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Meiotic Recombination, Aneuploidy Rate In The Sperm From Men With Non-obstructive Azoospermia
difference because the groups of the patients were nearly identical or identical in age and number of fertilized oocytes per patient. The prospective randomized trial is now underway to obtain a more irrevocable evidences of the impact of the sperm source on embryonic viability in cases of obstructive azoospermia. Supported by: None
Tuesday, October 18, 2005 4:30 p.m. O-189 Meiotic Recombination, Aneuploidy Rate In The Sperm From Men With Non-obstructive Azoospermia. S. Ma, S. Arsovska, K. Ferguson, M. Nigro, V. Chow. University of British Columbia, Vancouver, BC, Canada. OBJECTIVE: Recent developments of immunocytological techniques with newly identified recombination proteins have opened up a new avenue to study the early stages of meiosis during spermatogenesis. We utilized these techniques to study meiosis I spermatocytes and post-meiotic spermatozoa from testicular samples from azoospermic men in order to study the events of spermatogenesis. DESIGN: A preliminary analysis of synaptonemal complex (SC), recombination, and chromosomal constitution (meiotic and post-meiotic) in testicular tissue of azoospermic men. MATERIALS AND METHODS: Three men with non-obstructive azoospermia (NOA) and one fertile man (vasectomy reversal: control) underwent testicular sperm extraction in preparation for intracytoplasmic sperm injection (ICSI). Testicular tissues were obtained and processed for immunofluorescence analysis. Antibodies against SCP3 protein, which binds sister chromatid pairs, were used to visualise synaptonemal complexes (SC). Antibodies against the DNA mismatch repair protein, MLH1, were used to identify crossovers. CREST antiserum was used to identify centromeric regions. After the immunostaining assay, fluorescence in situ hybridisation (FISH) with probes for chromosomes X, Y and 18 was done on to same cell preparation, and spermatocytes and spermatozoa were assessed. Each of the three NOA patients was compared to the control patient with respect to percent of spermatocytes in various stages of meiosis I using two-sample z-tests of proportion. Further, each patient was compared to the control with respect to the mean number of MLHI foci using two-sample t-tests. FISH results were also analyzed for each of the three azoospermia patients (separate and combined) and compared with the control, again using two-sample z-tests of proportion. RESULTS: The number of autosomal recombination foci in each patient was not significantly different from control. The frequencies of XY bivalents with at least one recombination focus were statistically similar in the patients and control (74.2% vs. 82.6%, respectively). All observed cells in pachytene had normal XY constitutions. In spite of this, the rate of sex chromosome aneuploidy in spermatozoa (post-meiotic) was significantly higher in the patients compared to the control (1.89% vs. 0.83%, p ⫽ 0.002). CONCLUSION: The combination of immunocytological technology with FISH can add a level of precision in etiological investigation of severe male factor infertility. The hypothesis that reduced recombination may lead to the increased aneuploidy in the sperm is not indicated by our results from three NOA men: other factors occurring after recombination repair may yield aneuploid spermatozoa. Supported by: This study was supported by Canadian Institutes of Health Research (MaMOP-53067).
