review article the impact of biopsy on human embryo...

Review ArticleThe Impact of Biopsy on Human Embryo DevelopmentalPotential during Preimplantation Genetic Diagnosis

Danilo Cimadomo12 Antonio Capalbo13 Filippo Maria Ubaldi13 Catello Scarica12

Antonio Palagiano4 Rita Canipari2 and Laura Rienzi13

1GENERA Centre for Reproductive Medicine Clinica Valle Giulia Via G de Notaris 2b 00197 Rome Italy2Dipartimento di Scienze Anatomiche University of Rome ldquoLa Sapienzardquo Istologiche Medico Legali e dellrsquoApparato LocomotoreSezione Istologia ed Embriologia Medica Via Antonio Scarpa 16 00161 Rome Italy3GENETYX Molecular Biology Laboratory Via Fermi 1 36063 Marostica Italy4Seconda Universita di Napoli Via Antonio Vivaldi 43 81100 Caserta Italy

Correspondence should be addressed to Danilo Cimadomo cimadomogeneraromait

Received 8 September 2015 Revised 15 December 2015 Accepted 5 January 2016

Academic Editor Karla Hutt

Copyright copy 2016 Danilo Cimadomo et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Preimplantation Genetic Diagnosis and Screening (PGDPGS) for monogenic diseases andor numericalstructural chromosomalabnormalities is a tool for embryo testing aimed at identifying nonaffected andor euploid embryos in a cohort produced duringan IVF cycle A critical aspect of this technology is the potential detrimental effect that the biopsy itself can have upon the embryoDifferent embryo biopsy strategies have been proposed Cleavage stage blastomere biopsy still represents the most commonlyused method in Europe nowadays although this approach has been shown to have a negative impact on embryo viability andimplantation potential Polar body biopsy has been proposed as an alternative to embryo biopsy especially for aneuploidy testingHowever to date no sufficiently powered study has clarified the impact of this procedure on embryo reproductive competenceBlastocyst stage biopsy represents nowadays the safest approach not to impact embryo implantation potential For this reasonas well as for the evidences of a higher consistency of the molecular analysis when performed on trophectoderm cells blastocystbiopsy implementation is gradually increasing worldwide The aim of this review is to present the evidences published to date onthe impact of the biopsy at different stages of preimplantation development upon human embryos reproductive potential

1 Introduction

Preimplantation Genetic Diagnosis and Screening (PGDPGS) is a tool whose application in Assisted ReproductionTechniques (ART) has significantly grown in the last decades[1]The final aim of PGDPGS is to define whether an embryois affected by a monogenic disease andor chromosomalimpairments thus preventing the implantation of a symp-tomatic fetus andor limiting the risks underlying the transferof chromosomally abnormal embryos (mainly implantationfailures and miscarriages) In synthesis PGDPGS is a pow-erful tool to reach the goal of a pregnancy and attenuate itsadverse events In order to achieve this goal it is mandatorynot to significantly harm the embryo during the biopsy andto preserve its viability and reproductive potential First do

not harm is a dogma in clinical practice that perfectly appliesalso to this context This review will be an overview ofthe different stages of embryo preimplantation developmentthe biopsy procedure can be performed at The definitionof the technical drawbacks that could resolve in a negativeinfluence on reproductive potential will be provided Mainlythree different approaches will be examined namely polarbody (PB) biopsy from the mature oocyte andor the zygoteblastomere biopsy at the cleavage stage and trophectoderm(TE) biopsy at the blastocyst stage Recently morula stagebiopsy has also been proposed and will be briefly discussed

The ESHRE PGD consortium data referring to years2009-2010 [2] highlighted an uneven distribution in thenumber of biopsy procedures performed in Europe betweenthe three main strategies In particular blastomere biopsy

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 7193075 10 pageshttpdxdoiorg10115520167193075

2 BioMed Research International

alone accounted for almost 90 of the total while TE biopsyaccounted for less than 1 The same data referring to years2012-2013 [3] instead showed an impressive countertendencysince the rate of PB biopsy and cleavage stage biopsy proce-dures decreased to approximately 2 and 75 respectivelyBlastocyst stage biopsy implementation instead is graduallyincreasing in ART The reason for this dramatic changeis a review and meta-analysis published by Mastenbroekand colleagues in 2011 clearly showing that PGS as it wasconducted namely cleavage stage biopsy analyzed by 9-chromosome FISH is a harmful procedure [4] Howeverwhile 9-chromosome FISH use has been strongly reduced infavor of themore accurate and reliable Comprehensive Chro-mosome Screening (CCS) techniques blastomere biopsy isstill the method mainly adopted for PGD The use of PBbiopsy did not spread due to its intrinsic logistic clinical andtechnical drawbacks that compromise its accuracy especiallywhen compared to TE biopsy strategy

2 Cleavage Stage Biopsy

Cleavage stage biopsy is normally performed on day 3embryos with at least 6 blastomeres The zona pellucida isopened and Ca++Mg++-free medium is used in order toloosen cell-cell adhesion and facilitate selected blastomereremoval A less traumatic impact on the growing embryoshould theoretically not affect blastulation [5] Howeverthis is a controversial issue since several studies in mouseshowed that Ca++ depletion caused remodeling of the cellularcytoskeleton inevitably impacting compaction [6ndash9]

The biopsy is mainly conducted following 3 methods ofzona breaching namely laser-assisted [10] mechanical [11]and Tyrodersquos drilling [12] Use of the laser-assisted methodrepresents 75 of all biopsy procedures declared in theESHRE PGD consortium data collection XII [2] thus it isthe mostly used approach However apparently all the threemethods do not impact clinical outcomes as randomizedcontrolled trials (RCTs) on sibling embryos have shown [13ndash16] Probably then the reason for the prevalence of laser-assisted method resides in the standardization and repro-ducibility of the hole produced within the zona pellucidawhich is less operator-dependent than the use of acidifiedTyrode [14] Furthermore besides being more precise andless time-consuming laser-assisted hatching requires also ashorter training period In fact the hole created by acidifiedTyrode depends on variables such as the amount of solutiondeposited the time of exposure and the operatorrsquos skillson the contrary few preset firings are sufficient according tolaser-assisted method

It has been estimated that the temperature in the mediasurrounding the embryo increases to 60ndash80∘C depending onthe intensity of the laser beam [17] However Taylor andcolleagues [18] highlighted that even when varying the laserpulse to be between 20 and 400mW biopsy operators didnot cause any negative effect on both technical and clinicaloutcomes after biopsy

