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IMPACT OF ABERRANT ALF GENE METHYLATION ON OUTCOME OF TESTICULAR SPERM EXTRACTION AND INTRACYTOPLASMIC SPERM INJECTION
682
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were Trypan (+). Trypan (+) sperm demonstrated either no response (55%) or a weak repulsive response (45%) to DEP. Some Trypan (-) sperm (12%) demonstrated no response to the DEP. CONCLUSIONS: Results suggest that this modified DEP platform is capable of non-invasively identifying and sorting viable from non-viable sperm. Sorted sperm may be individually retrieved, for use with ICSI. Sensitivity to detect viability may be greater than with the Trypan Blue dye exclusion test.
Source of Funding: None
1888 ACETYLATED HISTONE H4K12 EXHIBITS DIFFERENT BINDING TO SPERM DNA BETWEEN FERTILE MEN AND INFERTILE PATIENTS Klaus Steger*, Agnieszka Paradowska, Sigrid Schumacher, HansChristian Schuppe, Marek Bartkuhn, Wolfgang Weidner, Giessen, Germany INTRODUCTION AND OBJECTIVES: Histone to protamine exchange, in men, is known to be incomplete, as sperm chromatin contains about 20% histones including histone H4 acetylated at lysine 12 (H4K12ac). As acetylated histones are normaly associated with transcriptional active genes and spermatozoa are known to represent transcriptional inactive cells, H4K12ac is thought to be involved in early gene expression during embryogenesis. In the present study, we identified genes associated with H4K12ac and analyzed differences in the binding patterns between fertile and infertile men. METHODS: After written informed consent, chromatin immunoprecipitation followed by promoter microarray analysis (ChIPon-chip) was performed on ejaculated sperm from fertile volunteers and patients that were unable to fertilize an oocyte when applying intracytoplasmic sperm injection. Sperm chromatin was incubated with a polyclonal antibody against H4K12ac. Subsequently, co-hybridization of enriched probe with cy5 and input material with cy3 was performed and the log2 ratio was measured (HG18, NimbleGene). RESULTS: In fertile volunteers, more than 500 target gene promoters could be identified for H4K12ac. 34 peaks with a false discovery rate <0.05 represented the highest confidence for binding sites. By contrast, only about 149 target gene promoters and a lower enrichment of each binding site could be identified in infertile patients. According to gene ontology classification, most of the H4K12ac interacting promoters are involved in DNA-dependent regulation of transcription (18%), signal transduction (9%), development (9%), metabolism (9%) and protein synthesis (9%), while in infertile patients, the majority of the genes were involved in metabolic process (21%). CONCLUSIONS: The aberrant acetylation pattern in infertile men might be responsible for insufficient sperm chromatin compaction resulting in male infertility and/or inappropriate transfer of epigenetic information to the zygote. Source of Funding: German Research Foundation (DFG)
Vol. 181, No. 4, Supplement, Wednesday, April 29, 2009
1889 IMPACT OF ABERRANT ALF GENE METHYLATION ON OUTCOME OF TESTICULAR SPERM EXTRACTION AND INTRACYTOPLASMIC SPERM INJECTION Kazuhiro Sugimoto*, Kouji Izumi, Kazutaka Narimoto, Sotaro Miwa, Yuji Maeda, Tohru Miyagi, Jiro Kanaya, Yasuhide Kitagawa, Yoshihumi Kadono, Hiroyuki Konaka, Atsushi Mizokami, Eitetsu Koh, Mikio Namiki, Kanazawa, Japan INTRODUCTION AND OBJECTIVES: It has been suggested that some cases of impaired spermatogenesis are caused by aberrant DNA methylation. However, there have been few studies of the impact of aberrant DNA methylation on the outcome of testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI). TFIIA-A/B-like factor, also known as ALF, is a germ cell-specific counterpart of the large (A/B) subunit of general transcription factor TFIIA, and plays an important role in spermiogenesis. The loss of ALF expression is associated with male infertility in humans. Previous studies in mice showed that transcriptional control of ALF in male germ cells involves DNA methylation. The present study was performed to examine whether aberrant DNA methylation of the human ALF gene is associated with impaired spermatogenesis. Furthermore, the impact of aberrant DNA methylation on the outcome of TESE-ICSI was also investigated. METHODS: The study population included 95 azoospermic or severe oligozoospermic patients, all of whom underwent testicular biopsy and were diagnosed histologically. Of these, 26 patients presented with Normal spermatogenesis and 17 with Hypospermatogenesis (all