). 5 and/or day 6 depending on embryo

). This technique
is cost-effective, because fewer embryos are tested, and it has been associated with increased chance of live birth in the last
decade (Elias M. Dahdouh 2015). Recently, TE biopsy has been shown to have no impact on blastocyst reproductive potential when compared with cleavage-stage biopsy, in which 39% reduction in implantation rate was reported (Elias M.
Dahdouh 2015). The paired RCT by Scott Jr. and colleagues is again the real
milestone that identified blastocyst stage biopsy as a procedure that does not
affect embryo viability and implantation potential. TE biopsy was reported to
have no impact, converse to what was found for blastomere biopsy. A possible
explanation for this difference is that a smaller proportion of the whole
cellular constitution of the embryo is removed, from a non-embryonic portion of
the blastocyst and at a stage of preimplantation development perhaps more
tolerant to manipulation (Danilo Cimadomo 2016). Although the LBR per transfer
may increase with this technique, it should be kept in mind that with extended
embryo culture, a higher rate of patients will not reach embryo transfer;
therefore couples should be carefully counselled about these technical
limitations and the procedure’s higher cost (Elias M. Dahdouh 2015). Two
different methods of TE biopsy have been published to date. The difference
between the two methods is the timing when the zona breach is carried out. It
can be done on day 3 and then the herniating cells are biopsied on day 5 or day
6 or AH can be carried out at the same time as the biopsy on day 5/6. No
significant differences across seven different operators from three IVF centres
in terms of both technical and clinical results were reported. In particular,
amplification rate, qPCR data concurrence, and estimated number of cells
retrieved, as well as ongoing implantation, biochemical, and miscarriage rates,
were comparable (Danilo Cimadomo 2016). Oocyte retrieval, fertilization and
culture of embryos should be undertaken in an establishment which has the
suitable laboratory premises, equipment and trained staff, in accordance in
accordance with the European Union Tissue and Cells directive or other local
laws (G. Harton 2010). The patients are subjected to a stimulation protocol
which consists of a Controlled Ovarian Hyperstimulation. The normally fertilised
oocytes are cultured in an incubator (e.g. Embryoscope) until day 6. PB biopsy
is carried out on day 1, cleavage stage biopsy is carried out on day 3 and TE
biopsy is carried out on day 5 and/or day 6 depending on embryo development.
Genetic counselling should be provided by a qualified clinical geneticist or
genetic counsellor. A specialist in reproductive medicine should provide
information regarding the IVF cycle to ensure that patients are fully informed
of all aspects of PGT before treatment starts (G. Harton 2010). Patients must
complete / sign the clinics consent forms for the relevant procedures of which
they will undertake. All patients undergoing treatment must have viral
screening in accordance with the local guidelines / regulations. Patients who
have a PB or a day 3 biopsy can have a fresh day 5 transfer once results show
that they have a euploid embryo suitable for transfer. For patients that have a
day 5 or 6 TE biopsy, they will have a subsequent Frozen Embryo Transfer (FET)
once results show that they have a euploid blastocyst suitable for transfer.
PGD / PGS is still relatively unregulated and lacks standardization compared
with other forms of diagnostic testing: however, more federal, state and local
governments are beginning to take an interest in PGD and some have begun
accrediting laboratories that offer PGD (Harper J. C. 2010a) (G. L. Harton
2010). The biopsy procedure is carried out in the IVF laboratory by a trained
and competent embryologist / biopsy practitioner. It is essential that the
embryologist has documented training records to deem them competent to carry
out the biopsy procedure. Laboratory staff performing clinical work should have
a recognised training and assessment programme (G. Harton 2010). It is
essential that the Standard Operating Procedure (SOP) is updated and adhered to
for the procedure. IVF clinics have a Quality Management System (QMS) in place
to ensure SOPs are reviewed and any deviations to the protocol is reported
through the QMS. Only embryos of sufficient cell number / quality will be
suitable to biopsy. If the biopsy is carried out on day 5 or 6 then the
blastocysts need to be cryopreserved for future use. This will require all the
necessary consumables, media and equipment for Vitrification. Most laboratories
carry out vitrification now instead of slow freezing as it has been documented
to achieve better results. Even if the biopsy is carried out at PB stage or
cleavage stage a process of cryopreservation needs to be available to freeze
surplus good quality embryos after the embryo transfer has taken place. It is
essential that all cumulus cells are completely removed at denudation for ICSI
or at the fertilization check for IVF to avoid DNA contamination. The PGD
Diagnosis laboratory carry out the amplification and the testing of the biopsy
samples. FISH was used more frequently in the past, however, FISH tests for a
limited number of chromosomes. The use of technology that allows for a more
comprehensive screening of chromosomes is used (array-based technology) –
clinics are applying array-comparative genomic hybridization (a-CGH) (Joyce
Harper 2010). The biopsy is mainly conducted following three methods of zona
breaching; (i) laser-assisted, (ii) mechanical and (iii) Tyrode’s drilling.
