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The Prognostic Value of Y Chromosome Microdeletions Involving the AZFb Region in Testicular Sperm Extraction
ethical issues prior to enrollment. Ovarian cortex was cryopreserved, transplanted in immunosuppressed mice and stimulated with gonadotropins. Fresh, frozen and transplanted tissues were histologically evaluated. Materials and methods: Ovarian tissue was surgically removed and collected in PBS containing hFSH. The outer 4 mm of the ovarian cortex was dissected into cubes of 13130.5 mm and frozen in straws or cryovials in Leibovitz medium L-15110% serum 11.5M ethylene glycol. Fresh and frozen-thawed ovarian cortex was transplanted into nude or NOD-SCID mice, which were stimulated with hFSH. Results: 17 patients (26 –39 yrs) were enrolled in this study. 12–100 ovarian fragments/patient were frozen. Follicle count decreased with patient age and a large variability in follicular number was observed between fragments, even in the same patient. Prior therapy did not preclude successful ovarian cryopreservation. The xenotransplantation of tissue from patients with good follicular counts resulted in follicular development and successful retrieval of immature oocytes. Conclusion: Oocyte retrieval from cryopreserved xenotransplanted human ovarian cortex is feasible, enabling evaluation of fertility potential as well as assessment of ovarian cryopreservation safety.
Wednesday, October 25, 2000 3:45 P.M. O-252 Y-Microdeletions in the Single Spermatozoon. N. Takeshita, T. Takeuchi, M. T. Kan, A. Chiu, Z. Rosenwaks, G. D. Palermo. The Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, New York, NY. Objective: Microdeletions on the long arm of the Y chromosome are frequently associated with abnormal spermatogenesis. In several studies of severely infertile men, the incidence of such microdeletions has been estimated to involve between 2.8 –55.5% of that population. Although severe male infertility has been successfully treated by intracytoplasmic sperm injection (ICSI), this approach has the potential to transmit the microdeletion, so perpetuating male infertility in the next generation. The aim of the study was to detect Y microdeletions in single spermatozoa. Design: Single spermatoza were obtained from normal semen analysis specimens. Single lymphocytes were isolated from consenting subjects. These cells were subjected to polymerase chain reaction (PCR) with complete genomic amplification. A second PCR cycle was used to examine 18 different Sequence Target Sites (STSs) in the AZF regions on the Y chromosome. Materials and Methods: Twenty single spermatozoa and 4 lymphocytes were isolated manually by micromanipulation and lysed with a standard lysis buffer. Single spermatozoa and lymphocytes were amplified with whole genomic degenerate oligonucleotide primer PCR (DOP-PCR). Genomic PCR products were divided into 52 PCR templates for a second PCR cycle specific for 18 STSs on the Y chromosome. These PCR products were analyzed by polyacrylamide gel electrophoresis. Results: STS fragments were identified in 29 of the 52 samples, and in 23 of the samples there was no specific STS detection. The highest incidence of DNA fragment detection was DAZ, SY208 (100%). The second highest fragment detected was DYS237, SY153 (73%). KAL-Y, SY182 and DYS218, SY127 were detected at rates of 56% and 50%, respectively. One sample was run with a multiplex of 8 different primer sets (compared to the standard 4 –5 primer sets/kit). In this sample, 6 out 8 STS fragments were detected from single sperm after DOP-PCR. Conclusion: 1) Detection of Y microdeletion loci was possible on single spermatozoa and on lymphocytes, using DOP-PCR with genomic amplification. 2) At low DNA concentrations, there was preferential amplification for different loci of the Y microdeletion. 3) Multiplex PCR was possible using 8 primer sets for Y microdeletions at the single cell level. It is likely that this technique can be applied to other immature germ cells, such as spermatogonia, primary or secondary spermatocytes as well as spermatids.
Wednesday, October 25, 2000 4:00 P.M. O-253 The Prognostic Value of Y Chromosome Microdeletions Involving the AZFb Region in Testicular Sperm Extraction. 1,2,3P. T. K. Chan, 3A.
