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Molecular basis for the mismatch repair deficiencies in non-obstructive azoospermia (NOA)
both type of azoospermia have pluripotency that can form teratoma. However, proliferating activity in SSC obtained from NOA was lower than those from OA. CONCLUSIONS: The present study provides information that existing SSCs in testicles of NOA patients can be isolated and well propagated in vitro by the highly efficient culture system. Therefore, this system could be used for treating NOA combined with human ART. Supported by: Supported by a grant from the Stem Cell Research Program (2006-04127) of the MEST, and KSF.
O-100 Tuesday, October 20, 2009 5:15 PM MOLECULAR BASIS FOR THE MISMATCH REPAIR DEFICIENCIES IN NON-OBSTRUCTIVE AZOOSPERMIA (NOA). S. Mukherjee, J. Addai, D. J. Lamb. Scott Department of Urology, Baylor College of Medicine, Houston, TX; Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX. OBJECTIVE: Mutations in mismatch repair (MMR) genes are associated with hereditary nonpolyposis colorectal cancer, a cancer susceptibility syndrome. Interestingly, MMR-deficient mice display sterility associated with abnormal chromosome pairing in meiosis, microsatellite instability and tumor predisposition. Testicular pathologies of these deficient mice are similar to men with NOA. We tested the hypothesis that mismatch repair deficiencies were present in NOA. DESIGN: A prospective study of 35 men with idiopathic NOA underwent testicular sperm extraction and intracytoplasmic sperm injection with IVF to attempt to conceive a pregnancy and provided tissue for fibroblast culture. Men with normal but obstructed spermatogenesis served as controls. MATERIALS AND METHODS: Mutation and gene expression studies, immunocytochemistry and Western blot of MLH1, MSH2 and MSH6 proteins were performed. RESULTS: Coimmunostaining with fluorescent-tagged antibodies against MLH1 and MSH2 defined the subcellular localization of the MMR proteins. MLH1 displayed diffuse cytoplasmic with a strong perinuclear staining in all patients, whereas, MSH2 showed a distinct punctuate nuclear staining. Two patients exhibited significantly decreased staining and one exhibited mislocalization of MMR proteins. Immunoprecipitation with MLH1 and MSH2 antibodies revealed a single band at 85kD and 100kD, respectively. Expression of MLH1 and MSH2 in these three patients was significantly decreased. Genomic DNA analysis of the 16 exons of MSH2, 19 exons of the MLH1 gene and 10 exons of the MSH6 genes by DHPLC reveal polymorphisms and genetic aberrations CONCLUSIONS: Mismatch repair proteins are deficient in some men with NOA. By elucidating the pathway for these combined genetic aberrations associated with infertility and cancer; one can investigate whether men with NOA are at increased risk for cancer development. Supported by: Supported in part by 2P01 HD 36289 and the NIH Cooperative Centers Program in Reproductive Research (U54 HD07495) from the National Institutes of Health.
O-101 Tuesday, October 20, 2009 5:30 PM CADMIUM AND ASSISTED REPRODUCTIVE TECHNOLOGIES: RELEVANCE TO PUBLIC HEALTH. S. H. Benoff, I. R. Hurley, G. M. Centola, R. Hauser, J. L. Marmar. Research, Feinstein Institute for Medical Research, Manhasset, NY; Clinical Laboratory, LifeCell Dx, Buffalo, NY; Environmental Health, Harvard School of Public Health, Boston, MA; Surgery, Cooper University Hospital, Camden, NJ. OBJECTIVE: Prior studies suggest that cadmium (Cd) may adversely affect sperm through a pro-oxidant mechanism generating reactive oxygen species (ROS) and oxidative stress. Infertile men have been shown to have higher levels of ROS. We tested the hypothesis that men requiring ICSI have increased Cd exposure. DESIGN: Analysis of the relationship between semen parameters, seminal plasma metal levels and fertilization rates among male partners of infertile couples undergoing ICSI (n ¼20; w/ varicoceles (VX], n¼7) and dose-compensated IVF inseminations (n¼96; w/ VX, n¼16) during the period 2/95 to 8/96. MATERIALS AND METHODS: Sperm concentration and motility were determined in duplicate specimens using WHO criteria and acrosome morphology was assessed in fixed, unstained sperm. Seminal plasma Cd, lead
