Preimplantation selecting a euploid embryo for transfer. Testing is

Preimplantation Genetic Testing (PGT) is a tool used in In
Vitro Fertilization (IVF) laboratories worldwide. It is used to identify if
embryos,prior to their use for transfer to the
uterus, are chromosomally normal (euploid) or abnormal (aneuploid), with the aim of selecting a euploid embryo for transfer. Testing is carried
out on cells extracted from the embryo by performing a biopsy. Embryo biopsy can take place atdifferent stages: Polar Body (PB) stage of first PB or both PBs, Cleavage stage of
one or two blastomeres on day
3 or blastocyst stage of Trophectoderm
(TE) cells on day 5 and/or day 6. Cleavage stage biopsy is
the most commonly used method in
Europe, although the use ofTE biopsy is increasing. Publications have found cleavage
stage to have a negative impact on embryo viability, even though it is a standardized
technique. PB biopsy is not commonly used as there is insufficient data
supporting its use and it suffers accuracy drawbacks. TE biopsy is proving to
be the safest approach. The aim of this review is to analyse the published
findings to date of embryo biopsy at the different developmental stages to
establish the optimal time to carry out the biopsy,
in order to achieve the most accurate result and sustain the viability and
reproductive potential of the embryo for future use. Preimplantation genetic
diagnosis (PGD) / preimplantation genetic screening (PGS) was as an
experimental procedure, developed in the 1980s, and is now an
established clinical option for couples undergoingIVF treatment worldwide. PGT is a tool whose application in assisted reproduction techniques (ART) has significantly grown in the last
decades (Danilo Cimadomo 2016). It is clear from the European IVF-Monitoring
ESHRE Consortium data that the number of reported PGD/PGS cycles increased from
6399 (2010) to 6824 (2011) and to 8433 (2012). Overall, 6095 PGS cycles were
reported in data collection XIV–XV (M. De Rycke 2017). It is generally accepted
that the most common reason for failed implantation in IVF is embryo aneuploid
(Hey-Joo Kang 2016). PGT is an early form of prenatal diagnosis (PND),
developed to help couples who are at risk of transmitting
an inherited disease to their offspring
(Elias M. Dahdouh 2015). If such couples wish to have a healthy
family, the main option to them is PND byamniocentesis or chorionic villus sampling (CVS) (Dale 2011). The final
aim of PGT is to define whether an embryo is affected by a monogenic disease
and/or chromosomal impairments, thus preventing the implantation of a
symptomatic fetus and/or limiting the risks underlying the transfer of chromosomally
abnormal embryos (mainly implantation failures and miscarriages) (Danilo
Cimadomo 2016). PGS has been carried out for infertile patients undergoing IVF with the aim of increasing the IVF pregnancy and delivery rates (G. L. Harton 2010). Cited indications for PGS include
advanced maternal age, repeated implantation failure, severe male factor and couples with normal karyotypes who
have experienced repeated miscarriages (G. L. Harton 2010). Other indications
were previous abnormal pregnancies, individuals with abnormal karyotypes,
including mosaicism for numerical chromosomal abnormalities and couples with
more than one indication (M. De Rycke 2017). The mean age of women undergoing
PGS was 39 years (M. De Rycke 2017). PGT is a powerful tool to reach the
goal of a pregnancy and attenuate its adverse events (A. C. Danilo
Cimadomo 2016). In order to achieve this goal, it is
mandatory not to significantly harm the embryo during the biopsy and to
preserve its viability and reproductive potential (Danilo Cimadomo 2016). A
consistent requirement for all PGT is the need to obtain DNA from the oocyte or
embryo (Katherine L. Scott 2013). There are different possible sources of
genetic material for testing in
the preimplantation window in patients undergoing an IVF
cycle:(i) the first and second polar bodies (PBs) (ii) one or two cells biopsied from 5-cell to 10-cell cleavage-stage
embryos on day 3 and (iii) several trophoblast cells (usually 5–10) sampled from the blastocyst (Antonio Capalbo 2013). Genetic analysis of
preimplantation developmental stages prior to replacement
