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This technique iscost-effective, because fewer embryos are tested, and it has been associated with increased chance of live birth in thelast decade (Elias M. Dahdouh 2015).

Recently, TE biopsy has been shown to have no impact on blastocyst reproductive potential when compared withcleavage-stage biopsy, in which 39%reduction in implantation rate was reported (Elias M. Dahdouh 2015). The pairedRCT by Scott Jr. and colleagues is again the real milestone that identifiedblastocyst stage biopsy as a procedure that does not affect embryo viabilityand implantation potential. TE biopsy was reported to have no impact, converseto what was found for blastomere biopsy. A possible explanation for thisdifference is that a smaller proportion of the whole cellular constitution ofthe embryo is removed, from a non-embryonic portion of the blastocyst and at astage of preimplantation development perhaps more tolerant to manipulation(Danilo Cimadomo 2016).

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Although the LBR per transfer may increase with thistechnique, it should be kept in mind that with extended embryo culture, ahigher rate of patients will not reach embryo transfer; therefore couplesshould be carefully counselled about these technical limitations and theprocedure’s higher cost (Elias M. Dahdouh 2015). Two different methods of TEbiopsy have been published to date.

The difference between the two methods isthe timing when the zona breach is carried out. It can be done on day 3 andthen the herniating cells are biopsied on day 5 or day 6 or AH can be carriedout at the same time as the biopsy on day 5/6. No significant differencesacross seven different operators from three IVF centres in terms of bothtechnical and clinical results were reported. In particular, amplificationrate, qPCR data concurrence, and estimated number of cells retrieved, as well asongoing implantation, biochemical, and miscarriage rates, were comparable(Danilo Cimadomo 2016). Oocyte retrieval, fertilization and culture of embryosshould be undertaken in an establishment which has the suitable laboratorypremises, equipment and trained staff, in accordance in accordance with theEuropean Union Tissue and Cells directive or other local laws (G. Harton 2010).

The patients are subjected to a stimulation protocol which consists of aControlled Ovarian Hyperstimulation. The normally fertilised oocytes arecultured in an incubator (e.g. Embryoscope) until day 6. PB biopsy is carriedout on day 1, cleavage stage biopsy is carried out on day 3 and TE biopsy iscarried out on day 5 and/or day 6 depending on embryo development. Geneticcounselling should be provided by a qualified clinical geneticist or geneticcounsellor. A specialist in reproductive medicine should provide informationregarding the IVF cycle to ensure that patients are fully informed of allaspects of PGT before treatment starts (G.

Harton 2010). Patients must complete/ sign the clinics consent forms for the relevant procedures of which they willundertake. All patients undergoing treatment must have viral screening inaccordance with the local guidelines / regulations. Patients who have a PB or aday 3 biopsy can have a fresh day 5 transfer once results show that they have aeuploid embryo suitable for transfer. For patients that have a day 5 or 6 TEbiopsy, they will have a subsequent Frozen Embryo Transfer (FET) once resultsshow that they have a euploid blastocyst suitable for transfer. PGD / PGS isstill relatively unregulated and lacks standardization compared with otherforms of diagnostic testing: however, more federal, state and local governmentsare beginning to take an interest in PGD and some have begun accreditinglaboratories that offer PGD (Harper J. C. 2010a) (G.

L. Harton 2010). Thebiopsy procedure is carried out in the IVF laboratory by a trained andcompetent embryologist / biopsy practitioner.

It is essential that theembryologist has documented training records to deem them competent to carryout the biopsy procedure. Laboratory staff performing clinical work should havea recognised training and assessment programme (G. Harton 2010). It isessential that the Standard Operating Procedure (SOP) is updated and adhered tofor the procedure. IVF clinics have a Quality Management System (QMS) in placeto ensure SOPs are reviewed and any deviations to the protocol is reportedthrough the QMS. Only embryos of sufficient cell number / quality will besuitable to biopsy. If the biopsy is carried out on day 5 or 6 then theblastocysts need to be cryopreserved for future use.

