Age related probability of having a chromosomally normal embryo for transfer following trophectoderm biopsy and comprehensive chromosome screening

Gynecology & Reproductive Sciences, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ; cGenetics, Rutgers-The State University of New Jersey, Piscataway, NJ. OBJECTIVE: Although it is known that mitochondria play a critical role in embryogenesis, the ability to characterize the sequence integrity of the entire mitochondrial genome in an embryo has yet to be developed. This study seeks to establish a novel NGS based methodology to characterize the entire mitochondrial genome sequence in human embryos for the first time. DESIGN: Blinded. MATERIALS AND METHODS: The mitochondrial genomes of 2 cell lines were sequenced using the ‘‘gold-standard’’ Sanger sequencing to identify bona fide differences between the 2 sequences. Controlled mixtures of two cell lines were made to produce known levels of mitochondrial heteroplasmy (0, 5, 10, 25, and 50% of one cell line to the other). NGS was performed on each mixture, beginning with either genomic DNA to prove the ability to detect heteroplasmy and then with 5-cell quantities to model the sensitivity required to analyze a trophectoderm (TE) biopsy. The validated assay was then applied to TE biopsies of 20 euploid blastocysts to establish applicability in a relevant tissue type. RESULTS: NGS based evaluation of genome-wide mitochondrial heteroplasmy in control mixtures of the 2 cell lines accurately ranked each level of heteroplasmy with 100% consistency and linearity in the genomic (r2¼96; P<0.0001) and 5 cell mixed samples (r2¼0.95; P<0.0001). Additionally, mitochondrial heteroplasmy in TE samples from 20 euploid blastocysts provided results with precision equivalent to the 5-cell model samples with all samples having 100% sequence coverage and an average depth of 8000x. CONCLUSION: This study has resulted in the successful development of an accurate NGS based methodology to characterize genome wide heteroplasmy in human embryos for the first time. Studies are ongoing to determine whether mitochondrial heteroplasmy as measured by this assay can be applied to predict embryonic reproductive potential, and to provide more insight into the cause of embryonic developmental arrest and the etiology of reproductive senescence.

O-197 Tuesday, October 23, 2012 05:15 PM DIMINISHED OVARIAN FUNCTION IS ASSOCIATED WITH DISTINCTIVE DNA METHYLOME CHANGES IN HUMAN OVARIAN GRANULOSA CELLS. B. Yu,a A. DeCherney,a J. Segars, Jr.,a A. Ignaszewski,b V. Russanova,c B. Howard.c aProgram in Reproductive and Adult Endocrinology, NICHD, NIH, Bethesda, MD; bEmbryology Laboratory, Shady Grove Fertility Reproductive Science Center, Rockville, MD; c Program in Genomics of Differentiation, NICHD, NIH, Bethesda, MD. OBJECTIVE: Diminished ovarian function is a primary cause for age-related decline in female fertility; however, its underlying mechanism remains unclear. This study investigated whether epigenetic mechanisms may underlie the ovarian aging process. DESIGN: Translational research. MATERIALS AND METHODS: Genomic DNA methylation patterns in ovarian granulosa cells were compared between two groups of women with differences in age and ovarian function. Group A consisted of young oocyte donors (mean age¼26 years) with a robust response to ovarian stimulation during ART (mean number of oocytes retrieved¼25). Group B comprised poor responders who were older (mean age ¼40 years) and responded poorly to ovarian stimulation during ART (oocytes retrieved %4 and peak estradiol level % 1000 pg/ml). Methylated DNA Capture followed by Next Generation Sequencing (MethylCap-seq) was used to compare DNA methylomes between the two groups. RESULTS: Two sets of experiments using ovarian granulosa cells from 20 individuals in each group revealed highly consistent differences in DNA methylome patterns between groups A and B. Compared to young women, ovarian granulosa cells from older women with diminished ovarian function showed reproducible focal hyper-methylation. Over 97% of regions that exhibited at least 5-fold enrichment and 2-fold change were more highly methylated in group B. These DNA methylation changes were selectively localized to the X chromosome, and this preferential localization became more striking in proximity (%1kb) to transcription start sites. CONCLUSION: Whole genome searches revealed focal hyper-methylation, especially on the X chromosome, in older women with diminished ovarian function, compared to young oocyte donors. Consistent differences in DNA methylation profiles between these two groups indicate that epigenetic changes in ovarian granulosa cells are associated with

