Time-lapse imaging of multinucleated embryos and the association with aneuploidy determined by preimplantation genetic screening

CONCLUSIONS: High rates of aneuploidy and mosaicism in donor oocyte cycles are consistent with the pregnancy rates of untested donor embryo transfers. The use of PGS with NGS can prevent transfer of aneuploid embryos in donor cycles.

P-120 Tuesday, October 18, 2016 DISCORDANCE AMONG SERIAL BIOPSIES OF MOSAIC R. H. Walmsley,a K. Bauckman,b EMBRYOS. G. Garrisi,a a c ca R. J. Mendola, P. Colls, S. Munne. Institute for Reproductive Medicine and Science at Saint Barnabas, Livingston, NJ; bReprogenetics, Highland Park, IL; cReprogenetics, Livingston, NJ.

Donor Oocyte NGS Results from Reference Genetics Laboratory

Total cycles Total Trophectoderm Biopsy Specimens for NGS Avg age of Donors (years) Avg age of Recipient (years) Avg Number Cycles per Center Avg Number Blastocysts Biopsied per Patient Euploid (%) Aneuploid(%) Mosaic (%) No Amplification (%) Rate of Degraded DNA (%)

268 2062 25.8
2.3 43.1
6.3 7.1
10.5 7.7
4.1 48.7
23.8 22.9
20.4 28.4
21.9 2.5
7.3 0.8
4.1

References: 1. Maxwell, S., et al. Why do euploid embryos miscarry? A retrospective study comparing aneuploidy rates within presumed euploid embryos resulting in miscarriage or live birth using next-generation sequencing (NGS)[abstract]. In: ESHRE; 2016 July 5-8; Helsinki, Finland. 2. Greco, E., et al. (2015). ‘‘Healthy Babies after Intrauterine Transfer of Mosaic Aneuploid Blastocysts.’’ N Engl J Med 373(21): 2089-2090.

OBJECTIVE: Some embryos determined to be mosaic have been shown to be capable of normal implantation and development, albeit with reduced implantation and increased miscarriage rates. This study correlates the original biopsy findings with details of the localization of aneuploid cells in mosaic blastocysts. DESIGN: Comparison of PGS results to inner cell mass (ICM) and several trophectoderm samples of the same embryo. MATERIALS AND METHODS: Embryos determined to be fully (90100%) aneuploid or instead to be mosaic (10-90% abnormal cells) during routine PGS cycles were reanalyzed with Next Generation Sequencing (NGS). An ICM sample, usually consisting of 5-10 cells, was initially isolated. Subsequently, 2-4 trophectodermal samples ranging in size from 10 to 50 cells were taken from each embryo. The NGS result from each specimen was compared to the original biopsy. Mosaics were sub-classified as complex mosaics (3 or more chromosome abnormalities), aneuploid mosaics (1-2 chromosomes being mosaic) or partial aneuploid mosaic (normal / partial aneuploidy). RESULTS: The results are shown in the table. Five embryos were normal in all retested samples (5/43, 11.6%). All partial mosaic embryos with normal ICMs had normal or mostly normal retested trophectoderm samples (8/8), compared to 2/8 with full aneuploid mosaicism. Distribution of Normal Biopsy Samples in Mosaic Embryos

