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On-going clinical pregnancies in couples with a sperm DNA fragmentation index (DFI) > 30%
embryos were diagnosed as abnormal on reanalysis. However, in these embryos, other abnormalities than the one originally assigned were discovered. Technical considerations such as overlapped or split FISH probe signals, hybridization failure, loss of nuclear materials during processing may account for some of the results. From this preliminary data we can see that PGD technologies may at times diagnose normal embryos as abnormal. Therefore we propose that embryos that are diagnosed as abnormal be evaluated day 4 and if appropriate, repeat biopsy should be considered as well as freezing those embryos that are deemed to be normal on re-biopsy. Supported by: None.
complete haplotypes were obtained in 71% (5– 45 sperm per patient). These sperm haplotypes were used for PGD in all tested embryos. CONCLUSION: Single sperm genotyping allowed identifying the required linked markers needed to avoid misdiagnosis in PGD for paternally derived mutations when no sufficient pedigree information is available and for couples with different mutations in the same gene. Sperm haplotypes analysis makes it possible not only to exclude the presence of the mutant allele in the embryos, but also to confirm the presence of the paternal normal gene, which is of particular importance in PGD for paternally derived dominant mutations. Supported by: None.
P-335 On-going clinical pregnancies in couples with a sperm DNA fragmentation index (DFI) > 30%. W. G. Kearns, R. Pen, A. Sagoskin, M. J. Levy. Shady Grove Center for Preimplantation Genetics, Rockville, MD; Shady Grove Fertility Reproductive Science Center, Rockville, MD.
P-334 Sperm DNA genotyping for preimplantation genetic diagnosis (PGD). I. Tur-Kaspa, S. Rechitsky, R. S. Ozen, T. Sharapova, K. Laziuk, Y. Verlinsky. Reproductive Genetics Institute, Chicago, IL. OBJECTIVE: IVF with PGD is currently used to avoid passing on a genetic disease to a child. Making DNA testing an accurate and reliable procedure, despite a high occurrence of allele-specific amplification failure (allele drop out, ADO) in single blastomeres PCR, two or more informative polymorphic linked markers should be tested with the causative gene. We describe single sperm DNA genotyping as a novel strategy for PGD when no sufficient pedigree information is available, for couples with different mutations in the same gene, and when a de novo paternal mutation occurred. DESIGN: Retrospective study. MATERIALS AND METHODS: Single sperms were isolated by micromanipulation followed by adequate cell lysis procedure to expose the DNA to the PCR reagents. The DNA regions surrounding the polymorphic sites were amplified so that sufficient PCR products can be generated for DNA genotyping. The sperm haplotypes were used for PGD of paternally derived alleles in all tested embryos. Nested and heminested multiplex PCR analyses of DNA sequences were performed for gene mapping and genetic disease diagnosis. RESULTS: Sperm samples of 22 males with 13 different single gene mutations were investigated. These men had either autosomal dominant mutation (Marfan’s syndrome, retinoblastoma, osteogenesis imperfecta, toberosclerosis I, dystonia, Von Hippel Lindau, and late onset cancer predisposition caused by p53 tumor suppressor gene mutations) or carried an autosomal recessive mutations (cystic fibrosis, sickle-cell disease, ␣- and -thalassemia, hypophosphatasia, and spinal muscular atrophy). 683 single sperms (range 10 –78 per patient) were isolated by micromanipulation. DNA was amplified from 74% of the sperm. Out of these DNA samples,
S254
