), 24, 80
omics technologies, 246 Human leukocyte antigen G (HLA-G), 247
EmbryoGlue®, 257 Hyperinsulinemia, 25
Endocrine and metabolic environment, 38–39 Hypogonadotropic hypogonadism, 142
Environmental insults, spermatogenesis
epigenetics, 145–146
gametes production, 134 I
male reproductive neuroendocrine function, 134 ICF, ATRX and Rubinstein–Taybi syndromes, 202
maternal and perinatal exposure Immunotech-Beckman assay, 60
maternal smoking and obesity, 135 Imprinting centers (ICs), 187
medullary region, 134 Intracytoplasmic morphologically selected sperm
offspring infertility, 135 injection (IMSI), 221
organochlorines, 136 acrosome reaction, 278
quinones, 137 “best group,” 268
TDS hypothesis, 135 classification scoring scale, 271
xenobiotics, 136, 137 DNA damage, 278
mature testis electron microscopy, 264
agriculture fertilizers, pesticides and embryo development and sperm nuclear defects,
herbicides, 140 283–284
alcohol consumption and drugs, 141–142 fertility potential, 278
cell phone and ionizing radiation, 144–145 ICSI, 264, 277
Index 293
implantation and pregnancy rates, 267 predictive marker

male gametes abnormalities, 277 qualitative ovarian response prediction, 70–71
microdroplets, 266 quantitative ovarian response prediction (see
motile sperm evaluation, 271 Quantitative ovarian response prediction)
MSOME (see Motile sperm organellar morphology progesterone
examination) age and ovarian reserve, 82
application, compromised semen parameters, bioadhesive gel preparation, 82
280–281 crinone, 82–83
high magnification, 278 endometrial maturation, 81
nuclear DNA content and organization damages, 265 estradiol to progesterone, luteal phase, 87–88
oligoasthenoteratozoospermia, 264, 268 follicular estrogen production and corpus
optics of Nomarski, 265 luteum, 80
phenotypic and genomic sperm anomalies, 284–287 frozen embryo transfer and donor oocyte recipient
reproductive prognosis assessment, 265 cycles, 84, 86–87
scoring and women’s age, 272–273 GnRH downregulation protocol, 83
semen impairment, 268 GnRH modulators, 80
semen parameters, 264 intramuscular injections, 81
sibling oocytes, 278 intravaginal vs. intramuscular progesterone,
spermatozoon’s morphology, 264 81–82
sperm characteristics, 271–272 luteal phase, 81
sperm classification, 270 odds ratio, 83
sperm DNA integrity, 278 optimal timing, 84, 85
sperm morphology, 277 pregnancy rates, 81
sperm nuclear abnormality, 284 retrospective studies, 80–81
sperm preparation technique, 268 serum levels monitoring, 86
sperm vacuoles, 279–280 vaginal gel, 82–83
subcellular organelles, 264, 265 vaginal progesterone, 84–85
vacuolated spermatozoa single embryo transfer, 15–16
DIC, 270 In vitro growth (IVG), 204
DNA integrity, 268 In vitro maturation (IVM), 204
female infertility factor, 269 IVF. See In vitro fertilization
FISH analysis, 270
morphological nuclear normalcy, 269
PSA-FITC, 270 L
sperm DNA fragmentation, 269 Lindane, 140
TUNEL assay, 269 Lipofuscin, 143
zona pellucida and oolemma, 264 Luteinizing hormone (LH), 118–119
Intracytoplasmic sperm injection (ICSI), 120, 167–168,
198, 204–205
Intramuscular progesterone, 81–83 M
Intrauterine insemination (IUI), 167 Male chromatin remodeling
In vitro fertilization (IVF), 27, 120 artificial reproductive techniques, 228
adoption of, 12–13 cytoplasmic and nuclear fusion, 228
ART, 167–168 DNA repair
classification and terminology, 12 dsDNA repair, 232
fertility treatment cost, 16–17 gammaHAX foci, 231–232
follicular flushing, 16 H2AX, 231, 232
mild IVF monopronuclear and tripronuclear, 232
aromatase inhibitors, 15 MPF, 231
clomiphene and gonadotropins, 15 NHEJ, 232, 233
GnRH antagonist, 12, 15 thymidine, 232
ovulation induction, 14 imprinting maintenance, zygote, 234
modified natural cycle male germline preparation, 228–229
clomiphene stimulation, 13 oocyte, embryonic development, 236–237
exogenous hormones, 12 paternal DNA demethylation, zygotic G1, 233
follicular scan, 13 paternal vs. maternal histones, 230–231
GnRH antagonists, 14 zygotic S-phase, 234–235
hCG injection, 13 Malonyldialdehide (MDA), 214
indomethacin, 13 Mass spectrometry (MS), 247
LH surge and oocyte retrieval methods, 13 Matrix associated regions (MARs), 231, 235
294 Index
Matrix attenuated laser desorption/ionization early embryonic loss, 105–106