P-119 Tuesday, October 18, 2016 IMPACT OF 24-CHROMOSOME ANEUPLOIDY TESTING ON THE OUTCOME OF PGD FOR SINGLE GENE DISORDERS. S. Rechitsky,a T. Pakhalchuk,a M. Prokhorovich,a a b c a G. San Ramon, Z. Zlatopolsky, A. Kuliev. Preimplanatation Molecular Genetics, Reproductive Genetic Innovations, Northbrook, IL; bCytogenetics, Reproductive Genetic Innovations, Northbrook, IL; cResearch, Reproductive Genetic Innovations, Northbrook, IL. OBJECTIVE: Although preimplantation 24-cromosomes aneuploidy testing (24-AT) is becoming an attractive option for ART patients of advanced reproductive age, it may be also useful as an additional test in the practice of PGD for single gene disorders (SGD). So the objective of this work is to investigate the impact of 24-AT on the reproductive outcome of PGD for SGD in couples of advanced reproductive age, based on our experience, representing the world’s largest PGD series for SGD. DESIGN: Retrospective Study MATERIALS AND METHODS: PGD for SGD was performed using the whole genome amplification (WGA) product, obtained in 24-AT procedure, allowing both PGD for SGD and concomitant 24-AT in the same biopsy material. A combined testing was performed in 762 (21.7%) cycles of a total of 4501 PGD cycles performed for SGD and HLA typing by the present time. This involved PGD for SGD combined with 24-AT for more than a hundred different conditions with or without preimplantation HLA typing. RESULTS: A total of 1835 unaffected or HLA matched embryos free of 24-chromosome aneuploidy were detected, from which 616 were transferred in 533 cycles (1.15 embryos per transfer, on the average), resulting in 375 (70.5%) unaffected pregnancies, 347 deliveries (92.3%) and birth of 382 healthy or HLA matched children. This is significantly different from the outcome of PGD without concomitant 24-AT (70.5% vs. 49.9%), suggesting a significantly improved pregnancy rate in a combined 24-AT, despite transferring 1.15 vs. 1.79 embryos on the average in PGD cycles without 24-AT. In addition, a two-fold reduction of spontaneous abortion rate was observed in the outcome of these pregnancies: 29 (7.7%) spontaneous abortions in 376 pregnancies, compared to 211 (14.8%) in 1424 PGD pregnancies without 24-AT, despite comparable maternal age. CONCLUSIONS: The results demonstrate significant improvement of reproductive outcome of PGD for SGD and HLA typing with introduction of concomitant 24-AT, including significant improvement of pregnancy rate from 49.9% to 70.5%, and reduction of spontaneous abortions rate from 14.8% to 7.7% .

FERTILITY & STERILITYÒ

Initial NGS Diagnosis Full Aneuploidy Full Partial Aneuploidy Complex Mosaic Partial Aneuploid Mosaic Mosaic Full Aneuploidy All Mosaics

Total Normal Embryos ICM

Normal Trophectoderm (Total of all samples) 4/70 (6%) 2/10 (20%)

19 3

0/19 (0%) 0/3 (0%)

12 15

2/12 (17%)a 8/15 (53%)b

14/44 (32%) 30/59 (51%)d

16

8/16 (50%)c

10/55 (18%)e

43

10/43 (23%) 54/158 (34%) a vs. b+c: p<0.05 d vs. e: p<0.01

CONCLUSIONS: A diagnostic biopsy of 5 trophectoderm cells may detect mosaicism that does not exist in the ICM and may be highly localized in the trophectoderm. In this group of 43 mosaic embryos, there were 12 (28%) with normal ICMs that also tested normal for all or several trophectoderm specimens. The presence of aneuploid cell lines only in limited areas of the trophectoderm suggests that the initial chromosomal malsegregation event occurred relatively close to the time of biopsy. The implantation potential of a blastocyst with a normal ICM and a mildly or highly mosaic trophectoderm remains unknown. Embryos with an initial diagnosis of complex mosaic are less likely to have a normal ICM. Embryos diagnosed as partial aneuploid mosaic may be more likely to possess both a normal ICM and substantially normal trophectoderm, compared to full aneuploid mosaics. These findings may aid the determination of mosaic embryos that are suitable for transfer. Depending on patient age, a mosaic embryo may have the same or lower chance of being compromised by aneuploidy as an undiagnosed embryo, and should be given the appropriate replacement priority if no euploid embryos are available. P-121 Tuesday, October 18, 2016 TIME-LAPSE IMAGING OF MULTINUCLEATED EMBRYOS AND THE ASSOCIATION WITH ANEUPLOIDY DETERMINED BY PREIMPLANTATION GENETIC SCREENING. L. R. Goodman, J. M. Goldberg, T. Falcone, N. Desai. Women’s Health Institute, Cleveland Clinic, Cleveland, OH.

