Accurate detection of segmental aneuploidy in preimplantation genetic screening using targeted next-generation DNA sequencing

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