Laser biopsy was the preferred method (80%); acidic Tyrode’s or mechanical zona
breaching was applied in 9% and 11% of cycles to PGS, respectively, declared in
the ESHRE PGD consortium data collection XIV-XV (M. De Rycke 2017). However,
apparently all the three methods do not impact clinical outcomes, as randomized
controlled trials (RCTs) on sibling embryos have shown (Danilo Cimadomo 2016).
Care should be taken when choosing the laser pulse so as not to damage the
cells. If the embryo has begun to blastulate and polarity exists, aim to breach
the zona opposite to the ICM, otherwise at a position where there is a large
PVS. Use a series of small laser pulses, moving from the outer to the inner ZP
to make a hole no bigger than the thickness of the ZP itself.  Probably
then the reason for the prevalence of laser assisted method resides in the
standardization and reproducibility of the hole produced within the ZP, which
is less operator-dependent than the use of acidified Tyrode (Danilo Cimadomo
2016). All biopsy procedures should be carried out on an inverted microscope
(e.g. Nikon) on a heated stage equipped with micromanipulation tools. Ensure
the laser (e.g. Hamilton Thorn) for AH is calibrated and validated. Assess each
available embryo for suitability for biopsy and ensure that any remaining
cumulus cells are washed in the culture media (HEPES buffered) dish (e.g. GTL,
Global, CSCM) prior to biopsy. Prepare biopsy dishes by aliquot drops of non
HEPES-buffered media (e.g. Sage, GMOPs plus), overlay with a mineral oil (e.g.
Sage, Microm) and place in non-gassed incubator to warm up before use. Move
suitable embryos to biopsy dish, ensuring all dishes are clearly labelled with
patient’s unique identifiers and the well number of the embryos. It is
essential for traceability that all steps are witnessed. Apply gentle suction
to attach the blastocyst firmly to the holding pipette (e.g. Research
Instruments, Vitrolife), ensuring you have good focus on the cells and
pipettes. Gently apply negative pressure with the TE biopsy pipette, and suck
5-10 cells into the lumen from the location where the initial zona breach was
carried out, where the TE cells are herniating. Gently release some pressure on
the biopsy pipette and stretch the TE cells until cell junctions can be seen. A
small laser pulse (e.g. 4.8µm) can be used to ablate the top and bottom of the
junction between the trophoblast cells. The blastocyst can now be released from
the holding pipette, whilst still being held by the biopsy pipette. The cells
can now be detached from the embryo by rubbing them against the holding pipette
with a single flick motion. A PB biopsy is performed by aspiration with a PB
aspiration pipette. It can be done simultaneously or sequentially by removing
the first PB and then second PB after fertilization. A cleavage stage biopsy is
performed on day 3 by the extrusion of one or two blastomeres, with the use of
a blastomere aspiration pipette. Biopsy on day 3 will be carried out on embryos
of >6 cells, or >8cells if two blastomeres are to be aspirated. This must
be carried out in a calcium-magnesium-free HEPES buffered media. Move embryos
back into culture for embryo transfer / vitrification using a witness. Number
each embryo individually to ensure they are frozen in the correct order. The
use of culture wells instead of droplets would decrease the possible mixing of
embryos in culture dishes due to possible movement of droplets during
handling (G. L. Harton 2010). The tubing of the biopsied calls / PBs is
performed on a dissecting microscope in a laminar airflow hood (LAF) to avoid
any DNA contamination, sterile gloves and surgical mask are worn. Appropriate
precautions should be taken both to prevent contamination of samples by
extraneous cells or DNA, by physical isolation (G. Harton 2010). Prepare tray
and PCR eppendorf tubes for biopsied cells, all clearly labelled and witnessed
with patient unique identifiers and embryo number. For every biopsy a
corresponding media blank sample should also be prepared. Aliquot lysis buffer
into each tube. A dish with aliquots of wash buffer is used to wash to cells
before being placed in the tubes. Cells are sent to the PGT laboratory on dry
ice where DNA is amplified and analysed. The successful cryopreservation of
excess embryos is an important component of assisted conception programs, with
vitrification widely recognised as the criterion standard method (Cara K.
