Caroline McCaffrey

Oocyte cryopreservation outcomes including pre-cryopreservation and post-thaw meiotic spindle evaluation following slow cooling and vitrification of human oocytes

OBJECTIVEnTo report our oocyte cryopreservation (OC) outcomes including meiotic spindle (MS) evaluation of metaphase II (MII) oocytes destined for OC and thaw.nnnDESIGNnRetrospective.nnnSETTINGnUniversity-based infertility center.nnnPATIENT(S)nWomen attempting pregnancy using cryopreserved oocytes.nnnINTERVENTION(S)nOC, MS evaluation.nnnMAIN OUTCOME MEASURE(S)nSurvival, two pronuclear (2PN) fertilization, achieving embryo quality suitable for transfer or refreezing, blastocyst formation.nnnRESULT(S)nThirty-two OC-thaw cycles resulted in 20 pregnancies, 18 either ongoing or delivered. In 26 cycles, MS evaluation was performed: 262/303 (86%) thawed/recovered oocytes survived, 218/262 (83%) achieved 2PN fertilization, 133/218 (61%) became suitable for day-3 and 122/218 (56%) for day-5 transfer. In total, 58 embryos were transferred resulting in a 62% pregnancy and a 41% implantation rate. Of oocytes evaluated before cryopreservation, 247 (82%) were spindle-positive; 96% of these were also spindle-positive after thawing. Blastocyst formation and suitability for day-5 transfer was achieved more often if a post-thaw spindle was visualized. Of all slow-cooled and vitrified oocytes, a higher percentage of those slow-cooled achieved 2PN fertilization and usability. MS evaluation of oocytes cryopreserved by either method was associated with similar outcomes.nnnCONCLUSION(S)nOC outcomes are improving. An MS was almost always exhibited both before cryopreservation and after thawing, suggesting that, with appropriate technique, OC presents minimal harm to the MII oocyte. A meiotic spindle evaluation might help to further OC technology.

What makes them split? Identifying risk factors that lead to monozygotic twins after in vitro fertilization

OBJECTIVEnTo identify the incidence, risk factors, and obstetric/perinatal outcomes associated with monozygotic twins (MZTs) after IVF.nnnDESIGNnNested case-control.nnnSETTINGnUniversity-based center.nnnPATIENT(S)nThe IVF cycles eventuating in pregnancy from 2000-2009.nnnINTERVENTION(S)nNone.nnnMAIN OUTCOME MEASURE(S)nThe MZT incidence, chorionicity/zygosity, pregnancy/neonatal outcome.nnnRESULT(S)nOf 6,223 gestations, 131 MZTs were diagnosed (2.1% incidence; 2.0% in autologous and 2.7% in donor IVF cycles), 10 were dichorionic, and 121 were monochorionic. Controlling for all risk factors, young oocyte age, extended culture (noncleavage embryos transferred on/after day 4), and year of IVF treatment cycle were significantly associated with MZT. When assessing factors associated with specific MZT placentation, day 3 assisted hatching correlated more with dichorionic MZT, whereas extended culture and advanced day 5 embryonic stage correlated with monochorionic MZT. Comparing monozygotic to dizygotic multigestation outcomes, MZT fared worse; however, once controlling for triplet gestation, only gestational age at delivery remained significantly compromised in the monozygotic group.nnnCONCLUSION(S)nAfter IVF the incidence of MZT is high, with young oocyte age, year of treatment, and extended culture (or embryo stage at transfer) conferring greatest risk. Regarding MZT type, assisted reproductive technology (ART) procedures may influence the timing of embryonic splitting (i.e., division in early embryonic development may be influenced by zona pellucida [ZP] manipulation whereas later splitting may occur during delayed implantation). Poor obstetric/perinatal outcome is significantly impacted by the presence of an extra fetus, as high-order multiple gestation concurrent with an MZT conveyed the worst prognosis.

