MCE5103

- 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).

Blastocyst trophectodem biopsy

This process can provide more material for genetic testing, potentially providing more accurate
diagnosis while without any interventions to the ICM which will develop to the true fetus (24). In
contrast to cleavage stage, blastocyst biopsy showed lower technical errors and less impacted by
mosaicism (1).

It is challenging to culture embryos until the blastocyst stage despite improvements in culture
systems, as data showed that up to 20% of cycles did not have embryos reaching blastocysts for
biopsy (48). However, it must be emphasized most of the embryos which fail to reach blastocyst
stage have chromosomal abnormalities; moreover, processing fewer embryos (but with higher
quality) is more cost-effective. Meanwhile, this problem still happens in cleavage stage biopsy if a
fresh embryo transfer is aimed (1), and Glujovsky, D., et al. (2012) also demonstrated that with a
current culture system higher live birth rates were seen in blastocyst culture than cleavage stage
transfers (52). On the other hand, biopsied blastocysts can only be cultured for maximum 24 hours
after biopsy; thus, insufficient time is allowed for genetic diagnosis, leading to the needs of
cryopreservation. Fortunately, vitrification in PGD programs illustrated interestingly high survival
rates of over 95% with blastocysts (1, 53, 54).

Interesting results on embryo aneuploidy were also reported, as the embryos, which were
diagnosed with aneuploidy with cleavage stage biopsy, then showed normal ploidy at the
blastocyst stage with day-5 biopsy, indicating that there may be a mechanism where aneuploidy
can be self-corrected before reaching blastocyst stage (55-57).

Regarding clinical outcomes, McArthur, S. J., et al. (2005) showed that after blastocyst biopsy,
fresh biopsied embryo transfers archived the implantation rate (confirmed by fetal heart) of 41%
and the rate for thawed biopsy embryos was 26% (48). Coming back to results published by Scott
Jr. et al (2013), not only did they show the impairment of cleavage stage biopsy on human
implantation potential, but the results also revealed that blastocyst biopsy did not impair this
potential, as no differences in the implantation rates between biopsied and non-biopsied groups
were observed (51% and 54%, respectively) (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).

EFFECTS OF OXIDATIVE STRESS AND AGEING ON EMBRYOS

Potential impacts of oxidative stress on embryo viability
Cellular respiration generates reactive oxygen species (ROS) such as superoxide aninon (O2-),
hydrogen peroxide (H2O2) and free radical hydroxyl (OH*-)(5). These chemicals are produced
continuously in mitochondria as a leakage of electons from the electron transport chain (4). In the
in vivo environment, ROS are balanced with antioxidants which act as scavenger to neutralize ROS
(5). When this balance is damage, the levels of ROS uncontrollably increase, leading to a condition
called oxidative stress (OS).

ROS have been known to damage mitochondrial lipids and protein (4), but mitochondrial DNA
(mtDNA) is actually the main target of this damage due to its short distance from the electron
transport chain while mtDNA does not have protective histones or DNA repair mechanism (4, 58).
Increasing damage to mitochondrial DNA, lipids and protein leads to abnormal mtDNA, which may
result in inhibition of mitochondria replication and an increase in mitochondrial membrane
permeability or a reduction of intact mitochondria hence leading to abnormal ATP production (4,
58). These mitochondrial damage also results in insufficient electron transport chain which will
produce more ROS, making OS more severe (4).

