First clinical application of simultaneous preimplantation mitochondrial DNA mutation load and 24 chromosome aneuploidy screening

P-110 Tuesday, October 26, 2010 IMPROVEMENT IN DIAGNOSTIC ACCURACY USING SINGLE NUCLEOTIDE POLYMORPHISM (SNP) MICROARRAYS VERSUS FLUORESCENCE IN SITU HYBRIDIZATION (FISH) IN PREIMPLANTATION GENETIC SCREENING (PGS) FOR ANEUPLOIDY. B. Yu, C. Chipko, K. S. Richter, E. A. Widra, A. DeCherney, W. G. Kearns. Program in Reproductive and Adult Endocrinology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD; Shady Grove Center for Preimplantation Genetics, Rockville, MD; Shady Grove Fertility Reproductive Science Center, Rockville, MD; The Johns Hopkins University School of Medicine, Baltimore, MD. OBJECTIVE: PGS for aneuploidy in women with recurrent pregnancy loss is controversial. One limitation of PGS using FISH is the limited number of chromosomes tested. SNP microarrays simultaneously test all 23 chromosomes from any embryonic cell. However, little data exists on the diagnostic improvement using SNP microarrays compared to FISH. The aim of this study was to test the hypothesis that using SNP microarrays increases diagnostic accuracy over 10-probe FISH. DESIGN: Retrospective study. MATERIALS AND METHODS: All patients referred for PGS from January 1 to April 15, 2010 with a history of R2 pregnancy losses were included. Four patients were tested by trophectoderm biopsy and all other patients had day-3 blastomere biopsy. 23-chromosome SNP microarray testing was used for each embryo. Prior to the availability of SNP microarray technology, we used 10-probe FISH for aneuploidy screening, testing chromosomes 13, 14, 15, 16, 17, 18, 21, 22, X, and Y. In order to measure the diagnostic improvement using SNP microarrays, we calculated the percentage of aneuploid embryos that would have been missed by 10-probe FISH. RESULTS: A total of 34 patients and 372 embryos were tested. Mean maternal age was 37  4.7 years and the average number of embryos tested was 10.9  6.1. Using SNP microarrays, 56% (210/372) of the embryos were diagnosed as abnormal and 3.4  2 euploid embryos were identified per patient. 13.3% of aneuploid embryos diagnosed by SNP microarrays would have been missed by 10-probe FISH. If 10-probe FISH had been used, 19.4% of FISH-diagnosed normal embryos would have been recommended for transfer when, in fact, they were aneuploid for untested chromosomes. In 23.5% of patients, FISH would have wrongly diagnosed 25% or more of the embryos as normal, with the highest error rate at 100%. CONCLUSION: PGS with SNP microarrays is diagnostically superior to 10-probe FISH. This improved accuracy of SNP microarrays may improve pregnancy outcomes. Supported by: In part by Program in Adult and Reproductive Endocrinology, NICHD, NIH. P-111 Tuesday, October 26, 2010 THE UNBELIEVABLE SHRINKING MOSAICISM PROBLEM OF PGD. T. Escudero, C. Gutierrez, P. Colls, D. Hill, K. Wiemer, M. Santi. Reprogenetics, Livingston, NJ; ART Reproductive Center, Beverly Hills, CA; Northwest Center for Reproductive Sciences, Kirkland, WA. OBJECTIVE: To determine the frequency of abnormal cells in mosaic embryos previously analyzed by PGD using array comparative genome hybridization (aCGH), as well as study mosaicism formation mechanisms. DESIGN: Reanalysis of non-replaced embryos on PGD patients. MATERIALS AND METHODS: Patients undergoing PGD using day 3 biopsy, aCGH, and day 5 replacement. Embryos were classified by aCGH as abnormal (75), undiagnosed (6), or normal but arrested (6). These embryos were fully analyzed by FISH using 19 probes (X,Y,1,2,7,8,9,10,12,13,14,15,16,17, 18,19,20,21,22). Mosaic mechanisms were defined as partial loss (2n/2n-1), balanced (2n/2n+1/2n-1), partial gain (2n/2n+1), chaotic, or partial polyploid (2n/ 4n). RESULTS: Of 87 embryos reanalyzed by FISH, 9 were mostly normal with 0-18% tetraploid cells (a normal feature in blastocysts); 1 had 18% XO cells and the rest were normal (classified by aCGH as abnormal), 2 had between 64% and 88% abnormal cells (both abnormal by aCGH), and the other 75 embryos had 100% abnormal cells, of which 30 were homogeneously aneuploid, 23 were homogeneously aneuploid for some chromosomes and mosaic for others, and 22 were mosaic 100% abnormal. In total 94% (45/48) mosaics were 100% abnormal, 2/48 had >50% abnormal cells, and 1/48 had <50% abnormal cells. On these 48 mosaics, the mechanisms were chaotic (25), partial loss (18), 2n/4n (7), balanced (7), and partial gain (4) (numbers do not add up to 44 because some embryos had more than one mechanism).

