Faculty Bibliography

PURPOSE: This multicentered retrospective study analyzed whether the quantity of euploid blastocysts in a given cohort after comprehensive chromosomal screening can be used to identify candidates for single embryo transfer. METHODS: Blastocysts from 437 patients underwent trophectoderm biopsy followed by array comparative genomic hybridization. Embryos were then selected for single or double embryo transfer. The number of euploid blastocysts produced and transferred for each patient was recorded, as was clinical pregnancy rate and multiple gestation rate. RESULTS: In patients with /= 4 euploid blastocysts, clinical pregnancy rate was not reduced with single embryo transfer was performed, whereas the multiple gestation rate was greatly reduced. CONCLUSIONS: Size of the euploid embryo cohort is a marker for success in single embryo transfer cycles. Patients who produce at least four euploid blastocysts are outstanding candidates for single embryo transer.

PURPOSE: To determine if Aneuploidy Risk Classification Models are predictive of euploidy/aneuploidy amongst IVF facilities. METHODS: We 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). RESULTS: The 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. CONCLUSIONS: The 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.

Preimplantation genetic diagnosis and screening improves the chances of achieving a viable pregnancy, not only free of undesired single-gene defects but also aneuploidy. In addition, improvements in vitrification provide an efficient means of preserving embryos (blastocysts). By combining trophectoderm biopsy with recent improvements in vitrification methods, only those embryos that have proved themselves viable and potentially more competent are tested. Using array comparative genomic hybridization (aCGH) to assess all 24 chromosomes, aneuploidy rates were compared between day-3 blastomere biopsy and day-5 trophectoderm biopsy. Of those 1603 embryos, 31% were euploid, 62% were aneuploid and 7% not analysable. A significantly larger proportion of embryos were euploid on day-5 biopsy (42%) compared with day-3 biopsy (24%, P<0.0001). The number of euploid embryos per patient was not significantly different. Combining extended culture, trophectoderm biopsy and aneuploidy assessment by aCGH and subsequent vitrification can provide a more efficient means of achieving euploid pregnancies in IVF. Preimplantation genetic diagnosis and screening improves the chances of achieving a viable pregnancy, not only free of undesired single-gene defects but also aneuploidy. In addition, improvements in vitrification provide an efficient means of preserving embryos (blastocysts). By combining trophectoderm biopsy with recent improvements in vitrification methods, only those embryos that have proved themselves viable and potentially more competent are tested. Using array comparative genomic hybridization to assess all 24 chromosomes, aneuploidy rates were compared between day-3 blastomere biopsy and trophectoderm biopsy. Of those 1603 embryos, 31% were euploid, 62% were aneuploid and 7% not analysable. A significantly larger proportion of embryos were euploid with trophectoderm biopsy (42%) compared with blastomere biopsy (24%, P<0.0001). The number of euploid embryos per patient was not significantly different. Combining extended culture, trophectoderm biopsy and aneuploidy assessment by array comparative genomic hybridization and subsequent vitrification can provide a more efficient means of achieving euploid pregnancies in IVF.

Objective: Next generation sequencing (NGS), a class of methods involving the production of vast quantities of DNA sequence data, is revolutionising genetic diagnostics. However, NGS has not been applied to research or diagnostics involving human embryos due to technical obstacles that prevent analysis single cells. We aimed to create new methods overcoming these limitations, allowing clinical application of NGS technology for the identification of viable/ healthy embryos. Methods: Whole genome amplification and NGS (Ion Torrent) methods were optimised. Data was analysed with novel tools developed by our laboratory. Results: In pre-clinical work, NGS correctly diagnosed abnormalities in 100% of single cells isolated from aneuploid cell lines. Additionally, 45/45 aneuploid embryos donated for research were also successfully diagnosed (verified by blinded analysis using arrayCGH). NGS accurately detected single gene disorders, correctly diagnosing cystic fibrosis in single cells from an affected individual. Moreover, a mitochondrial DNA mutation was successfully detected and accurately quantified in single fibroblasts from a heteroplasmic patient with mitochondrial disease. Ultimately, NGS was employed clinically for the purpose of selecting chromosomally normal blastocysts for uterine transfer, resulting in a pregnancy. Conclusions: A powerful NGS technique applicable to single cells was successfully developed, allowing simultaneous testing for aneuploidy, single gene mutations and mitochondrial disease with exceptional accuracy. NGS also provided a DNA fingerprint for the sample, assisting embryo identification. It was possible to complete NGS analysis in a timescale compatible with a fresh embryo transfer. Furthermore, the cost was significantly less than existing methods for embryo testing, suggesting this approach may ultimately bring preimplantation diagnosis for serious genetic disorders within the reach of a much larger number of patients. Following rigorous pre-clinical validation, the novel protocol was applied clinically, resulting in a healthy ongoing pregnancy. This represents the first report detailing a pregnancy achieved after using an NGS-based embryo screening protocol