S94
Abstracts
Mielnik, 2C.-A. Cook, 2D. Liotta, 2Z. Ye, 2L. L. Veeck. 1Department of Urology and 2Cornell Institute for Reproductive Medicine, New YorkCornell Medical Center, 3The Population Council, New York, NY. Objectives: Testicular sperm extraction (TESE) for intracytoplasmic sperm injection (ICSI) is an effective treatment for men with non-obstructive azoospermia. Overall, approximately 35% of these men have no spermatozoa found in their testicular tissue. The success of TESE cannot be predicted based on preoperative hormonal profiles (FSH, testosterone), nor based on testicular volume. Even a prior diagnostic testis biopsy does not absolutely predict the success of TESE, due to the heterogeneous nature of sperm maturation within seminiferous tubules of the testicle. Preliminary data in recent literature suggest a correlation between microdeletions involving the AZFb of the Y-chromosome and the absence of spermatozoa with TESE (Brandell et al., Human Reprod., 1998). We hereby report our expanded experience of the relationship between microdeletions involving AZFb and TESE results in men with non-obstructive azoospermia. Design: A retrospective analysis of azoospermic men who underwent TESE from 1996 to 2000 was performed. The outcomes of men with AZFb microdeletion of the Y-chromosome were analyzed. Materials and Methods: All TESE were performed under local or general anesthesia by one surgeon. TESE were performed with either multiple biopsies or with a single large incision with multiple samples of the seminiferous tubules under microscopic vision (microdissection TESE). The procedures were performed in conjunction with planned in-vitro fertilization cycles with ICSI for the female partner. Y-chromosomal microdeletion was tested in all patients using DNA extracted from peripheral leukocytes with a series of 35 sequence-tagged sites on Yq using polymerase chain reaction. Results: Among 181 patients who undergone TESE, 10 (6%) were found to have microdeletion involving the AZFb regions and 5 of them (3%) have microdeletion in AZFb region only. For men with non-obstructive azoospermia and no detectable deletions of the AZFb region, 114/171 (67%) men had sperm retrieved with TESE, whereas none of the 11 patients with microdeletions involving the AZFb region had sperm found with TESE (p,0.001). The average duration of TESE in patients with deletions involving the AZFb region was 3.5 hours. Conclusions: Genetic studies may provide important prognostic information in the management of patients with severe male infertility, as illustrated by our observation that the presence of an AZFb deletion is a significantly adverse prognostic finding for TESE. When counseling men with AZFb deletions, clinicians should apprise them of these results before attempting TESE-ICSI.
Wednesday, October 25, 2000 4:15 P.M. O-254 Effective Cell Sorting After Flow Cytometric Sperm Sample Analysis in an Azoospermic Patient with Polycystic Kidney Disease. A. Akerman, M. Goldstein, J. J. Hariprashad, M. T. Kan, Z. Rosenwaks, G. D. Palermo. The Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, New York, NY. Objective: Isolation of haploid cells from non-obstructive azoospermic patients for successful use in ICSI, represents a major challenge. The aim of the study was to isolate haploid cells from an enzymatically digested testicular biopsy sample, using flow cytometry analysis followed by cell sorting. In addition we attempted to compare flow cytometry outcome to histopathological diagnosis. Design: A testicular biopsy was obtained from an azoospermic patient with polycystic kidney disease and multiple epdidymal cysts. Isolated testicular spermatogenic cells were stained for flow cytometric analysis. The ploidy of the cells was confirmed by cytogenetic analysis. Materials and Methods: Testicular germ cells were obtained from a consenting patient undergoing diagnostic testicular biopsy. Sections of intact biopsied tissue were prepared for histopathological analysis. The remaining biopsy sample was treated by collagenase digestion, then stained and processed for flow cytometric analysis and additional cell sorting. Diagnostic correlations were performed blinded. For flow cytometric analysis, cells were classified as haploid, diploid (using human peripheral lymphocytes as a control), and tetraploid. After DNA quantification, cells
Vol. 74, No. 3, Suppl. 1, October 2000
Wednesday, October 25, 2000 3:45 P.M. O-252 Y-Microdeletions in the Single Spermatozoon. N. Takeshita, T. Takeuchi, M. T. Kan, A. Chiu, Z. Rosenwaks, G. D. Palermo. The Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, New York, NY. Objective: Microdeletions on the long arm of the Y chromosome are frequently associated with abnormal spermatogenesis. In several studies of severely infertile men, the incidence of such microdeletions has been estimated to involve between 2.8 –55.5% of that population. Although severe male infertility has been successfully treated by intracytoplasmic sperm injection (ICSI), this approach has the potential to transmit the microdeletion, so perpetuating male infertility in the next generation. The aim of the study was to detect Y microdeletions in single spermatozoa. Design: Single spermatoza were obtained from normal semen analysis specimens. Single lymphocytes were isolated from consenting subjects. These cells were subjected to polymerase chain reaction (PCR) with complete genomic amplification. A second PCR cycle was used to examine 18 different Sequence Target Sites (STSs) in the AZF regions on the Y chromosome. Materials and Methods: Twenty single spermatozoa and 4 lymphocytes were isolated manually by micromanipulation and lysed with a standard lysis buffer. Single spermatozoa and lymphocytes were amplified with whole genomic degenerate oligonucleotide primer PCR (DOP-PCR). Genomic PCR products were divided into 52 PCR templates for a second PCR cycle specific for 18 STSs on the Y chromosome. These PCR products were analyzed by polyacrylamide gel electrophoresis. Results: STS fragments were identified in 29 of the 52 samples, and in 23 of the samples there was no specific STS detection. The highest incidence of DNA fragment detection was DAZ, SY208 (100%). The second highest fragment detected was DYS237, SY153 (73%). KAL-Y, SY182 and DYS218, SY127 were detected at rates of 56% and 50%, respectively. One sample was run with a multiplex of 8 different primer sets (compared to the standard 4 –5 primer sets/kit). In this sample, 6 out 8 STS fragments were detected from single sperm after DOP-PCR. Conclusion: 1) Detection of Y microdeletion loci was possible on single spermatozoa and on lymphocytes, using DOP-PCR with genomic amplification. 2) At low DNA concentrations, there was preferential amplification for different loci of the Y microdeletion. 3) Multiplex PCR was possible using 8 primer sets for Y microdeletions at the single cell level. It is likely that this technique can be applied to other immature germ cells, such as spermatogonia, primary or secondary spermatocytes as well as spermatids.