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Abstracts
(Pb) and zinc (Zn) levels were quantified by atomic absorption. Fertilization rates were defined by standard protocols. RESULTS: Sperm concentration, motility, morphology and fertilization rates were all reduced in ICSI patients as compared to IVF (P<0.01). Although Zn and Pb levels did not differ between the two groups, Cd was significant elevated in ICSI patients as compared to IVF (P<0.004). ICSI fertilization was positively correlated with sperm motility (Spearman correlation [SC], r¼0.38, P<0.05) and Zn (SC, r¼0.73, P<0.001). The proportion of men with VX treated with ICSI was higher than those treated with IVF (35% vs. 17%). In the ICSI group, Cd was higher in subjects with VX than without VX (P<0.02) or with VX treated with IVF (P<0.003). CONCLUSIONS: The data suggest that seminal plasma Cd levels are elevated in men requiring ICSI, with or without VX. Both sperm motility and seminal plasma Zn levels positively correlated with ICSI fertilization rates. Cd may increase sperm ROS production with associated sperm membrane damage and decreased motility. Zn has the potential to be an inexpensive therapeutic antioxidant. Supported by: Grants (SB) ES06100, ES10496 from NIEHS, NIH.
THE SOCIETY FOR REPRODUCTIVE ENDOCRINOLOGY AND INFERTILITY O-102 Tuesday, October 20, 2009 4:15 PM ENDOMETRIOTIC STROMAL CELLS EXPRESS A MOLECULAR PATTERN CONSISTENT WITH DECREASED RETINOID UPTAKE, METABOLISM AND ACTION. M. E. Pavone, E. Pearson, M. Milad, Y. H. Cheng, S. E. Bulun. Obstetrics and Gynecology, Northwestern University, Chicago, IL. OBJECTIVE: To investigate the expression of genes involved in the retinoic acid (RA) signaling pathway, including genes involved in retinol uptake, metabolism, transport and transcriptional activation in endometrial stromal cells from disease-free women and endometriotic stromal cells from ovarian endometriomas. DESIGN: Laboratory study of human endometrial and endometriotic stromal cells. MATERIALS AND METHODS: Stromal and epithelial cells from normal endometrium and endometriosis were isolated. Real-time reverse transcription-polymerase chain reaction was used to measure mRNA levels. Western blotting was used to evaluate protein expression. To investigate the expression of nuclear receptors, nuclear extracts were obtained. RESULTS: STRA6, the gene responsible for retinol uptake into cells, was preferentially expressed in endometrial stromal as opposed to epithelial cells. There was a significantly decreased mRNA expression of the major genes involved in the retinoic acid pathway, including STRA6, cellular retinol binding protein-1, aldehyde dehydrogenase 1A2, cellular retinoic acid binding protein-2 and cytochrome p450 (CYP26A1) in endometriotic stromal cells. Nuclear extracts from stromal cells showed that RAR-alpha and RXR-alpha were significantly under-expressed in endometriotic stromal cells compared to normal endometrial stromal cells. Differences in protein were confirmed by western blotting. CONCLUSIONS: We have previously demonstrated that epithelial HSD17B2, the enzyme which catalyzes the conversion of estradiol to the less potent estrone, is not induced in endometriosis because of a defect in the secretion of paracrine factors made by stromal cells, one of which was found to be RA. In this study, we have demonstrated that this ‘‘defect’’ in endometriotic stromal cells involves the uptake, metabolism, transport and action of retinoids. These molecular differences ultimately result in the higher levels of estradiol present in endometriotic lesions. Supported by: NIH (U54-HD40093), Friends of Prentice.