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OBJECTIVE: To determine if embryo multinucleation visualized by continuous time-lapse imaging is associated with an increased risk of aneuploidy DESIGN: Prospective cohort study MATERIALS AND METHODS: A series of patients under the age of 38 years undergoing in-vitro fertilization for unexplained infertility were recruited to participate when at least 20% of their embryos exhibited evidence of multinucleation (MU) on day two of continuous time-lapse culture in the EmbryoScopeTM. All expanded blastocysts underwent trophectoderm (TE) biopsy on day 5 or 6 for genetic screening. Un-biopsied early blastocysts, as well as embryos arrested at cleavage/morula stage, were also sent for analysis. Continuous variables were compared with the student’s t-test, categorical with chi-squared and logistic regression was performed to account for confounding variables. RESULTS: A total of 133 embryos from nine couples were evaluated for genetic analysis. The average age of patients was 31.9 +/- 3.2 years, who had 15.3 +/- 7.3 embryos with an average of 56.3 +/- 18.9% exhibiting MU. Of the 133 embryos, 72 (54.1%) developed to the expanded blastocyst stage and were able to undergo TE biopsy. There was no difference in embryo development to the expanded blastocyst stage between MU embryos and those without evidence of MU (56.9 vs. 43.1%; p ¼ 0.37). When all embryos were evaluated, 57 (42.9%) were euploid, 49 (36.8%) exhibited aneuploidy, 22 (16.5%) were resulted as nonconcurrent and the remaining 5 (3.8%) had no amplification. There was no difference aneuploidy rate between MU and non-MU embryos (44.4 vs. 47.1%; p ¼ 0.83). When accounting for confounding factors, MU embryos were equally as likely to be genetically normal (OR 1.08 95% CI 0.48 - 2.46; p ¼ 0.85) and to make it to the blastocyst stage (OR 0.69 95% CI 0.31 - 1.48; p ¼ 0.34) as those without evidence of MU. Of the TE biopsied embryos with diagnosis (n ¼ 53), the rate of aneuploidy was also found to be similar between MU and non-MU embryos (45.5 vs. 35.0%; p ¼ 0.57). When evaluating kinetic parameters available though continuous time-lapse imaging, none of the timing of developmental milestones differed by MU. Stage of arrest was also similar between the two groups. CONCLUSIONS: In this study, there was no increase in risk of aneuploidy in multinucleated embryos. Additional data is needed to corroborate these findings. Supported by: The Foundation for Embryonic Competence. P-122 Tuesday, October 18, 2016 ACCURATE DETECTION OF SEGMENTAL ANEUPLOIDY IN PREIMPLANTATION GENETIC SCREENING USING TARGETED NEXT-GENERATION DNA SEQUENCING. M. A. Umbarger, K. Germain, A. Gore, B. Breton, L. C. Walters-Sen, T. Mullen, N. Faulkner. Good Start Genetics, Cambridge, MA. OBJECTIVE: To evaluate the sensitivity and specificity of segmental aneuploidy detection as a function of segmental copy number variant (CNV) size using EmbryVu, a targeted next-generation DNA sequencing (NGS) assay. DESIGN: We have previously described a targeted NGS-based method for preimplantation genetic screening (PGS) that has been validated for accurate detection of full chromosome aneuploidy (Gole et al., 2016). The EmbryVu test leverages the FAST-SeqS NGS technology to target tens of thousands of sites across the genome and therefore has the potential to detect sub-chromosomal or segmental aneuploidy. To measure the specificity and sensitivity of this test with respect to segmental aneuploidy, we first tested cell line-derived samples known to contain specific segmental CNVs of varying sizes as well as known negative samples. Next, to evaluate performance with respect to segmental CNVs falling in parts of the genome for which samples were not available, we developed and implemented a simulation strategy that enables genome-wide assessment of automated segmental calling sensitivity and specificity. MATERIALS AND METHODS: Cell-line derived genomic DNA from 138 cell lines with known segmental duplication/deletions ranging in size from 0.1 to 80 Mb and 170 known normal samples were run through the EmbryVu assay and the resulting molecular karyotype calls were compared to the previously reported karyotypes. Next, using data derived from trophectoderm biopsies from embryos that were classified as normal on both EmbryVu and microarray comparative genomic hybridization, we simulated segmental duplications of varying sizes across the entire genome and evaluated the frequency at which these duplications were called. RESULTS: For the cell-line derived samples, 112 of the 115 expected segmental copy number variants of size at least 10 Mb were called, yielding

