Embryo Biopsy: Mce5103 - Literature Review
Embryo Biopsy: Mce5103 - Literature Review
Embryo Biopsy: Mce5103 - Literature Review
- LITERATURE REVIEW
EMBRYO BIOPSY
IN THE CONTEXT OF
AGE-RELATED OXIDATIVE STRESS AND MELATONIN AS AN ANTIOXIDANT
Thanh Son Nguyen
MCE student 2017
Student ID 27715884
INTRODUCTION
PGS and PGD have been developed as a powerful tool in ART as the main aim of the testing is to
detect and prevent the transfer of genetically and/or chromosomally abnormal embryos (1, 2).
Embryo biopsy is required to obtain material for PGS/PGD. There are three stages of the embryos
at which biopsy can be performed (1). The biopsy process includes two main steps: zona pellucida
breaching and cell removal (3). Different stages of biopsy revealed different effects on embryos
and ART outcomes (3).
High levels of reactive oxygen species (ROS) reduce oocyte quality resulting in poor quality of
embryos (1, 4). In addition, advanced maternal age may be an indication of PGS/PGD (1).
Therefore, embryos created by aged oocytes, which are spontaneously of poor quality, then
receive more damage from biopsy process. Based on this logic, it is concerned that embryos of
increased maternal age may be affected more than the younger group. Not only is the embryo the
source of ROS, but the ART setting is also the exogenous cause of oxidative stress (5). To overcome
the issue of ROS, melatonin was introduced as a promise antioxidant with certain effectiveness in
improving embryo.
Therefore, the relationship of embryo biopsy and oxidative stress especially in aged women and
effectiveness of melatonin capture this writers attention. Discussed in this review is embryo
biopsy during PGS/PGD including three methods of zona pellucida drilling, three main stages of
embryo biopsy and impacts of different stage of biopsy on embryos and ART outcomes. Another
aim of this review is to discuss oxidative stress in ART particularly age-related oxidative stress and
effects of melatonin on embryos development. The literature gap between embryo biopsy and
oxidative stress is also discussed at the end.
EMBRYO BIOPSY
Preimplantation genetic diagnosis and screening
Preimplantation genetic testing has been divided into preimplantation genetic diagnosis (PGS) and
preimplantation genetic screening (PGS). In fact, PGD refers to the testing to detect a specific
genetic disease, while PGS is used to the detect aneuploidy and DNA mutation and/or
rearrangement (6).
PGD have been used to detect such sex-linked conditions as hemophilia, Rett syndrome, vitamin
D-resistant rickets and pseudohyperparathyroidsm (1). Single-gene disorders such as cystic
fibrosis, sickle cell disease, beta-thalassemia, Fragile X syndrome and Huntingtons disease are also
detected by PGD (3, 7). Moreover, PGD has been done effectively to detect chromosomal
rearrangements including deletions, inversions and translocations (7). In addition, an expanding
indication for PGD is mitochondria diseases (2) such as severe Leigh syndrome (8-10), where cells
are found to have a mix of normal and mutated mitochondrial DNA (mtDNA).
The indications for PGS have not been listed. This screening is often considered in special cases.
Advanced maternal age increase the risk of aneuploid embryos due to abnormal meiosis in the
oocyte (11, 12), which were observed in most spontaneous miscarriages (13, 14). Although routine
PGS does not improve pregnancy rates in in vitro fertilization (IVF) with increased maternal age
(15, 16), this testing showed a bias to a reduced risk of miscarriage and increased chance of live
birth (16). Repeated pregnancy loss may also act as an indication of PGS because 80% of
spontaneous abortion occurring in the first-trimester is caused by fetal aneuploidy (17). In IVF
treatment, repeated failure may also lead to a consideration of doing PGS because this failure is
associated with aneuploid embryos (18). PGS may also be considered in the case of severe male
infertility due to the high risk of chromosomally abnormal sperm (19-21) such as Klinefelter
syndrome, Y chromosome microdeletions, and Robertsonian translocations (21). Another aim of
PGS in ART treatment is sex selection for various reasons, of which prevention of sex-linked
disorders is legal. Recently, sex selection may be required to prevent non-Mendelian disorders
often referring to polygenic disorders and having unbalance incidences between sexes (22, 23).
Stages of embryo biopsy
Biopsy can be conducted at different developmental stages of the embryos, including polar body
biopsy, cleavage stage biopsy, and blastocyst biopsy (1) (figure 1 and table 1).
Table 1. Comparison among different stages of embryo biopsy (adopted from Ermanno G. et al.
2013) (24)
(1)
(2)
(3)
Figure 1. Embryo biopsy (adopted from Ermanno G. et al. 2013) (24)
(1) polarbody biopsy.
(2) Cleavage stage biopsy
(3) Blastocyst trophectoderm biopsy
Polar body biopsy
The polar body is biopsied and obtained from MII oocytes or zygotes (25). The first polar body can
be used for PGS while PGD requires the second polar body (25). The two polar bodies can be
obtained in the same biopsy process of in subsequent processes (24).