O-103 Tuesday, October 20, 2009 4:30 PM ASSOCIATION OF BRCA1 GENE MUTATIONS WITH DIMINISHED OVARIAN RESERVE: A NOVEL CONNECTION BETWEEN DNA REPAIR AND OVARIAN AGING IN HUMANS. K. Oktay, J. Y. Kim, D. Barad, S. Babayev. Obstetrics & Gynecology/Institute for Fertility Preservation, New York Medical College, Valhalla, NY; Institute for Fertility Preservation, CHR, NY, NY. OBJECTIVE: Recent knock out models suggested a relationship between DNA repair gene function and germ cell reserve. Our aim was to determine if
Vol. 92., No. 3, Supplement, September 2009
O-100 Tuesday, October 20, 2009 5:15 PM MOLECULAR BASIS FOR THE MISMATCH REPAIR DEFICIENCIES IN NON-OBSTRUCTIVE AZOOSPERMIA (NOA). S. Mukherjee, J. Addai, D. J. Lamb. Scott Department of Urology, Baylor College of Medicine, Houston, TX; Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX. OBJECTIVE: Mutations in mismatch repair (MMR) genes are associated with hereditary nonpolyposis colorectal cancer, a cancer susceptibility syndrome. Interestingly, MMR-deficient mice display sterility associated with abnormal chromosome pairing in meiosis, microsatellite instability and tumor predisposition. Testicular pathologies of these deficient mice are similar to men with NOA. We tested the hypothesis that mismatch repair deficiencies were present in NOA. DESIGN: A prospective study of 35 men with idiopathic NOA underwent testicular sperm extraction and intracytoplasmic sperm injection with IVF to attempt to conceive a pregnancy and provided tissue for fibroblast culture. Men with normal but obstructed spermatogenesis served as controls. MATERIALS AND METHODS: Mutation and gene expression studies, immunocytochemistry and Western blot of MLH1, MSH2 and MSH6 proteins were performed. RESULTS: Coimmunostaining with fluorescent-tagged antibodies against MLH1 and MSH2 defined the subcellular localization of the MMR proteins. MLH1 displayed diffuse cytoplasmic with a strong perinuclear staining in all patients, whereas, MSH2 showed a distinct punctuate nuclear staining. Two patients exhibited significantly decreased staining and one exhibited mislocalization of MMR proteins. Immunoprecipitation with MLH1 and MSH2 antibodies revealed a single band at 85kD and 100kD, respectively. Expression of MLH1 and MSH2 in these three patients was significantly decreased. Genomic DNA analysis of the 16 exons of MSH2, 19 exons of the MLH1 gene and 10 exons of the MSH6 genes by DHPLC reveal polymorphisms and genetic aberrations CONCLUSIONS: Mismatch repair proteins are deficient in some men with NOA. By elucidating the pathway for these combined genetic aberrations associated with infertility and cancer; one can investigate whether men with NOA are at increased risk for cancer development. Supported by: Supported in part by 2P01 HD 36289 and the NIH Cooperative Centers Program in Reproductive Research (U54 HD07495) from the National Institutes of Health.
O-101 Tuesday, October 20, 2009 5:30 PM CADMIUM AND ASSISTED REPRODUCTIVE TECHNOLOGIES: RELEVANCE TO PUBLIC HEALTH. S. H. Benoff, I. R. Hurley, G. M. Centola, R. Hauser, J. L. Marmar. Research, Feinstein Institute for Medical Research, Manhasset, NY; Clinical Laboratory, LifeCell Dx, Buffalo, NY; Environmental Health, Harvard School of Public Health, Boston, MA; Surgery, Cooper University Hospital, Camden, NJ. OBJECTIVE: Prior studies suggest that cadmium (Cd) may adversely affect sperm through a pro-oxidant mechanism generating reactive oxygen species (ROS) and oxidative stress. Infertile men have been shown to have higher levels of ROS. We tested the hypothesis that men requiring ICSI have increased Cd exposure. DESIGN: Analysis of the relationship between semen parameters, seminal plasma metal levels and fertilization rates among male partners of infertile couples undergoing ICSI (n ¼20; w/ varicoceles (VX], n¼7) and dose-compensated IVF inseminations (n¼96; w/ VX, n¼16) during the period 2/95 to 8/96. MATERIALS AND METHODS: Sperm concentration and motility were determined in duplicate specimens using WHO criteria and acrosome morphology was assessed in fixed, unstained sperm. Seminal plasma Cd, lead
S30
Abstracts
(Pb) and zinc (Zn) levels were quantified by atomic absorption. Fertilization rates were defined by standard protocols. RESULTS: Sperm concentration, motility, morphology and fertilization rates were all reduced in ICSI patients as compared to IVF (P<0.01). Although Zn and Pb levels did not differ between the two groups, Cd was significant elevated in ICSI patients as compared to IVF (P<0.004). ICSI fertilization was positively correlated with sperm motility (Spearman correlation [SC], r¼0.38, P<0.05) and Zn (SC, r¼0.73, P<0.001). The proportion of men with VX treated with ICSI was higher than those treated with IVF (35% vs. 17%). In the ICSI group, Cd was higher in subjects with VX than without VX (P<0.02) or with VX treated with IVF (P<0.003). CONCLUSIONS: The data suggest that seminal plasma Cd levels are elevated in men requiring ICSI, with or without VX. Both sperm motility and seminal plasma Zn levels positively correlated with ICSI fertilization rates. Cd may increase sperm ROS production with associated sperm membrane damage and decreased motility. Zn has the potential to be an inexpensive therapeutic antioxidant. Supported by: Grants (SB) ES06100, ES10496 from NIEHS, NIH.