Why do euploid embryos miscarry? A case-control study comparing the rate of aneuploidy within presumed euploid embryos that resulted in miscarriage or live birth using next-generation sequencing

OBJECTIVEnTo determine whether undetected aneuploidy contributes to pregnancy loss after transfer of euploid embryos that have undergone array comparative genomic hybridization (aCGH).nnnDESIGNnCase-control study.nnnSETTINGnUniversity-based fertility center.nnnPATIENT(S)nCases included 38 patients who underwent frozen euploid ET as determined by aCGH, resulting in miscarriage. Controls included 38 patients who underwent frozen euploid ET as determined by aCGH, resulting in a live birth.nnnINTERVENTION(S)nNext-generation sequencing (NGS) protocols were internally validated. Saved amplified DNA samples from the blastocyst trophectoderm biopsies previously diagnosed as euploid by aCGH were reanalyzed using NGS. Cytogenetic reports of the products of conception for 20 of the pregnancies resulting in miscarriage were available for comparison.nnnMAIN OUTCOME MEASURE(S)nThe incidence of aneuploidy and mosaicism using NGS within embryos resulting in miscarriage and live birth.nnnRESULT(S)nOf euploid embryos analyzed by aCGH resulting in miscarriage, 31.6% were mosaic and 5.2% were polyploid by NGS. The rate of chromosomal abnormalities was significantly higher in embryos resulting in miscarriage (36.8%) than in those resulting in live births (15.8%). The rate of mosaicism was twice as high among embryos resulting in miscarriage than those resulting in live birth, but this was not statistically significant. Next-generation sequencing detected more cases of mosaicism than cytogenetic analysis of products of conception.nnnCONCLUSION(S)nUndetected aneuploidy may increase the risk of first trimester pregnancy loss. Next-generation sequencing may detect mosaicism and triploidy more frequently than aCGH, which could help to identify embryos at high risk of miscarriage. Mosaic embryos, however, should not be discarded as some can result in live births.

Oocyte efficiency: does live birth rate differ when analyzing cryopreserved and fresh oocytes on a per-oocyte basis?

OBJECTIVEnTo compare the efficiency of oocyte cryopreservation (OC) and IVF using the metric live births per mature oocyte retrieved.nnnDESIGNnRetrospective analysis.nnnSETTINGnUniversity-based fertility center.nnnPATIENT(S)nForty women who underwent OC with thaw attempt between 2004 and 2010; 25 autologous and 15 donor-oocyte treatments were included. One thousand nine hundred eight women underwent their first, fresh conventional IVF treatment between 2004 and 2010; 1,392 used autologous oocytes, and 516 used donor oocytes. Autologous and donor-oocyte cycles were analyzed separately. All oocytes were obtained from women <age 40 years old.nnnINTERVENTION(S)nNone.nnnMAIN OUTCOME MEASURE(S)nLive-birth rate per mature oocyte retrieved (primary outcome), live-birth rate per ET, and blastocyst formation rate.nnnRESULT(S)nWhen OC/thaw cycles were compared with fresh IVF attempts, there was no significant difference in live-birth rate per mature oocyte retrieved (2.7% vs. 4.2%, respectively) or live-birth rate per ET (45.8% vs. 51.9%). Significantly more oocytes were harvested in the OC versus fresh IVF cycles (21 vs. 16); however, fewer blastocysts developed (3.9 vs. 6.3). Thus, we noted an age-independent approximate twofold decrease in blastocyst formation in the OC group.nnnCONCLUSION(S)nOur data suggest that while assisted reproductive technology remains an inefficient process, OC may be approaching fresh IVF when live birth is the primary consideration. However, OC may negatively impact the potential for blastocyst formation.

Assessing morphokinetic parameters via time lapse microscopy (TLM) to predict euploidy: are aneuploidy risk classification models universal?

PurposeTo determine if Aneuploidy Risk Classification Models are predictive of euploidy/aneuploidy amongst IVF facilities.MethodsWe retrospectively applied key time lapse imaging events of embryos (Campbell et al.[5, 6]) to stratify embryos into 3 groups: low, medium and high risk of aneuploidy. The actual ploidy results (from array comparative genomic hybridization) were compared with expectations [5, 6]. Sources of variability in morphokinetic parameters were determined using Analysis of Variance (ANOVA).ResultsThe model failed to segregate euploid embryos from aneuploid embryos cultured at our facility. Further analysis indicated that the variability of embryos among patients was too great to allow selection of euploid embryos based on simple morphokinetic thresholds. Clinical selection of embryos based on morphokinetics alone is unlikely to identify euploid embryos accurately for transfer or yield higher rates of live delivery.ConclusionsThe use of non-invasive morphokinetics is unlikely to discriminate aneuploid from euploid embryos. Further, it does not approach the accuracy of preimplantation genetic screening with array comparative genomic hybridization.