Nevertheless zona pellucida breaching itself can impactsubsequent processes along preimplantation development up

to the blastocyst stage In particular several studies high-lighted the impairment of the blastocyst hatching processwhose seriousness depends on number and size of holesproduced [19ndash21] The severity of this issue is exacerbatedby the concurrent removal of one blastomere [22 23] Inparticular Kirkegaard and colleagues [23] compared thedevelopment to blastocyst stage of biopsied cleavage stageembryos versus control nonbiopsied embryos through time-lapse microscopy (TLM) They highlighted that the blasto-cysts obtained from biopsied embryos showed delayed com-paction process and hatched in a nonphysiological fashionbypassing the prolonged period of zona pellucida thinningThis translated to smaller blastocysts with thicker zonapellucida

Blastomere biopsy is also affected by problems associatedwith single cell analysis both technical (eg high rate ofamplification failure) [24 25] and biological In particularchromosomal mosaicism namely the presence of cells withdifferent karyotypes within the same embryo seems to reachits highest level at this stage of preimplantation development[26ndash29] In order to compensate for this a two-blastomerebiopsy strategy has been proposed However this strategycould involve a depletion of the embryonicmass of about 25and in turn impact clinical outcomes [30 31]On the contraryinitial evidences did not demonstrate a detrimental impactof blastomere(s) loss (after biopsy andor cryopreservation)on deriving embryo development and implantation potential[32ndash35] In fact ESHRE guidelines in 2010 suggested thatthis procedure could be safely applied when embryos arecomposed of ge6 cells with less than 30 of fragmentation[36]

In 2013 strong evidence against this approach arose fromthe pairedRCTby Scott Jr and colleagues [37] and completelychanged the previous scenario In particular here the twobest quality embryos produced within a single cohort duringan IVF cycle were selected for transfer either at the cleavageor at the blastocyst stage One embryo was randomized forbiopsy without aneuploidy testing and the other was insteadused as paired control Both embryos were then transferredIn case a single embryo implanted the biopsy fragment wassubmitted to aSNP-based fingerprinting as also either fetalDNA frommaternal blood or buccal DNA from the newbornafter delivery Matching results indicated that the implantedembryo was the biopsied one whereas nonmatching thatthe control embryo was the one that led to the delivery Adramatic 39 relative reduction in implantation rate wasreported when cleavage stage biopsy was conducted withrespect to control

An interesting theory is that embryos at this stageof preimplantation development are relatively fragile sinceEmbryonic Genome Activation and cell differentiation pro-cesses have not occurred yetThus downstreamdevelopmen-tal processes can be irreparably compromised by removing acell from the embryo Such an impact in fact reflects also in alower blastocyst rate after cleavage stage biopsy with respectto undisturbed embryos as reported in several papers [38ndash40]

Given the number of studies showing the ineffective-ness and potential impairment of cleavage stage biopsy it

BioMed Research International 3

is not surprising that Mastenbroek and colleagues [4] intheir review and meta-analysis highlighted the failure ofPGS when conducted by 9-chromosome FISH on biopsiedblastomere(s)

However many embryologists and molecular biologistsremained confident about the potential of aneuploidy testingin IVF Thus novel strategies to achieve this objective arebeing pursued At present RCTs to assess the clinical valueof blastomere biopsy when associated with CCS rather thanFISH are in the pipeline (NCT01571076 NCT01950104 reg-istered in httpswwwclinicaltrialsgov) Moreover novelbiopsy strategies are being investigated by moving eitherbackwards or onwards along the preimplantation develop-ment timeline

3 Polar Body Biopsy

Polar body (PB) biopsy on MII oocytes andor zygotes wasencouraged as a valuable alternative to blastomere biopsy[41 42] In some countries this was mainly due to legalreasons since embryo biopsy is not allowed PB biopsy ispotentially less invasive than any other stage of preimplan-tation development since it entails the removal of wasteproducts ofmeiosisHowever the applicability of this strategyhas always been under debate as mirrored by the ESHREPGD Consortium data In fact PB biopsy has been used inonly 10ndash15 of all the procedures performed in Europe in thelast decade Nowadays this rate is further decreasing probablydue to the number of studies that highlighted technicaleconomical biological and clinical deficiencies underlyingthe approach For instance Capalbo and colleagues [43]reported high false positive and negative error rates whenadopting this biopsy strategy In this regard mitotic andpaternally derived aneuploidies cannot in fact be detectedThe authors underlined also that both PBs from all theMII oocytes andor zygotes are needed regardless of theirdevelopmental potential thus making the procedure time-consuming This in turn increases the lab workload anotherimportant drawback related to this strategy

The methods for zona drilling are the same as previ-ously described for blastomere biopsy Several papers inthe literature described their application for PBs biopsyand reported no negative effects upon quality parameterssuch as oocytes lysis and activation rates development aftercalcium ionophore treatment embryo chromosome break-age incidence after Tarkowski preparation andor embryodevelopment [44ndash47] Neonatal outcomes after PB biopsy-based PGD are also comparable to those obtained withcleavage stage-based approach [13] On the contrary Levinand colleagues [48] reported a higher fragmentation ratea lower embryo quality a higher cleavage arrest rate anda lower mean number of blastomeres in day 3 when PBbiopsy is performed with respect to control However theauthors did not evaluate embryo implantation potentialafter this biopsy approach a limitation shared by all thepapers dealing with this topic No sufficiently powered well-controlled studies have been published that report a lackof PB biopsy impact upon embryo implantation potentialIn light of this absence the safety of the procedure still

remains an arguable assumption [49] To this regard thereare two ongoing studies currently registered on the websitehttpswwwclinicaltrialsgov that deal with PB biopsy theESHRE ESTEEM RCT (NCT01532284) and a study by theWeill Medical College (Cornell University) (NCT01574404)Hopefully they will report powerful data about the clinicalefficiency of PB biopsy to resolve the remaining issues

A last operative concern regards whether to follow asequential or simultaneous biopsy approach Specificallyaccording to the former the PBs are retrieved at differenttimes while according to the latter both are retrieved atonce Even though guidelines regarding the proper timing forbiopsy have not been established it should occur between8 and 14 hours after fertilization By performing the biopsybefore this time range there is a risk of enucleation dueto spindle remnants in the second PB (Figure 1) while PBdisintegration or degeneration might occur if the biopsy isperformed later [42 50] Following a simultaneous biopsyrather than a sequential one a double exposure to suboptimalenvironmental conditions can also be preventedHowever noclinical data have been produced up to date to solve this issue

To conclude the application of PB biopsy has beengradually reduced in favor of TE biopsy due to the absenceof reliable supporting data and the possibility of diagnosticinaccuracy

