- Email: [email protected]
Impact of the number of previous miscarriages on the clinical outcome of recurrent miscarriage couples undergoing comprehensive chromosome screening (CCS)
Associates of New Jersey, Basking Ridge, NJ; bReproductive Endocrinology and Infertility, Robert Wood Johnson Medical School, Basking Ridge, NJ. OBJECTIVE: Preimplantation genetic testing (PGT) is shifting to TE biopsy (Bx) to attain improved safety, reliability, and accuracy. Unlike blastomere Bx where the number of cells is clear, TE Bx involves removing a small segment where the range of cells obtained as well as variability between centers is not known. This study seeks to characterize the cellularity of TE Bx’s from a large cohort of human blastocysts from multiple centers using a novel assessment methodology. DESIGN: Prospective observational. MATERIALS AND METHODS: A standard curve of quantitative real time (q)PCR-based mean threshold cycles (CT) from a 96-plex reaction was established by analyzing the results of assays on known numbers of cells (1, 2, 3, 4, 5, 10, 15, and 20) from cell lines that are easily counted and loaded. The curve was then used to interpolate cell numbers using the CT data from analysis of TE Bx’s undergoing aneuploidy screening. TE Bx qPCR data from 8 centers were analyzed. Data were stratified based on the day of development on which the Bx was performed (Days 5 vs 6). Pearson correlation for the relationship between cell number and data quality (copy number concordance) was evaluated. RESULTS: Data from 10,106 blastocyst Bx’s included 3,229 day 5 and 6,877 day 6 samples. The calculated number of cells per Bx was equivalent amongst the 8 centers. Day 5 biopsies averaged 6.2 1.9 cells (95% CI, 2 to 10), while day 6 averaged 7.2 2.6 cells (95% CI, 2 to 12) (P¼3 X 10-79). Only 0.07% of the biopsies were predicted to contain a single cell. Better data quality was obtained with increasing cell number (P<0.0001). CONCLUSION: In this large multi-center cohort of blastocyst Bx’s, the number of cells varied within a narrow range. The difference based on day of Bx indicates that the developmental status of the embryo may be the most important variable. Consistency across centers confirms that sample sufficiency is widely attainable. New PGT assays for TE may be safely designed and validated on 2 or more cells.
P-171 Tuesday, October 15, 2013 IMPACT OF THE NUMBER OF PREVIOUS MISCARRIAGES ON THE CLINICAL OUTCOME OF RECURRENT MISCARRIAGE COUPLES UNDERGOING COMPREHENSIVE CHROMOSOME SCREENING (CCS). N. Al-Asmar,a C. Rubio,b,d,e V. Peinado,b I. Campos-Galindo,b S. Garcia-Herrero,b C. Simon.a,b,c,d,e aIVIGen, Miami, FL; bIVIOMICS, Paterna, Valencia, Spain; cInstituto Universitario IVI, Valencia, Spain; dINCLIVA, Valencia University, Valencia, Spain; eFundacion Instituto Valenciano de Infertilidad (FIVI), Paterna, Valencia, Spain. OBJECTIVE: To assess the impact of the number of previous miscarriages in couples with R2 miscarriages of unknown etiology. DESIGN: Retrospective observational study. MATERIALS AND METHODS: A total of 132 CCS cycles were included in women %38 years of age, from January 2010 to December 2012. Day-3 embryo biopsies were performed and embryo transfer was scheduled on day-5. A single blastomere from each embryo underwent Whole Genome Amplification (WGA) with Sureplex DNA Amplification System (BlueGnome Ltd, Cambridge, UK). WGA products and genomic DNA used as control were labeled with Cy3 and Cy5 fluorophores. Both labeled DNAs were co-hybridized overnight in the 24sure platform (BlueGnome Ltd, Cambridge, UK). Slides were scanned and analyzed by BlueFuse Multi software (BlueGnome Ltd, Cambridge, UK). Pearson’s correlation (p<0.05) was performed using SPSS Statistics for Windows, Version 17.0 (Chicago: SPSS Inc.). RESULTS: Results according to the number of previous miscarriages are shown in the table. No correlation was observed for the described variables. 2 3 4 R5 miscarriages miscarriages miscarriages miscarriages No. of cycles % Transfers % Abnormal embryos % Complex aneuploidies* % Partial aneuploidies % Chaotic pattern Pregnancy rate/transfer Miscarriage rate Implantation rate Pregnancy rate/cycle