In vitro fertilization with preimplantation genetic screening improves implantation and live birth in women age 40 through 43

PurposeIn Vitro Fertilization is an effective treatment for infertility; however, it has relatively low success in women of advanced maternal age (>37) who have a high risk of producing aneuploid embryos, resulting in implantation failure, a higher rate of miscarriage or birth of a child with chromosome abnormalities. The purpose of this study was to compare the implantation, miscarriage and live birth rates with and without preimplantation genetic screening (PGS) of embryos from patients aged 40 through 43xa0years.MethodsThis is a retrospective cohort study, comparing embryos screened for ploidy using trophectoderm biopsy and array comparative genomic hybridization to embryos that were not screened. We compared pregnancy outcomes for traditional fresh IVF cycles with day 5 embryo transfers, Frozen Embryo Transfer (FET) cycles without PGS and PGS-FET (FET of only euploid embryos) cycles of patients with maternal ages ranging from 40 to 43xa0years, undergoing oocyte retrievals during the period between 1/1/2011 and 12/31/2012.ResultsThe implantation rate of euploid embryos transferred in FET cycles (50.9xa0%) was significantly greater than for unscreened embryos transferred in either fresh (23.8xa0%) or FET (25.4xa0%) cycles. The incidence of live birth per transferred embryo for PGS-FET (45.5xa0%) was significantly greater than for No PGS fresh (15.8xa0%) or No PGS FET (19.0xa0%) cycles. The incidences of live birth per implanted sac for PGS FET cycles (89.3xa0%), No PGS fresh cycles (66.7xa0%) and No PGS FET cycles (75.0xa0%) were not significantly different.ConclusionsThe present data provides evidence of the benefits of PGS with regard to improved implantation and live birth rate per embryo transferred.

Mitochondrial DNA quantification as a tool for embryo viability assessment: retrospective analysis of data from single euploid blastocyst transfers

STUDY QUESTIONnDoes the amount of mitochondrial DNA (mtDNA) in blastocyst biopsy specimens have the potential to serve as a biomarker of euploid embryo implantation ability, independent of morphology?nnnSUMMARY ANSWERnThe results of this study strongly suggest that elevated mtDNA levels, above a previously defined threshold, are strongly associated with blastocyst implantation failure and represent an independent biomarker of embryo viability.nnnWHAT IS KNOWN ALREADYnImproved methods of embryo selection are highly desirable in order to increase the efficiency of IVF treatment. At present, even the transfer of chromosomally normal embryos of high morphological grade cannot guarantee that a pregnancy will follow. Recently, it has been proposed that the quantity of mtDNA in embryonic cells may be an indicator of developmental potential, with higher levels of mtDNA associated with reduced implantation. However, thus far reported data sets have been relatively small and in some cases have lacked appropriate validation.nnnSTUDY DESIGN, SIZE, DURATIONnThis large, blinded, retrospective study involved the analysis of relative mtDNA levels in 1505 euploid blastocysts obtained from 490 couples undergoing preimplantation genetic testing for aneuploidy. Implantation outcomes were compared to mtDNA levels in order to determine the capacity of the method to predict viability and to assess the validity of previously established thresholds.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnDNA from blastocyst biopsy samples was amplified and then subjected to aneuploidy analysis using next generation sequencing or array comparative genomic hybridization. Only those embryos classified as chromosomally normal had their mtDNA levels assessed. This analysis was undertaken retrospectively using quantitative real-time PCR, without knowledge of the outcome of embryo transfer. Predictions of implantation failure, based upon mtDNA levels were subsequently compared to the observed clinical results. All cycles involved the transfer of a single embryo.nnnMAIN RESULTS AND THE ROLE OF CHANCEnOf all blastocysts analyzed, 9.2% (139/1505) contained mtDNA levels above a previously established viability threshold and were therefore predicted to have reduced chances of implantation. To the date of analysis, 282 euploid blastocysts had been transferred with an overall implantation rate of 65.6% (185/282). Of the transferred embryos, 249 contained levels of mtDNA in the normal range, 185 of which produced a pregnancy, giving an implantation rate of 74.3% for euploid embryos with normal quantities of mtDNA. However, 33 of the transferred embryos were determined to have elevated mtDNA quantities. None of these led to a pregnancy. Therefore, the negative predictive value of mtDNA assessment in this cohort was 100% (33/33). The difference between the implantation rates for embryos with normal and elevated mtDNA levels was highly significant (P < 0.0001). The mtDNA thresholds, used for classification of embryos, were unaffected by female age or the clinic in which the IVF was undertaken. The probability of an embryo having elevated levels of mtDNA was not influenced by variation in embryo morphology.nnnLIMITATIONS, REASONS FOR CAUTIONnThis study provides strong evidence that mtDNA quantification can serve as a valuable tool to assist the evaluation of blastocyst viability. However, to determine the true extent of any clinical benefits, other types of investigations, such as non-selection studies and randomized controlled trials, will also be necessary.nnnWIDER IMPLICATIONS OF THE FINDINGSnThe results of this study suggest that mtDNA quantity can serve as an independent biomarker for the prediction of euploid blastocyst implantation potential. Prospective studies should now be undertaken to confirm these results. Additionally, investigations into the underlying biological cause(s) of elevated mtDNA levels and an enhanced understanding of how they relate to diminished implantation potential would be invaluable.nnnSTUDY FUNDING/COMPETING INTEREST(S)nThis study was supported by funding provided by Reprogenetics. None of the authors have any competing interests.