4 Morula Stage Biopsy

Recently morula stage biopsy has been proposed [51] Fewdata have been produced to evaluate its actual feasibilityhowever it is technically similar to cleavage stage biopsyand thus it shares its same drawbacks (eg the need forCa++Mg++-free buffer to loosen compaction) The mainadvantage of morula stage biopsy is instead shared withTE biopsy approach namely the number of cells retrievedThe analysis of more than a single cell leads in fact to amore robust downstreammolecular investigation which setsamong the reasons that prompted blastocyst stage biopsystrategy

5 Blastocyst Stage Biopsy

Blastocyst stage biopsy strategy was an important break-through in modern IVF It was reported for the first timeby de Boer and colleagues in 2004 [52] and the first livebirths following this approach were reported in 2005 byKokkali and colleagues [53] and by McArthur and colleagues[30] respectively Several preclinical and clinical studies soonrecognized its value so that at present it is gradually replacingboth cleavage stage and PB biopsy approaches

The power of TE biopsy resides in its higher technical andbiological robustnessThis approach in fact entails both lowerinfluence of procedural errors and lower impact ofmosaicismon the molecular analysis

However high standards are required for blastocystculture and cryopreservation which is an important limitingfactor for the widespread implementation of this strategyNevertheless once a proper culture system is set blastocyst


(a) (b) (c) (d)

(a998400) (b998400) (c998400) (d998400)

Figure 1 Oocyte first polar body biopsy prior to fertilization displayed through polarized light microscopy Polarized light microscopy allowsidentification of the chromosome meiotic spindle (indicated by the white arrows in all figures) (a)ndash(d) Metaphase II oocyte first polar bodybiopsy with no damage to the meiotic spindle (a1015840)ndash(d1015840) Telophase I oocyte first polar body biopsy with enucleation of the oocyte whosemeiotic spindle remains attached to the polar body during aspiration

culture itself elicits higher live birth rate per embryo transferthan cleavage stage This aspect in particular was highlightedby Glujovsky and colleagues in their Cochrane review of 12RCTs [54] Moreover it is important to underline that alsocleavage stage biopsy subtended culture to the blastocyst stageif aiming at performing a fresh embryo transfer of euploidembryos [37ndash39] Thus if any risk derived from the culturesystem it would be shared by both biopsy approaches

In order not to lose precious euploid blastocysts afterwarming also an excellent vitrification program is requiredIn this regard several papers in literature reported noblastocyst degeneration after biopsy [30 55 56] and a survivalrate after warming always higher than 95 [30 57 58]

Following a cycle segmentation and SET policy euploidcryopreserved blastocyst transfer also prevents hyperstimu-lation syndrome and multiple pregnancy [59 60] a furtherimportant advantage

We have recently evaluated our clinical practice across a4-year period during which blastocyst stage PGDPGS pluseuploid SET policy was gradually implemented especially foradvanced maternal age patients Such a clinical setting didnot affect the global efficacy of our treatments namely thepregnancy rate per oocyte retrieval was kept constant withrespect to the past but increased their global efficiency Inparticular a significant increase in the overall pregnancy rateper transfer and the reduction in the multiple pregnancy rateafter the implementation of this novel setting were reported[59] In this scenario Forman et al [61] also demonstratedthat single euploid blastocyst transfer equals the implantationrate of double untested blastocyst transfer but it elicits betterobstetrical and perinatal outcomes [62]

Finally the paired RCT by Scott Jr and colleagues [37]is again the real milestone that identified blastocyst stage

biopsy as a procedure that does not affect embryo viabilityand implantation potential TE biopsy was reported to haveno impact converse to what was found for blastomere biopsyA possible explanation for this difference is that a smallerproportion of the whole cellular constitution of the embryois removed from a nonembryonic portion of the blastocystand at a stage of preimplantation development perhaps moretolerant to manipulation

This evidencewas pivotal for the growing implementationof the novel TE biopsy CCS-based PGS policy worldwideDahdouh and colleagues recently underlined in a meta-analysis and in a review the positive clinical predictivevalue underlying this policy [63 64] Several RCTs arealso in the pipeline and they will provide additional evi-dences during the next years (NCT01219283 NCT02032264NCT02268786 NCT01977144 and NCT01917240 registeredin httpswwwclinicaltrialsgov ISRCTN81216689 regis-tered in httpwwwisrctncom) In particular blastocyststage biopsy will be evaluated together with next-generationsequencing or quantitative polymerase chain reaction analy-ses The additional predictive value of the blastocyst culturesystem adopted and of euploid blastocyst morphology will bealso investigated

The systematic review by Lee and colleagues [65] sum-marized all the observational prospective analyses and RCTspublished to date However here the authors underlined animportant limitation of many studies specifically comparingthe clinical outcomes between TE and blastomere biopsyapproaches In particular even if a higher ongoingclinicalpregnancy rate was always reported through the formerapproach no correction for patient andor embryo char-acteristics was provided and no prospective randomizationwas applied The prospective double-blinded nonselectionstudy by Scott Jr and colleagues [66] is then at least to


+

(1)

(2)

ICM

ICM + TE

TE

(a)

+

ICM

TEICM

ICM

(b)

Figure 2 Schematic comparison between two different blastocyst biopsy approaches (a) Day 3 hatching-based blastocyst biopsy entailingthe production of a hole in the zona pellucida at the cleavage stage and the biopsy of hatching trophectoderm cells from that hole Severalpitfalls can derive a thicker zona pellucida and a smaller blastocyst since being composed by fewer and bigger trophectoderm cells Hatchingcan either occur far from the ICM (a1) or involve the ICM itself impairing the procedure (a2) (b) zona opening with simultaneous blastocystbiopsy approach leaves the embryo undisturbed throughout its in vitro development up to the fully expanded blastocyst stage Simultaneouslyopening the zona and retrieving the fragment allow the operator to choose the area and the amount of cells to biopsy Circle with inner crossindicates laser pulse TE trophectoderm ICM Inner Cell Mass

our knowledge the only paper properly designed to conductthis investigation Here by comparing the positive clinicalpredictive values (rate of embryos actually leading to anongoing pregnancy after a CCS-based euploid diagnosis) ofthe two approaches they confirm the higher reliability ofblastocyst stage analysis with respect to cleavage stage one(482 versus 292 119901 = 00016)

