Reanalysis of failed vitrified euploid blastocyst transfer cycles using next-generation sequencing

from a second biopsy for each embryo. Phase 2 involved clinical utilization of combined SGD and CCS testing with follow-up. RESULTS: Workup time approximated 1 month for each case. Phase 1 testing examined 152 embryos and demonstrated 99% concordance with reference lab data with all discrepancies confirmed as an error with the reference labs results. Phase 2 involved clinical application of these methods in 43 patients (304 embryos). A definitive result was reported for 99.7% (303/304) embryos, with 0.3% (1/304) having an inconclusive result likely due to recombination. All cases for which a sample was available in a newborn child confirmed the PGD result. In patients receiving a transfer with follow-up, clinical outcomes were excellent with an implantation rate of 75% (12/16), with 92% of patients achieving pregnancy. CONCLUSIONS: This methodology has demonstrated excellent concordance with current methods of SGD and provides a unique opportunity to avoid pitfalls of WGA when targeting additional loci in the genome in parallel with CCS. Additional advantages of the method include the ability to manage microdeletion and duplication cases and the potential for a 4 hour turnaround time. In addition, it is well established that SNP markers are denser than STRs thus generally reducing the distance of markers from mutations and the potential impact of recombination. P-506 Wednesday, October 21, 2015 THE CLINICAL OUTCOME OF PREIMPLANTATION GENETIC DIAGNOSIS (PGD) CYCLES UNDER PRIVATELY FUNDED AND PUBLICLY FUNDED PERIODS IN ONE CENTER. A. Ao,a X. Zhang,b L. Zhang,b W. Buckett.b aObstetrics and Gynecology, McGill Univerisity, Montreal, QC, Canada; bMcGill University, Montreal, QC, Canada. OBJECTIVE: To compare the overall clinical outcome of IVF-PGD cycles during privately funded period and publicly funded period in one center. DESIGN: A single center retrospective study. MATERIALS AND METHODS: All couples that underwent IVF-PGD cycles within specific study periods were divided under two groups: Privately funded period (PRP) where the IVF and PGD treatment cycles were privately funded and publicly funded period (PUP), where the treatment cycles were funded by the provincial government healthcare system. Under Privately funded period, 298 cycles (195 PGS and 42 translocation and 61 SGD) and under publicly funded period, 221 cycles (59 PGS and 72 translocation and 90 SGD) underwent IVF-PGD procedure. The outcomes were assessed for pregnancy rate and live birth rate. RESULTS: The average age of female patients for both private and publicly funded periods were similar (35 vs 36 years old). The fertilization rate (73% vs 72%), embryo biopsy rate (80% vs 80%) and genetic diagnosis rate (89% vs 94%) were not significantly different between these two groups. More embryos were transferred per cycle during PRP than PUP (2.34 vs 1.12, p<0.05) but did not have an effect on clinical pregnancy rate per embryo transfer cycle (CPR/ET), 38.7% vs 36.6%. The Implantation rate was however significantly higher in PUP group (23% vs 32%, p<0.05). The live birth rate was similar in both groups (30% vs 31%). There was no difference in miscarriage rate (22% vs 23%). A total of 116 babies were born during PRP (27 twins, 2 triplets and 56 singletons) compared to 51 babies born (49 singletons, 1 twins) during PUP where all but one pregnancy was twins and 4 ongoing singleton pregnancies. CONCLUSIONS: Restricted number of embryo transfer in publicly funded period had positive effect of reducing multiple pregnancy rate from 35% to 1.9% without compromising CPR and Live birth rate. There may be multiple factors which influenced a higher number of embryo transfer in privately funded period group in addition to the fact that patients were fully responsible for the cost of IVF-PGD treatment cycle.influenced a higher number of embryo transfer in privately funded period group in addition to the fact that patients were fully responsible for the cost of IVF-PGD treatment cycle. P-507 Wednesday, October 21, 2015 REANALYSIS OF FAILED VITRIFIED EUPLOID BLASTOCYST TRANSFER CYCLES USING NEXT-GENERATION SEQUENCING. S. Zozula,a M. C. Schiewe,a J. Blazek,b R. E. Anderson,a A. Harutunian,c M. Large.b aSCIRS/FPG Labs, Newport Beach, CA; bGenesis Genetics, Houston, TX; cGenesis Genetics, Milford, MI. OBJECTIVE: In 2014, our VFET/PGS cycles produced cumulative implantation and ongoing pregnancy rates of 82% and 78%, respectively. We