Center, Charlestone, WV; Clinical and Research Center for Human Reproduction Roger Abdelmassih, Sao Paulo, Brazil. OBJECTIVE: There is accumulating evidence that embryos from cases of severe oligoasthenoteratozoospermia (OAT) have lower developmental potential then from cases with normal sperm. The lower implantation in part is due to the increased number of chromosomal aberrations. However, even if the rate of chromosomal aberrations were increased 100 times, this would result in only 3-5% of chromosomally abnormal sperm cells and would not explain the 20-30% reduction in implantation rates. Therefore, it is likely that some post-meiotic changes in spermatozoa are implicated. Supporting this hypothesis are experimental and clinical data showing for example, that extended in vitro incubation of normal sperm significantly increases embryonic mortality. Furthermore, there is clinical evidence that implantation rates in OAT patients can be significantly improved by collecting ejaculate directly into the culture media. Also, given the increase in DNA’s packaging abnormalities in OAT on the one hand and an increased level of reactive oxygen species on the other, the elevated risk of sperm DNA damage could be expected. Therefore, at least in theory, OAT patients could benefit from having spermatozoa for ICSI extracted from the testicle. However, there are no clinical data that would show that IVF/ICSI outcomes of cases using testicular spermatozoa are better than that of cases when ejaculated sperm is used. DESIGN: Cases of TESA with obstructive azoospermia were matched by age, number of fertilized oocytes and number of embryos transferred with severe OAT cases. MATERIALS AND METHODS: Obstructive azoospermia TESA cases performed from January 1st 2001 through March 30th 2005 were matched with cases of severe OAT, performed during the same time-frame, by age, number of fertilized oocytes and number of embryos transferred . Cases involving oocyte donation were excluded. RESULTS: 1,2 p⫽0.003
CONCLUSION: To the best of our knowledge this is the first casecontrolled study comparing the outcomes of TESA with ejaculated sperm in cases of severa OAT. Since the TESA group consisted of patients with obstructive azoospermia, our findings must be treated with caution as the testicular spermatogenesis in OAT patients is disturbed. However, our observation is highly clinically relevant and strengthens the case for prospective randomized trial that would compare the outcomes using ejaculated and testicular spermatozoa in cases of severe OAT. Supported by: None
Tuesday, October 18, 2005 5:15 p.m. O-191
Tuesday, October 18, 2005 4:45 p.m. O-190 Testicular Spermatozoa From Obstructive Azoospermia Patients Have Higher Developmental Potential Than Ejaculated Spermatozoa In Cases Of Severe Oligoasthenoteratozoospermia: Case Controlled Study. D. I. Dozortsev, R. Neme, F. Oliver, V. Abdelmassih, S. Abdelmassih, R. Abdelmassih. Clinical and Research Center for Human Reproduction Roger Abdelmassih, Sao Paulo, Brazil, Charlestone Area Medical
S78
Abstracts
Outcomes of High-magnification ICSI in Cases With Low, Moderate and High Degrees of Sperm DNA Fragmentation. A. Hazout, P. Cohen Bacrie, A. Junca, J. Tesarik. ART Unit Eylau Muette, Paris, France; Laboratoire d’Eylau, Paris, France; MAR and Gen, Molecular Assisted Reproduction and Genetics, Granada, Spain. OBJECTIVE: High-magnification ICSI, which is characterized by sperm selection at a magnification of x6000 instead of x400 usual in conventional ICSI, has previously been reported to improve clinical outcomes in cases of repeated ICSI failures caused by the paternal factor. Sperm DNA fragmentation is one of the possible causes of such failures. The aim of this study
Vol. 84, Suppl 1, September 2005
Tuesday, October 18, 2005 4:30 p.m. O-189 Meiotic Recombination, Aneuploidy Rate In The Sperm From Men With Non-obstructive Azoospermia. S. Ma, S. Arsovska, K. Ferguson, M. Nigro, V. Chow. University of British Columbia, Vancouver, BC, Canada. OBJECTIVE: Recent developments of immunocytological techniques with newly identified recombination proteins have opened up a new avenue to study the early stages of meiosis during spermatogenesis. We utilized these techniques to study meiosis I spermatocytes and post-meiotic spermatozoa from testicular samples from azoospermic men in order to study the events of spermatogenesis. DESIGN: A preliminary analysis of synaptonemal complex (SC), recombination, and chromosomal constitution (meiotic and post-meiotic) in testicular tissue of azoospermic men. MATERIALS AND METHODS: Three men with non-obstructive azoospermia (NOA) and one fertile man (vasectomy reversal: control) underwent testicular sperm extraction in preparation for intracytoplasmic sperm injection (ICSI). Testicular tissues were obtained and processed for immunofluorescence analysis. Antibodies against SCP3 protein, which binds sister chromatid pairs, were used to visualise synaptonemal complexes (SC). Antibodies against the DNA mismatch repair protein, MLH1, were used to identify crossovers. CREST antiserum was used to identify centromeric regions. After the immunostaining assay, fluorescence in situ hybridisation (FISH) with probes for chromosomes X, Y and 18 was done on to same cell preparation, and spermatocytes and spermatozoa were assessed. Each of the three NOA patients was compared to the control patient with respect to percent of spermatocytes in various stages of meiosis I using two-sample z-tests of proportion. Further, each patient was compared to the control with respect to the mean number of MLHI foci using two-sample t-tests. FISH results were also analyzed for each of the three azoospermia patients (separate and combined) and compared with the control, again using two-sample z-tests of proportion. RESULTS: The number of autosomal recombination foci in each patient was not significantly different from control. The frequencies of XY bivalents with at least one recombination focus were statistically similar in the patients and control (74.2% vs. 82.6%, respectively). All observed cells in pachytene had normal XY constitutions. In spite of this, the rate of sex chromosome aneuploidy in spermatozoa (post-meiotic) was significantly higher in the patients compared to the control (1.89% vs. 0.83%, p ⫽ 0.002). CONCLUSION: The combination of immunocytological technology with FISH can add a level of precision in etiological investigation of severe male factor infertility. The hypothesis that reduced recombination may lead to the increased aneuploidy in the sperm is not indicated by our results from three NOA men: other factors occurring after recombination repair may yield aneuploid spermatozoa. Supported by: This study was supported by Canadian Institutes of Health Research (MaMOP-53067).