Bradley 2017). Vitrification simplifies and frequently improves
cryopreservation because it eliminates mechanical injury from ice and
eliminates the need to find optimal cooling and warming rates (Wowk. 2014). In
a paper published by Chen et al 2017, according to their findings, optimal
vitrification time >3 hours to enable blastocysts to reach ¾ re-expansion
but not fully expansion or full re-expansion or hatching provides improved
implantation and pregnancy rates after FER (Hsiu-Hui Chen 2017). The proportion
of embryos not found on warming and embryos degenerated after warming mainly
reflect operator skill. In the Alpha survey, the median competence values were
3% and 10% for not-found and degenerated embryos, respectively. It was
mentioned that clinicians should be aware that although laboratories strive for
100% recovery, not all embryos submitted to PGT will be recovered after warming
 (Medicine. 2017). Implantation rates following a randomized paired
analysis of the effects of cleavage and blastocyst stage biopsies on embryo
reproductive potential. Sustained implantation and delivery of the biopsied
embryo were significantly reduced compared with its control sibling when biopsy
was performed on day 3 at the cleavage stage. A similar paired analysis
demonstrated that the developmental potential of embryos undergoing TE biopsy
at the blastocyst stage was equivalent to the non-biopsied control siblings. (Richard
T. Scott 2013)   The most commonly used strategy to conduct PGT in Europe
still entails cleavage stage rather than TE biopsy. The perception of the
former as less operator dependent and more reproducible possibly underlies this
tendency. In particular, embryos reach cleavage stage synchronously and are
similar in terms of morphological quality and a single biopsy protocol has been
described in literature. Blastocyst stage biopsy instead is characterized by a
heterogeneous cohort of embryos in terms of both morphology and developmental
rate (Danilo Cimadomo 2016). It was the consensus that the implantation rate
for blastocysts biopsied for PGS should exceed that expected for the
age-matched patient population in the same clinic. From the literature, a meta-analysis
reported an improvement of 30% sustained implantation rate after the transfer
of PGS-selected blastocysts relative to controls (Medicine. 2017). If the
embryos arrest before day 5 or 6, given today’s greatly improved laboratory
conditions, it probably means that, in that particular cycle, they were not
viable and not destined to become a live birth. The recent Cochrane literature
supports improved pregnancy rates per transfer with blastocyst as opposed to
day 3 transfers (Silber. 2017). TE biopsy has been shown to be safer and more
accurate than cleavage stage blastomere biopsy. Embryos that have undergone TE
biopsy have been demonstrated to have a higher implantation rate (47.6%)
compared with those that have undergone blastomere biopsy (26.7%) (Ariel
Weissman 2017). Richard Scott et al, see Figure IV, has published showing
sustained implantation and delivery of the biopsied embryo were significantly
reduced compared with its control sibling when biopsy was performed on day 3 at
the cleavage stage. The accuracy and reliability of the diagnosis is increased
by analysing two blastomeres of the embryo, however, the removal of two
blastomeres might have an effect on the implantation capacity of the embryo
(Hilde Van de Velde 2000). Levin and colleagues reported a higher fragmentation
rate, a lower embryo quality, a higher cleavage arrest rate, and a lower mean
number of blastomeres in day 3 when PB biopsy is performed with respect to
control (Danilo Cimadomo 2016). Amplification rate in particular is an
important parameter since a second biopsy would be needed in case of a
non-conclusive result. Importantly, all the papers where TE based CCS analysis
was adopted reported always less than 3.0% of undiagnosed blastocysts. This
point represents a further advantage of this approach with respect to the
previous single cell-based ones (Danilo Cimadomo 2016). Despite the initial
enthusiasm, subsequent RCTs and a meta-analysis indicated that PGS using FISH
failed to show improved reproductive outcomes. Subsequently, professional
societies discouraged the use of PGS in this form, and its use declined (Ariel
Weissman 2017).To complicate matters even further, these advanced techniques,
particularly NGS, have unveiled the phenomenon of embryonic mosaic aneuploidy (Ariel
Weissman 2017) . In the field of PGT there is an ongoing debate in relation to
the optimal time to carry out the biopsy procedure. Each stage presents with
specific diagnostic advantages as well as critical limitations that relate to
aneuploidy genesis during both meiosis and the preimplantation period of embryo
development (Antonio Capalbo 2013). As discussed throughout this review paper
there are advantages and drawbacks published for all stages of embryo biopsy.