(MALDI), 247 “vanishing twin” syndrome
Maturation promoting factor (MPF), 231 definition, 106
Microsomal ethanol-oxidizing system (MEOS), 141 embryo reduction, 110–111
MIS. See Mullerian inhibiting substance incidence in ART, 106–107
Monoamniotic pregnancy, 104, 105 perinatal results, 108–110
Monochorionic, monoamniotic (MC/MA) twins, 100 symptoms, 108
Monochorionic twin pregnancies, 103–105 trisomy 21 screening, 110
Mono (2-ethylhexyl) phthalate (MEHP), 139 ultrasound diagnosis, 107–108
Monozygotic twinning and perinatal outcomes
ART, 92
DC/DA twins, 94–95 N
determining amnionicity/chorionicity, 93–94 Non homologous end joining (NHEJ), 232, 233
embryology, 92–93 Nucleoplasmin 2 (NPM2), 230
monochorionic, diamniotic (MC/DA) twins Nucleotide excision repair (NER), 232, 233
anomalous cofetus, 99–100
clinical management, 95
MC/MA twins, 100 O
risk assessment, 95 1-o-alkyl–2-acetyl-sn-glycero–3-phosphocholine
sIUGR, 97–98 (PAF), 246
TRAP sequence, 98–99 Obesity
TTTS (see Twin-twin transfusion syndrome) ART therapy, 39
perinatal risks, 93 chronic disease, 36
triplet and higher order multiple gestations, 92, 93 definitions, 36
twin gestations, 92 diet and adverse reproductive outcomes, 38
Motile sperm organellar morphology examination endometrial vs. oocyte factors, 38–39
(MSOME) prenatal growth and PCOS, 38
application, compromised semen parameters, reproductive health
280–281 female, 37–38
criteria and evaluation procedure, 266 male, 36–37
morphological anomalies visualization, 267 reproductive spectrum, 36
regional nuclear shape malformation, 266 Odds ratio, 83
sperm analysis, 265 OHSS. See Ovarian hyperstimulation syndrome
spermatozoa, 266 Oligozoospermia, 142
sperm evaluation, 266 Ovarian dysfunction
sperm nucleus normalcy, 267 autoimmune process, 62, 63
Mullerian inhibiting substance (MIS) clomiphene citrate, 64
clinical diagnostic marker, 71 follicular ovarian pool, 62
GnRH agonist treatment, 72 hypogonadotropic amenorrhea, 62
heteromeric receptor system, 58 oligomenorrheic women, 64
homodimeric disulfide-linked glycoprotein, 58 PCOS, 63–64
IVF predictive marker (see In vitro fertilization, POI, 62
predictive marker) polycystic ovaries, 63
ovarian ageing, 64–66 Ovarian folliculogenesis, 58–59
ovarian dysfunction (see Ovarian dysfunction) Ovarian hyperstimulation syndrome (OHSS), 17,
ovarian folliculogenesis, 58–60 69–70
ovarian reserve markers, 72 Ovarian-stimulated cycles
ovarian stimulation regimens, 72 endometrial fluid, 44
type I & II receptor, 58 endometrial receptivity
women circulating biopsies, 50, 51
current assays, 60 embryonic implantation, 51
factors modulating serum, 60–62 gene expression, 50
Multiple pregnancy gene ontology, 51
chorionicity and amnionicity menstrual cycle, 51, 52
diamniotic pregnancies, 104 primary cell culture, 53
intertwin vascular anastomoses, 104 stromal cells, 53
monoamniotic pregnancy, 104, 105 glandular and luminal epithelium, 44
monozygotic and dizygotic twins, 104 hormonal regulation, 45–46
spontaneous twin pregnancies, 103 human endometrium
zygosity, 104 agonists vs. antagonists, 48–49
Index 295
natural cycles, 46–47 germ cells, 122