This will require all thenecessary consumables, media and equipment for Vitrification. Most laboratoriescarry out vitrification now instead of slow freezing as it has been documentedto achieve better results. Even if the biopsy is carried out at PB stage orcleavage stage a process of cryopreservation needs to be available to freezesurplus good quality embryos after the embryo transfer has taken place. It isessential that all cumulus cells are completely removed at denudation for ICSIor at the fertilization check for IVF to avoid DNA contamination. The PGDDiagnosis laboratory carry out the amplification and the testing of the biopsysamples. FISH was used more frequently in the past, however, FISH tests for alimited number of chromosomes. The use of technology that allows for a morecomprehensive screening of chromosomes is used (array-based technology) –clinics are applying array-comparative genomic hybridization (a-CGH) (JoyceHarper 2010).

The biopsy is mainly conducted following three methods of zonabreaching; (i) laser-assisted, (ii) mechanical and (iii) Tyrode’s drilling.Laser biopsy was the preferred method (80%); acidic Tyrode’s or mechanical zonabreaching was applied in 9% and 11% of cycles to PGS, respectively, declared inthe ESHRE PGD consortium data collection XIV-XV (M. De Rycke 2017). However,apparently all the three methods do not impact clinical outcomes, as randomizedcontrolled trials (RCTs) on sibling embryos have shown (Danilo Cimadomo 2016).

Care should be taken when choosing the laser pulse so as not to damage thecells. If the embryo has begun to blastulate and polarity exists, aim to breachthe zona opposite to the ICM, otherwise at a position where there is a largePVS. Use a series of small laser pulses, moving from the outer to the inner ZPto make a hole no bigger than the thickness of the ZP itself.  Probablythen the reason for the prevalence of laser assisted method resides in thestandardization and reproducibility of the hole produced within the ZP, whichis less operator-dependent than the use of acidified Tyrode (Danilo Cimadomo2016). All biopsy procedures should be carried out on an inverted microscope(e.g. Nikon) on a heated stage equipped with micromanipulation tools. Ensurethe laser (e.

g. Hamilton Thorn) for AH is calibrated and validated. Assess eachavailable embryo for suitability for biopsy and ensure that any remainingcumulus 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 nonHEPES-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. Movesuitable embryos to biopsy dish, ensuring all dishes are clearly labelled withpatient’s unique identifiers and the well number of the embryos. It isessential for traceability that all steps are witnessed. Apply gentle suctionto attach the blastocyst firmly to the holding pipette (e.g. ResearchInstruments, Vitrolife), ensuring you have good focus on the cells andpipettes.

Gently apply negative pressure with the TE biopsy pipette, and suck5-10 cells into the lumen from the location where the initial zona breach wascarried out, where the TE cells are herniating. Gently release some pressure onthe biopsy pipette and stretch the TE cells until cell junctions can be seen. Asmall laser pulse (e.g. 4.8µm) can be used to ablate the top and bottom of thejunction between the trophoblast cells. The blastocyst can now be released fromthe holding pipette, whilst still being held by the biopsy pipette. The cellscan now be detached from the embryo by rubbing them against the holding pipettewith a single flick motion.

A PB biopsy is performed by aspiration with a PBaspiration pipette. It can be done simultaneously or sequentially by removingthe first PB and then second PB after fertilization. A cleavage stage biopsy isperformed on day 3 by the extrusion of one or two blastomeres, with the use ofa blastomere aspiration pipette. Biopsy on day 3 will be carried out on embryosof >6 cells, or >8cells if two blastomeres are to be aspirated. This mustbe carried out in a calcium-magnesium-free HEPES buffered media. Move embryosback into culture for embryo transfer / vitrification using a witness.

Numbereach embryo individually to ensure they are frozen in the correct order. Theuse of culture wells instead of droplets would decrease the possible mixing ofembryos in culture dishes due to possible movement of droplets duringhandling (G. L.

