130 E.S. Surrey and W.B. Schoolcraftand embryologists obtain as much information aspossible about the developmental and implanta-tion potential of embryos considered for transfer.The shift to more widespread transfer of blas-tocyst as opposed to cleavage stage embryos ingood prognosis patients (including oocyte donorrecipients) has represented one of the key factorsin improving outcomes. Indeed, the debate inoocyte donation has shifted from the question ofwhether blastocyst stage transfer is feasible towhether blastocyst stage transfer should be stan-dard and cleavage stage transfer the exception.We will provide evidence to support this conten-tion in this chapter.Why Blastocyst Stage Transfer?There are a host of potential advantages to the useof blastocyst stage embryo transfer in the oocytedonation model (Table 10.1 and Fig. 10.1).Perhaps the most important is the fact that theembryo can be transferred into the uterus at theappropriate developmental stage. The tubal envi-ronment to which the cleavage stage embryo isexposed in vivo is signiﬁcantly different withregard to nutrients and pH than the uterus towhich the blastocyst stage embryo is exposed,and therefore, transfer at an earlier developmen-tal stage may inhibit embryonic development .Secondly, uterine contractility progressivelydecreases in the luteal phase from the day of hCGadministration with the most profound declineoccurring between 4 and 7 days . This wouldtheoretically result in a more quiescent state atthe time of blastocyst transfer which could aidimplantation. Thirdly, it appears that full activa-tion of the genome of the embryo does not occuruntil after the cleavage stage . Extendingembryo culture would allow identiﬁcation ofembryos with an inherent developmental block.The beneﬁts of extended embryo culture areclearly dependent on the culture system. Thedevelopment of highly speciﬁc sequential andnonsequential systems as well as meticulousattention to air quality and laboratory techniquehas allowed for routine successful developmentof embryos to the blastocyst stage in vitro [3, 6].Perhaps the most compelling reason in favorof blastocyst transfer is the signiﬁcantly higherpregnancy and implantation rates achieved incomparison to cleavage transfer. The bulk of evi-dence has been obtained from IVF cycles employ-ing autologous oocytes, which shall be presentedﬁrst. However, one can only assume that out-comes obtained from oocytes derived fromyounger women without inherent fertility problemsTable 10.1 Advantages of blastocyst culture and transferin oocyte donationEnhanced synchrony with uterine environmentTransfer into a more quiescent uterusEnhanced developmental informationIncreased implantation ratesFull activation of embryonic genomeabFig. 10.1 (a) Photograph of high-quality eight-cellembryo derived from an oocyte donor 3 days after oocyteaspiration. (b) Photograph of high-quality expanded blas-tocyst derived from an oocyte donor 5 days after oocyteaspiration
13110 Blastocyst Versus Cleavage Stage Embryo Transfer: Maximizing Success Rates(oocyte donors) would only be higher which isconﬁrmed by the small number of trials address-ing this speciﬁc population.Blastocyst Transfer: IVF OutcomesTwo studies both published in 2004 evaluatingelective single embryo transfer (eSET) in goodprognosis patients are illustrative of the potentialadvantage of blastocyst transfer. Thurin et al. ran-domized 611 women less than 36 years of agewith at least two good-quality embryos to eSETor double embryo transfer, of which 97.2 %underwent transfer on day 2 or 3 (the majority onday 2) . The implantation rate for the ﬁrsteSET was 33.6 %. In contrast, Gardner et al. ran-domized 48 women with similar baseline charac-teristics and at least 10 follicles >12 mm indiameter on the day of hCG administration toelective single or double day 5 blastocyst stageembryo transfers . In this case, the implanta-tion rate for the single blastocyst transfer groupwas 60.9 %.A host of prospective randomized trials havecompared cleavage to extended stage embryotransfer, the majority of which demonstratedimproved outcomes with the latter [9–24]. One ofthe few trials which reported lower live birth rateswith blastocyst transfer noted similar implanta-tion rates for both groups . Interestingly, allblastocyst transfers in this study were performedon day 6, which may be a confounding variable.Indeed, others have demonstrated that day 5 