into the uterus inevitably involves removal of some cellular material from one
of these stages (Steirtegem. 2001). The majority of biopsies (67%) were
performed at cleavage stage; blastocyst biopsy was carried out in only 4% of cycles (M. De Rycke 2017). Despite originating from the same zygote, not all embryonic cells share
identical chromosomal complements. Mitotic errors during embryo development can result in chromosomally distinct
populations; these are termed mosaic embryos. Mosaicism can occur as early as the 2-cellstage, although detection at the blastocyst stage is more common because more TE
cell can be simultaneously analysed (Rubio. 2017). This is an important factor
to consider when deciding on the most appropriate time
to carry out the biopsy procedure. Current PGS
practice, employs comprehensive chromosomal screening of all 24 chromosomes
using (aCGH), single-nucleotide polymorphism (SNP) microarrays, quantitative or
real-time polymerase chain reaction
(RT-PCR), and next-generation sequencing (NGS) (Ariel Weissman 2017). Embryo development
is a dynamic process in which embryo
morphology changes significantly over a time span of even a few hours (J.G. Lemmen 2008) (Mar?´a Cruz 2012). Table I shows the different embryo developmental stages. In nature, fertilization occurs only after both the oocyte and the
spermatozoon have completed their final stage of maturation. Sperm – oocyte
interaction is a complex process of cell – cell interaction that requires
species – specific recognition and binding of the two cells (Dale
2011). The optimal fertilized oocyte should bespherical, and have two PBs, with two centrally located, juxtaposed pronuclei (PN) that are
even-sized, with distinct membranes (Group 2011). It was noted that oocytes undergo both nuclear and cytoplasmic maturation and
that these processes are neither the same nor necessarily even synchronous (Group 2011). In the laboratory, two methods of
in – vitro fertilization are available – IVF and Intra cytoplasmic sperm injection (ICSI). Regardless of the fertilisation procedure used,
it is important to denude the oocytes of excess cumulus cells
that may be attached to the zona pellucida (ZP), also if IVF is the method of
fertilization used, sperm attached to the ZP may influence the biopsy results.
So any sperm attached to the ZP needs to be removed. The phenomenon of early
cleavage and its impact on pregnancy rates in humans was reported for the
first time by Edwards et al (1984), who clearly showed that embryos
cleaving more rapidly had a greater chance of
implanting. In other studies, the timing of the first zygotic division has been successfully used in human IVF programmes to identify embryos of superior quality (Mar?´a Cruz 2012). After completing
fertilization, the zygote undergoes its first mitotic division and then continues to divide by mitosis into a number of smaller cells known as
blastomeres (Dale 2011). The consensus was that the currentexpected observation for embryo development is 4-cells on day 2 and
8-cells on day 3, depending on the time elapsed post
insemination (Group 2011). A number of parameters are evaluated – cell number,
symmetry and granularity, type and percentage of fragmentation, multinucleated
blastomeres and degree of compaction, as described in Alikani et al (2000)
(Mar?´a Cruz 2012). The first event that determines the directed development of
previously undifferentiated blastomeres
is compaction. The embryo undergoes compaction to form a Morula. This is a calcium dependent process. With compaction, the blastomeres flatten against each other and begin to form junctionsbetween them so that boundaries between blastomeres can no longer be distinguished (Dale 2011).
Between the 16-cell and 32-cell stage, a second morphological change occurs, known as Cavitation. Activation of Na+, K+ ATP-ase systems
in the TE cells results in energy-dependent active transport of sodium pumped into the central area of the
embryo, followed by osmotically driven passive movement of water to form a
fluid-filled cavity called the Blastocoele. Blastocoele formation and expansion
is critical for further development, as it is
essential for further differentiation of the inner cell mass (ICM) (Dale 2011).