FERTILITY & STERILITYÒ

strive to gain insight as to why some women do not succeed using euploid embryos. The aim of this study was to determine whether Next-Generation Sequencing (NGS) of euploid blastocysts (BL) offers greater sensitivity in aneuploidy detection than array Comparative Genomic Hybridization (aCGH) and thus may improve future outcomes. DESIGN: 53 DNA samples from aCGH determined euploid BL used in 49 VFET cycles that failed to implant (n¼34) or establish a viable pregnancy (n¼15) were re-karyotyped using NGS. MATERIALS AND METHODS: BL were previously cultured in GlobalÒ medium (LifeGlobal; 25 ml droplets under oil) + 7.5% SPS and tri-gas MCO5M Panasonic incubators. Trophectoderm (TE) biopsies were performed on day 5 and 6 before microSecure vitrification (mS-VTF) in DMSO-free VTF solutions (ICE). Euploid BL were subsequently rapidly warmed, eluted and equilibrated for 2-4hr prior to single embryo transfer (ET; n¼45) or dual ET (n¼4). Whole genome amplified DNA samples from TE biopsies previously determined to be euploid were analyzed by NGS for copy number variation using the VeriSeq-PGS platform (Illumina). The copy number variant (CNV) profiles obtained from sequencing were compared to the CNV profiles originally obtained using aCGH technology, using CAP validated calling protocols, to assess for the presence of mosaicism. Differences in outcomes were analyzed by the Chi-square test (p<0.05). Failed VFET cycles that could not be explained by an aneuploid NGS diagnosis were further investigated for potential uterine factors and BMI issues. RESULTS: NGS detected an increase (p<0.05) in TE mosaicism (26.4%; n¼14) of the embryos retested ranging in value from 25-60% mosaic, of which 17% were reclassified as aneuploid embryos based on the level of mosaicism detected in the TE biopsy. No overt correlations to BMI or uterine lining thickness issues were detected in failed VFET. CONCLUSIONS: Since 2011, approximately 75% of our VFET/PGS cycles using aCGH determined euploid BL typically implant and maintain viable pregnancies. Due to increased coverage and sensitivity, NGS was able to detect 9 additional aneuploid BL containing mosaicism in approximately 30-50% of the cells obtained during biopsy. Another 5 embryos were deemed potentially viable mosaics. Research still needs to be conducted to determine the significance and presence of mosaicism in the TE and its effect on successful pregnancies. Secondly, we were unable to explain, by genetic causes, why 74% of our other failed PGS cycles failed to achieve live births. Overall, based on the increased sensitivity and accuracy of NGS shown in this study, we could anticipate a 5-6% increase in viable implantations in future VFET/PGS cycles. Supported by: This study was conducted with the scientific collaboration of Genesis Genetics.

P-508 Wednesday, October 21, 2015 PREIMPLANTATION GENETIC DIAGNOSIS (PGD) FOR INHERITED DISORDERS USING SINGLE NUCLEOTIDE POLYMORPHISM (SNP) ARRAYS: CLINICAL OUTCOMES FROM 300 CYCLES. S. Jaroudi,a,b R. Prates,a R. E. Cabey,a D. Goldberg-Strassler,a W. Chang,c A. N. Beltsos,d C. A. Benadiva,e S. Anderson,f D. I. Hoffman,g M. Konstantinidis.a aReprogenetics, Livingston, NJ; bReprogenetics Middle East, Al Ain, United Arab Emirates; cSouthern California Reproductive Center, Beverly Hills, CA; dFertility Centers of Illinois, Chicago, IL; eUniversity of Connecticut, Farmington, CT; fMain Line Fertility, Bryn Mawr, PA; gIVF Florida Reproductive Associates, Margate, FL. OBJECTIVE: Assessment of a universal SNP array based protocol, known as Karyomapping, as a PGD treatment strategy for patients requesting embryo testing for inherited disorders. DESIGN: Karyomapping utilizes a universal protocol, applicable to virtually all patients, for linkage-based PGD. It eliminates the need to develop and optimize patient-specific protocols, drastically reducing the waiting time for initiation of IVF treatment. This technology was previously validated using the gold-standard polymerase chain reaction methodology (with 100% concordance) and has proven to be highly efficient for even the most complex of cases, including those not feasible using conventional PGD methods.Among the 300 Karyomapping cases performed between December 2013 and May 2015, clinical outcome and follow-up data was collected for 223 PGD cycles with transferable embryos. MATERIALS AND METHODS: Patients were referred from over 50 IVF centers in the USA. Embryos were biopsied at the blastocyst stage and frozen for transfer in subsequent cycle(s). Samples were whole genome amplified and analyzed using Karyomapping combined with direct mutation detection as needed and/or independent comprehensive chromosome screening (CCS) when requested (84% cases).

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