Center, Charlestone, WV; Clinical and Research Center for Human Reproduction Roger Abdelmassih, Sao Paulo, Brazil. OBJECTIVE: There is accumulating evidence that embryos from cases of severe oligoasthenoteratozoospermia (OAT) have lower developmental potential then from cases with normal sperm. The lower implantation in part is due to the increased number of chromosomal aberrations. However, even if the rate of chromosomal aberrations were increased 100 times, this would result in only 3-5% of chromosomally abnormal sperm cells and would not explain the 20-30% reduction in implantation rates. Therefore, it is likely that some post-meiotic changes in spermatozoa are implicated. Supporting this hypothesis are experimental and clinical data showing for example, that extended in vitro incubation of normal sperm significantly increases embryonic mortality. Furthermore, there is clinical evidence that implantation rates in OAT patients can be significantly improved by collecting ejaculate directly into the culture media. Also, given the increase in DNA’s packaging abnormalities in OAT on the one hand and an increased level of reactive oxygen species on the other, the elevated risk of sperm DNA damage could be expected. Therefore, at least in theory, OAT patients could benefit from having spermatozoa for ICSI extracted from the testicle. However, there are no clinical data that would show that IVF/ICSI outcomes of cases using testicular spermatozoa are better than that of cases when ejaculated sperm is used. DESIGN: Cases of TESA with obstructive azoospermia were matched by age, number of fertilized oocytes and number of embryos transferred with severe OAT cases. MATERIALS AND METHODS: Obstructive azoospermia TESA cases performed from January 1st 2001 through March 30th 2005 were matched with cases of severe OAT, performed during the same time-frame, by age, number of fertilized oocytes and number of embryos transferred . Cases involving oocyte donation were excluded. RESULTS: 1,2 p⫽0.003
CONCLUSION: To the best of our knowledge this is the first casecontrolled study comparing the outcomes of TESA with ejaculated sperm in cases of severa OAT. Since the TESA group consisted of patients with obstructive azoospermia, our findings must be treated with caution as the testicular spermatogenesis in OAT patients is disturbed. However, our observation is highly clinically relevant and strengthens the case for prospective randomized trial that would compare the outcomes using ejaculated and testicular spermatozoa in cases of severe OAT. Supported by: None
Tuesday, October 18, 2005 5:15 p.m. O-191
Tuesday, October 18, 2005 4:45 p.m. O-190 Testicular Spermatozoa From Obstructive Azoospermia Patients Have Higher Developmental Potential Than Ejaculated Spermatozoa In Cases Of Severe Oligoasthenoteratozoospermia: Case Controlled Study. D. I. Dozortsev, R. Neme, F. Oliver, V. Abdelmassih, S. Abdelmassih, R. Abdelmassih. Clinical and Research Center for Human Reproduction Roger Abdelmassih, Sao Paulo, Brazil, Charlestone Area Medical
S78
Abstracts
Outcomes of High-magnification ICSI in Cases With Low, Moderate and High Degrees of Sperm DNA Fragmentation. A. Hazout, P. Cohen Bacrie, A. Junca, J. Tesarik. ART Unit Eylau Muette, Paris, France; Laboratoire d’Eylau, Paris, France; MAR and Gen, Molecular Assisted Reproduction and Genetics, Granada, Spain. OBJECTIVE: High-magnification ICSI, which is characterized by sperm selection at a magnification of x6000 instead of x400 usual in conventional ICSI, has previously been reported to improve clinical outcomes in cases of repeated ICSI failures caused by the paternal factor. Sperm DNA fragmentation is one of the possible causes of such failures. The aim of this study
Vol. 84, Suppl 1, September 2005