Nonetheless the most commonly used strategy to con-duct PGDPGS in Europe still entails cleavage stage ratherthan TE biopsyThe perception of the former as less operator-dependent and more reproducible possibly underlies thistendency In particular embryos reach cleavage stage syn-chronously and are similar in terms of morphological qualityand a single biopsy protocol has been described in litera-ture Blastocyst stage biopsy instead is characterized by aheterogeneous cohort of embryos in terms of both mor-phology and developmental rate and two different methods

to perform it have been published up to date The firstmethod has been described by McArthur and colleagues[30] and entails a hole in the zona pellucida performedin day 3 of preimplantation development A consequentnonphysiological hatching at the blastocyst stage makes theprocedure easier but it also exposes the embryo to a potentialstress along preimplantation development [23] The secondmethod instead was described by Capalbo and colleagues[67] and entails simultaneous zona opening and TE biopsyThe embryo is thus left undisturbed up to day 5 day 6or even day 7 and it is biopsied exclusively after reachingfull expansion (Figure 2) We investigated the accuracy andthe reproducibility of this second approach associated withqPCR-basedCCSNo significant differences across 7 differentoperators from 3 IVF centers in terms of both technicaland clinical results were reported In particular amplificationrate qPCR data concurrence and estimated number of cells


No evidence to date of degeneration after biopsy

Standardized technique

High risk of amplification failure FPFN results exclusion of paternal genome

Generally more accepted from a legal point of view

No impact on implantation potential and cryopreservation

Accurate reliable and reproducible

Removal of a nonembryonic portion of the blastocyst

Removal of a low proportion of total blastocystrsquos cell number

No meaningful data about impact on implantation

Risk of enucleation or PB degeneration

Impact on embryo development

Biopsy of both PBs needed

Risk of amplification failure and high rate of technical artefacts

Removal of a portion of the embryo regardless of its future destiny

Significant decrease in implantation potential

High embryonic mass depletion


(a)

(b)

(c)

Figure 3 Comparison between different biopsy stages Despite the fact that trophectoderm-based blastocyst biopsy approach (c) is not sucha widespread method as cleavage stage one (b) several preclinical and clinical evidences recognized its value and highlighted its advantageswith respect to the latter as also in comparison with polar body approach (a) Black arrows indicate negative evidences described in literaturewhite arrows indicate positive evidences described in literature question marks indicate still controversial aspects PB polar body TEtrophectoderm FP false positive FN false negative

retrieved as well as ongoing implantation biochemical andmiscarriage rates were comparable [68]

Amplification rate in particular is an important param-eter since a second biopsy would be needed in case of anonconclusive result Importantly all the papers where TE-based CCS analysis was adopted reported always less than30 of undiagnosed blastocysts [56 58 62 67 68] Thispoint represents a further advantage of this approach withrespect to the previous single cell-based ones

6 Future Perspectives

An ideal outcome would be to bypass the embryo biopsystep and predict euploidy andor reproductive competencethrough noninvasive methods For instance static mor-phological evaluation TLM criteria and geneticproteomicmetabolomic screening of spent IVF culture media were allproposed as promising tools Unfortunately several papersdefined conventional parameters of embryo evaluation as just

mildly correlated with aneuploidy rate [67 69 70] TLM asa limited tool to predict euploidyimplantation [71ndash73] andspent IVF culture media analysis as a fascinating theory thatdid not provide clinical benefit to date [74 75]

The current perception is that noninvasive techniquescould provide novel parameters to enhance our predictivepower upon implantation beyond the level guaranteed byPGDPGS rather than trying to replace this tool

7 Conclusion

Amniocentesis and chorionic villus sampling are methodsavailable to detect vital chromosomal syndromes in the fetusduring gestation however these approaches expose womento a high risk of miscarriage ranging between 1 and 3 [76ndash80] Chan and colleagues [81] underlined the importanceof safety for patients by reporting that pregnant Chinesewomen are more prone to accept a moderate risk of undiag-nosed aneuploidies than a procedure-relatedmiscarriage risk


ge1 Combined with the possibility of significantly reducingimplantation failure andor miscarriage rates through PGS itis mandatory to adopt a safe biopsy method Similarly thisneed applies to PGD for monogenic mutations a techniquewhich requires further costs due to molecular probes con-struction for embryo diagnosis

The evidence produced in the last decades extensivelyhighlights the drawbacks of the cleavage stage approach inPGDPGS A significant decrease in clinical outcomes derivesfrom the use of such a harmful biopsy strategy

Sufficiently powered studies highlighting a similar neg-ative impact for PB biopsy are still missing However thisstrategy suffers from important diagnostic issues leading tohigh false positive and false negative error rates [43]

The blastocyst approach instead ensures accuracy relia-bility and reproducibility and importantly shows no impactupon embryo viability and reproductive potential The com-parison of TE biopsy strategy to previous ones (summarizedin Figure 3) strongly suggests that it is the most promisingapproach in PGDPGS

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] E CoonenMDeRycke G Kokkali et alData from the ESHREPGD Consortium ESHRE Abstract Book Annual Meeting2015

[2] C Moutou V Goossens E Coonen et al ldquoESHRE PGDConsortium data collection XII cycles from January to Decem-ber 2009 with pregnancy follow-up to October 2010rdquo HumanReproduction vol 29 no 5 pp 880ndash903 2014

[3] J Traeger-Synodinos E C Coonen M De Rycke et al Datafrom the ESHRE PGD Consortium ESHRE Abstract BookAnnual Meeteing 2014

[4] S Mastenbroek M Twisk F van der Veen and S ReppingldquoPreimplantation genetic screening a systematic review andmeta-analysis of RCTsrdquo Human Reproduction Update vol 17no 4 pp 454ndash466 2011

[5] J C Dumoulin M Bras E Coonen J Dreesen J P Geraedtsand J L Evers ldquoEffect of Ca2+Mg2+-free medium on thebiopsy procedure for preimplantation genetic diagnosis and fur-ther development of human embryosrdquo Human Reproductionvol 13 no 10 pp 2880ndash2883 1998

[6] H P M Pratt C A Ziomek W J D Reeve and M HJohnson ldquoCompaction of the mouse embryo an analysisof its componentsrdquo Journal of Embryology and ExperimentalMorphology vol 70 pp 113ndash132 1982

[7] L Clayton S V Stinchcombe andM H Johnson ldquoCell surfacelocalisation and stability of uvomorulin during early mousedevelopmentrdquo Zygote vol 1 no 4 pp 333ndash344 1993

[8] M Sefton M H Johnson L Clayton and J M L McConnellldquoExperimental manipulations of compaction and their effectson the phosphorylation of uvomorulinrdquo Molecular Reproduc-tion and Development vol 44 no 1 pp 77ndash87 1996