Cleavage stage biopsy
In humans, cleavage stage biopsy may be performed on day 3 with 6-10 blastomeres (1, 26), as
biopsy at the 4-cell stage will alter the ratio of inner cell mass (ICM) to trophectoderm cells
(TE)(24); meanwhile, the 8-cell stage showed totipotency and higher mitotic index [47].
Blastocyst trophectoderm biopsy
Blastocyst trophectoderm biopsy can be performed on day 5 or 6 in humans (24), two to nine TE
cells are biopsied and used for PGS/PGD, hence the ICM or the fetus is not affected (24).
Zona pellucida opening
Mechanical zona dissection is the traditional method used in polar body biopsy, using a glass
microneedle to create 20-40x2 um slits on the zona pellucida for the biopsy pipette to approach
the polar body (27, 28). This method is also be used for cleavage stage biopsy as these slits are
thought to be able to close after removing the pipette, which can improve premature hatching
embryos (29).
In the acid tyrodes method, a controlled stream of acid tyrodes at the pH level of 2.2-2.6 is used to
directly dissolute the zona pellucida to create the hole of 15-20um (30). This method used to be
the most commonly used in cleavage stage biopsy (31).
In laser-assisted drilling method, a wide range of laser lights have been investigated and the near
infra-red 1.48um diode laser is now widely accepted, as this light is absorbed by the zona pellucida
but not embryo DNA or culture media(30, 32).
Optimal application of those three methods as described above has shown no differences in
clinical outcomes in ART (30, 33, 34). The application of laser assistance to drill the zona pellucida
has been suggested in all stages of embryo biopsy and has been performed in 75% of biopsy
performances (35) as this method is precise, less time consuming and easy to train (1).
Impacts of biopsy on embryos and important considerations
Polar body biopsy
Since the polar body is the meiotic waste product and not necessary, performing biopsy at this
stage has been thought not to have any deleterious effects on the embryos (1). When performing
polar body for MII oocytes using laser drilling, the oocytes showed no differences in oocyte lysis
rates, oocyte activation rates, oocyte development rates and embryo chromosomal fragmentation
(36). When comparing neonatal outcomes after PB biopsy to those of cleavage stage biopsy, the
results were not different (33). In contrast, when considering the quality of the embryos, laser-
assisted PB biopsy was shown to cause higher rates of day 2-embryo fragmentation, lower rates of
quality embryos at cleavage stage, higher rates of cleavage arrest and lower numbers of
blastomeres on day3-embryos (37). Unfortunately, the implantation potential of embryos after PB
biopsy has not been investigated (1).
This process provides material for diagnosis of maternally originated aneuploidy, translocations
and single-gene defects (25). Nevertheless, the material cannot be used to detect paternally
inherited diseases and chromosomal abnormalities occurring after zygotic events (1). Moreover,
this biopsy process was also reported to have more false positive results and negative errors (38).
Therefore, although this method has been used in only 15% of all biopsy performances, the
number appears to be continuously declining (35).
The diagnostic limitations of this process can be solved with cleavage stage and blastocyst
trophectoderm biopsy.
Cleavage stage biopsy
Performing biopsy at the cleavage stage allows adequate time for genetic testing, as the embryo
then can be transferred as a blastocyst on day 2 or 3 post-biopsy, hence avoiding cryopreservation
of biopsied embryos, which may have certain risk of damage (24, 39).
After opening the zona pellucida, the embryos are treated with Ca2+/Mg2+-free media to
decompact the blastomeres (1). In the mouse model, a lack of Ca2+ lead to alterations in network
of surface and intracytoplasmic protein tubules, hence affecting recompaction of blastomeres (40-
43). When considering hatching rates of the blastocysts, the size of the hole negatively affects
blastocyst hatching in both mice and humans (44). This effect is more detrimental when
concurring with removal of one blastomere (45, 46). In fact, biopsied embryos showed late
compaction and hatching compared to non-biopsied embryos (46).
Another issue in this biopsy process is chromosomal mosaicism (47) which may lead to false
positive results (1). To deal with this problem, two blastomeres may be biopsied to use for two
independent assessments, resulting in a loss of at least 25% of embryonic cells, which may affect
clinical outcomes (48, 49), but current evidence in humans is not indicating harmful effects of this
cell loss on embryo development and implantation (43, 50).
In the mouse model, Sugawara, A. and M. A. Ward (2013) showed that cleavage stage biopsy
reduced mouse embryo developmental competency in both pre-implantation and post-
implantation on the C57BL/6 mouse strain, which is the model of infertile couples seeking ART
treatment.
Stronger evidence against cleavage stage biopsy, from a randomized and paired clinical trial, was
introduced by Scott Jr. et al (2013). In fact, the results showed that only 30% of biopsied embryos
could successfully implant and result in live birth, compared to the rate of 50% in non-biopsied
embryos, indicating that cleavage biopsy reduce the implantation potential in humans (51).
These days, when the single embryo transfer policy is followed, in conjunction with successful
vitrification and high effectiveness of blastocyst biopsy, this strategy is now steadily taking place of
the cleavage stage biopsy and polar body biopsy (1).