Particularly in ovarian oocytes, the cells that have the largest number of mitochondria (59),
despite meiotic arrest, they are still have active metabolic activity, hence ROS are still produced,
putting the oocyte under OS (4). Therefore, in aged women, long time exposure to ROS damage
oocyte mtDNA, leading to dysfunctional mitochondrial activity (59). Meanwhile, oocyte
mitochondria play a vital role in the embryo as embryonic mitochondria are solely maternally
inherited (59) because sperm mitochondria are degraded and removed by oocyte proteasome
(60). In addition, embryonic mitochondria do not replicate until the embryos have hatched; thus
energy demand from the mature oocyte, zygote to early stages of the embryo is met by maternal
mitochondria (61) and mitochondria oxidative phosphorylation is critical in this provision (62).
Therefore, advanced maternal age, a leading factor to dysfunctional mitochondira, has
detrimental influence on preimplantation embryo development (59). To be more specific,
inadequate ATP production in maternal mitochondria can cause aneuploidy due to a lack of
energy for chromosomal segregation (59). Furthermore, low ATP contents may affect mitosis,
blastomere compaction, and the formation of blastocoel (4) and decrease the proportion of
embryos breaching the hatching or hatched blastocyst stage (63). Additionally, ATP content was
reported to associate with embryo implantation potential (63). Based on this logic, OS with high
levels of ROS in aged oocytes may result in poor quality embryos with less development and
implantation potential.

Mitochondrial dysfunction in aged oocytes is not the only way that OS and ROS can affect
embryos. Moreover, ROS has been known to be able to damage cell membrane and nuclear DNA
(4) leading to embryonic defects and retardation (64, 65). In addition, OS can also trigger apoptosis
causing embryo fragmentation which limits the implantation potential (66). Therefore, putting
embryos under OS with exposure to increased levels of ROS can impair embryo development and
increase miscarriage rates (67).

In the ART setting, despite efforts, an in vitro setting cannot mimic the exact in vivo condition (5),
hence embryos appear to be exposed to more OS than in the in vivo condition due to a lack of
antioxidants while facing sources of ROS which can be divided into internal and external (5).

Sources of oxidative stress in ART
Internal ROS source
The embryo itself is a source of ROS. As discussed above, embryos mainly generate ROS via
mitochondrial oxidative phosphorylation, and in vitro cultured mouse embryos showed higher
levels of ROS than in vivo (68). In addition, ROS are also generated via xanthine oxidase and
NADPH systems (65) which needs more studying.
External ROS source
Oxygen can activate enzymes involving to production of superoxide radical (5). Culturing embryos
in low oxygen concentration showed lower OS levels, higher blastulation rates, and higher live
birth rates (69). Metallic cations such as Fe2+ and Cu2+ can increase ROS levels but can be solved by
the addition of metal chelators to culture media (5, 65). Visual light can trigger photodynamic
stress causing oxidative damage to unsaturated lipids on cholesterols on the cell membrane (5,
68). Spermatozoa are an important source of ROS, as these cells have no ROS scavenging systems
(5, 59). Serum in culture media contains amine oxidase which can accelerate the oxidant load and
increase ROS levels (5). In order to deal with these sources of ROS and reduce its influences,
interventions in the ART setting have been applied (figure 2).

Figure 2. Actions to control OS in the ART setting (adopted from Sajal et al. 2010) (5)

Melatonin as an antioxidant in ART
Melatonin in humans is a hormone secreted by the pineal gland (70). Melatonin can act as an
antioxidant with such functions as directly scavenging free radicals, stimulating antioxidative
enzymes, increasing efficient mitochondrial activity, reducing electron leakage and increase
effectiveness of other antioxidants (70). Tamura, H., et al. 2008 showed that oral supplementation
of melatonin can reduce OS in follicular fluid and improve oocyte quality as the number of
degenerating oocytes decreased while the fertilization rates marginally increased (71). Eryilmaz, O.
G., et al. (2011) again showed that the dose of 3mg could improve the number of retrieved
oocytes, the MII oocyte rate and the proportion of Grade I embryos but did not improve the
fertilization, implantation and clinical pregnancy rates (72).

Culture of mouse embryos in melatonin at the concentration of 100-1000 nM increased
fertilization rates, and embryo developmental rates (73). Asgari, Z., et al. in 2012 also showed that
embryos cultured in melatonin of 10-100nM had higher developmental rates, higher numbers of
total cells, TE and ICM and improved implantation rates (74). In 2014, Niknafs, B., et al.
demonstrated that melatonin could improve developmental rates and prevent induced apoptosis
(75).

