Development and validation of next-generation sequencing (NGS) based PGD

MATERIALS AND METHODS: Included were patients referred to a PGD lab for array CGH with a diagnosis of idiopathic RPL. Pregnancy history and outcomes were obtained from 12 referring centers. Clinical pregnancy was defined as a gestational sac on ultrasound and ongoing pregnancy as a pregnancy past 2nd trimester of gestation. All cycles reaching biopsy were included. Miscarriage rates were compared to those expected for RPL according to the literature. RESULTS: Data on 181 cycles with follow up were included. The average maternal age was 37.2 with a range of 25-45 years. Patients having no normal embryos were significantly older (39.2 vs 36.7y, P<0.001) and had fewer embryos biopsied (4.2 vs 8.3, P<0.001) than patients having at least one normal embryo for transfer. See Table 1 for pregnancy results. CONCLUSION: Previous studies have showed that the miscarriage rate in idiopathic RPL patients over age 35 is 30-40%. The present results show a significantly lower miscarriage rate of 9% to 3% in couples conceiving after PGD with aCGH (P<0.001). Ongoing pregnancy rate per IVF-PGD cycle was higher in patients undergoing D5 biopsy compared to D3 biopsy despite having a similar number of chromosomally normal embryos in both groups. However, older patients and those with fewer eggs had a higher chance of not having euploid embryos. O-178 Tuesday, October 23, 2012 04:30 PM DEVELOPMENT AND VALIDATION OF NEXT-GENERATION SEQUENCING (NGS) BASED PGD. A. Fedick,a,b X. Tao,a B. Devkota,a D. Taylor,a,c R. T. Scott, Jr.,a,c N. R. Treff.a,c aReproductive Medicine Associates of New Jersey, Morristown, NJ; bMolecular Genetics, Microbiology and Immunology, UMDNJ- Robert Wood Johnson Medical School, Piscataway, NJ; cObstetrics, Gynecology, and Reproductive Sicences, UMDNJRobert Wood Johnson Medical School, New Brunswick, NJ. OBJECTIVE: NGS is an emerging technology which provides an unprecedented high-throughput, and highly parallel and base pair resolution data for genetic analysis. In order to investigate its applicability to PGD, this study aims to begin developing and validating NGS based technology for analysis in human embryos. DESIGN: Blinded. MATERIALS AND METHODS: Parental and blastocyst DNA were obtained from a couple at risk of transmitting Cystic Fibrosis (CF) to their offspring via inheritance of compound heterozygosity of the delta F508 and I507 mutations. Embryonic CF genotypes were obtained from 3 independent sources; a major reference laboratory, internal TaqMan allelic discrimination, and NGS based genotyping. CF testing results were compared across each protocol to evaluate the consistency of NGS. 96 invariant loci were also evaluated using copy number analysis for aneuploidy screening. NGS was performed using an Ion Torrent Personal Genome Machine. RESULTS: NGS provided 100% equivalent PGD diagnoses when compared to both a reference lab and internal qPCR based analyses (n¼16). In contrast to conventional PCR strategies, NGS provided a unique ability to identify compound heterozygosity due to the highly parallel single stranded nature of the sequence data. Furthermore, both single gene disorder and aneuploidy screening could be performed in parallel. CONCLUSION: This study has demonstrated that NGS can provide blastocyst PGD results with a high level of consistency to established methodologies while allowing simultaneous evaluation of a single gene disorder and aneuploidy from the same biopsy. Although it remains costly and time consuming, trends in cost reduction and development of vitrification may soon result in the practical application of NGS technology to PGD. O-179 Tuesday, October 23, 2012 04:45 PM 23-CHROMOSOME SINGLE NUCLEOTIDE POLYMORPHISM (SNP) MICROARRAY PREIMPLANTATION GENETIC SCREENING (PGS) FOR RECURRENT PREGNANCY LOSS (RPL) IN 687 IN VITRO FERTILIZATION (IVF) CYCLES AND 5871 EMBRYOS. K. J. Tobler,a P. R. Brezina,a A. T. Benner,b L. Du,b B. Boyd,b W. G. Kearns.a,b aDepartment of Gynecology and Obstetrics, Division of Reproductive Endocrinology and Infertility, Johns Hopkins Medical Institutions, Baltimore, MD; bGenetics, Center for Preimplantation Genetics, LabCorp, Rockville, MD. OBJECTIVE: We determined the pregnancy and miscarriage rates of patients undergoing IVF and dense microarray PGS for RPL. DESIGN: Retrospective review. MATERIALS AND METHODS: A retrospective review was conducted of all embryos that underwent PGS by 23 chromosome SNP microarrays from January 2010 to April 2012. Patients underwent standard IVF and PGS primarily due to a history of R 2 spontaneous miscarriages. Embryo biopsy

