| Background and Aims:Male infertility which is set by the World Health Organization (WHO) refers to couples living more than1year and does not use any contraceptive measures, causing the women’s infertility due to male factors. Male infertility brings a huge inconvenience to people’s lives, causing some burden to individuals and society. In the15%lower fertility couples of reproductive age of the world, male factor infertility accounts for50%. Male infertility mainly display in spermatogenic failure. There are so many definite factors that causes male infertility, including the varicocele, obstruction, and reproductive system infections, endocrine or autoimmune diseases, environmental toxicology, and genetic factors. There are so many important genetic factors. The large number of studies in recent years declears that chromosomal abnormalities, Y chromosome deletions and gene mutations are all related to male dyszoospermia. In1976, Tiepol et al. found distal microdeletion of the long arm of Y-chromosome, and conjectured there may be some spermatogenesis-related genes in this region, then called this region as AZF. Later, some scientists conjectured microdeletions in AZF may have important relationship wich spermatogenesis.In2008,L. Lazaros et al. did a study about the Greek population and found that there is the relationship between the androgen receptor gene (CAG) n SHBG gene (TAAAA) n repeat number and sperm and semen quality. In2010,Mohammad Reza Safarinejad carried a research about the Finnish population and found a gene which is related to male infertility:SHBG (Sex hormone binding globulin) gene has the association with men oligospermia and asthenozoospermia. In addition, the mutation at the rs6259(Asp327Asn) loci of this gene lead to an increase of an additional carbohydrate chain, which led to the reduction of metabolic clearance rate of SHBG, an increase of SHBG half-life, and therefore increased the SHBG level, by changing the androgen and estrogen bioavailability and behavior, to change the biological activity of the androgen and estrogen, and further lead to male infertility. Mei-Tsz did a study on the han population in Taiwan about the estrogen-related genes and found that the rs1799941loci and rs6259site of SHBG gene is related with male spermatogenic failure and is a newly discovered candidate gene of male infertility. This study selected two sites of SHBG gene. There were studies have found that the rs6259site has relationship with the male infertility of the Finnish population and the Han population in Taiwan, but there is no study illustrate that the rs6259site has relationship with the China’s Henan Han population of male infertility, while there is no study illustrate that the rs727428site has relationship with the male infertility at home and broad.Our study use the method of multiplex polymerase chain reaction (Mutiplex Polymerase chain reaction Mutiplex PCR) and polymerase chain reaction-restriction fragment length polymorphism (Polymerase chain reaction restriction-the fragment length polymorphism by PCR-RFLP). First we exclude the Y chromosome missing, then we exam the rs6259and rs727428site of SHBG gene polymorphism and the distribution in the Henan Han population, and have the further study of relationship between these two loci and male infertility. Thus provides a theoretical basis for the clinical detection and treatment of male infertility.Study population:1Patients The study population consisted of208infertility patients (mean age±SD:32.4±6.4)from the Reproductive Center and inspection of outpatient treatment of First Affiliated Hospital of Zhengzhou University from March to August in2011. Patients had3routine semen analysis and diagnosed with azoospermia or severe oligozoospermia (sperm concentration<20×106/ml) according to the World Health Organization criteria.Patients with orchitis, liver and kidney diseases, chromosomal abnormalities, endocrine diseases, genital diseases or other illnesses, were excluded in this study. Idiopathic azoospermia in48cases,160cases of oligozoospermia.2Controls The control group were consisted of more than183healthy individuals(mean age±SD:32.8±6.7) who were randomly selected and had given birth.All people were Henan population and had similar diets.Signed consent form was obtained from each participant.Methods:Genomic DNA was extracted from1mL semen cells by using the phenol/chloroform method. Design and synthetize primers, and obtain target fragment through amplication by PCR instrument.The target fragment was digested by using the Hinfl restriction enzyme and respectively using3%and2%agarose gel electrophoresis to detect the results of multiplex PCR, PCR and PCR-RFLP observed by UV analysis of gel imager and recorded results.Statistical analysis:All the statistical tests were performed using SPSS17.0software for Windows. P values less than0.05were considered as statistically significant.Gene frequency using mathematical notation. Differences in the allele and genotype frequencies between infertility group and control group were calculated using the Chi-square test.Testing for deviation of genotype distribution from Hardy-weinberg equilibrium and haplotype analysis were performed using the SHEsis software. Odds ratio (OR) and95%confidence intervals (95%CI) were calculated to assess the association between gene mutation and male infertility.Results:1. Microdeletions were detected in12.02%(25/208) infertile men with idiopathic azoospermia or oligozoospermia, showed a statistically significant differences between male infertility group and the control group (P<0.05).2. There is a statistically significant differences between men with idiopathic azoospermia and oligozoospermia3. The AZFc microdeletion of the Y chromosome can result in different phenotypes in different individuals.4. In Henan Han population, the genotype of rs6259and rs727428of SHBG gene in line with the Hardy-weinberg equilibrium (P>0.05), which shows that it has a good population representation.5. In Henan Han population, differences of allele frequency of rs6259locus between male infertility group and control group was statistically significant (P<0.05).6. Stratified analysis of rs6259according to age showed a statistically significant differences between25-35years old male infertility group and the control group (P<0.05).7. Density packet analysis of rs6259locus show that:the GG and GA genotypes were significantly different between the mild oligozoospermia group and very severe less refined group (P<0.05).8. In Henan Han population, CC genotype frequency of rs727428locus were statistically significant between male infertility group and control group (P<0.05).9. In Henan Han population, rs6259A/G and rs727428C/T haplotype analysis, we found that the AT haplotype type difference was statistically significant between the male infertility group and control group (P<0.05).10. Rs727428polymorphism is the existence of racial differences.Conclusion: 1. Come to the Y chromosome AZF region microdeletions is an important factor leading to spermatogenic failure.2. AZFc microdeletion has different clinical phenotypes.3. Risk analysis found that the A allele of rs6259of SHBG gene may be a genetic risk factor of male infertility in Henan Han population especially in man aged25-36.4. The relative risk analysis showed that the CC genotype of rs727428maybe a genetic protective factor of male infertility in Henan Han population, which may decrease the risk of male infertility.5. The AT haplotype of SHBG gene may be a genetic risk factor of male infertility in Henan Han population. |
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