Long-term cryopreservation of human oocytes does not increase embryonic aneuploidy

OBJECTIVEnTo determine if long-term cryopreservation of human oocytes affects oocyte developmental competence, blastocyst euploidy, or live-birth rates.nnnDESIGNnRetrospective cohort study.nnnSETTINGnUniversity-based fertility center.nnnPATIENT(S)nA total of 33 patients with cryopreserved oocytes underwent oocyte thaw, blastocyst culture, trophectoderm biopsy, and 24-chromosome preimplantation genetic screening (PGS) with array comparative genomic hybridization between December 2011 and July 2014; subjects were compared with 2:1 age-matched controls with fresh oocytes whose embryos underwent trophectoderm biopsy and PGS during the same period.nnnINTERVENTION(S)nNone.nnnMAIN OUTCOME MEASURE(S)nRates of fertilization, blastulation, euploidy, implantation, and live birth.nnnRESULT(S)nThirty-three patients (mean age 36.2 ± 3.8xa0y) thawed 475 oocytes that had been cryopreserved for a median of 3.5xa0years. Compared with 66 age-matched controls who underwent inxa0vitro fertilization and PGS with fresh oocytes, embryos derived from cryopreserved oocytes demonstrated compromised blastocyst formation (54.5% vs. 66.2%) despite no impairment in fertilization (72.8% vs. 73.2%). Results showed no difference in the number of euploid blastocysts (1.7 ± 1.9 vs. 2 ± 2.5), percentage of euploid blastocysts (44.5% vs. 47.6%), rate of implantation (65% vs. 65%), or rate of live birth and ongoing pregnancy (62.5% vs. 55%) after 24-chromosome PGS with cryopreserved or fresh oocytes.nnnCONCLUSION(S)nEmbryos derived from cryopreserved oocytes demonstrate impaired blastulation but equivalent rates of euploidy, implantation, and live birth compared with blastocysts derived from fresh oocytes, supporting the safety and efficacy of oocyte cryopreservation.

Impact of developmental stage at cryopreservation and transfer on clinical outcome of frozen embryo cycles.