P-170 Tuesday, October 15, 2013 ANEUPLOIDY PREVALENCE IS NO DIFFERENT BETWEEN EMBRYOS BIOPSIED ON DAY 5 VS DAY 6. J. K. Park,a S. Singh,a S. Berchuck,b G. Couchman,a W. Meyer.a aCarolina Conceptions, Raleigh, NC; bBiostatistics, University of North Carolina at Chapel Hill, Chapel Hill, NC. OBJECTIVE: Aneuploidy screening of blastocysts is being used to improve live birth rates and reduce the number of embryos for transfer. Some embryos cannot be biopsied on day 5 of culture due to slower development but can undergo biopsy on day 6. We compared the euploidy rate of day 5 vs day 6 embryos with the hypothesis that slower developing day 6 embryos were less likely to be euploid. DESIGN: Retrospective analysis. MATERIALS AND METHODS: A retrospective analysis was performed for all preimplantation genetic screening (PGS) cases via trophectoderm biopsy. Samples were analyzed with array comparative genomic hybridization for all chromosomes. The following information was collected from these cycles: female age, indication for PGS, number of embryos biopsied, number biopsied on day 5, number biopsied on day 6, and whether embryos were euploid or aneuploid. Linear regression was performed to determine whether the percentage of euploid embryos was different between embryos biopsied on day 5 as compared to day 6. RESULTS: There were 37 cases of aneuploidy screening, with patients ranging from 28 to 41 years. Out of 208 embryos biopsied, 101 were euploid (48.6%). Out of 147 biopsied on day 5, 70 were euploid (47.6%). Out of 61 biopsied on day 6, 31 were euploid (46.3%). There was no significant difference in the percentage of euploid embryos between those biopsied on day 5 compared to those biopsied on day 6 (p¼0.7). The difference in the percentage of euploid embryos remained non-significant after analyzing women <35 yo and women 35 yo and over. Increasing age, however, was significantly associated with a lower percentage of euploid embryos (p<0.5). The presence of a parental translocation was also associated with a lower percentage of euploid embryos (p<0.5). CONCLUSION: Slower developing embryos that are biopsied on day 6 of culture do not have a significantly different euploidy rate compared to embryos biopsied on day 5 of culture.
FERTILITY & STERILITYÒ
*
50 88.0 56.6
53 77.3 66.4
23 69.6 65.2
6 100 50.0
11.1
16.3
21.5
20.8
7.3
4.8
5.9
0
13.9
18.3
10.4
20.8
54.5
58.5
68.7
66.7
4.2 50.7 48.0
20.8 42.4 45.3
0 66.7 55.0
12.5 40.0 66.7
Embryos with aneuploidies for more than one chromosome.
CONCLUSION: High pregnancy and implantation rates were achieved with CCS in couples experiencing repeated miscarriages of unknown etiology. No correlation was found with the number of previous miscarriages and the percentage and type of chromosome abnormalities. Reproductive outcome was not affected by the number of previous miscarriages. P-172 Tuesday, October 15, 2013 PREIMPLANTATION GENETIC SCREENING BY MASSIVELY PARALLEL SEQUENCING ON A MISEQ USING TRANSPOSASET. Royce,a BASED LIBRARY PREPARATION. F. Kaper,a B. Klotzle,a X. Lee,b L. Wu,b Z. Zhang.b aIllumina, Inc., San Diego, CA; b State Key Laboratory of Medical Genetics, Changsha, Hunan, China. OBJECTIVE: Preimplantation genetic screening (PGS) to detect aneuploid embryos prior to implantation is becoming common practice. Currently used methods include fluorescent in situ hybridization (FISH), array comparative genomic hybridization, and single nucleotide polymorphism array analysis of a single cell or a 3-5 cell biopsy of either the cleavage-stage embryo or blastocyst respectively. We aimed to use a massively parallel sequencing (MPS) approach.