Harton 2010). The tubing of the biopsied calls / PBs isperformed on a dissecting microscope in a laminar airflow hood (LAF) to avoidany DNA contamination, sterile gloves and surgical mask are worn. Appropriateprecautions should be taken both to prevent contamination of samples byextraneous cells or DNA, by physical isolation (G. Harton 2010). Prepare trayand PCR eppendorf tubes for biopsied cells, all clearly labelled and witnessedwith patient unique identifiers and embryo number. For every biopsy acorresponding media blank sample should also be prepared. Aliquot lysis bufferinto each tube.

A dish with aliquots of wash buffer is used to wash to cellsbefore being placed in the tubes. Cells are sent to the PGT laboratory on dryice where DNA is amplified and analysed. The successful cryopreservation ofexcess embryos is an important component of assisted conception programs, withvitrification widely recognised as the criterion standard method (Cara K.Bradley 2017). Vitrification simplifies and frequently improvescryopreservation because it eliminates mechanical injury from ice andeliminates the need to find optimal cooling and warming rates (Wowk. 2014). Ina paper published by Chen et al 2017, according to their findings, optimalvitrification time >3 hours to enable blastocysts to reach ¾ re-expansionbut not fully expansion or full re-expansion or hatching provides improvedimplantation and pregnancy rates after FER (Hsiu-Hui Chen 2017). The proportionof embryos not found on warming and embryos degenerated after warming mainlyreflect operator skill.

In the Alpha survey, the median competence values were3% and 10% for not-found and degenerated embryos, respectively. It wasmentioned that clinicians should be aware that although laboratories strive for100% recovery, not all embryos submitted to PGT will be recovered after warming (Medicine. 2017). Implantation rates following a randomized pairedanalysis of the effects of cleavage and blastocyst stage biopsies on embryoreproductive potential. Sustained implantation and delivery of the biopsiedembryo were significantly reduced compared with its control sibling when biopsywas performed on day 3 at the cleavage stage.

A similar paired analysisdemonstrated that the developmental potential of embryos undergoing TE biopsyat the blastocyst stage was equivalent to the non-biopsied control siblings. (RichardT. Scott 2013)   The most commonly used strategy to conduct PGT in Europestill entails cleavage stage rather than TE biopsy. The perception of theformer as less operator dependent and more reproducible possibly underlies thistendency. In particular, embryos reach cleavage stage synchronously and aresimilar in terms of morphological quality and a single biopsy protocol has beendescribed in literature.

Blastocyst stage biopsy instead is characterized by aheterogeneous cohort of embryos in terms of both morphology and developmentalrate (Danilo Cimadomo 2016). It was the consensus that the implantation ratefor blastocysts biopsied for PGS should exceed that expected for theage-matched patient population in the same clinic. From the literature, a meta-analysisreported an improvement of 30% sustained implantation rate after the transferof PGS-selected blastocysts relative to controls (Medicine. 2017). If theembryos arrest before day 5 or 6, given today’s greatly improved laboratoryconditions, it probably means that, in that particular cycle, they were notviable and not destined to become a live birth. The recent Cochrane literaturesupports improved pregnancy rates per transfer with blastocyst as opposed today 3 transfers (Silber. 2017).

TE biopsy has been shown to be safer and moreaccurate than cleavage stage blastomere biopsy. Embryos that have undergone TEbiopsy have been demonstrated to have a higher implantation rate (47.6%)compared with those that have undergone blastomere biopsy (26.7%) (ArielWeissman 2017). Richard Scott et al, see Figure IV, has published showingsustained implantation and delivery of the biopsied embryo were significantlyreduced compared with its control sibling when biopsy was performed on day 3 atthe cleavage stage.