stages of spermatogenesis present but proportionate reduction at each level). Genomic DNA and total RNAs were isolated from the testes of all patients. Quantitative methylation analysis of the ALF promoter CpG island was performed using MassARRAY (Sequenom) methylation analysis based on matrix-assisted laser desorption/ionization time-offlight mass spectrometry. The mRNA expression was analyzed by realtime PCR. Clinically, sperm retrieval rate (SRR), fertilization rate (FR), and pregnancy rate (PR) were evaluated. RESULTS: Methylation analysis indicated promoter hypomethylation in all 26 patients with Normal spermatogenesis. Only 5 of 17 patients with Hypospermatogenesis showed promoter hypermethylation (PH group), while the remaining 12 showed promoter hypomethylation (non-PH group). The levels of ALF mRNA expression in PH were significantly lower than in non-PH (P=0.020). With regard to clinical outcome, SRR was similar between PH and non-PH (100% vs. 100%). However, both FR and PR were higher in PH than in non-PH (100% vs. 67% and 100% vs. 67%, respectively). CONCLUSIONS: The results of the present study suggested that aberrant DNA methylation of the human ALF gene promoter is involved in Hypospermatogenesis. However, TESE-ICSI may overcome the impaired spermatogenesis associated with aberrant DNA methylation. Source of Funding: None
1890 PLURIPOTENT STEM CELLS FROM THE ADULT HUMAN TESTIS. Nina Kossack, Juanito Meneses, Palo Alto, CA; Shai Shefi, Tel Hashomer, Israel; Ha Nam Nguyen, Shawn Chavez, Cory Nicholas, Joerg Cromoll, Renee A Reijo Pera, Palo Alto, CA; Paul J Turek*, San Francisco, CA INTRODUCTION AND OBJECTIVES: Pluripotent cells, derived from spermatogonial stem cells, have been reported from both neonatal and adult mice testes. The derivation of these stem cells may involve reprogramming of endogenous spermatogonia in culture. Based on a similar principle, we sought to derive human pluripotent germline stem cells from testis biopsies. METHODS: Testis biopsies were obtained from healthy, adult men and mechanically and enzymatically dissociated with collagenase and DNase. Cells were then cultured on gelatin coated dishes in MEM-A supplemented with 10% FBS and allowed to proliferate until distinct colonies formed on top of the testicular stromal monolayer. These colonies were manually transferred to mouse embryonic fibroblasts (MEFs) and
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were Trypan (+). Trypan (+) sperm demonstrated either no response (55%) or a weak repulsive response (45%) to DEP. Some Trypan (-) sperm (12%) demonstrated no response to the DEP. CONCLUSIONS: Results suggest that this modified DEP platform is capable of non-invasively identifying and sorting viable from non-viable sperm. Sorted sperm may be individually retrieved, for use with ICSI. Sensitivity to detect viability may be greater than with the Trypan Blue dye exclusion test.
Source of Funding: None
1888 ACETYLATED HISTONE H4K12 EXHIBITS DIFFERENT BINDING TO SPERM DNA BETWEEN FERTILE MEN AND INFERTILE PATIENTS Klaus Steger*, Agnieszka Paradowska, Sigrid Schumacher, HansChristian Schuppe, Marek Bartkuhn, Wolfgang Weidner, Giessen, Germany INTRODUCTION AND OBJECTIVES: Histone to protamine exchange, in men, is known to be incomplete, as sperm chromatin contains about 20% histones including histone H4 acetylated at lysine 12 (H4K12ac). As acetylated histones are normaly associated with transcriptional active genes and spermatozoa are known to represent transcriptional inactive cells, H4K12ac is thought to be involved in early gene expression during embryogenesis. In the present study, we identified genes associated with H4K12ac and analyzed differences in the binding patterns between fertile and infertile men. METHODS: After written informed consent, chromatin immunoprecipitation followed by promoter microarray analysis (ChIPon-chip) was performed on ejaculated sperm from fertile volunteers and patients that were unable to fertilize an oocyte when applying intracytoplasmic sperm injection. Sperm chromatin was incubated with a polyclonal antibody against H4K12ac. Subsequently, co-hybridization of enriched probe with cy5 and input material with cy3 was performed and the log2 ratio was measured (HG18, NimbleGene). RESULTS: In fertile volunteers, more than 500 target gene promoters could be identified for H4K12ac. 34 peaks with a false discovery rate <0.05 represented the highest confidence for binding sites. By contrast, only about 149 target gene promoters and a lower enrichment of each binding site could be identified in infertile patients. According to gene ontology classification, most of the H4K12ac interacting promoters are involved in DNA-dependent regulation of transcription (18%), signal transduction (9%), development (9%), metabolism (9%) and protein synthesis (9%), while in infertile patients, the majority of the genes were involved in metabolic process (21%). CONCLUSIONS: The aberrant acetylation pattern in infertile men might be responsible for insufficient sperm chromatin compaction resulting in male infertility and/or inappropriate transfer of epigenetic information to the zygote. Source of Funding: German Research Foundation (DFG)