OBJECTIVE: To determine what percentage of couples where the male partner has fair to poor fertility potential according to DNA fragmentation studies achieve a healthy pregnancy following in vitro fertilization (IVF) protocols. DESIGN: Case report. MATERIALS AND METHODS: Clinical studies reported by SCSA Diagnostics (Brookings, SD) suggest that males with a DNA fragmentation index above 30 % are in a group of sub-fertile men that demonstrate a longer period to establish a pregnancy, more required IVF/ intracytoplasmic sperm injection cycles, and are at increased risk of spontaneous abortion or no pregnancy (fair to poor fertility potential). SCSA Diagnostics suggests that men with a DFI ⱕ 15% have excellent fertility potential and men between 15 and 29.9% have good to fair fertility potential. We performed a preliminary study to determine the clinical outcome of couples undergoing IVF where the male had a DFI ⱖ 30 %. DNA fragmentation studies were performed by SCSA Diagnostics (Brookings, SD). Fifty-eight couples were enrolled in the study. Female inclusion criteria are women ⬍ 37, those with regular ovulatory cycles, and normal ovarian function (⬍ 10 mIU/ml FSH day 3 and ⬍ 80 pg/ml E2). RESULTS: 31% (18 / 58) of men exhibited excellent fertility potential (mean ⫽ 10.1 %, range: 4.8 ⫺ 14.4 %). 48.3 % (28 / 58) of men had good to fair fertility potential (mean ⫽ 21.4 %, range: 15 ⫺ 29.2 %), whilst 20.7% (12 / 58) of studied men had fair to poor fertility potential (mean ⫽ 44.6 %, range: 31.4 ⫺ 58.2 %). 25 % (3 / 12) of couples where the male partner has fair to poor fertility potential have successful ongoing pregnancies. Each pregnancy is ⱖ 10 weeks gestation. One couple has undergone 3 unsuccessful IVF cycles and 3 unsuccessful frozen embryo transfers (FET). The female is 35 and the male partner has normal conventional semen parameters. His DFI is 58.2%. They have a healthy singleton pregnancy. The second couple has one previous unsuccessful IVF cycle, her age is 35 and the male partner has abnormal semen parameters. His DFI is 31 %. The couple has a healthy ongoing twin pregnancy. The third couple has 3 previous unsuccessful intrauterine inseminations. Her age is 31 and he has abnormal semen parameters. His DFI is 32%. The couple has an ongoing twin pregnancy. CONCLUSION: While a DNA fragmentation index ⱖ 30% may play a significant role in unexplained male infertility, it does not preclude the chance of a couple achieving a healthy pregnancy. Supported by: None.
P-336 The fate of frozen/thawed embryos derived from cycles with preimplantation genetic diagnosis. I. Tur-Kaspa, J. Cieslak, N. Ilkevitch, A. Bernal, S. Rechitsky, Y. Verlinsky. Reproductive Genetics Institute, Chicago, IL. OBJECTIVE: To determine the achievability of embryo cryopreservation after preimplantation genetic diagnosis (PGD) for a future frozen embryo transfer (FET) cycle. DESIGN: Retrospective analysis of 66 clinical FET cycles (163 embryos) in which PGD was performed prior to cryopreservation. MATERIALS AND METHODS: Cell sampling for PGD was accom-
Vol. 82, Suppl. 2, September 2004
complete haplotypes were obtained in 71% (5– 45 sperm per patient). These sperm haplotypes were used for PGD in all tested embryos. CONCLUSION: Single sperm genotyping allowed identifying the required linked markers needed to avoid misdiagnosis in PGD for paternally derived mutations when no sufficient pedigree information is available and for couples with different mutations in the same gene. Sperm haplotypes analysis makes it possible not only to exclude the presence of the mutant allele in the embryos, but also to confirm the presence of the paternal normal gene, which is of particular importance in PGD for paternally derived dominant mutations. Supported by: None.
P-335 On-going clinical pregnancies in couples with a sperm DNA fragmentation index (DFI) > 30%. W. G. Kearns, R. Pen, A. Sagoskin, M. J. Levy. Shady Grove Center for Preimplantation Genetics, Rockville, MD; Shady Grove Fertility Reproductive Science Center, Rockville, MD.