The accuracy and reliability of the diagnosis is increasedby analysing two blastomeres of the embryo, however, the removal of twoblastomeres might have an effect on the implantation capacity of the embryo(Hilde Van de Velde 2000). Levin and colleagues reported a higher fragmentationrate, a lower embryo quality, a higher cleavage arrest rate, and a lower meannumber of blastomeres in day 3 when PB biopsy is performed with respect tocontrol (Danilo Cimadomo 2016). Amplification rate in particular is animportant parameter since a second biopsy would be needed in case of anon-conclusive result. Importantly, all the papers where TE based CCS analysiswas adopted reported always less than 3.0% of undiagnosed blastocysts. Thispoint represents a further advantage of this approach with respect to theprevious single cell-based ones (Danilo Cimadomo 2016). Despite the initialenthusiasm, subsequent RCTs and a meta-analysis indicated that PGS using FISHfailed to show improved reproductive outcomes. Subsequently, professionalsocieties discouraged the use of PGS in this form, and its use declined (ArielWeissman 2017).

To complicate matters even further, these advanced techniques,particularly NGS, have unveiled the phenomenon of embryonic mosaic aneuploidy (ArielWeissman 2017) . In the field of PGT there is an ongoing debate in relation tothe optimal time to carry out the biopsy procedure. Each stage presents withspecific diagnostic advantages as well as critical limitations that relate toaneuploidy genesis during both meiosis and the preimplantation period of embryodevelopment (Antonio Capalbo 2013).

As discussed throughout this review paperthere are advantages and drawbacks published for all stages of embryo biopsy.Some more significant than others. PB and cleavage biopsy allow ample time tohave the genetic testing complete and have the results so the patient can havea fresh transfer in that cycle. TE biopsy requires extended blastocyst cultureto day 5/6 so it does not permit this and vitrification needs to take placeafter the biopsy and the patients will have a FER in the future. This is notnecessary seen as an disadvantage for TE biopsy as many papers have data sayingthat FER and fresh transfer have very similar implantation rates.

Euploid cryopreservedblastocyst transfer prevents hyperstimulation syndrome and multiple pregnancy,a further important advantage (Danilo Cimadomo 2016). Forman et al, alsodemonstrated that single euploid blastocyst transfer equals the implantationrate of double untested blastocyst transfer, but it elicits better obstetricaland perinatal outcomes (Danilo Cimadomo 2016). It has been suggested thatembryo culture to the blastocyst stage could naturally select the most viableembryos, as grossly abnormal embryos would fail to develop to this stage;therefore, evaluating the ability of a zygote to reach the blastocyst stage invitro can be considered equivalent to natural selection of embryos (Mar?´a Cruz2012). Morphologic grading should be used to help in the selection amongeuploid blastocysts (Mohamad Irani 2017). PB biopsy is an excellent source ofmaternal origin, however it provides no information on mutations of paternalorigin. Although PB analysis provides important prognostic information forcouples about the origin of aneuploidies, there is still ongoing debate on theneed to perform this type of biopsy (Elias M. Dahdouh 2015).

It does avoidlegal and ethical concerns in countries where embryo biopsy is not permitted.It entails a large workload in the laboratory due to the increased number ofoocytes to be tested, also both PBs need to be biopsied either together or atdifferent times. There is a risk of enucleation due to spindle remnants in thesecond PB, while PB disintegration or degeneration might occur if the biopsy isperformed too late (Danilo Cimadomo 2016). There is a query on its effect onembryo development and it offers no meaningful data about the impact onimplantation. Compared to PB biopsy, cleavage stage and TE biopsy result in alower number of embryos to test. Cleavage stage biopsy is a relativelystandardised technique while TE biopsy does require a highly trained level ofexpertise. However, extended culture leads to more cancellations in treatmentwhen no suitable blastocyst develops (Mar?´a Cruz 2012).  It is accurate,reliable and reproducible with no impact on implantation potential.

Cleavagestage biopsy has evidenced a significant decrease in implantation potential.With cleavage stage biopsy, a portion of the embryo is removed regardless ofthe knowledge of its future destiny. From a day 3 embryo of e.g.