OBJECTIVE: To assess the relationship between maternal age, chromosome abnormality, implantation, and pregnancy loss. DESIGN: Multicenter retrospective study. SETTING: IVF centers in the United States. PATIENT(S): IVF patients undergoing chromosome screening. INTERVENTION(S): Embryo biopsy on day 3 or day 5/6 with preimplantation genetic diagnosis (PGD) by array comparative genomic hybridization. MAIN OUTCOME MEASURE(S): Aneuploidy, implantation, pregnancy, and loss rates. RESULT(S): Aneuploidy rates increased with maternal age from 53% to 93% for day 3 biopsies and from 32% to 85% for blastocyst biopsies. Implantation rates for euploid embryos for ages <35-42 years did not decrease after PGD: ranges 44%-32% for day 3 and 51%-40% for blastocyst. Ongoing pregnancy rates per transfer did not decrease for maternal ages <42 years after PGD with day 3 biopsy (48.5%-38.1%) or blastocyst biopsy (64.4%-54.5%). Patients >42 years old had implantation rates of 23.3% (day 3), 27.7% (day 5/6), and the pregnancy rate with day 3 biopsy was 9.3% and with day 5 biopsy 10.3%. CONCLUSION(S): Selective transfer of euploid embryos showed that implantation and pregnancy rates were not significantly different between reproductively younger and older patients up to age 42 years. Some patients who start an IVF cycle planning to have chromosome screening do not have euploid embryos available for transfer, a situation that increases with advancing maternal age. Mounting data suggests that the dramatic decline in IVF treatment success rates with female age is primarily caused by aneuploidy.

Our objective is to describe a successful live birth from oocyte vitrification followed by thaw, fertilization, blastocyst culture, trophectoderm biopsy, vitrification, and subsequent thaw. Fifteen mature oocytes were frozen from a patient with uterine factor infertility. Thirteen oocytes survived the thaw, and five underwent trophectoderm biopsy and were refrozen. Three euploid embryos were obtained. A single euploid embryo was transferred in the second thaw cycle to a known recipient leading to the delivery of a normal male infant. This case report is proof of the concept that preimplantation screening and diagnosis is an option for fertility preservation patients.

OBJECTIVE: To compare cycle and clinical outcomes between natural or medicated FET cycles. DESIGN: Retrospective study in an academic institution. MATERIALS AND METHODS: There were 1177 FET cycles from 2005- 2012. FET included embryos created from autologous or donor oocytes. Embryo transfers from oocyte thaws were excluded. Groups were analyzed for % donor cycles, % vitrification, % preimplantation genetic diagnosis/ screening (PGD), number of monitoring days, embryos transferred (ET), embryos thawed, day of embryo transfer, maximal endometrial (EM) thickness before transfer, peak estradiol (E2) and progesterone (P4) in follicular cycle, clinical pregnancy rate (CPR), spontaneous abortion rate (SABR) and ongoing/live birth rate (OP/LBR). CPR was defined by fetal cardiac activity on 1st-trimester ultrasound. SABR was calculated per pregnancy with a sac. T-tests were performed with p<0.05. RESULTS: Subset analysis was performed excluding PGD cycles, confirming no difference between natural and medicated cycles except for days of monitoring, peak E2 and peak P4. CONCLUSION: There can be advantages to both natural FET (lack of medication costs and injections) and medicated cycles (flexibility). Natural and medicated FET are equally effective in terms of pregnancy outcomes and should be offered based on patient preference and suitability (Table Presented)