[9] R Pey C Vial G Schatten and M Hafner ldquoIncrease of intra-cellular Ca2+ and relocation of E-cadherin during experimental

decompaction of mouse embryosrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 95 no22 pp 12977ndash12982 1998

[10] W Feichtinger H Strohmer P Fuhrberg et al ldquoPhotoablationof oocyte zona pellucida by erbium-YAG laser for in-vitrofertilisation in severe male infertilityrdquo The Lancet vol 339 no8796 p 811 1992

[11] J Cohen C Elsner H Kort H Malter J Massey and MP Mayer ldquoImmunosuppression supports implantation of zonapellucida dissected human embryosrdquo Fertility and Sterility vol53 no 4 pp 662ndash665 1990

[12] J Cohen ldquoAssisted hatching of human embryosrdquo Journal of InVitro Fertilization and Embryo Transfer vol 8 no 4 pp 179ndash190 1991

[13] T Eldar-Geva N Srebnik G Altarescu et al ldquoNeonataloutcome after preimplantation genetic diagnosisrdquo Fertility andSterility vol 102 no 4 pp 1016ndash1021 2014

[14] A De Vos and A Van Steirteghem ldquoAspects of biopsy pro-cedures prior to preimplantation genetic diagnosisrdquo PrenatalDiagnosis vol 21 no 9 pp 767ndash780 2001

[15] S Geber R Bossi C B Lisboa M Valle and M SampaioldquoLaser confers less embryo exposure than acid tyrode forembryo biopsy in preimplantation genetic diagnosis cycles arandomized studyrdquo Reproductive Biology and Endocrinologyvol 28 no 9 p 58 2011

[16] A E Jones G Wright H I Kort R J Straub and Z PNagy ldquoComparison of laser-assisted hatching and acidifiedTyrodersquos hatching by evaluation of blastocyst development ratesin sibling embryos a prospective randomized trialrdquoFertility andSterility vol 85 no 2 pp 487ndash491 2006

[17] K Rink G Delacretaz R P Salathe et al ldquoNon-contact micro-drilling of mouse zona pellucida with an objective-delivered148 120583m diode laserrdquo Lasers in Surgery and Medicine vol 18 no1 pp 52ndash62 1996

[18] T H Taylor J W Gilchrist S V Hallowell et al ldquoThe effects ofdifferent laser pulse lengths on the embryo biopsy procedureand embryo development to the blastocyst stagerdquo Journal ofAssisted Reproduction and Genetics vol 27 no 11 pp 663ndash6672010

[19] H E Malter and J Cohen ldquoBlastocyst formation and hatchingin vitro following zona drilling of mouse and human embryosrdquoGamete Research vol 24 no 1 pp 67ndash80 1989

[20] J Cohen and D Feldberg ldquoEffects of the size and numberof zona pellucida openings on hatching and trophoblast out-growth in the mouse embryordquo Molecular Reproduction andDevelopment vol 30 no 1 pp 70ndash78 1991

[21] F Schmoll H Schneider M Montag K Wimmers K Rinkand K Schellander ldquoEffects of different laser-drilled openingsin the zona pellucida on hatching of in vitro-produced cattleblastocystsrdquo Fertility and Sterility vol 80 supplement 2 pp714ndash719 2003

[22] F E Duncan P Stein C J Williams and R M Schultz ldquoTheeffect of blastomere biopsy on preimplantation mouse embryodevelopment and global gene expressionrdquo Fertility and Sterilityvol 91 no 4 pp 1462ndash1465 2009

[23] K Kirkegaard J J Hindkjaer and H J Ingerslev ldquoHumanembryonic development after blastomere removal a time-lapseanalysisrdquo Human Reproduction vol 27 no 1 pp 97ndash105 2012

[24] D Brodie C E Beyer E Osborne V Kralevski S Rasi and TOsianlis ldquoPreimplantation genetic diagnosis for chromosomerearrangementsmdashone blastomere biopsy versus two blastomere


biopsyrdquo Journal of Assisted Reproduction and Genetics vol 29no 8 pp 821ndash827 2012

[25] G Harton J Traeger-Syndinos and V Goossens ldquoData fromthe ESHRE PGD consortiumrdquo Human Reproduction vol 27supplement 2 p ii58 2012

[26] S Munne K M Sultan H U Weier J A Grifo J Cohen andZ Rosenwaks ldquoAssessment of numeric abnormalities of X Y18 and 16 chromosomes in preimplantation human embryosbefore transferrdquo American Journal of Obstetrics and Gynecologyvol 172 no 4 part 1 pp 1191ndash1199 1995

[27] D Wells and J D A Delhanty ldquoComprehensive chromoso-mal analysis of human preimplantation embryos using wholegenome amplification and single cell comparative genomichybridizationrdquo Molecular Human Reproduction vol 6 no 11pp 1055ndash1062 2000

[28] L Voullaire H Slater R Williamson and L Wilton ldquoChro-mosome analysis of blastomeres from human embryos by usingcomparative genomic hybridizationrdquoHuman Genetics vol 106no 2 pp 210ndash217 2000

[29] M Bielanska S L Tan and A Ao ldquoChromosomal mosaicismthroughout human preimplantation development in vitroincidence type and relevance to embryo outcomerdquo HumanReproduction vol 17 no 2 pp 413ndash419 2002

[30] S JMcArthurD Leigh J TMarshall KADeBoer andR P SJansen ldquoPregnancies and live births after trophectoderm biopsyand preimplantation genetic testing of human blastocystsrdquoFertility and Sterility vol 84 no 6 pp 1628ndash1636 2005

[31] J Cohen D Wells and S Munne ldquoRemoval of 2 cells fromcleavage stage embryos is likely to reduce the efficacy ofchromosomal tests that are used to enhance implantation ratesrdquoFertility and Sterility vol 87 no 3 pp 496ndash503 2007

[32] J Cieslak-Janzen I Tur-Kaspa Y Ilkevitch A Bernal RMorris and Y Verlinsky ldquoMultiple micromanipulations forpreimplantation genetic diagnosis do not affect embryo devel-opment to the blastocyst stagerdquo Fertility and Sterility vol 85 no6 pp 1826ndash1829 2006

[33] K Hardy K L Martin H J Leese R M L Winston and AH Handyside ldquoHuman preimplantation development in vitrois not adversely affected by biopsy at the 8-cell stagerdquo HumanReproduction vol 5 no 6 pp 708ndash714 1990