LITERATURE GAP BETWEEN EMBRYO BIOPSY AND OXIDATIVE STRESS IN AGEING
OOCYTES

Does biopsy increase oxidative stress on embryos?
It is clear that biopsy put physical and biological stress on embryos as the embryo integrity is to
some extent damaged. Among different kinds of stress that the embryos are confronted, oxidative
stress seems the most considerable and dangerous. Although numerous studies have been done
to have a better understanding on oxidative stress in terms of the molecular mechanisms with the
roles of ROS and antioxidants. In ART, a list of endogenous and exogenous factors leading to
increased levels of ROS has been established with strong evidence. There are also various methods
to measure OS, such as using OS biomarkers namely malondialdehyde (MDA) and 8-hydroxy-2'-
deoxyguanosine (8-OHdG)(5, 76) or metabolic profiling which is a new, rapid, non-invasive method
to measure OS (77). However, there have not been any studies investigating the OS status of the
embryo after biopsy. Therefore, the question of whether or not embryo biopsy processes put the
embryos under oxidative stress has not been answered.

Regardless of biopsy technique, biopsy creates a wound to the embryos, which needs to
increase metabolism and ATP production to heal up, hence probably increasing ROS. Moreover,
since the current focus is turning to blastocyst biopsy using laser-assisted drilling method due to
the evident benefits, laser treatment might act as an exogenous leading factor of ROS. This idea is
based on the fact that although the laser beam which is currently used is safe to humans, this laser
increases the temperature in media surrounding embryos to 60-80oC (78) while there is a
connection between heat shock and oxidative stress (79).

Does biopsy have the same effects on embryos created from aged and young oocytes?
Oocyte quality decreases with age due to long exposure to ROS, hence affecting the quality of
resultant embryos. It must also be emphasized that abnormal mitochondrial activity in the
embryos produces more ROS than normal, worsening the embryos created by aged oocytes.
Moreover, in the ART setting which impose extra stress on the embryos, aged-oocyte-created
embryos appear to confront with more OS or ROS compared to young oocyte counterparts. In
other words, embryos created from aged oocytes may have less potential of development and
implantation and archive lower ART outcomes. If biopsy also puts embryos under OS, then the
impacts of OS on embryos in the aged oocyte group might be significantly larger than in the
younger group.

However, when accessing the impacts of biopsy on embryos, studies were less likely to involve
maternal age. In fact, most the evidence saying blastocyst biopsy using laser drilling did not
mention the role of maternal age. For instance, evidence introduced by Scott Jr. et al (2013) on
effects of cleavage stage and blastocyst biopsy on ART clinical outcomes, which is considered a
strong evidence to advocate blastocyst biopsy, fail to put these effects in the context of age. The
only results on implantation rates involving maternal age in the experimental design that this
writer could find is the article published by McArthur, S. J., et al. 2005. In their study, only patients
under 44 years old were selected (48). Although the involved age is quite high, it did not reflect
ageing population as literature showed that ROS and OS severely affect maternal mitochondria
over 45 years (4). There might be other studies get maternal age involved in the biopsy impacts
but the number may still insufficient.

CONCLUSION
Different zona pellucida opening methods are proven not to have different effects on embryos if
optimal techniques are performed, but laser-assisted drilling is the most widely used due to
technical advantages. On the other hand, cleavage stage biopsy has been proven to have negative
impacts on embryos. In contrast blastocyst biopsy seems to be more practical with less adverse
effects and the current trend is towards this process although its effects still need more research
to provide stronger evidence for more widely implementation. At the moment, there is a lack of
connection between blastocyst biopsy and oxidative stress particularly ages-related oxidative,
which is the main cause of the reduction in oocyte and embryo quality. Oxidative stress, in
general, is a contributing factor to ART failure. Therefore, effects of blastocyst should be
investigated in the context of advanced maternal age and in relation of ROS sources in the ART
setting. Moreover, interventions should also be applied to overcome the influence of oxidative
stress in ART, of which melatonin is a potential candidate to study.

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