P-334 Sperm DNA genotyping for preimplantation genetic diagnosis (PGD). I. Tur-Kaspa, S. Rechitsky, R. S. Ozen, T. Sharapova, K. Laziuk, Y. Verlinsky. Reproductive Genetics Institute, Chicago, IL. OBJECTIVE: IVF with PGD is currently used to avoid passing on a genetic disease to a child. Making DNA testing an accurate and reliable procedure, despite a high occurrence of allele-specific amplification failure (allele drop out, ADO) in single blastomeres PCR, two or more informative polymorphic linked markers should be tested with the causative gene. We describe single sperm DNA genotyping as a novel strategy for PGD when no sufficient pedigree information is available, for couples with different mutations in the same gene, and when a de novo paternal mutation occurred. DESIGN: Retrospective study. MATERIALS AND METHODS: Single sperms were isolated by micromanipulation followed by adequate cell lysis procedure to expose the DNA to the PCR reagents. The DNA regions surrounding the polymorphic sites were amplified so that sufficient PCR products can be generated for DNA genotyping. The sperm haplotypes were used for PGD of paternally derived alleles in all tested embryos. Nested and heminested multiplex PCR analyses of DNA sequences were performed for gene mapping and genetic disease diagnosis. RESULTS: Sperm samples of 22 males with 13 different single gene mutations were investigated. These men had either autosomal dominant mutation (Marfan’s syndrome, retinoblastoma, osteogenesis imperfecta, toberosclerosis I, dystonia, Von Hippel Lindau, and late onset cancer predisposition caused by p53 tumor suppressor gene mutations) or carried an autosomal recessive mutations (cystic fibrosis, sickle-cell disease, ␣- and -thalassemia, hypophosphatasia, and spinal muscular atrophy). 683 single sperms (range 10 –78 per patient) were isolated by micromanipulation. DNA was amplified from 74% of the sperm. Out of these DNA samples,
S254
OBJECTIVE: To determine what percentage of couples where the male partner has fair to poor fertility potential according to DNA fragmentation studies achieve a healthy pregnancy following in vitro fertilization (IVF) protocols. DESIGN: Case report. MATERIALS AND METHODS: Clinical studies reported by SCSA Diagnostics (Brookings, SD) suggest that males with a DNA fragmentation index above 30 % are in a group of sub-fertile men that demonstrate a longer period to establish a pregnancy, more required IVF/ intracytoplasmic sperm injection cycles, and are at increased risk of spontaneous abortion or no pregnancy (fair to poor fertility potential). SCSA Diagnostics suggests that men with a DFI ⱕ 15% have excellent fertility potential and men between 15 and 29.9% have good to fair fertility potential. We performed a preliminary study to determine the clinical outcome of couples undergoing IVF where the male had a DFI ⱖ 30 %. DNA fragmentation studies were performed by SCSA Diagnostics (Brookings, SD). Fifty-eight couples were enrolled in the study. Female inclusion criteria are women ⬍ 37, those with regular ovulatory cycles, and normal ovarian function (⬍ 10 mIU/ml FSH day 3 and ⬍ 80 pg/ml E2). RESULTS: 31% (18 / 58) of men exhibited excellent fertility potential (mean ⫽ 10.1 %, range: 4.8 ⫺ 14.4 %). 48.3 % (28 / 58) of men had good to fair fertility potential (mean ⫽ 21.4 %, range: 15 ⫺ 29.2 %), whilst 20.7% (12 / 58) of studied men had fair to poor fertility potential (mean ⫽ 44.6 %, range: 31.4 ⫺ 58.2 %). 25 % (3 / 12) of couples where the male partner has fair to poor fertility potential have successful ongoing pregnancies. Each pregnancy is ⱖ 10 weeks gestation. One couple has undergone 3 unsuccessful IVF cycles and 3 unsuccessful frozen embryo transfers (FET). The female is 35 and the male partner has normal conventional semen parameters. His DFI is 58.2%. They have a healthy singleton pregnancy. The second couple has one previous unsuccessful IVF cycle, her age is 35 and the male partner has abnormal semen parameters. His DFI is 31 %. The couple has a healthy ongoing twin pregnancy. The third couple has 3 previous unsuccessful intrauterine inseminations. Her age is 31 and he has abnormal semen parameters. His DFI is 32%. The couple has an ongoing twin pregnancy. CONCLUSION: While a DNA fragmentation index ⱖ 30% may play a significant role in unexplained male infertility, it does not preclude the chance of a couple achieving a healthy pregnancy. Supported by: None.
P-336 The fate of frozen/thawed embryos derived from cycles with preimplantation genetic diagnosis. I. Tur-Kaspa, J. Cieslak, N. Ilkevitch, A. Bernal, S. Rechitsky, Y. Verlinsky. Reproductive Genetics Institute, Chicago, IL. OBJECTIVE: To determine the achievability of embryo cryopreservation after preimplantation genetic diagnosis (PGD) for a future frozen embryo transfer (FET) cycle. DESIGN: Retrospective analysis of 66 clinical FET cycles (163 embryos) in which PGD was performed prior to cryopreservation. MATERIALS AND METHODS: Cell sampling for PGD was accom-
Vol. 82, Suppl. 2, September 2004