| Background and Objective:As the provisions by the World Health Organization (WHO), a couple may be considered infertile if, after one year or more of regular sexual intercourse, without contraception, the woman has not become pregnant. The male infertility refers to the infertility caused by the male factors. In recent years, the proportion of infertility has been increasingly high in married couples of the appropriate age, and infertility has brought many negative effects to the society. Male infertility has many and quite complex causes, such as trauma, testicular dysplasia, endocrine or autoimmune diseases, infarction, administration of hormone drugs or cancer drugs, environmental toxins, bad habits, and genetic factors, etc. A few cases of male infertility can not be reasonably explained based on the molecular level, called idiopathic male infertility, in which about50%are caused by genetic factors. In recent years, a large number of studies have found that chromosomal abnormalities, Y chromosome microdeletions and mutations in related genes are associated with the changes in male reproductive capacity.An increasing number of scholars have verified the important relationship between the microdeletions distal to the AZF region on the long arm of Y chromosome and spermatogenesis dysfunction. Based on this conclusion, the effect of Y-chromosome AZF microdeletions must be first ruled out to study the correlation between the related gene polymorphism and male reproductive capacity.In1996, Mosselman et al. first cloned another subtype of estrogen receptor from the testicular tissue, which was later called ESRβ or ESR2. It was found that the rs4986938G/A locus of ESR2gene was located at nucleotide1730in the3’ untranslated region of exon8; the rs1256049G/A locus of ESR2gene was located at nucleotide1082in the ligand binding domain of exon5. In2010, Mohammad Reza Safarinejad et al. conducted a research on the correlation between the polymorphism of these two loci of ESR2gene in Iranian population and male infertility and found that the haplotype was related to male infertility. In2011, Taiwan scholar Wen Lee et al. found that the polymorphism of rs4986938and rs1256049loci of ESR2gene was related to the semen concentration; and the polymorphism of rs1256049locus was also related to the sperm motility. In2012, Japanese scholars Tsutomu Ogata et al. conducted a research on the correlation between the polymorphism of multiple SNPs loci of ESR2gene and male infertility and found that the haplotype "TGTAGA" might be a risk haplotype of male infertility. In this study, two loci of ESR2gene (rs4986938and rs1256049) were selected to verify the correlation between the ESR2gene polymorphism and male infertility in the Han population in Henan province. These two loci were meaningless in the studies of Iranian and Japanese populations, but were related to the sperm concentration and sperm motility in the research by Taiwan scholars. And these loci had not been reported at home.In this study, we first conducted a detection of microdeletions in Y-chromosomal AZF region by using multiplex polymerase chain reaction (Mutiplex PCR) in the case group. After exclusion of AZF microdeletions, the resting objects of study in the case group were then given polymorphic analysis on rs4986938and rs1256049loci of ESR2gene in the Han population in Henan province by using the polymerase chain reaction-fragment length polymorphism (PCR-RFLP) technique. The distribution of locus polymorphism in the Han population in Henan province and its correlation with male reproductive capacity were also researched, so as to provide the relevant theoretical basis for clinical detection and treatment of male infertility.Objects of Study:1. Case groupThe semen specimens of397patients with infertility admitted in the Outpatient Laboratory Department of the First Affiliated Hospital of Zhengzhou University from November2012to March2013were collected. The outpatients were aged between22and42years (mean age of29.4±6.1years). Patients were all given routine semen analysis referring to the criteria of World Health Organization (WHO). And they were diagnosed as azoospermia and oligozoospermia by three consecutive tests (sperm count was less than20×10-6/ml). In addition, patients underwent genital surgery, with orchitis, varicocele, endocrine disorders, liver and kidney diseases, chromosomal abnormalities, and genital abnormalities were all excluded.2. Control group347cases of healthy men with at least a healthy child through the normal means were randomly selected as the control group, who were aged between22and42years (mean age of31.7±2.6years).All objects of study in the case group and the control group were from Henan region. They were with the roughly same diet and all provided with informed consent.Methods:The semen was collected for extraction of genomic DNA by classical phenol-chloroform method. The synthesized primers were used for amplifying the target gene with the PCR instrument, which was then digested by Rsa I and Alu I restriction enzymes. Finally, the2.5%agarose gel electrophoresis were used for genotype detection of the amplified and digested fragments, which were observed and recorded with UV gel imaging system.Statistical Analysis:SHEsis online software and SPSS17.0statistical software were used for analysis of all data. The inspection level was α=0.05. Both the case group and control group were given Hardy-Weinberg (HWE) tests by using SHEsis online software in order to estimate the representativeness of population of the selected data. SPSS17.0software was used to calculate OR values and95%confidence interval and estimate the risk of genetic mutation to the incidence of male infertility.Results:1. Among397cases of patients in the case group,50cases of Y chromosome microdeletions were detected.2. In the Han population in Henan province, the genotype frequencies of ER2gene on rs4986938and rs1256049loci were consistent with Hardy-Weinberg equilibrium, indicating that it has a good representativeness of the population.3. In this study, the genotype and allele frequency were statistically significant in the comparison of rs4986938locus between the case group and the control group (P<0.05).4. The density packet analysis on rs4986938locus showed that GG genotype is significantly different between the severe and mild oligospermia groups (P<0.05).5.In this study, the comparison of rsl256049locus between two groups showed that the genotype and allele frequency were significantly different (P<0.05).6. The density packet analysis on rs1256049loci showed that GG genotypes were significantly different between the severe oligozoospermia group, extremely severe oligozoospermia group and azoospermia group (P<0.05).7. The haplotype analysis of rs4986938G/A and rs1256049G/Aloci in the Han population in Henan province between the male infertility group and the normal control group showed that A-G, G-A and G-G haplotypes were significantly different between two groups (P<0.05).Conclusion:1%Risk analysis found that the A allele of rs4986938and the A allele of rs1256049of ESR2gene may be a genetic risk factor of male infertility in Henan Han population.2、The A-G/G-A haplotype of ESR2gene may be a genetic risk factor of male infertility in Henan Han population but the G-G haplotype may be a genetic protect factor of male infertility in Henan Han population.3、ESR2gene polymorphisms may be associations with male infertility in Henan Han population. |
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