Although several early IVF successes were achieved after transferring fully formed blastocysts, the majority of embryos replaced worldwide over the past 30 years have been at the cleavage stage. The programme at this study centre has previously found that delaying an embryo transfer to day 5 can reduce the chance for a high-order multiple gestation without compromising the pregnancy rate because fewer embryos can be replaced. To evaluate the impact of transfer day and embryonic stage at cryopreservation on cycle outcome, 6069 fresh and 706 frozen transfers from 2000-2006 performed at this study centre were retrospectively analysed. Approximately half of the fresh transfers were performed on day 3, with a shift to day-5 transfer over the study period with no change in cryopreservation incidence. Implantation, clinical pregnancy and live birth rates were significantly higher following day-5 transfer. When frozen-thawed embryos (2-cell to day-6 blastocysts) were transferred, acceptable pregnancy and live birth rates were achieved at all stages but thawed embryos transferred as day-5 blastocysts generated consistently higher clinical pregnancy and live birth rates. Transfer of embryos frozen on day 6 had the highest miscarriage and lowest live birth rates. Barring government regulation, an IVF programmes day for cryopreservation generally depends on its management of and success with fresh embryo transfer.

Clinical implications of mitochondrial DNA quantification on pregnancy outcomes: a blinded prospective non-selection study.

STUDY QUESTIONnCan quantification of mitochondrial DNA (mtDNA) in trophectoderm (TE) biopsy samples provide information concerning the viability of a blastocyst, potentially enhancing embryo selection and improving IVF treatment outcomes?nnnSUMMARY ANSWERnThis study demonstrated that euploid blastocysts of good morphology, but with high mtDNA levels had a greatly reduced implantation potential.nnnWHAT IS KNOWN ALREADYnBetter methods of embryo selection leading to IVF outcome improvement are necessary, as the transfer of chromosomally normal embryos of high morphological grade cannot guarantee the establishment of an ongoing pregnancy. The quantity of mtDNA in embryonic cells has been proposed as a new biomarker of viability-higher levels of mtDNA associated with reduced implantation potential.nnnSTUDY DESIGN, SIZE, DURATIONnmtDNA was quantified in 199 blastocysts, previously biopsied and shown to be chromosomally normal using preimplantation genetic testing for aneuploidy (PGT-A). These were generated by 174 couples (average female age 37.06 years). All patients underwent IVF in a single clinic. The study took place in a blinded, non-selection manner-i.e. mtDNA quantity was not known at the time of single embryo transfer. The fate of the embryos transferred was subsequently compared to the mtDNA levels measured.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnEmbryos were biopsied at the blastocyst stage. The TE samples obtained were subjected to whole genome amplification followed by comprehensive chromosome analysis via next generation sequencing. The same biopsy specimens were also tested using quantitative PCR, allowing highly accurate mtDNA quantification. After blastocyst transfer, the code used for blinding was broken and analysis undertaken to reveal whether the amount of mtDNA had any association with embryo implantation.nnnMAIN RESULTS AND THE ROLE OF CHANCEnmtDNA analysis of the 199 blastocysts revealed that 9 (5%) contained unusually high levels of mtDNA. All embryo transfers involved a single chromosomally normal blastocyst of good morphology. Of these, 121 (60%) led to ongoing pregnancies, 11(6%) led to biochemical pregnancies, and 10 (5%) spontaneously miscarried. All (100%) of these blastocysts had mtDNA levels considered to be normal/low. The remaining 57 (29%) blastocysts failed to implant. Among these non-viable embryos there were 9 (16%) with unusually high levels of mtDNA. This meant that the ongoing pregnancy rate for morphologically good, euploid blastocysts, with normal/low levels of mtDNA was 64% (121/190). In contrast, the ongoing pregnancy rate for the same type of embryos, but with elevated mtDNA levels, was 0/9 (0%). This difference was highly statistically significant (P < 0.0001).nnnLIMITATIONS REASONS FOR CAUTIONnTo determine the true extent of any clinical benefits a randomized clinical trial will be necessary. Research is needed to improve understanding of the biology of mtDNA expansion.nnnWIDER IMPLICATIONS OF THE FINDINGSnThis is the first investigation to evaluate the clinical impact of increased mtDNA in a prospective blinded manner. Results confirm that embryos with elevated mtDNA rarely implant, supporting its use as a viability biomarker. A total of 64% of euploid blastocysts with normal/low mtDNA implanted versus 60% for the cohort as a whole.nnnSTUDY FUNDING/COMPETING INTEREST(S)nThis study was supported by institutional funding (Reprogenetics UK and Reprogenetics). DW is supported by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Programme. None of the authors have any competing interests.