Vol. 181, No. 4, Supplement, Wednesday, April 29, 2009
1889 IMPACT OF ABERRANT ALF GENE METHYLATION ON OUTCOME OF TESTICULAR SPERM EXTRACTION AND INTRACYTOPLASMIC SPERM INJECTION Kazuhiro Sugimoto*, Kouji Izumi, Kazutaka Narimoto, Sotaro Miwa, Yuji Maeda, Tohru Miyagi, Jiro Kanaya, Yasuhide Kitagawa, Yoshihumi Kadono, Hiroyuki Konaka, Atsushi Mizokami, Eitetsu Koh, Mikio Namiki, Kanazawa, Japan INTRODUCTION AND OBJECTIVES: It has been suggested that some cases of impaired spermatogenesis are caused by aberrant DNA methylation. However, there have been few studies of the impact of aberrant DNA methylation on the outcome of testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI). TFIIA-A/B-like factor, also known as ALF, is a germ cell-specific counterpart of the large (A/B) subunit of general transcription factor TFIIA, and plays an important role in spermiogenesis. The loss of ALF expression is associated with male infertility in humans. Previous studies in mice showed that transcriptional control of ALF in male germ cells involves DNA methylation. The present study was performed to examine whether aberrant DNA methylation of the human ALF gene is associated with impaired spermatogenesis. Furthermore, the impact of aberrant DNA methylation on the outcome of TESE-ICSI was also investigated. METHODS: The study population included 95 azoospermic or severe oligozoospermic patients, all of whom underwent testicular biopsy and were diagnosed histologically. Of these, 26 patients presented with Normal spermatogenesis and 17 with Hypospermatogenesis (all stages of spermatogenesis present but proportionate reduction at each level). Genomic DNA and total RNAs were isolated from the testes of all patients. Quantitative methylation analysis of the ALF promoter CpG island was performed using MassARRAY (Sequenom) methylation analysis based on matrix-assisted laser desorption/ionization time-offlight mass spectrometry. The mRNA expression was analyzed by realtime PCR. Clinically, sperm retrieval rate (SRR), fertilization rate (FR), and pregnancy rate (PR) were evaluated. RESULTS: Methylation analysis indicated promoter hypomethylation in all 26 patients with Normal spermatogenesis. Only 5 of 17 patients with Hypospermatogenesis showed promoter hypermethylation (PH group), while the remaining 12 showed promoter hypomethylation (non-PH group). The levels of ALF mRNA expression in PH were significantly lower than in non-PH (P=0.020). With regard to clinical outcome, SRR was similar between PH and non-PH (100% vs. 100%). However, both FR and PR were higher in PH than in non-PH (100% vs. 67% and 100% vs. 67%, respectively). CONCLUSIONS: The results of the present study suggested that aberrant DNA methylation of the human ALF gene promoter is involved in Hypospermatogenesis. However, TESE-ICSI may overcome the impaired spermatogenesis associated with aberrant DNA methylation. Source of Funding: None
1890 PLURIPOTENT STEM CELLS FROM THE ADULT HUMAN TESTIS. Nina Kossack, Juanito Meneses, Palo Alto, CA; Shai Shefi, Tel Hashomer, Israel; Ha Nam Nguyen, Shawn Chavez, Cory Nicholas, Joerg Cromoll, Renee A Reijo Pera, Palo Alto, CA; Paul J Turek*, San Francisco, CA INTRODUCTION AND OBJECTIVES: Pluripotent cells, derived from spermatogonial stem cells, have been reported from both neonatal and adult mice testes. The derivation of these stem cells may involve reprogramming of endogenous spermatogonia in culture. Based on a similar principle, we sought to derive human pluripotent germline stem cells from testis biopsies. METHODS: Testis biopsies were obtained from healthy, adult men and mechanically and enzymatically dissociated with collagenase and DNase. Cells were then cultured on gelatin coated dishes in MEM-A supplemented with 10% FBS and allowed to proliferate until distinct colonies formed on top of the testicular stromal monolayer. These colonies were manually transferred to mouse embryonic fibroblasts (MEFs) and