Some more significant than others. PB and cleavage biopsy allow ample time to
have the genetic testing complete and have the results so the patient can have
a fresh transfer in that cycle. TE biopsy requires extended blastocyst culture
to day 5/6 so it does not permit this and vitrification needs to take place
after the biopsy and the patients will have a FER in the future. This is not
necessary seen as an disadvantage for TE biopsy as many papers have data saying
that FER and fresh transfer have very similar implantation rates. Euploid cryopreserved
blastocyst transfer prevents hyperstimulation syndrome and multiple pregnancy,
a further important advantage (Danilo Cimadomo 2016). Forman et al, also
demonstrated that single euploid blastocyst transfer equals the implantation
rate of double untested blastocyst transfer, but it elicits better obstetrical
and perinatal outcomes (Danilo Cimadomo 2016). It has been suggested that
embryo culture to the blastocyst stage could naturally select the most viable
embryos, as grossly abnormal embryos would fail to develop to this stage;
therefore, evaluating the ability of a zygote to reach the blastocyst stage in
vitro can be considered equivalent to natural selection of embryos (Mar?´a Cruz
2012). Morphologic grading should be used to help in the selection among
euploid blastocysts (Mohamad Irani 2017). PB biopsy is an excellent source of
maternal origin, however it provides no information on mutations of paternal
origin. Although PB analysis provides important prognostic information for
couples about the origin of aneuploidies, there is still ongoing debate on the
need to perform this type of biopsy (Elias M. Dahdouh 2015). It does avoid
legal and ethical concerns in countries where embryo biopsy is not permitted.
It entails a large workload in the laboratory due to the increased number of
oocytes to be tested, also both PBs need to be biopsied either together or at
different times. There is a risk of enucleation due to spindle remnants in the
second PB, while PB disintegration or degeneration might occur if the biopsy is
performed too late (Danilo Cimadomo 2016). There is a query on its effect on
embryo development and it offers no meaningful data about the impact on
implantation. Compared to PB biopsy, cleavage stage and TE biopsy result in a
lower number of embryos to test. Cleavage stage biopsy is a relatively
standardised technique while TE biopsy does require a highly trained level of
expertise. However, extended culture leads to more cancellations in treatment
when no suitable blastocyst develops (Mar?´a Cruz 2012).  It is accurate,
reliable and reproducible with no impact on implantation potential. Cleavage
stage biopsy has evidenced a significant decrease in implantation potential.
With cleavage stage biopsy, a portion of the embryo is removed regardless of
the knowledge of its future destiny. From a day 3 embryo of e.g. >6 cells
there is high embryonic mass depletion when one to two cell are removed. TE
biopsy allows the removal of a lower portion of total blastocysts cell number
as there are more cells available and it is the removal of non-embryonic
portion. The evidence produced in the last decades extensively highlights the
drawbacks of the cleavage stage approach in PGT. A significant decrease in
clinical outcomes derives from the use of such a harmful biopsy strategy.
Sufficiently powered studies highlighting a similar negative impact for PB
biopsy are still missing. However, this strategy suffers from important
diagnostic issues leading to high false positive and false negative error rates
(Danilo Cimadomo 2016).