blas-tocysts may be better synchronized with endome-trial development than more slowly developingembryos transferred on day 6, resulting in higherpregnancy rates with day 5 transfer [25, 26].Perhaps more telling are the results of pro-spective randomized trials comparing electivesingle cleavage to blastocyst stage embryo trans-fer. Papanikolaou et al. randomly assigned 351women under 36 years of age to transfer of a sin-gle cleavage stage (day 3) or blastocyst stage (day5) embryo . The study was terminated afteran interim analysis demonstrated signiﬁcantlyhigher ongoing pregnancy rates (58 % vs. 41 %,P=0.02; 95 % CI 1.06–2.66) and live birth rates(56 % vs. 38 %, P=0.01; 95 % CI 1.09–2.18) perembryo transfer procedure in the blastocystgroup. Subsequently, Zech and coworkers per-formed a similar study of 227 women £36 yearsof age undergoing a ﬁrst or second IVF cycle,resulting in ³5 fertilized oocytes . Asigniﬁcantly higher implantation rate per embryotransfer was achieved with blastocyst transfer(35.6 % vs. 23.7 %, P<0.05). Guerif and cowork-ers recently completed a prospective study of 478couples assigned to day 2 eSET or single blasto-cyst transfer on day 5 or 6 . It is important tonote that patients were assigned on a “voluntarybasis” which represents a confounding variable.Nevertheless, the delivery rate per fresh embryotransfer was again signiﬁcantly higher after sin-gle blastocyst transfer (36.7 % vs. 25.1 %,P<0.01) (Table 10.2). It is interesting to note thata recent meta-analysis of live birth rates afterelective single cleavage stage embryo transfer inprospective randomized trials described a livebirth rate of 26.7 % .Two recent meta-analyses addressing thisissue with different designs and reaching differ-ent conclusions have been published. An updatedCochrane review evaluated randomized trials ofearly cleavage (day 2/3) versus blastocyst (day5/6) stage transfers . Sixteen of the 45identiﬁed trials met inclusion criteria and wereanalyzed. Interestingly, there was no differencein live birth rates per couple in seven randomizedclinical trials (day 2/3: 34.3 % vs. day 5/6:35.4 %; OR 1.16, 95 % CI 0.74–1.44). This phe-nomenon held true for “good prognosis” patientsas well. There was also a greater likelihood ofhaving no embryos to transfer in the blastocystTable 10.2 Comparative implantation rates (IR) result-ing in live birth after elective single cleavage (eSET) orblastocyst stage (eBT) embryo transferFirst author (Ref.) eSet eBT PNIR/ET(%) NIR/ET(%)Papanikolaou  176 43 176 58 0.04Zech  99 23.2 128 32.8 <0.05Zech a86 25.6 76 40.8 <0.05Guerif  243 25.1 235 36.7 <0.01aExcellent-quality embryos only
132 E.S. Surrey and W.B. Schoolcraftgroup, although this phenomenon was notsigniﬁcantly different for good prognosis patients.This analysis did not evaluate implantation ratesper se.In a more recent meta-analysis, eight random-ized trials met stricter inclusion criteria of trulyrandomized design, transfer of equal numbers ofembryos between the two groups and includedonly studies which had been previously publishedas full text in a peer review publication . Inthis analysis, live birth rates were signiﬁcantlyhigher after blastocyst versus cleavage stagetransfers (OR 1.39, 95 % CI 1.10–1.76, P=0.005).Given the design of this meta-analysis with equalnumbers of embryos transferred in each group,these data would more closely approximate anassessment of relative implantation potential.Clearly, there are weaknesses with both analy-ses. The most critical of which for the purpose ofthis discussion is the fact that neither address out-comes of oocyte donor cycles. Even subset analy-sis of “good” prognosis patients cannot becompared to oocyte donors . The average ageof oocyte donors would be presumably less thanthat of IVF patients, and more importantly, oocytedonors would have no underlying history of infer-tility. In addition, outcomes from day 5 and 6blastocyst transfer were typically combined,which represents a confounding variable as pre-viously described .Blastocyst Transfer: Oocyte DonationOutcomesAs previously mentioned, the outcome data forblastocyst versus cleavage stage embryo transferin the oocyte donation model is limited. We areaware of no prospective randomized trialsspeciﬁcally addressing this patient subset.Schoolcraft and Gardner reported a retrospec-tive series of 229 patients undergoing oocytedonation at the Colorado Center for ReproductiveMedicine, of whom 116 