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P-171 Tuesday, October 15, 2013 IMPACT OF THE NUMBER OF PREVIOUS MISCARRIAGES ON THE CLINICAL OUTCOME OF RECURRENT MISCARRIAGE COUPLES UNDERGOING COMPREHENSIVE CHROMOSOME SCREENING (CCS). N. Al-Asmar,a C. Rubio,b,d,e V. Peinado,b I. Campos-Galindo,b S. Garcia-Herrero,b C. Simon.a,b,c,d,e aIVIGen, Miami, FL; bIVIOMICS, Paterna, Valencia, Spain; cInstituto Universitario IVI, Valencia, Spain; dINCLIVA, Valencia University, Valencia, Spain; eFundacion Instituto Valenciano de Infertilidad (FIVI), Paterna, Valencia, Spain. OBJECTIVE: To assess the impact of the number of previous miscarriages in couples with R2 miscarriages of unknown etiology. DESIGN: Retrospective observational study. MATERIALS AND METHODS: A total of 132 CCS cycles were included in women %38 years of age, from January 2010 to December 2012. Day-3 embryo biopsies were performed and embryo transfer was scheduled on day-5. A single blastomere from each embryo underwent Whole Genome Amplification (WGA) with Sureplex DNA Amplification System (BlueGnome Ltd, Cambridge, UK). WGA products and genomic DNA used as control were labeled with Cy3 and Cy5 fluorophores. Both labeled DNAs were co-hybridized overnight in the 24sure platform (BlueGnome Ltd, Cambridge, UK). Slides were scanned and analyzed by BlueFuse Multi software (BlueGnome Ltd, Cambridge, UK). Pearson’s correlation (p<0.05) was performed using SPSS Statistics for Windows, Version 17.0 (Chicago: SPSS Inc.). RESULTS: Results according to the number of previous miscarriages are shown in the table. No correlation was observed for the described variables. 2 3 4 R5 miscarriages miscarriages miscarriages miscarriages No. of cycles % Transfers % Abnormal embryos % Complex aneuploidies* % Partial aneuploidies % Chaotic pattern Pregnancy rate/transfer Miscarriage rate Implantation rate Pregnancy rate/cycle
P-170 Tuesday, October 15, 2013 ANEUPLOIDY PREVALENCE IS NO DIFFERENT BETWEEN EMBRYOS BIOPSIED ON DAY 5 VS DAY 6. J. K. Park,a S. Singh,a S. Berchuck,b G. Couchman,a W. Meyer.a aCarolina Conceptions, Raleigh, NC; bBiostatistics, University of North Carolina at Chapel Hill, Chapel Hill, NC. OBJECTIVE: Aneuploidy screening of blastocysts is being used to improve live birth rates and reduce the number of embryos for transfer. Some embryos cannot be biopsied on day 5 of culture due to slower development but can undergo biopsy on day 6. We compared the euploidy rate of day 5 vs day 6 embryos with the hypothesis that slower developing day 6 embryos were less likely to be euploid. DESIGN: Retrospective analysis. MATERIALS AND METHODS: A retrospective analysis was performed for all preimplantation genetic screening (PGS) cases via trophectoderm biopsy. Samples were analyzed with array comparative genomic hybridization for all chromosomes. The following information was collected from these cycles: female age, indication for PGS, number of embryos biopsied, number biopsied on day 5, number biopsied on day 6, and whether embryos were euploid or aneuploid. Linear regression was performed to determine whether the percentage of euploid embryos was different between embryos biopsied on day 5 as compared to day 6. RESULTS: There were 37 cases of aneuploidy screening, with patients ranging from 28 to 41 years. Out of 208 embryos biopsied, 101 were euploid (48.6%). Out of 147 biopsied on day 5, 70 were euploid (47.6%). Out of 61 biopsied on day 6, 31 were euploid (46.3%). There was no significant difference in the percentage of euploid embryos between those biopsied on day 5 compared to those biopsied on day 6 (p¼0.7). The difference in the percentage of euploid embryos remained non-significant after analyzing women <35 yo and women 35 yo and over. Increasing age, however, was significantly associated with a lower percentage of euploid embryos (p<0.5). The presence of a parental translocation was also associated with a lower percentage of euploid embryos (p<0.5). CONCLUSION: Slower developing embryos that are biopsied on day 6 of culture do not have a significantly different euploidy rate compared to embryos biopsied on day 5 of culture.
FERTILITY & STERILITYÒ
*
50 88.0 56.6
53 77.3 66.4
23 69.6 65.2
6 100 50.0
11.1
16.3
21.5
20.8
7.3
4.8
5.9
0
13.9
18.3
10.4
20.8
54.5
58.5
68.7
66.7
4.2 50.7 48.0
20.8 42.4 45.3
0 66.7 55.0
12.5 40.0 66.7
Embryos with aneuploidies for more than one chromosome.
CONCLUSION: High pregnancy and implantation rates were achieved with CCS in couples experiencing repeated miscarriages of unknown etiology. No correlation was found with the number of previous miscarriages and the percentage and type of chromosome abnormalities. Reproductive outcome was not affected by the number of previous miscarriages. P-172 Tuesday, October 15, 2013 PREIMPLANTATION GENETIC SCREENING BY MASSIVELY PARALLEL SEQUENCING ON A MISEQ USING TRANSPOSASET. Royce,a BASED LIBRARY PREPARATION. F. Kaper,a B. Klotzle,a X. Lee,b L. Wu,b Z. Zhang.b aIllumina, Inc., San Diego, CA; b State Key Laboratory of Medical Genetics, Changsha, Hunan, China. OBJECTIVE: Preimplantation genetic screening (PGS) to detect aneuploid embryos prior to implantation is becoming common practice. Currently used methods include fluorescent in situ hybridization (FISH), array comparative genomic hybridization, and single nucleotide polymorphism array analysis of a single cell or a 3-5 cell biopsy of either the cleavage-stage embryo or blastocyst respectively. We aimed to use a massively parallel sequencing (MPS) approach.
S197