THE SOCIETY FOR REPRODUCTIVE ENDOCRINOLOGY AND INFERTILITY O-102 Tuesday, October 20, 2009 4:15 PM ENDOMETRIOTIC STROMAL CELLS EXPRESS A MOLECULAR PATTERN CONSISTENT WITH DECREASED RETINOID UPTAKE, METABOLISM AND ACTION. M. E. Pavone, E. Pearson, M. Milad, Y. H. Cheng, S. E. Bulun. Obstetrics and Gynecology, Northwestern University, Chicago, IL. OBJECTIVE: To investigate the expression of genes involved in the retinoic acid (RA) signaling pathway, including genes involved in retinol uptake, metabolism, transport and transcriptional activation in endometrial stromal cells from disease-free women and endometriotic stromal cells from ovarian endometriomas. DESIGN: Laboratory study of human endometrial and endometriotic stromal cells. MATERIALS AND METHODS: Stromal and epithelial cells from normal endometrium and endometriosis were isolated. Real-time reverse transcription-polymerase chain reaction was used to measure mRNA levels. Western blotting was used to evaluate protein expression. To investigate the expression of nuclear receptors, nuclear extracts were obtained. RESULTS: STRA6, the gene responsible for retinol uptake into cells, was preferentially expressed in endometrial stromal as opposed to epithelial cells. There was a significantly decreased mRNA expression of the major genes involved in the retinoic acid pathway, including STRA6, cellular retinol binding protein-1, aldehyde dehydrogenase 1A2, cellular retinoic acid binding protein-2 and cytochrome p450 (CYP26A1) in endometriotic stromal cells. Nuclear extracts from stromal cells showed that RAR-alpha and RXR-alpha were significantly under-expressed in endometriotic stromal cells compared to normal endometrial stromal cells. Differences in protein were confirmed by western blotting. CONCLUSIONS: We have previously demonstrated that epithelial HSD17B2, the enzyme which catalyzes the conversion of estradiol to the less potent estrone, is not induced in endometriosis because of a defect in the secretion of paracrine factors made by stromal cells, one of which was found to be RA. In this study, we have demonstrated that this ‘‘defect’’ in endometriotic stromal cells involves the uptake, metabolism, transport and action of retinoids. These molecular differences ultimately result in the higher levels of estradiol present in endometriotic lesions. Supported by: NIH (U54-HD40093), Friends of Prentice.
O-103 Tuesday, October 20, 2009 4:30 PM ASSOCIATION OF BRCA1 GENE MUTATIONS WITH DIMINISHED OVARIAN RESERVE: A NOVEL CONNECTION BETWEEN DNA REPAIR AND OVARIAN AGING IN HUMANS. K. Oktay, J. Y. Kim, D. Barad, S. Babayev. Obstetrics & Gynecology/Institute for Fertility Preservation, New York Medical College, Valhalla, NY; Institute for Fertility Preservation, CHR, NY, NY. OBJECTIVE: Recent knock out models suggested a relationship between DNA repair gene function and germ cell reserve. Our aim was to determine if
Vol. 92., No. 3, Supplement, September 2009