[34] V Goossens M De Rycke A De Vos et al ldquoDiagnosticefficiency embryonic development and clinical outcome afterthe biopsy of one or two blastomeres for preimplantationgenetic diagnosisrdquoHuman Reproduction vol 23 no 3 pp 481ndash492 2008

[35] L Rienzi FUbaldiM Iacobelli et al ldquoDevelopmental potentialof fully intact and partially damaged cryopreserved embryosafter laser-assisted removal of necrotic blastomeres and post-thaw culture selectionrdquo Fertility and Sterility vol 84 no 4 pp888ndash894 2005

[36] G L Harton M C Magli K Lundin M Montag J Lemmenand J C Harper ldquoESHRE PGD ConsortiumEmbryology Spe-cial Interest Groupmdashbest practice guidelines for polar body andembryo biopsy for preimplantation genetic diagnosisscreening(PGDPGS)rdquo Human Reproduction vol 26 no 1 pp 41ndash462011

[37] R T Scott Jr K M Upham E J Forman T Zhao and NR Treff ldquoCleavage-stage biopsy significantly impairs humanembryonic implantation potential while blastocyst biopsy doesnot a randomized and paired clinical trialrdquo Fertility andSterility vol 100 no 3 pp 624ndash630 2013

[38] C Staessen W Verpoest P Donoso et al ldquoPreimplantationgenetic screening does not improve delivery rate in womenunder the age of 36 following single-embryo transferrdquo HumanReproduction vol 23 no 12 pp 2818ndash2825 2008

[39] S Debrock C Melotte C Spiessens et al ldquoPreimplantationgenetic screening for aneuploidy of embryos after in vitrofertilization in women aged at least 35 years a prospectiverandomized trialrdquo Fertility and Sterility vol 93 no 2 pp 364ndash373 2010

[40] T Hardarson C Hanson K Lundin et al ldquoPreimplantationgenetic screening in women of advanced maternal age causeda decrease in clinical pregnancy rate a randomized controlledtrialrdquoHuman Reproduction vol 23 no 12 pp 2806ndash2812 2008

[41] E Fragouli M Katz-Jaffe S Alfarawati et al ldquoComprehensivechromosome screening of polar bodies and blastocysts fromcouples experiencing repeated implantation failurerdquo Fertilityand Sterility vol 94 no 3 pp 875ndash887 2010

[42] M Montag M Koster T Strowitzki and B Toth ldquoPolar bodybiopsyrdquo Fertility and Sterility vol 100 no 3 pp 603ndash607 2013

[43] A Capalbo S Bono L Spizzichino et al ldquoSequential compre-hensive chromosome analysis on polar bodies blastomeres andtrophoblast insights into female meiotic errors and chromo-somal segregation in the preimplantation window of embryodevelopmentrdquoHuman Reproduction vol 28 no 2 pp 509ndash5182013

[44] M Montag K van der Ven B Rosing and H van der VenldquoPolar body biopsy a viable alternative to preimplantationgenetic diagnosis and screeningrdquo Reproductive BioMedicineOnline vol 18 no 1 pp 6ndash11 2009

[45] M CMagli MMontagM Kster et al ldquoPolar body array CGHfor prediction of the status of the corresponding oocyte Part IItechnical aspectsrdquoHumanReproduction vol 26 no 11 pp 3181ndash3185 2011

[46] M Montag K van der Ven G Delacretaz K Rink and H vander Ven ldquoLaser-assisted microdissection of the zona pellucidafacilitates polar body biopsyrdquo Fertility and Sterility vol 69 no3 pp 539ndash542 1998

[47] I Hammoud D Molina-Gomes M Albert et al ldquoAre zonapellucida laser drilling and polar body biopsy safe for invitro matured oocytesrdquo Journal of Assisted Reproduction andGenetics vol 27 no 7 pp 423ndash427 2010

[48] I Levin B Almog T Shwartz et al ldquoEffects of laser polar-bodybiopsy on embryo qualityrdquo Fertility and Sterility vol 97 no 5pp 1085ndash1088 2012

[49] K L Scott KHHong andR T Scott Jr ldquoSelecting the optimaltime to perform biopsy for preimplantation genetic testingrdquoFertility and Sterility vol 100 no 3 pp 608ndash614 2013

[50] L Albricci S Romano R Maggiulli et alUse of Polarized LightMicroscopi to Assess Meiotic Spindle Configuration Prior to 1PBBiopsy for PGD ESHRE Abstract Book Annual Meeting 2009

[51] E E Zakharova V V Zaletova and A S KrivokharchenkoldquoBiopsy of human morula-stage embryos outcome of 215IVFICSI cycles with PGSrdquo PLoS ONE vol 9 no 9 Article IDe106433 2014

[52] K A de Boer J W Catt R P S Jansen D Leigh and SMcArthur ldquoMoving to blastocyst biopsy for preimplantationgenetic diagnosis and single embryo transfer at Sydney IVFrdquoFertility and Sterility vol 82 no 2 pp 295ndash298 2004

[53] G Kokkali C Vrettou J Traeger-Synodinos et al ldquoBirth of ahealthy infant following trophectodermbiopsy fromblastocystsfor PGD of 120573-thalassaemia major case reportrdquo Human Repro-duction vol 20 no 7 pp 1855ndash1859 2005


[54] D Glujovsky D Blake C Farquhar and A Bardach ldquoCleavagestage versus blastocyst stage embryo transfer in assisted repro-ductive technologyrdquoCochraneDatabase Systematic Reviews vol11 no 7 Article ID CD002118 2012

[55] W B Schoolcraft E Fragouli J Stevens S Munne M GKatz-Jaffe and DWells ldquoClinical application of comprehensivechromosomal screening at the blastocyst stagerdquo Fertility andSterility vol 94 no 5 pp 1700ndash1706 2010

[56] S J McArthur D Leigh J T Marshall A J Gee K A DeBoer and R P S Jansen ldquoBlastocyst trophectoderm biopsyand preimplantation genetic diagnosis for familial monogenicdisorders and chromosomal translocationsrdquoPrenatal Diagnosisvol 28 no 5 pp 434ndash442 2008

[57] A Cobo M J de los Santos D Castello P Gamiz P Camposand J Remohı ldquoOutcomes of vitrified early cleavage-stageand blastocyst-stage embryos in a cryopreservation programevaluation of 3150 warming cyclesrdquo Fertility and Sterility vol98 no 5 pp 1138e1ndash1146e1 2012

[58] A Capalbo N R Treff D Cimadomo et al ldquoComparison ofarray comparative genomic hybridization and quantitative real-time PCR-based aneuploidy screening of blastocyst biopsiesrdquoEuropean Journal of Human Genetics vol 23 no 7 pp 901ndash9062015