underwent day 3 transferand 113 underwent day 5 transfer . Meanages of donors and of recipients were similarbetween the groups. The average blastocystdevelopment rate was 58.7 %. Implantation ratesresulting in documented fetal cardiac activity perembryo transfer were signiﬁcantly higher inpatients receiving a blastocyst transfer (65.0 %vs. 41.6 %, P<0.01). Clinical pregnancy ratesper retrieval were also signiﬁcantly higher afterblastocyst transfer (87.6 % vs. 75.0 %, P<0.05)despite transferring a signiﬁcantly lower meannumber of embryos (Table 10.3 and Fig. 10.2).These results were conﬁrmed by Shapiro and col-leagues who reported a mean implantation rate of52.8 % with a 66.7 % ongoing pregnancy rate in47 donor cycles after blastocyst transfer on eitherday 5 or 6 .Table 10.3 Oocyte donation: day 3 versus day 5 embryotransfer: cycle characteristicsDay 3 Day 5 PNo. of donor cycles 116 113 –Donor age(mean±SEM)28.8±0.44 27.8±0.41 NSRecipient age(mean±SEM)39.9±0.43 41.3±0.41 NSBlastocyst develop-ment (%)– 58.2 –Embryos transferred(mean±SEM)3.2±0.05 2.1±0.04 <0.01Embryos frozen(mean±SEM)5.2±0.59 5.6±0.43 NSAdapted from Schoolcraft and Gardner P < 0.05P < 0.01Clinicalpregnancy/retrievalImplantation/embryotransfer (+ fetal heart)PercentOocyte donation cycle outcomesday 3 versus day 5 transferDay 3 Day 5010090807060504030201075 %87.6 %41.6 %65 %Fig. 10.2 Oocyte donation cycle outcomes comparingday 3 versus day 5 embryo transfer in a large retrospectiveseries (Adapted from Schoolcraft and Gardner )
13310 Blastocyst Versus Cleavage Stage Embryo Transfer: Maximizing Success RatesA more contemporary review of all oocytedonation cycles performed at the Colorado Centerfor Reproductive Medicine from 2004 through2009 revealed that the implantation rate from 236day 3 transfers was 45.4 % and that of 828 day 5transfers was 72.5 %. The ongoing pregnancyrate after cleavage stage transfers was 70.3 % incomparison to 87.4 % after blastocyst transfer.A recent retrospective analysis compared cleav-age (day 3) to blastocyst (day 6) stage embryotransfer in 93 consecutive oocyte donation cycles. Once again, signiﬁcantly higher implantationrates (64±6 % vs. 27±7 %, P<0.01) and clinicalpregnancy rates (73 % vs. 40 %, P<0.01) wereobtained after blastocyst transfers. Even afteroocyte vitriﬁcation, implantation rates in oocytedonor cycles after blastocyst development andtransfer were extremely encouraging .In contrast, Soderström-Anttila and Vilskareported upon a 5-year experience with electivecleavage stage embryo transfer in both anony-mous and non-anonymous oocyte donation cycles. An implantation rate of 43.2 % per embryotransfer was reported. Previously, Mirkin et al.reported a 22 % implantation rate with day 3transfers in oocyte donation cycles .There are several important confounding vari-ables in the aforementioned trials. The lack ofappropriately designed prospective randomizedtrials is a weakness. However, given the retro-spective data from oocyte donors and prospec-tive trials derived from good prognosis IVFpatients, there is little to suggest that day 3 trans-fer is more advantageous in the oocyte donationmodel given an appropriate embryology labora-tory setting. The combination of outcomes fromday 5 and 6 blastocyst embryo transfers in thesetrials remains problematic. Although Shapiroet al. have demonstrated that clinical pregnancyrates from day 5 blastocyst transfers are superiorto day 6 transfers in autologous IVF cycles, theynoted the opposite phenomenon with oocytedonor cycles . This may reﬂect a higherdegree of synchrony between embryo and endo-metrium based on the speciﬁc endometrial prep-aration protocol employed. These data have notbeen conﬁrmed, and one would remain con-cerned that transfer of more slowly expandingblastocysts may also reﬂect compromiseddevelopmental potential.A third confounding variable, which has notbeen addressed in any of the aforementioned tri-als, is the impact of male age. It can be assumedthat in the average oocyte donation cycle, pater-nal age would be elevated in comparison to “goodprognosis” IVF cycles. Several studies have sug-gested that increasing paternal age (particularly>50 years) is associated with an adverse outcomein oocyte donor cycles [39, 40]. Both trials dem-onstrated a deleterious effect on blastocyst devel-opment rate. However, this ﬁnding has not beenuniversally demonstrated .The Case Against Blastocyst TransferGiven the