Conventional blastocyst grading systems include the following three
morphologic parameters: degree of blastocoel expansion, ICM, and TE cells. These parameters are good predictors of live birth rate (LBR) after
fresh and frozen-thawed embryo transfer (FET) cycles (Mohamad Irani 2017). Optimal or good-morphology blastocysts were defined as the embryos that were characterized by a cohesive TE composed of numerous
sickle-shaped cells as well as a tightly packed ICM (Mar?´a Cruz 2012). The
consensus for a blastocyst scoring system was that there should be a
combination of stage and score (Group 2011). Data by Irani et al 2017 show that euploid embryos graded as excellent are associated
with statistically significantly higher
implantation and ongoing pregnancy rates than euploid embryos graded as good,
average or poor (Mohamad Irani 2017). Figure I shows the stages of human embryo development in culture. B shows a fertilized oocyte on day 1, the arrows indicating to the PN, with the two PBs at the 12oclock position. C shows a 2-cell embryo with small fragmentation seen at 12oclock
position, the blastomeres are
relatively symmetrical in size. D shows a 4-cell embryo on day 2. E shows
an 8-cell embryo on day 3. F shows an embryo entering compaction, it is
difficult to count the number of blastomeres as the cell membranes of the
blastomeres are difficult to distinguish. G is a morula on day 4. H and J show blastocysts, the TE and ICM are
clearly visible. The embryo throughout all stages of development is surrounded by the protective ZP. The estimation of embryo
viability prior to transfer is notoriously subjective (Mar?´a Cruz 2012). The biopsy procedure is carried out on oocytes / embryos of sufficient quality
so it is important that all the biopsy practitioners are grading the oocytes /
embryos the same. An embryo of insufficient quality will be less likely to survive the biopsy procedure and
less likely to achieve a pregnancy. Both blastocysts of good and poor morphology have almost the same
probability of carrying chromosomal abnormalities (Antonio Capalbo 2013). The
outcome of PGS programmes faced two difficulties; (i) the use of fluorescent
in situ hybridization (FISH) was not sufficient to
detect the segregation errors that occur in chromosomes that FISH was not equipped to detect and (ii) the type of cells biopsied (Antonio Capalbo
2013). This review will focus on the type of cells to biopsy. Biopsy can be performed by different
methods: removal of one or two
PBs from the unfertilized oocyte or the zygote, removal of one or
two blastomeres at the cleavage stage or removal of several cells at the blastocyst stage (G. L. Harton 2010). Figure II illustrates
the advantages and disadvantages of each method. Cleavage stage blastomere biopsy still represents the most commonly used method in Europe nowadays, although this approach has been shown to have a
negative impact on embryo viability and implantation potential (Danilo Cimadomo 2016). Harton et al 2010 published that cleavage-stage biopsy remains the most widely practised form of embryo biopsy worldwide, accounting for approx.
90% of all reported PGD cycles (Harper et al 2010c). PB biopsy has been
proposed as an alternative to embryo biopsy especially for aneuploidy testing.
However, to date no sufficiently
powered study has clarified the impact of this procedure
on embryo reproductive competence (Danilo Cimadomo 2016). Blastocyst stage biopsy represents nowadays
the safest approach not to impact embryo implantation potential. For this reason, as well as for the evidences of
a higher consistency of themolecular analysis when performed
on TE cells, blastocyst biopsy implementation is gradually increasing worldwide (Danilo Cimadomo 2016). The use of PB biopsy did not spread due to its intrinsic logistic, clinical, and technical drawbacks that compromise its
accuracy,especially when compared to TE biopsy strategy (Danilo Cimadomo 2016).