[59] F M Ubaldi A Capalbo S Colamaria et al ldquoReduction ofmultiple pregnancies in the advanced maternal age populationafter implementation of an elective single embryo transferpolicy coupled with enhanced embryo selection pre- and post-intervention studyrdquo Human Reproduction vol 30 no 9 pp2097ndash2106 2015

[60] B S Shapiro S T Daneshmand H Restrepo F C GarnerM Aguirre and C Hudson ldquoMatched-cohort comparison ofsingle-embryo transfers in fresh and frozen-thawed embryotransfer cyclesrdquo Fertility and Sterility vol 99 no 2 pp 389ndash3922013

[61] E J FormanKHHongKM Ferry et al ldquoIn vitro fertilizationwith single euploid blastocyst transfer a randomized controlledtrialrdquo Fertility and Sterility vol 100 no 1 pp 100ndash107e1 2013

[62] E J Forman K H Hong J M Franasiak and R T Scott JrldquoObstetrical and neonatal outcomes from the BEST Trial singleembryo transfer with aneuploidy screening improves outcomesafter in vitro fertilization without compromising delivery ratesrdquoAmerican Journal of Obstetrics and Gynecology vol 210 no 2pp 157e1ndash157e6 2014

[63] E M Dahdouh J Balayla and J A Garcıa-Velasco ldquoCompre-hensive chromosome screening improves embryo selection ameta-analysisrdquo Fertility and Sterility vol 104 no 6 pp 1503ndash1512 2015

[64] E M Dahdouh J Balayla and F Audibert ldquoTechnical updatepreimplantation genetic diagnosis and screeningrdquo Journal ofObstetrics and Gynaecology Canada vol 37 no 5 pp 451ndash4632015

[65] E Lee P Illingworth L Wilton and G M Chambers ldquoTheclinical effectiveness of preimplantation genetic diagnosis foraneuploidy in all 24 chromosomes (PGD-A) systematic reviewrdquoHuman Reproduction vol 30 no 2 pp 473ndash483 2015

[66] R T Scott Jr K Ferry J Su X Tao K Scott and N R TreffldquoComprehensive chromosome screening is highly predictive ofthe reproductive potential of human embryos a prospectiveblinded non-selection studyrdquo Fertility and Sterility vol 97 no4 pp 870ndash875 2012

[67] A Capalbo L Rienzi D Cimadomo et al ldquoCorrelationbetween standard blastocystmorphology euploidy and implan-tation an observational study in two centers involving 956screened blastocystsrdquo Human Reproduction vol 29 no 6 pp1173ndash1181 2014

[68] A Capalbo F M Ubaldi D Cimadomo et al ldquoConsistentand reproducible outcomes of blastocyst biopsy and aneuploidyscreening across different biopsy practitioners a multicentrestudy involving 2586 embryo biopsiesrdquo Human Reproductionvol 31 no 1 pp 199ndash208 2015

[69] Z Yang J Liu G S Collins et al ldquoSelection of single blastocystsfor fresh transfer via standard morphology assessment aloneand with array CGH for good prognosis IVF patients resultsfrom a randomized pilot studyrdquo Molecular Cytogenetics vol 5no 1 p 24 2012

[70] S Alfarawati E Fragouli P Colls et al ldquoThe relationshipbetween blastocyst morphology chromosomal abnormalityand embryo genderrdquo Fertility and Sterility vol 95 no 2 pp520ndash524 2011

[71] Z Yang J Zhang S A Salem et al ldquoSelection of competentblastocysts for transfer by combining time-lapse monitoringand array CGH testing for patients undergoing preimplantationgenetic screening a prospective study with sibling oocytesrdquoBMCMedical Genomics vol 7 article 38 2014

[72] L Rienzi A Capalbo M Stoppa et al ldquoNo evidence ofassociation between blastocyst aneuploidy and morphokineticassessment in a selected population of poor-prognosis patientsa longitudinal cohort studyrdquo Reproductive BioMedicine Onlinevol 30 no 1 pp 57ndash66 2015

[73] D J Kaser and C Racowsky ldquoClinical outcomes followingselection of human preimplantation embryos with time-lapsemonitoring a systematic reviewrdquoHuman Reproduction Updatevol 20 no 5 Article ID dmu023 pp 617ndash631 2014

[74] K Kirkegaard A S P Svane J S Nielsen J J HindkjaeligrN C Nielsen and H J Ingerslev ldquoNuclear magnetic reso-nance metabolomic profiling of Day 3 and 5 embryo culturemedium does not predict pregnancy outcome in good prog-nosis patients a prospective cohort study on single transferredembryosrdquo Human Reproduction vol 29 no 11 pp 2413ndash24202014

[75] T Hardarson A Ahlstrom L Rogberg et al ldquoNon-invasivemetabolomic profiling of Day 2 and 5 embryo culture mediuma prospective randomized trialrdquo Human Reproduction vol 27no 1 pp 89ndash96 2012

[76] M S Verp ldquoPrenatal diagnosis of genetic disordersrdquo in Princi-ples and Practice of Medical Therapy in Pregnancy N GleicherEd pp 159ndash170 Appleton and Lange Norwalk Conn USA2nd edition 1992

[77] G Schemmer and A Johnson ldquoGenetic amniocentesis andchorionic villus samplingrdquo Obstetrics amp Gynecology Clinics ofNorth America vol 20 no 3 pp 497ndash521 1993

[78] A B Caughey LMHopkins andMENorton ldquoChorionic vil-lus sampling comparedwith amniocentesis and the difference inthe rate of pregnancy lossrdquo Obstetrics and Gynecology vol 108no 3 part 1 pp 612ndash616 2006

[79] A Tabor C H F Vestergaard and Oslash Lidegaard ldquoFetal lossrate after chorionic villus sampling and amniocentesis an11-year national registry studyrdquo Ultrasound in Obstetrics andGynecology vol 34 no 1 pp 19ndash24 2009


[80] L G Jackson J M Zachary S E Fowler et al ldquoA randomizedcomparison of transcervical and transabdominal chorionic-villus samplingrdquoThe New England Journal of Medicine vol 327no 9 pp 594ndash598 1992