aforementioned evidence in favor ofblastocyst transfer in the oocyte donation model,there remain several arguments which have beenhistorically made in opposition to this approach:1. A high percentage of otherwise viable embryoson day 3 fail to develop to the blastocystin vitro and would be “lost” for transfer.2. Cryopreservation of supernumerary blastocyststage embryos results in lower survival ratesthan at earlier developmental stages, resultingin a decline in overall cycle efﬁciency.3. Transfer of embryos at the blastocyst stagemay be associated with an increased risk ofmonozygotic twinning.We will address each of these issues.The contention that viable day 3 embryos willnot survive in vitro to the blastocyst stage andwould have a greater likelihood of surviving in theuterus clearly cannot be directly tested since thesame embryo cannot be evaluated in two places atonce. The failure of embryos to develop in vitromay indeed be secondary to a suboptimal labora-tory environment. However, in an optimal labora-tory setting, this phenomenon may also be due toembryos with inherent genetic and metabolicimpairment leading to arrested development. Otherfactors to consider would be those of advancedpaternal age, severe sperm abnormalities, and theimpact of cycles with an older (typically known)donor. In their meta-analysis, Blake et al. reported
134 E.S. Surrey and W.B. Schoolcraftthat the likelihood that couples would have noembryos to transfer is signiﬁcantly higher for blas-tocyst versus cleavage stage embryos .However, when these investigators limited theiranalysis to good prognosis IVF patients, this dif-ference was not statistically signiﬁcant (OR 1.58;95 % CI 0.65–3.82). These trials did not includeoocyte donation cycles, a situation with a presum-ably better prognosis than “best case” autologousIVF patients. Indeed, we had previously reportedthat 58 % of fertilized donor oocytes undergoingextended culture in sequential medium reached theblastocyst stage, of which 84 % were felt to be ofhigh quality .It is not necessary to commit to blastocysttransfer in all cycles without exception, however.Evaluation of embryo quality and number at thepronuclear and, perhaps more importantly, at thecleavage stage may serve as an imperfect predic-tor for blastocyst development potential. Neuberet al. reported a high correlation between pronu-clear symmetry, early cleavage, and subsequentblastocyst development . Dessolle andcoworkers created a predictive model for failedblastocyst development based on fertilizationtechnique, female age, as well as number andquality of day 3 embryos . This view has notbeen uniformly accepted in that others havesuggested that morphologic assessment ofembryos at the pronuclear or cleavage stage ispoorly predictive of the likelihood of blastocystdevelopment [44, 45].The ability to efﬁciently cryopreserve super-numeraryembryosenhancestheoverallefﬁciencyof any given oocyte aspiration procedure. If out-comes with blastocyst stage cryopreservationwere signiﬁcantly compromised compared topronuclear or cleavage stage freezing, thenbeneﬁts of fresh blastocyst transfer would beneutralized. Meta-analyses have reported that therate of embryo freezing was higher at days 2–3versus days 5–6 [31, 32]. However, these reportsonly suggest that more embryos were availablefor cryopreservation at earlier developmentalstages, as would be expected, but not that out-comes were enhanced from subsequent transfers.Guerif and coworkers previously noted that intheir program, fresh elective single blastocysttransfer pregnancy rates were higher than elec-tive cleavage stage embryo transfers, but oncefrozen embryo transfers were included, cumula-tive delivery rates were not signiﬁcantly differentbetween the two groups .However, outcomes from blastocyst cryo-preservation are not consistent among laborato-ries, and published reports cannot be universallyapplied. In addition, it is important to note thatresults from earlier studies may not reﬂect cur-rently employed techniques. Veeck et al. reporteda 76.3 % survival rate of blastocysts cryopre-served using slow-freeze techniques with anongoing clinical pregnancy rate of 59.2 % .In a retrospective analysis from this same group,clinical pregnancy rates (64.2 % vs. 37.4 % vs.42.1 %, P<0.05) and implantation rates (38.5 %vs. 15.2 % vs. 17.1 %, P<0.05) per transfer weresigniﬁcantly higher after transfer of thawed blas-tocysts in comparison to