Genetic testing was carried out on either one
blastomere (43% ofcycles to PGD) or two blastomeres per embryo (37% of cycles to PGD). In
14% of cycles a mixture of
one and two blastomeres was applied. PBs or TE cells were used in the remaining 6% of cycles (M. De Rycke 2017). The biopsied
sample should be representative of the embryos’ chromosomal constitution and
viability after transfer (Antonio Capalbo 2013). Even in the hands of the most
experienced embryologists and geneticists, reading are not always 100% of the
time. Therefore, it is possible that the report would reveal a ‘no result’. In this particular instance, the patients are left with a cryopreserved embryo that
has no genetic result (Tyl H. Taylor 2014). A benchmark for the tubing rate of
95% was reported in the Alpha survey (Medicine. 2017). Comparison between different biopsy
stages. Black arrows indicate negative evidences; white arrows indicate
positive evidences; question marks indicate still controversial aspects (Danilo
Cimadomo 2016). This biopsy technique is usually performed in countries where
embryo biopsy is considered illegal (e.g. Italy, Germany, Austria). PB removal
requires access to the perivitelline space (PVS) of the oocyte by creating an opening of the ZP, which
can be accomplished by mechanical or laserdissection. This procedure can be done sequentially by removing the
first and second PBs at separate times or by a simultaneous approach in
which both PBs are removed concurrently (Elias M. Dahdouh 2015). It has recently been shown that distinction
between PB1 and PB2 by morphology alone is only 63% consistent when biopsied simultaneously (Antonio Capalbo 2013). Even though
guidelines regarding theproper timing for biopsy have not
been established, it should occur between 8h and 14h after
ICSI (Danilo Cimadomo 2016). A recent study showed that both first and second PB are
prone to meiotic errors. Unfortunately, this technique carries drawbacks
when used during PGD, especially its limitation
to diagnosis of genetic or chromosomal abnormalities carried by maternal DNA
alone. In PGS, PB biopsy is still a matter for debate because of questions pertaining to its cost effectiveness (the high number of oocytes needed to be
tested), the high incidence of post-meiotic chromosome abnormalities that
cannot be detected by PB biopsy approach, and the questionable diagnostic
accuracy of PB biopsy given the possible self-correction of meiotic aneuploidy
(Elias M. Dahdouh 2015). PB biopsy is potentially less invasive than any other
stage of preimplantation development, since it entails the removal of waste products of meiosis.
However, the applicability of this strategy has always been under debate, as mirrored by the
ESHRE PGD Consortium data. In fact, PB biopsy has been used in only 10–15% of
all the procedures performed in Europe in the last decade. Capalbo and
colleagues reported high false positive and negative error rates when adopting
this biopsy strategy. In this regard, mitotic and paternally derived
aneuploidies cannot in fact be detected (Danilo Cimadomo 2016). To conclude,
the application of PB biopsy has been gradually reduced in favour of TE biopsy
due to the absence of reliable supporting data and the possibility of
diagnostic inaccuracy (Danilo Cimadomo 2016). PB screening for aneuploidy.
There are two fundamental types of errors that occur in maternal meiosis
I: non-disjunction and Premature Separation of Sister Chromatids (PSSC). That means
that for each chromosome pair there are five unique outcomes that may occur
during meiosis I: normal, non-disjunction with either loss or gain, and PSSC
with either loss or gain. Assuming no additional errors in meiosis II, there are seven possible outcomes by the time maternal meiosis is
complete. This reflects that there are two possible outcomes in meiosis II after PSSC in meiosis I (Katherine L. Scott 2013).   Blastomere
biopsy is also affected by problems associated with single cell analysis, both
technical (e.g. high rate of amplification failure) and biological. In
particular, chromosomal mosaicism, namely, the presence of cells with different
karyotypes within the same embryo, seems to reach its highest level at this stage of
preimplantation development. In order to compensate for this, a two
blastomerebiopsy strategy
has been proposed. However, this strategy could involve a depletion of the embryonic mass of about
25% and in turn impact clinical outcomes (Danilo Cimadomo 2016). Biopsies
performed earlier (4-cells) may alter the ratio of ICM to TE, which may be
detrimental to embryo development (Dale 2011).