stimulated cycles, 47 gonadal function, 120
human uterus, 44 gonadotoxic effects, 118
oocyte, 44 hematologic malignancies, 120
pseudostratification, 45 interleukins and tumor necrosis factor, 120
stromal edema, 45 IVF and ICSI, 128
leukemia, 122
Leydig cells, 122
P lymphoma, 121–122
Persistent Müllerian duct syndrome, 58 multimodal therapy, 125–126
Phthalate esters, 139–140 radiation therapy, 125
Pisum sativum agglutinin-Fluorescein isothiocyanate semen and endocrine parameters, 118
(PSA-FITC), 270 solid tumors, 122
Polycystic ovarian syndrome (PCOS) spermatogenesis
androgen concentrations, 25 definition, 118
antithyroid antibodies, 6 downregulation, 127
gonadotropins, 26 intratesticular testosterone, 119
insulin resistance, 25–26 LH and FSH, 118–119
pathophysiology, 24 Sertoli-cell function, 119
serum concentrations, 24–25 Type A and Type B spermatogonia, 118
Polymerase chain reaction (PCR), 47 surgery, 123
Polyunsaturated fatty acids (PUFA), 138, 139 testicular cancer, 120–121
Post translational modification (PTM), 230, 231, 234 Sperm chromatin dispersion (SCD) test, 161
Preimplantation embryos, 188–189 Sperm chromatin structure assay (SCSA), 156,
Preimplantation genetic diagnosis (PGD), 198 160–161
Premature ovarian insufficiency (POI), 62 Sperm DNA damage
Primordial germ cells (PGCs), 188–189, 203 abortive apoptosis, 158
PTM. See Post translational modification andrological examination, Adam, 156
assessment methods
annexin V binding ability assay, 162
R anti-/pro-apoptotic proteins evaluation, 162
Radiofrequency electromagnetic waves (RF-EMW), AOT, 161
144–145 CMA3, 162
Reactive oxygen species (ROS), 158–159 COMET assay, 160
Receiver operating characteristic (ROC) curves, 66 oxidized deoxynucleoside, 162
Recombinant follicle stimulating hormone (rFSH), 48 SCD test, 161
Reproductive autoimmune failure syndrome, 4 SCSA, 156, 160–161
Retinoblastoma (RB), 201 TdT and TUNEL, 160
Ring finger 8 (RNF8), 184 tolouidine blue test, 161
Rubinstein-Taybi syndrome, 202 caspases and endonucleases activation, 159
chromatin modelling, spermiogenesis
process, 158
S DFI, 156, 172
SCSA. See Sperm Chromatin Structure Assay hormonal status and tubal patency,
Selective intrauterine growth restriction (sIUGR), Linda, 156
97–98 human sperm chromatin, 157–158
Sertoli and Leydig cells, 135 lifestyle and environmental factors, 169
Single embryo transfer, 15–16 ROS, 158–159
Singleton pregnancy, 105 semen quality assessment, 156–157
Smads, 21 sperm DNA integrity
Soluble HLA-G (sHLA-G), 247 and cancer, 169
Sperm, cancer therapies chance of spontaneous pregnancy, 164–165
chemotherapy, 123–125 clinical threshold values, 172
fertility human spermatozoa, 172
after cancer treatment, 126 intra-individual variation, 168–169
