The Association Study Between SEMG1,SEPT12 And Asthenozoospermia

Part1 The Gene Expression Analysis of SEMG1 and SEPT12Objective: To investigate the transcriptional expression on m RNA levels of Semenogelin I(SEMG1)and Septin 12(SEPT12)in asthenozoospermia group and normal control group,and understand the correlation between SEMG1,SEPT12 gene and asthenozoospermia.Methods: 40 adult semen samples of asthenozoospermia group and normal control group were collected respectively to separate and purify by Percoll gradient centrifugation.After purification,the sperm RNA was extracted and reverse transcribed into c DNA.We used Real-time quantitative reverse transcription polymerase chain reaction(q RT-PCR)method to detect SEMG1 and SEPT12 gene m RNA levels with corresponding gene-specific amplification primers in case and control groups.Results: After the amplification of objective genes and reference gene by q RT-PCR,we obtained the corresponding specific amplification bands.SEMG1 gene target fragment was 163 bp and SEPT12 gene target fragment was 167 bp,while reference gene GAPDH fragment was 189 bp.The subsequent q RT-PCR amplification curve and melting curve showed strong amplification specificity of SEMG1 gene,SEPT12 gene and reference gene GAPDH.Differences in quantitative variables between two groups were analyzed by using the Student's t-test.The results showed that,1.SEMG1 gene expression was up-regulated in asthenozoospermia group,and its relative expression level was significantly higher than normal control group(1.98±0.24 vs.1.05±0.35,P<0.05).2.SEPT12 gene expression was down-regulated in asthenozoospermia group,and its relative expression level was significantly lower than normal control group(0.36±0.14 vs.1.03±0.25,P<0.05).Conclusion: 1.The mRNA expression level of SEMG1 gene was significantly higher in the asthenozoospermia group than that in normal control group,which may mediate the causation of asthenozoospermia.2.The m RNA expression level of SEPT12 gene was significantly lower in the asthenozoospermia group,which may be connected with the caustion of asthenozoospermia.Part 2 Study on SEMG1 and SEPT12 Gene Polymorphisms in Asthenozoospermia PatientsObjective: To analyse the polymorphisms of rs16989820 and rs2301366 loci of SEMG1 gene,rs759991 and rs6500634 loci of SEPT12 gene by KASP? genotyping assays,and explore the association between these four loci polymorphisms and asthenozoospermia in Henan,and provide a theoretical basis for the clinical detection ofasthenozoospermia.Method: 276 adult semen samples of normal control group andpatients with asthenozoospermia were collected respectively to extract sperm DNA.For SEMG1 and SEPT12 gene,genotyping was performed by KASP? genotyping assays.The PCR amplification products of different homozygote genotypes were randomly selected and verified by DNA sequencing.Results: 1.The distribution of frequencies of rs16989820,rs2301366 loci in SEMG1 gene and rs759991 and rs6500634 loci in SEPT12 gene in the asthenozoospermia and normal control groups were consistent with Hardy-Weiberg equilibrium(The P value were all above 0.05).2.There were no differences in allele and genotype frequencies distribution between the asthenozoospermia group and normal control group of both rs16989820 and rs2301366 loci(P>0.05).3.The distribution of allele and geneotype frequencies of rs759991 locus in SEPT12 gene between the asthenozoospermia group and normal control group showed a significant difference(P<0.05).The T allele and TT genotype were significantly associated with an increased risk of asthenozoospermia in Henan(P=0.007,OR=1.413,95%CI: 1.098-1.818;P=0.021,OR=1.891,95%CI: 1.097-3.261).4.The distribution of allele frequency of rs6500634 locus in SEPT12 gene between the asthenozoospermia group and normal control group showed a significant difference(P<0.05).The T allele was significantly associated with adecreased risk of asthenozoospermia in Henan(P=0.030,OR=0.661,95%CI: 0.454-0.963).5.Through haplotype analysis of rs759991 and rs6500634 loci in SEPT12 gene,we found that C-T haplotype was associated with a decreased risk for asthenozoospermia(P=0.006,OR=0.431,95%CI: 0.233-0.798),while T-C haplotype was connected with an increased risk for asthenozoospermia(P=0.002,OR=1.549,95%CI: 1.180-2.033).Conclusion: 1.SEPT12 gene rs759991 T allele and TT genotype were potential risk factors for asthenozoospermia in Henan Han population,which may increase the occurrence of asthenozoospermia.2.SEPT12 geners6500634 T allele was potential protective factor for asthenozoospermia in Henan Han population,which may decrease the occurrence of asthenozoospermia.3.The C-T haplotype of SEPT12 gene may be a genetic porotective factor of asthenozoospermia while T-C haplotype may be a genetic risk factor of asthenozoospermia in Henan.