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ASRM Abstracts

a sensitivity estimate of 0.974 (0.926,0.991). A total of 6 samples were falsely classified as aneuploid, yielding a specificity estimate of 0.965 (0.925,0.984), an estimate that was very similar to that obtained in our embryo-based simulation studies - 0.968 (0.909, 0.989). In addition, our simulations illustrate that while there do exist positions in the genome where segmental detection sensitivity is reduced, these regions often coincide with heterochromatic and/or highly repetitive regions that are likely problematic with most PGS technologies. CONCLUSIONS: The EmbryVu PGS test, using the FAST-SeqS targeted NGS technology, is capable of accurately detecting sub-chromosomal changes of 10 Mb and larger with an analytic sensitivity of >97% and a specificity that is comparable to that noted in our previous validation study. Comprehensive genome-wide simulations further expand upon our understanding of the performance of this test. References: 1. Gole, J. et al. Analytical validation of a novel next-generation sequencing based preimplantation genetic screening technology. Fertility and Sterility 2016;105: e25. Supported by: This study was funded by Good Start Genetics, Inc. P-123 Tuesday, October 18, 2016 SEGMENTAL ANEUPLOIDY IN PREIMPLANTATION GENETIC SCREENING STRATIFIED BY AGE AND CLINICAL INDICATION USING TARGETED NEXT-GENERATION DNA SEQUENCING. N. Faulkner, L. C. Walters-Sen, B. Breton, A. Gore, M. Zhu, K. Robinson, S. E. Hallam. Good Start Genetics, Cambridge, MA. OBJECTIVE: To present the clinical experience of evaluating >5,000 patient embryos for segmental aneuploidy using a targeted next-generation DNA sequencing (NGS) assay. DESIGN: The EmbryVu assay is a targeted NGS-based method for preimplantation genetic screening (PGS; initially validated for accurate detection of full chromosome aneuploidy). The method targets tens of thousands of sites across the genome and therefore has the potential to detect segmental aneuploidy. We recently validated the sensitivity and specificity of this test with a modified analysis algorithm. Here we share data from a retrospective analysis of clinical patient data reprocessed through the modified algorithm. MATERIALS AND METHODS: Trophectoderm biopsies containing 5-10 cells from blastocyst-stage embryos scheduled for FET were submitted for clinical PGS analysis. All samples were analyzed using the EmbryVu assay and associated bioinformatics pipeline. Data was then de-identified, except for egg age and clinical indication for testing, and re-analyzed using the modified algorithm to identify sub-chromosomal abnormalities. RESULTS: Over 5,000 embryo biopsies from over 1,000 patient cycles were retrospectively studied. Segmental changes were observed in each age and clinical indication group, including donor eggs. 5-42% of aneuploid embryos contained a segmental aneuploidy which was inversely correlated with age, as expected. Segmental changes were seen in both embryos with additional whole chromosome aneuploidies and in isolation as the only abnormal finding. When evaluating all embryo biopsies submitted, we identified sub-chromosomal abnormalities in 5-16% of total embryos which concurs with prior publications. Other than known familial balanced translocations, there was no significant correlation between clinical indication and rate of segmental aneuploidy. However, the clinical utility of specifically assessing the presence or absence of sub-chromosomal aneuploidy was apparent in a number of families with previously unidentified parental balanced rearrangements. CONCLUSIONS: Segmental gains and losses of chromosomal material have been reported in the literature to occur in approximately 2-15% of all blastocyst stage embryos. Our data supports this rate across all age groups for segmental aneuploidies, unlike whole chromosome aneuploidy that has a marked increase correlated with egg age. The EmbryVu assay with the updated algorithm is a low cost-to-consumer PGS test that can detect segmental aneuploidy and is beneficial to all ART patients including young and donor populations. Supported by: This study was funded by Good Start Genetics, Inc. P-124 Tuesday, October 18, 2016 MISCARRIAGE AND ANEUPLOIDY RATES ARE HIGHER IN VERY YOUNG DONORS. J. LLACER, J. Guerrero, B. Lledo, J. Ortiz, J. Ten, R. Bernabeu. Reproductive Medicine, Instituto Bernabeu, Alicante, Spain.

Vol. 106, No. 3, Supplement, September 2016