On the contrary, initial evidences did not demonstrate a detrimental impact of
blastomere(s) loss (after biopsy and/or cryopreservation) on deriving embryo development and implantation potential. In fact, ESHRE guidelines in 2010 suggested that this procedure could
be safely applied when embryos are composed of ?6 cells with less than 30% of
fragmentation (Danilo Cimadomo 2016). An interesting theory is that embryos at
this stage of preimplantation development are relatively fragile since
Embryonic Genome Activation and cell differentiation processes have not
occurred yet. Thus, downstream developmental processes can be irreparably
compromised by removing a cell from the embryo. Such an impact in fact reflects
also in a lower blastocyst rate after cleavage stage biopsy with respect to
undisturbed embryos, as reported in several papers (Danilo Cimadomo 2016). More
recently, several randomised controlled studies produced evidence that day 3
biopsy with PGS is detrimental to IVF outcomes, partially due to its invasive
nature and to embryo mosaicism (Hey-Joo Kang 2016). Harper et al 2010 discusses the lack of positive outcome from 11 RCTs (10 using cleavage stage biopsy and one using blastocyst
biopsy) that can be explained by the likelihood that the tested blastomere
is not representative for the whole embryo. Indeed, high levels
of chromosomal mosaicism have been observed
in blastomeres from cleavage stage embryos evaluate by FISH for a limited
number of chromosomes in
infertile women or by array technology for all chromosomes in fertile women (Joyce
Harper 2010). Extracting two blastomeres has
been previously shown to have detrimental effects on embryo development and thus should be avoided. Themajor drawback of blastomere biopsy is the risk of mosaicism, which
might be responsible for the false-positive or false-negative results
encountered with PGT.
However, this technique is compatible with fresh embryo transfer on day 5 to day 6 of embryo development, given that genetic results will usually be available one to two days after
blastomere biopsy (Elias M. Dahdouh 2015). Hilde Van de
Velde et al evaluated the implantation of embryos after the removal of one, two or three cells in 188 PGD
cycles where a transfer was done and theyadvised analysing two
cells from a ?7-cell stage embryo in order to render the diagnosis more accurate and reliable (Hilde Van de
Velde 2000). A Ca++/Mg ++- free medium is used in order to loosen the cell-cell adhesion and facilitate
blastomere removal (A. C. Danilo Cimadomo 2016). Blastocyst stage biopsy strategy was an important breakthrough in modern IVF, first reported by de Boer and colleagues in 2004 (Danilo Cimadomo 2016). TE biopsy has both its advantages and
disadvantages. It is not possible to have the testing carried out intime for a fresh transfer as most IVF clinics do not have
their own genetic laboratory and are required to
send the biopsy samples to an external laboratory for testing. This means that vitrification is necessary for
all biopsied samples while the fertility clinic awaits the results. Evidence suggests that vitrified
– warmed embryo transfers have equivalent or higher
pregnancy rates and improved neonatal
outcomescompared with fresh embryo transfers, which is
hypothesized to be due to avoidance of deleterious effects from hormone stimulation on endometrial
preparation and receptivity (Cara K. Bradley 2017). The power
of TE biopsy resides in its higher technical and biological robustness. This
approach in fact entails both lower influence of procedural errors and lower impact of mosaicism on the
molecular analysis (Danilo Cimadomo 2016). Retrieval of 5 to 10 TE cells from a 100-cell or 150-cell blastocyst corresponds
with a lower
proportion of cell loss (3.3% to 10%) than the removal of one
or two blastomeres from a 6-cell to 8-cell embryo, which reduces the cell
content by 12.5% to 33%. Blastocyst biopsy also provides more starting DNA
templates than day 3 biopsy, which would theoretically lead to improved sensitivity and specificity of PGD and is associated
with lower rates of mosaicism (Elias M. Dahdouh 2015).