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Abstracts

CONCLUSION: The vast majority of mosaic embryos have 100% abnormal cells thus resulting in only 2% (1/48) of mosaics causing errors, for a 1.2% error rate (1/81) after single cell biopsy on day 3, and aCGH analysis. Higher reported error rates in the past were due to incomplete analysis of the embryo and/or technical artifacts. The major mechanism of mosaicism is not balanced non-disjunction but chaotic divisions and anaphase lag (partial loss), both most probably resulting from defective spindles.

P-112 Tuesday, October 26, 2010 FIRST CLINICAL APPLICATION OF SIMULTANEOUS PREIMPLANTATION MITOCHONDRIAL DNA MUTATION LOAD AND 24 CHROMOSOME ANEUPLOIDY SCREENING. X. Tao, J. Campos, K. M. Ferry, B. Levy, N. R. Treff, R. T. Scott, Jr. Reproductive Medicine Associates of New Jersey, Morristown, NJ; UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ. OBJECTIVE: Mitochondrial Encephalomyopathy, Lactic Acidosis with Stroke-Like Episodes (MELAS) syndrome is caused by mutations in mitochondrial DNA. There is no curative treatment for this fatal disease. This study developed, validated, and applied PGD to reduce the risk of inheritance of MELAS. DESIGN: Case report. MATERIALS AND METHODS: Single lymphocytes from a 3243A>G MELAS mutation carrier patient and her affected daughter with known mutation loads were used to validate a quantitative real-time PCR (qPCR) mutation load assay. qPCR analysis of single cells accurately assigned mutation load using the formula: proportion of mutant ¼1/(1+1/2DCT). Preclinical validation involved evaluation of mutation load in oocytes and embryos from the patient which were unsuitable for transfer. Trophectoderm biopsies from blastocysts suitable for transfer were evaluated for MELAS mutation load and screened for 24 chromosome aneuploidy by a previously validated qPCR method. RESULTS: A variety of mutation loads were observed in oocytes (n¼8; 9 to 89%) and embryos (n¼16; 7 to 91%), indicating the potential to select low MELAS mutation load embryos by PGD. Variation of mutation loads within multiple biopsies (n¼41) from individual arrested embryos (n¼7) were also low (from 0.5 to 2.9%), indicating faithful representation of the entire embryo by a single biopsy. A euploid male blastocyst displayed a mutation load of 12% and was designated eligible for transfer. The low mutation load decreases the probability of an effected child and selection of a male prevents transmission of MELAS to future generations. CONCLUSION: This study established: (i) a qPCR test that accurately quantifies the 3243A>G mutation load in single cells, (ii) that oocytes and embryos from the MELAS mutation carrier posses a variety of mutation loads, (iii) that cells within the same embryo provide consistent results, and (iv) the ability to simultaneously assess mutation load and 24 chromosome aneuploidy providing the lowest aggregate risk for genetic disease.

P-113 Tuesday, October 26, 2010 FIRST PREGNANCIES AFTER BLASTOCYST BIOPSYAND RAPID 24 CHROMOSOME ANEUPLOIDY SCREENING ALLOWING A FRESH TRANSFER WITHIN FOUR HOURS OF BIOPSY. N. R. Treff, X. Tao, D. Taylor, K. M. Ferry, R. T. Scott, Jr. Reproductive Medicine Associates of New Jersey, Morristown, NJ; UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ. OBJECTIVE: Trophectoderm (TE) biopsy on blastocysts may represent a safer and more reliable time to perform embryo biopsy. TE biopsies include only extraembryonic cells, involve a reduction in the fraction of the embryo biopsied, and provide multiple cells which improves precision in most analyses. To attain these putative advantages, it has previously been necessary to cryopreserve the blastocysts after biopsy to accommodate the prolonged time interval needed for microarray or CGH based 24 chromosome aneuploidy screening (24chr-AS). This study describes the initial clinical experience of the first 24chr-AS technology rapid enough to allow for TE analysis with fresh transfer within 4 hours of biopsy. DESIGN: Prospective observational. MATERIALS AND METHODS: Patients with recurrent loss, advanced maternal age, or recurrent implantation failure were recruited. All embryos underwent TE biopsy on day 5 of culture. Real-time PCR based 24chr-AS was performed. This technology displayed >98% accuracy in preclinical validation. Results were available within 4 hours. A karyotype was assigned to

Vol. 94., No. 4, Supplement, September 2010