biologic and psychosocial consideration, 127 Molecular Human Reproduction, 172
congenital malformation, 128 Practice Committee of the American Society
ethical considerations, 128–129 for Reproductive Medicine, 172
post-treatment options, 127–128 SCSA testing, 172
pre-treatment options, 127 testing and ART, 165–168
296 Index
Sperm epigenome T
cellular epigenome, 181–182 TdT-mediated 2’-deoxyuridine 5’-triphosphate-nick end
chromatin remodeling, spermatogenesis labelling (TUNEL) assay, 160
BRDT, 184 Teratozoospermia, 141
CDY and HAT, 184 Terminal deoxynucleotidyl transferase (TdT), 160
cell cycle vs. developmental transcription Testicular dysgenesis syndrome (TDS), 135
factors, 185 Testicular sperm extraction (TESE), 127
H2A/H2B ubiquination, 182 Time-of-flight (TOF), 248
histone-protamine transition, 182 Tolouidine blue (TB) test, 161
H3K4me and H3k27me, 185 Topoisomerase II (TopoII), 229
IVF/ICSI, 184 Tripronuclear (3PN) zygotes, 230
nucleosomes, 186 Turner syndrome, 63
P1/P2 ratio, 184 Twin reversed arterial perfusion (TRAP) sequence,
reprogramming efficiency, 185 98–99
RNF8, 184 Twin-twin transfusion syndrome (TTTS)
spermatids, 182 birthweights and neonatal hemoglobin, 96
spermatogonial stem cell, 186 clinical management, 97
zygotic transcription, 186 donor and recipient twins, 96
DNA methylation, germline, 186 MC/DA twin pregnancies, 95
genome-wide reprogramming, 188 pathophysiology, 96–97
imprinted genes, 187 Quintero staging system, 96
Sperm selection ultrasound criteria, 96
excessive semen ROS production, 215–216
hyaluronic acid binding
biochemical sperm markers and diminished sperm U
function, 212–213 Ultrasound-guided embryo transfer
chromosomal aneuploidies, 217 catheter placement, 256
dysmature spermatozoa, 218 CCRM experience, 257–258
high DNA integrity, 219 ectopic pregnancy, 256
“physiologic” sperm selection, 218 fundal endometrium, 256
solid state HA, 218–219 in vitro fertilization, 255–256
sperm dysmaturity and persistent histones, 219 optimization, 256–257
Tygerberg normal sperm, 218 ovarian hyperstimulation, 255
ICSI, 212 pregnancy rates, 256
IMSI, 221 randomized trials vs. meta-analysis, 257
infertility definition, 212
SCSA issues and usefulness, 213–214
semen analysis, 212 V
source, assisted reproduction, 216 Vitamin C, 141, 142
spermatozoa de-selection, apoptotic marker proteins, Vitamin E, 141, 142
217
sperm biochemical markers, 212–213
sperm charge properties, 220–221 W
sperm chromatin maturation, 216–217 Window of implantation (WOI), 47, 48
sperm nuclear DNA integrity, 213 World Health Organization, 36
sperm preparation, gradient centrifugation, 214–215
Steroidogenic cell autoimmunity, 62
Surface-enhanced laser desorption/ionization X
(SELDI), 248 Xylene, 140