FERTILITY & STERILITYÒ

the age-related decline in ovarian function. Supported by: In part by Intramural Programs PRAE and PGD, NICHD, NIH. O-198 Tuesday, October 23, 2012 05:30 PM AGE RELATED PROBABILITY OF HAVING A CHROMOSOMALLY NORMAL EMBRYO FOR TRANSFER FOLLOWING TROPHECTODERM BIOPSY AND COMPREHENSIVE CHROMOSOME SCREENING. D. A. Kelk, J. Lo, K. Reyes, M. P. Leondires, J. M. Hurwitz, C. M. Murdock. Reproductive Medicine Associates of Connecticut, Norwalk, CT. OBJECTIVE: It is well documented that aneuploidy rates increase considerably with maternal age. This study examines the age related probability of having euploid embryos available for transfer following trophectoderm (TE) biopsy and comprehensive chromosomal screening (CCS). DESIGN: Retrospective review of chromosomal data on 567 blastocysts from 124 CCS cycles. MATERIALS AND METHODS: Embryos were group cultured in 50ml drops of Global culture media in 5-well dishes in MINC incubators with a 6.5% CO2/5.0% O2 gas mixture until TE biopsy was performed on Day 5 or 6. Blastocysts were placed in individual 50ml drops in individual wells in a 5-well dish following biopsy. RESULTS: A total of 567 blastocysts were biopsied for 124 patients with maternal age ranging from 29 to 47 years of age. Of the 567 TE biopsies performed, 55.7% were performed on Day 5 and 44.3% on Day 6. The no diagnosis rate was 1.9%. The average number of blastocysts biopsied and percent of aneuploid embryos by maternal age as well as the percent of patients with euploid embryos available for transfer are displayed in Table 1. Aneuploidy Rates by Patient Age

Maternal # Patients Avg # Blastocysts % % Patients with Age with Biopsy Biopsied (n) Aneuploid Euploid for ET <35 35-37 38-40 41-42 43+ All

14 20 59 22 9 124

8.3 (116) 5.5 (110) 4.0 (236) 3.7 (82) 2.5 (23) 4.6 (567)

21.4% 40.0% 58.9% 72.8% 72.7% 50.2%

100% 95.0% 74.6% 59.1% 33.3% 75.0%

CONCLUSION: As expected, the rate of aneuploidy increased with maternal age. In patients <35, the aneuploidy rate was 21.4% but increased to greater than 70% for patients over 40. There was an age-related decrease in the percentage of patients with euploid embryos for transfer. Only 33% of patients >43 had a euploid blastocyst, while 100% of patients <35 had euploid embryos available for ET. Although high pregnancy rates/ETare achieved using CCS, age remains the limiting factor in obtaining a euploid embryo for transfer. These data provide valuable information for counseling all patients considering ART.

O-199 Tuesday, October 23, 2012 05:45 PM MOLECULAR KARYOTYPE ANALYSIS OF SINGLE NUCLEOTIDE POLYMORPHISM MICROARRAY FOR EARLY SPONTANEOUS MISCARRIAGE AFTER ASSISTED REPRODUCTIVE TECHNOLOGY. G. Li, Y. Liu, L. Hu, Y. Guo, Y. Su, Y. Sun. Reproductive Medical Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China. OBJECTIVE: To detect molecular karyotype of early spontaneous abortion tissue after assisted reproductive technology (ART) utiliting single nucleotide polymorphism microarray (SNP array). DESIGN: Retrospective. MATERIALS AND METHODS: IRB approval was obtained. Miscarriage tissues underwent DNA extraction and 23-chromosome SNP microarray analysis using HumanCytoSNP-12 DNA beadchips and GenomeStudio software. RESULTS: 81 patients were enrolled. 16 experienced natural conception (NC) and 65 were pregnant by ART. Of the 65 cases, 4 underwent artificial insemination (AI), 32 fresh in vitro fertilization-embryo transfer (IVF-ET),

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