thawed cleavage or pro-nuclearstageembryos.Thereisdisagreementamong investigators as to whether there are dif-ferences in outcomes from blastocysts cryopre-served on day 5 versus day 6 when transferred toan appropriately prepared endometrium [26, 48].An alternative approach for clinics uncomfort-able with blastocyst slow-freeze techniques is tofreeze supernumerary embryos at the pronuclearor cleavage stage and then allow subsequentlythawed embryos to grow to the blastocyst stagebefore transfer. Employing this approach withoocyte donors, Shapiro and colleagues reportedsimilar implantation and pregnancy rates as withfresh transfers . The disadvantage of thisapproach is the inability to select embryos forfresh transfer from the full cohort of embryoswhich could have a deleterious impact on thesuccess of the fresh embryo transfer.The introduction of successful blastocystvitriﬁcation has signiﬁcantly improved theefﬁciency of cryopreservation and enhanced out-comes due to the elimination of intracellular icecrystal formation [50, 51]. In a recent review andmeta-analysis, Loutradi et al. reported a post-thaw blastocyst survival rate that was signiﬁcantlyhigher using vitriﬁcation as opposed to slow-freeze techniques (OR 2.2, 95 % CI 1.53–3.16). At the Colorado Center for Reproductive
13510 Blastocyst Versus Cleavage Stage Embryo Transfer: Maximizing Success RatesMedicine, we have reported a 97.8 % survivalrate after blastocyst vitriﬁcation even after tro-phectoderm biopsy . In fact, some investiga-tors have reported signiﬁcantly higher pregnancyand implantation rates in nondonor IVF cyclesafter transfer of vitriﬁed and warmed blastocyststhan after fresh transfer . This may be due tothe presence of a more receptive endometrium inthe prepared frozen embryo transfer cycle.However, in the case of oocyte donation cycles,endometrial preparation of the recipient would besimilar for a fresh or frozen transfer cycle makingthis issue less relevant.The ﬁnal concern which has been raisedregarding blastocyst transfer is the question ofwhether prolonged culture is associated with anyinherent increased pregnancy risks. Several inves-tigators have suggested that the incidence ofmonozygotic twinning may be increased afterblastocyst versus cleavage stage embryo transfer[55–57]. This has been attributed to a possibleincrease in the hardness of the zona pellucida dueto prolonged in vitro embryo culture. It is inter-esting to note that in two more recent studies, theincidence of monozygotic twinning was no dif-ferent between blastocyst and cleavage stagetransfers [58, 59]. This change may be reﬂectiveof advances in culture medium. In addition, thesedata are not derived from oocyte donation cycles,and therefore, we are forced to extrapolate to thatmodel.Blastocyst Selection: Is MorphologyEnough?Although it would appear from the evidence pro-vided that implantation rates with blastocysttransfer are signiﬁcantly enhanced over day 3transfer in both autologous and donor IVF cycles,the results remain imperfect. In an effort to maxi-mize success while minimizing multiple preg-nancies, elective single embryo transfer clearly isideal. Thus, enhancing the accuracy of embryoselection techniques is critical to achieving thisgoal.Assessments of morphology and developmen-tal rate have been the mainstays of this approach.We have previously discussed the merits of day 5versus day 6 fresh blastocyst transfers. Employinga morphologic grading system based on thedegree of blastocyst expansion along with thedevelopment and architecture of both the innercell mass and trophectoderm, Gardner et al. dem-onstrated a relationship between blastocyst gradeand implantation . When two top-qualityblastocysts were transferred (³3AA) (69 % ofpatients), the implantation rates were signiﬁcantlyhigher than the 15 % of patients who had onlylower-scoring blastocysts (<3AA) transferred(69.9 % vs. 78.1 %).In the setting of oocyte donor cycles, the pre-dictive value of morphologic assessment is evenless clear. In reviewing all oocyte donor cyclesperformedattheColoradoCenterforReproductiveMedicine from 2004 to 2009, we noted thatimplantation rates after transfer of expanded butnot perfect blastocysts were similar to thosetransferred which were felt to be perfect in qual-ity and not dramatically different than in the smallnumber of patients with only morulae availableto transfer (Table 10.4).In the best of circumstances, blastocyst