>6 cellsthere is high embryonic mass depletion when one to two cell are removed. TEbiopsy allows the removal of a lower portion of total blastocysts cell numberas there are more cells available and it is the removal of non-embryonicportion. The evidence produced in the last decades extensively highlights thedrawbacks of the cleavage stage approach in PGT. A significant decrease inclinical outcomes derives from the use of such a harmful biopsy strategy.Sufficiently powered studies highlighting a similar negative impact for PBbiopsy are still missing. However, this strategy suffers from importantdiagnostic issues leading to high false positive and false negative error rates(Danilo Cimadomo 2016). Cleavage biopsy has a risk of amplification failure andhigh rate of technical artefacts, and a high incidence of mosaicism and alleledrop out while TE biopsy suffers from a decrease rate of mosaicism.

Furtherresearch into the phenomenon of mosaicism is necessary to deal with the issue.The comparison of TE biopsy strategy to previous ones strongly suggests that itis the most promising approach in PGT (Danilo Cimadomo 2016). Another proposedmethod of embryo selection by Poli et al is the investigation of theapplication of proteomic measurements that could improve success rates inclinical embryology. They describe a procedure that allows the identificationand quantification of proteins of embryonic origin, present in the blastocoel withnegligible risk of harm to the living embryo. By using targeted proteomics,they demonstrated the feasibility of quantifying multiple proteins in samplesderived from single blastocoels and that such measurements correlate withaspects of embryo viability, such as chromosomal (ploidy) status (Maurizio Poli2015). An ideal outcome would be to bypass the embryo biopsy step and predicteuploidy and/or reproductive competence through non-invasive methods (DaniloCimadomo 2016). However, according to current trials in women undergoing ART,there is insufficient evidence to show that metabolomics assessment ofembryos before implantation has any meaningful effect on rates of live birth,ongoing pregnancy, or miscarriage rates.

The existing evidence varied from verylow to low-quality (Siristatidis C. S. 2017). Also unfortunately, severalpapers defined conventional parameters of embryo evaluation as just mildlycorrelated with aneuploidy rate (Danilo Cimadomo 2016). Current technologyallows for test performance in the range of 98-99% accuracy (G. Harton 2010).The patients should understand that a misdiagnosis is possible and the optionsthat are available to them should a pregnancy occurs, including prenataltesting and genetic diagnosis (G.

Harton 2010). The transfer of mosaicaneuploid embryos has been reported to result in the occasional birth ofhealthy children, but there is a great deal of uncertainty about the transferof such embryos and the circumstances and conditions for doing so (ArielWeissman 2017). The disadvantage of PND is that if the diagnosis shows thefetus to be affected, the couple have to decide whether they wish to terminatethe pregnancy or carry on with the knowledge that their child is going to beaffected by the genetic disease (Dale 2011). However, these approaches exposewomen to a high risk of miscarriage ranging between 1 and 3% (Danilo Cimadomo2016). PGD offers some of these couples an alternative, as the diagnosis isperformed on the preimplantation embryo and only unaffected embryos aretransferred to the patient.

The pregnancy is therefore initiated with theknowledge that the fetus is free from the disease (Dale 2011). Over the lastseveral years, blastocyst biopsy with analysis of all chromosomes hassupplanted day 3 analysis via FISH (G. Harton 2010). Three randomizedcontrolled trials have shown a beneficial role of PGS using comprehensivechromosome screening technology on TE cells. In general, PGS allows for adecrease in embryo transfer number, higher implantation rates per transfer, anda lower rate of miscarriage once implantation occurs for older patientsundergoing IVF (Mohamad Irani 2017). To finalise, PGT is an establishedpractice in IVF laboratories worldwide, however, there is a need to have aregulated, standardised technique to ensure the embryos survival, viability andproductive potential is preserved.

Cleavage stage biopsy remains the mostcommon method in Europe. Even though t has been found to have a negative impacton embryo viability. TE biopsy is not such a widespread method but it isincreasing as several preclinical and clinical evidences recognise its valueand its advantages compared to cleavage and PB biopsy.  

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