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was performed at either the cleavage or blastocyst stage. Sample DNA was amplified and analyzed using HumanCytoSNP-12 DNA beadchips and GenomeStudio and KaryoStudio software. Embryos derived from parents with known translocations or inversions were excluded from the study. Data was split into four patient groups based on maternal age (<35, 35-37, 38-40, >40). Microarray data was also compared to previously published fluorescence in situ hybridization (FISH) PGS data from our laboratory (previously published). Binomial confidence intervals for proportions were calculated. RESULTS: A total of 5871 molecular karyotypes were obtained from 687 IVF cycles. The overall pregnancy rate per IVF cycle for women in this trial undergoing 23-chromosome SNP microarray PGS for RPL was 57% with a miscarriage rate of 9%. In blastocyst stage PGS, the pregnancy rate, 75%, was significantly (P<0.001) higher than the cleavage stage pregnancy rate, (47%). Miscarriage rates were significantly (P¼0.002) higher in the cleavage stage (12%) than the blastocyst stage (5%). Both cleavage and blastocyst stage PGS yielded significantly (P<0.001) higher pregnancy rates than FISH PGS. Pregnancy rates decreased and miscarriage rates increased in all groups with advancing maternal age. CONCLUSION: PGS using 23 chromosome SNP microarrays in women with RPL was superior to FISH PGS. Furthermore, performing the biopsy at the blastocyst stage was superior to cleavage stage biopsy. The application of dense microarray 23 chromosome PGS at the blastocyst stage may substantially improve pregnancy outcomes in couples suffering from RPL. O-180 Tuesday, October 23, 2012 05:00 PM HIGH RATE OF MICRODELETIONS IN MISCARRIAGE PRODUCTS OF CONCEPTION (POC) SAMPLES SUGGESTS ROLE FOR PREIMPLANTATION GENETIC DIAGNOSIS (PGD). M. Rabinowitz, S. Sigurjonsson, J. Keller, M. Maisenbacher, Z. Demko, B. Pettersen. Natera, Inc., San Carlos, CA. OBJECTIVE: Evaluate rate of deletions and duplications (del/dup) including microdel/dups (1-10MB) found in POC samples to determine potential value of routine screening during PGD. DESIGN: Retrospective analysis. MATERIALS AND METHODS: Review of 2,397 consecutive POC samples sent from 244 clinics to a single reference lab. POC and maternal blood or buccal sample was collected by referring clinic and shipped overnight to the reference lab where chorionic villi were sorted/cleaned using a standardized technique. Genotyping was performed using Illumina CytoSNP-12 microarrays and an informatics technique (Parental Support) that also identifies maternal cell contamination and parental source of aneuploidy. RESULTS: 5.25% of samples (98) contained del/dup. 3.4% of samples (64) contained at least one microdel/dup, 8 of which contained 2 microdel/dups for a total of 72 microdel/dups. 26 of the 72 (36%) microdel/dups seen were of significant size or location to be classified as possibly clinically significant, resulting in a total of 25 cases (1.1%) with microdel/dups of possible/known clinical significance. These microdel/dups are unlikely to cause miscarriage and can lead to birth defects or a genetic syndrome in the born child. Results Number % Total excluding of cases % Total MCC Miscarriage samples tested 2,397 46,XX maternal cell contamination 529 Normal fetal (46,XX or XY) 753 Full chromosome aneuploidy and/or UPD 947 Triploidy 114 Tetraploidy (3:1 segregation) 4 del/dup 98 del/dup 1-10MB 64 Of possible/known clinical significance 25

100% N¼1868 22% 31.4% 40.3% 39.5% 50.7% 4.8% 6.1% 0.17% 0.21% 4.1% 5.25% 2.67% 3.4% 1.1% 1.34%

CONCLUSION: PGD has been used to help prevent conception of a child at high risk for genetic disease. At 1.1%, frequency of possibly clinically significant microdel/dups at birth is significantly higher than incidence of Down syndrome at 1/600. It would have substantial clinical value if emerging PGD technology platforms such as sequencing could target common microdel/dup syndromes for routine screening. O-181 Tuesday, October 23, 2012 05:15 PM BENEFITS OF PREIMPLANTATION GENETIC SCREENING (PGS) IN ADVANCED MATERNAL AGE (AMA) WOMEN

Vol. 98, No. 3, Supplement, September 2012