Wednesday, October 25, 2000 4:00 P.M. O-253 The Prognostic Value of Y Chromosome Microdeletions Involving the AZFb Region in Testicular Sperm Extraction. 1,2,3P. T. K. Chan, 3A.
S94
Abstracts
Mielnik, 2C.-A. Cook, 2D. Liotta, 2Z. Ye, 2L. L. Veeck. 1Department of Urology and 2Cornell Institute for Reproductive Medicine, New YorkCornell Medical Center, 3The Population Council, New York, NY. Objectives: Testicular sperm extraction (TESE) for intracytoplasmic sperm injection (ICSI) is an effective treatment for men with non-obstructive azoospermia. Overall, approximately 35% of these men have no spermatozoa found in their testicular tissue. The success of TESE cannot be predicted based on preoperative hormonal profiles (FSH, testosterone), nor based on testicular volume. Even a prior diagnostic testis biopsy does not absolutely predict the success of TESE, due to the heterogeneous nature of sperm maturation within seminiferous tubules of the testicle. Preliminary data in recent literature suggest a correlation between microdeletions involving the AZFb of the Y-chromosome and the absence of spermatozoa with TESE (Brandell et al., Human Reprod., 1998). We hereby report our expanded experience of the relationship between microdeletions involving AZFb and TESE results in men with non-obstructive azoospermia. Design: A retrospective analysis of azoospermic men who underwent TESE from 1996 to 2000 was performed. The outcomes of men with AZFb microdeletion of the Y-chromosome were analyzed. Materials and Methods: All TESE were performed under local or general anesthesia by one surgeon. TESE were performed with either multiple biopsies or with a single large incision with multiple samples of the seminiferous tubules under microscopic vision (microdissection TESE). The procedures were performed in conjunction with planned in-vitro fertilization cycles with ICSI for the female partner. Y-chromosomal microdeletion was tested in all patients using DNA extracted from peripheral leukocytes with a series of 35 sequence-tagged sites on Yq using polymerase chain reaction. Results: Among 181 patients who undergone TESE, 10 (6%) were found to have microdeletion involving the AZFb regions and 5 of them (3%) have microdeletion in AZFb region only. For men with non-obstructive azoospermia and no detectable deletions of the AZFb region, 114/171 (67%) men had sperm retrieved with TESE, whereas none of the 11 patients with microdeletions involving the AZFb region had sperm found with TESE (p,0.001). The average duration of TESE in patients with deletions involving the AZFb region was 3.5 hours. Conclusions: Genetic studies may provide important prognostic information in the management of patients with severe male infertility, as illustrated by our observation that the presence of an AZFb deletion is a significantly adverse prognostic finding for TESE. When counseling men with AZFb deletions, clinicians should apprise them of these results before attempting TESE-ICSI.
Wednesday, October 25, 2000 4:15 P.M. O-254 Effective Cell Sorting After Flow Cytometric Sperm Sample Analysis in an Azoospermic Patient with Polycystic Kidney Disease. A. Akerman, M. Goldstein, J. J. Hariprashad, M. T. Kan, Z. Rosenwaks, G. D. Palermo. The Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, New York, NY. Objective: Isolation of haploid cells from non-obstructive azoospermic patients for successful use in ICSI, represents a major challenge. The aim of the study was to isolate haploid cells from an enzymatically digested testicular biopsy sample, using flow cytometry analysis followed by cell sorting. In addition we attempted to compare flow cytometry outcome to histopathological diagnosis. Design: A testicular biopsy was obtained from an azoospermic patient with polycystic kidney disease and multiple epdidymal cysts. Isolated testicular spermatogenic cells were stained for flow cytometric analysis. The ploidy of the cells was confirmed by cytogenetic analysis. Materials and Methods: Testicular germ cells were obtained from a consenting patient undergoing diagnostic testicular biopsy. Sections of intact biopsied tissue were prepared for histopathological analysis. The remaining biopsy sample was treated by collagenase digestion, then stained and processed for flow cytometric analysis and additional cell sorting. Diagnostic correlations were performed blinded. For flow cytometric analysis, cells were classified as haploid, diploid (using human peripheral lymphocytes as a control), and tetraploid. After DNA quantification, cells
Vol. 74, No. 3, Suppl. 1, October 2000