mor-phology is not completely predictive of outcome.New tools, the details of which are beyond thescope of this chapter, may add additional infor-mation regarding the embryo in order to enhancethe selection process. Aneuploidy screening mayrepresent one of these approaches. The incidenceof aneuploid embryos increases signiﬁcantlywith age, a phenomenon which one wouldassume would be negated with the use of a youngoocyte donor. Indeed, Fragouli et al. noted a lowaneuploidy rate (3 %) using comparative genomichybridization techniques after polar body biopsyof oocytes derived from donors with an averageage of 22 years . However, these data do notreﬂect the impact of advanced paternal age,which is more commonly associated with oocytedonation cycles and may play a role in increas-ing the incidence of aneuploid embryos despite ahigh percentage of euploid oocytes. Excitingnew validated techniques allowing for compre-hensive chromosomal screening of blastocyststage embryos have been shown to increase implan-tation rates by 50 % compared to contemporary
136 E.S. Surrey and W.B. Schoolcraftautologous IVF cycles [62, 63, 64]. These tech-niques have primarily been employed in coupleswith recurrent implantation failure, unexplainedrecurrent pregnancy loss, and advanced maternalage and not in the oocyte donation model or inthe average younger infertile patient. However,if the value of this approach is conﬁrmed inappropriately designed prospective randomizedtrials, a case could be made for future investiga-tion of this technique in other models as well.Indeed, analysis of blastocyst gene expressionmay take us beyond simple aneuploidy screen-ing in creating a proﬁle which predicts implanta-tion potential .In addition, noninvasive approaches to theassessment of the viability of embryos whichmay be morphologically similar are areas ofintense investigation. Proteomic and metabolo-mic assessments of spent culture medium mayrepresent dynamic means of creating a uniqueproﬁle of biomarkers to predict blastocyst viabil-ity [66, 67]. If validated, these approaches wouldcertainly have application in the oocyte donationmodel as well.SummaryOocyte donation represents the most consistentlysuccessful therapy in the assisted reproductivetechnologies. As success rates have improved,multiple pregnancy rates have also increased. Asa result, the need to more effectively select a sin-gle embryo for transfer without compromisingefﬁcacy has become a critical issue. Extendingembryo development to the blastocyst stage hasrepresented a clear advance in this regard.Reﬁnements in culture medium and laboratorytechniques as well as increased competence invitriﬁcation technology serve to reinforce thisapproach. New technologies in genomics, pro-teomics, and metabolomics will allow cliniciansand embryologists to create a proﬁle of theimplantation potential of an embryo whichextends beyond an assessment of morphologyalone. The question, therefore, has shifted fromwhich situations would be appropriate for blasto-cyst transfer to which situations, if any, would notbe appropriate for blastocyst transfer in oocytedonation cycles.Table 10.4 Oocyte donation outcomes and blastocyst quality at Colorado Center for Reproductive Medicine(2004–2009)Stage Cycles Total embryos transferred Ongoing pregnancy (%) Implantation (%)Only morulae 7 21 100 71.4Only early (1,2,2/3)a29 66 69 53Only advanced but not perfect(AB, BA, BB)a93 189 84.9 72Only perfect (AA)a453 866 91.6 77.6aBased on scale described by Gardner et al. Editor’s CommentaryBlastocysts have taken center stage in eggand embryo donation since the ﬁrst reportof a birth in 1984. John Buster, M.D. atHarbor/UCLA, noted during the early uter-ine lavage experiments that only recipientsof recovered blastocysts became pregnantand that the implantation and pregnancyrates of these transferred embryos wereremarkably high. Of course, the efﬁciencyof uterine lavage and natural cycles pre-vented the development of embryo trans-fers along these lines. Development of thein vitro methods proceeded, and for thenext two decades, cleavage stage embryosbecame the focus and the practice in bothconventional IVF and oocyte donation.By the mid-1990s, it was becoming increas-ingly clear that we had a multiple birthproblem in our recipients. Remarkably, andlooking back in retrospect, at that time, thestandard was still to transfer up to ﬁve
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