[81] YM Chan D S Sahota O K Chan T Y Leung and T K LauldquoMiscarriage after invasive prenatal diagnostic procedures howmuch risk our pregnant women are willing to takerdquo PrenatalDiagnosis vol 29 no 9 pp 870ndash874 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


alone accounted for almost 90 of the total while TE biopsyaccounted for less than 1 The same data referring to years2012-2013 [3] instead showed an impressive countertendencysince the rate of PB biopsy and cleavage stage biopsy proce-dures decreased to approximately 2 and 75 respectivelyBlastocyst stage biopsy implementation instead is graduallyincreasing in ART The reason for this dramatic changeis a review and meta-analysis published by Mastenbroekand colleagues in 2011 clearly showing that PGS as it wasconducted namely cleavage stage biopsy analyzed by 9-chromosome FISH is a harmful procedure [4] Howeverwhile 9-chromosome FISH use has been strongly reduced infavor of themore accurate and reliable Comprehensive Chro-mosome Screening (CCS) techniques blastomere biopsy isstill the method mainly adopted for PGD The use of PBbiopsy did not spread due to its intrinsic logistic clinical andtechnical drawbacks that compromise its accuracy especiallywhen compared to TE biopsy strategy

2 Cleavage Stage Biopsy

Cleavage stage biopsy is normally performed on day 3embryos with at least 6 blastomeres The zona pellucida isopened and Ca++Mg++-free medium is used in order toloosen cell-cell adhesion and facilitate selected blastomereremoval A less traumatic impact on the growing embryoshould theoretically not affect blastulation [5] Howeverthis is a controversial issue since several studies in mouseshowed that Ca++ depletion caused remodeling of the cellularcytoskeleton inevitably impacting compaction [6ndash9]

The biopsy is mainly conducted following 3 methods ofzona breaching namely laser-assisted [10] mechanical [11]and Tyrodersquos drilling [12] Use of the laser-assisted methodrepresents 75 of all biopsy procedures declared in theESHRE PGD consortium data collection XII [2] thus it isthe mostly used approach However apparently all the threemethods do not impact clinical outcomes as randomizedcontrolled trials (RCTs) on sibling embryos have shown [13ndash16] Probably then the reason for the prevalence of laser-assisted method resides in the standardization and repro-ducibility of the hole produced within the zona pellucidawhich is less operator-dependent than the use of acidifiedTyrode [14] Furthermore besides being more precise andless time-consuming laser-assisted hatching requires also ashorter training period In fact the hole created by acidifiedTyrode depends on variables such as the amount of solutiondeposited the time of exposure and the operatorrsquos skillson the contrary few preset firings are sufficient according tolaser-assisted method

It has been estimated that the temperature in the mediasurrounding the embryo increases to 60ndash80∘C depending onthe intensity of the laser beam [17] However Taylor andcolleagues [18] highlighted that even when varying the laserpulse to be between 20 and 400mW biopsy operators didnot cause any negative effect on both technical and clinicaloutcomes after biopsy

Nevertheless zona pellucida breaching itself can impactsubsequent processes along preimplantation development up

to the blastocyst stage In particular several studies high-lighted the impairment of the blastocyst hatching processwhose seriousness depends on number and size of holesproduced [19ndash21] The severity of this issue is exacerbatedby the concurrent removal of one blastomere [22 23] Inparticular Kirkegaard and colleagues [23] compared thedevelopment to blastocyst stage of biopsied cleavage stageembryos versus control nonbiopsied embryos through time-lapse microscopy (TLM) They highlighted that the blasto-cysts obtained from biopsied embryos showed delayed com-paction process and hatched in a nonphysiological fashionbypassing the prolonged period of zona pellucida thinningThis translated to smaller blastocysts with thicker zonapellucida

Blastomere biopsy is also affected by problems associatedwith single cell analysis both technical (eg high rate ofamplification failure) [24 25] and biological In particularchromosomal mosaicism namely the presence of cells withdifferent karyotypes within the same embryo seems to reachits highest level at this stage of preimplantation development[26ndash29] In order to compensate for this a two-blastomerebiopsy strategy has been proposed However this strategycould involve a depletion of the embryonicmass of about 25and in turn impact clinical outcomes [30 31]On the contraryinitial evidences did not demonstrate a detrimental impactof blastomere(s) loss (after biopsy andor cryopreservation)on deriving embryo development and implantation potential[32ndash35] In fact ESHRE guidelines in 2010 suggested thatthis procedure could be safely applied when embryos arecomposed of ge6 cells with less than 30 of fragmentation[36]

In 2013 strong evidence against this approach arose fromthe pairedRCTby Scott Jr and colleagues [37] and completelychanged the previous scenario In particular here the twobest quality embryos produced within a single cohort duringan IVF cycle were selected for transfer either at the cleavageor at the blastocyst stage One embryo was randomized forbiopsy without aneuploidy testing and the other was insteadused as paired control Both embryos were then transferredIn case a single embryo implanted the biopsy fragment wassubmitted to aSNP-based fingerprinting as also either fetalDNA frommaternal blood or buccal DNA from the newbornafter delivery Matching results indicated that the implantedembryo was the biopsied one whereas nonmatching thatthe control embryo was the one that led to the delivery Adramatic 39 relative reduction in implantation rate wasreported when cleavage stage biopsy was conducted withrespect to control

An interesting theory is that embryos at this stageof preimplantation development are relatively fragile sinceEmbryonic Genome Activation and cell differentiation pro-cesses have not occurred yetThus downstreamdevelopmen-tal processes can be irreparably compromised by removing acell from the embryo Such an impact in fact reflects also in alower blastocyst rate after cleavage stage biopsy with respectto undisturbed embryos as reported in several papers [38ndash40]

Given the number of studies showing the ineffective-ness and potential impairment of cleavage stage biopsy it




3 Polar Body Biopsy













(a) (b) (c) (d)

(a998400) (b998400) (c998400) (d998400)











+

(1)

(2)

ICM

ICM + TE

TE

(a)

+

ICM

TEICM

ICM

(b)























(a)

(b)

(c)








7 Conclusion









References




























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















3 Polar Body Biopsy













(a) (b) (c) (d)

(a998400) (b998400) (c998400) (d998400)











+

(1)

(2)

ICM

ICM + TE

TE

(a)

+

ICM

TEICM

ICM

(b)























(a)

(b)

(c)








7 Conclusion









References




























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b) (c) (d)

(a998400) (b998400) (c998400) (d998400)











+

(1)

(2)

ICM

ICM + TE

TE

(a)

+

ICM

TEICM

ICM

(b)























(a)

(b)

(c)








7 Conclusion









References




























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















+

(1)

(2)

ICM

ICM + TE

TE

(a)

+

ICM

TEICM

ICM

(b)























(a)

(b)

(c)








7 Conclusion









References




























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































(a)

(b)

(c)








7 Conclusion









References




























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























References




























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















review article the impact of biopsy on human embryo...

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