The Study Of The Screening For Candidate Genes Of Male Infertility And The Moleculor Mechanisms

Objectives To evaluate the association of PRM1, PRM2, TEX15, KIT, KITLG andPRDM9genes variants with susceptibility of severely defective spermatogenesis, weperformed a case-control study in Chinese Han population.Methods A cohort of309patients (199cases with idiopathic non-obstructiveazoospermia and110cases with idiopathic severe oligozoospermia) and377controlswhich have normal sperm parameters were recruited in the Chinese Han population.Fourty six single nucleotide polymorphisms (SNPs) of the6candidate genes weregenotyped by Sequenom iplex. SHEsis software and SPSS13.0softwares wereemployed for statistical analysis, P<0.05was considered statistically significant.Bonferroni analysis was performed to correct the P value.Results The results showed that PRM1variant rs35576928(p. R34S) was significantlyassociated with severe oligozoospermia and played a protective role against the disease(P=0.0032, Bonferroni correction, OR=0.43). Haplotype analysis of7SNPs sites ofPRM1and PRM2genes in combination exhibited that haplotype TACCGGC played asignificant protective role against the occurrence of oligozoospermia comparing with the controls (P=0.002, Bonferroni correction, OR=0.602,95%CI=0.432-0.838).Haplotype TACCTGC was strongly associated with the risk of the clinical phenotype ofsevere oligozoospermia (P=0.002, Bonferroni correction, OR=2.716,95%CI=1.398-5.273). Two SNPs of TEX15gene rs323346(p. I1035V) and rs323347(p. A104C) wereidentified to be significantly associated with spermatogenesis disorder. Furtherhaplotype analysis showed that the haplotype GC(rs323347、rs323346) conferred asignificantly increased risk to spermatogenesis disorder (P=0.04，OR=1.624，95%CI：1.010-2.610)。While33SNPs of PRDM9，KIT�'�KITLG genes had no significantlydifferent between the spermatogenesis disorder patients and the controls.Conclusion Our findings indicated that PRM1and TEX15genes variants might beassociated with the spermatogenesis disorder in the Chinese Han population. Objectives To explore the distrupted expression of TEX15in testicular specimen ofspermatogenesis disorder patients and the possible regulatory mechanism, as well asmeiotic synapsis and genetic recombination in spermatocytes of patients.Methods By immunohistochemistry and realtime-PCR, we examined the expressionlevel of TEX15in35cases of NOA patients and25controls testicular tissue. Immuno-fluorescence staining revealed meiotic abnormalities in patients with decreased TEX15 level. Application of the miRNAs biological information database predicted thepotential regulatory miRNAs, followed by luciferase reporter gene analysis to detect thebinding capacity of miRNAs with TEX15mRNA3’UTR. SPSS13.0software wasemployed for statistical analysis, P <0.05was considered statistically significant.Results Both realtime-PCR and immunohistochemistry showed that TEX15expressionlevel significantly decreased in the35cases of the NOA patients (P=0.0006, OR:0.3389,95%CI:0.1619-0.5160). Luciferase reporter gene analysis suggests thatmiR-183is a possible TEX15regulator, by affecting TEX15post-transcriptionalexpression. The spermatocytes immunofluorescence staining find that NOA patientspossess much less genetic recombination loci in each spermatocytes, as well as themuch more discontinuous region (gap) and non-synaptic region (split) in pachytenespermatocytes.Conclusions miR-183regulates TEX15gene by directly combining to the3’UTR region,causing the post-transcriptional decreasing of TEX15and the abnormal geneticrecombination during meiosis, eventually leading to the occurrence of spermatogenesisdisorder. Objectives PRDM9is histone methyltransferase involved in the combination of generecombination hotspots and chromosome recombination.Our preliminary studies found that PRDM9gene polymorphism might not be associated with spermatogenesis disorder,this part of study aims to explore whether the PRDM9genes involved inspermatogenesis disorder through CpG methylation in promoter region.Methods Applying sodium bisulfite sequencing (bisulfite genomic sequencing PCR,BSP) to directly detect PRDM9gene promoter region CpG sites methylation status in25cases of NOA patients and14control subjects.Results PRDM9gene promoter region (5’UTR+577～+829) CpG were in methylationstatus in both cases and controls, only occasionally non-methylated sites were detected.Methylation degree in control group were93.1%(81out of87CpG sites), Methylationdegree in NOA group were96.2%(152out of158CpG sites), no statisticallysignificant difference between two groups(P=0.282). The methylation degree of theeach CpG sites had no statistically significant different in the two groups either.Conclusions DNA methylation of PRDM9promoter region is not associated withspermatogenesis disorder.