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Biennial Review of Infertility

ISBN 978-1-4419-8455-5 e-ISBN 978-1-4419-8456-2

Library of Congress Control Number: 2011928231

© Springer Science+Business Media, LLC 2011

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

The initial volume of Biennial Review of Infertility was published in 2009.

Boston, MA, USA Catherine Racowsky

Part I Female Infertility

1 Autoimmunity and Female Infertility: Fact vs. Fiction............ 3

4 The Role of Obesity in Reproduction......................................... 35

Part II Male Infertility

10 The Effect of Cancer Therapies on Sperm:

11 Environmental Insults on Spermatogenesis............................... 133

Part III Assisted Reproduction Techniques

14 ART and Epigenetic Disorders:

Part IV Evolving Controversies in Contemporary

19 IMSI as a Valuable Tool for Sperm Selection

Ashok Agarwal, PhD, HCDL Center for Reproductive Medicine,

Dimitrios G. Goulis, MD, PhD Section of Reproductive Medicine,

Akanksha Mehta, MD Division of Urology, Rhode Island Hospital,

Mark Sigman, MD Division of Urology, Rhode Island Hospital,

C. Racowsky et al. (eds.), Biennial Review of Infertility: Volume 2, 3

fertilized ova will generate a live birth and 50%

1.2.1.1 Clinical Criteria Antiphospholipid antibodies have been found

Table 1.1 Associations of autoantibodies with female infertility

C. Racowsky et al. (eds.), Biennial Review of Infertility: Volume 2, 11

with or without concomitant GnRH antagonist

This term is used to define scenarios in which

2.1.2 Modified Natural Cycle IVF 2.2 Adoption of Minimal

Table 2.1 Different protocols of minimal stimulation IVF

C. Racowsky et al. (eds.), Biennial Review of Infertility: Volume 2, 19

3.1 Introduction r­ eference to current clinical applications of serum

CAGAC, with a relatively low specificity or with

The human AMH gene has a length of 2,750 base-

3.2.4 Serum Concentrations During

3.2.5 Clinical Applications women with PCOS [66]. In mice, exogenous

resistance [91], while long-term metformin

3.3.5 AMH and Gonadotropins 3.4.1 Ovulation Induction

that basal AMH measurement works as well as

C. Racowsky et al. (eds.), Biennial Review of Infertility: Volume 2, 35

4.1 Introduction 4.2 Definitions

survive to reproduce, their reproductive experi- develop high dehydroepiandrosterone sulfate

C. Racowsky et al. (eds.), Biennial Review of Infertility: Volume 2, 43

Others researchers have elucidated biochemical

Fig. 5.1 Profile of expression of the hormones during the menstrual cycle

endometria precisely according to their transcrip- by microarray technology have to be added to

Day in Day in stimulated Paired # Genes

In the ovary, Mullerian-Inhibiting Substance (MIS) is produced by the

C. Racowsky et al. (eds.), Biennial Review of Infertility: Volume 2, 57

causes of Persistent Müllerian Duct Syndrome in

6.3.2 Factors Modulating Serum therefore, probably of minor significance when

of MIS were able to discriminate between incipi-

6.6 MIS as Predictive Marker in IVF with a greater number of retrieved oocytes. In

Table 6.1 Studies on AMH as marker of ovarian response to COH

Table 6.5 Comparison of characteristics of the most widely used markers of ovarian

10. Ingraham HA, Hirokawa Y, Roberts LM, Mellon SH,

E.H. Yanushpolsky (*)

C. Racowsky et al. (eds.), Biennial Review of Infertility: Volume 2, 79

preparation, which is the most convenient and

Boston, MA, USA Catherine Racowsky

Part IV Evolving Controversies in Contemporary

1.2.1.1 Clinical Criteria Antiphospholipid antibodies have been found

2.1.2 Modified Natural Cycle IVF 2.2 Adoption of Minimal

3.1 Introduction r eference to current clinical applications of serum

3.2.4 Serum Concentrations During

3.2.5 Clinical Applications women with PCOS [66]. In mice, exogenous

3.3.5 AMH and Gonadotropins 3.4.1 Ovulation Induction

4.1 Introduction 4.2 Definitions

6.3.2 Factors Modulating Serum therefore, probably of minor significance when

6.6 MIS as Predictive Marker in IVF with a greater number of retrieved oocytes. In

8.6 Monochorionic, Diamniotic 8.6.2 Overall Clinical Management

8.6.6 Anomalous Cofetus

twins developed congestive heart failure [39]. 8.6.6.1 Clinical Management

(week 5) to 6 mm (week 10). Nomograms of 9.3.4 Symptoms

10.1 Introduction 10.2 Overview of Spermatogenesis

normozoopermia in pretreatment analysis, with 10.3.4 Other Solid Tumors

10.6.1 Pretreatment Options 10.6.2 Posttreatment Options

11.3.2.1 Smoking A recent study showed that motility is one of

looser human sperm chromatin packaging as 12.4.2 Abortive Apoptosis

12.4.3 Reactive Oxygen Species

easure of the amount of DNA damage in the

12.5.3 Sperm Chromatin Structure

12.5.7 Oxidised Deoxynucleoside 12.5.10 Evaluation of Anti- or

12.7 The Effects of Lifestyle 12.8 Sperm DNA Integrity

spermatogenesis and reproductive potential. H2A s ubsequently replaced by protamines, which

14.6.2 Sperm from Infertile Men 14.6.5 Intracytoplasmic Sperm

15.1 Introduction 15.2 Beyond Routine Sperm

h istones [71–75]. Accordingly, based on the

19.1 Introduction assessment of sperm morphology, limited by its

19.2.2 IMSI Step-by-Step

s uspended by adding 3 mL of sperm culture

19.3.3 Establishment of Classification r eal-time, might increase embryo quality in terms

f ertilization and pregnancy outcomes [4]. ICSI