| Background and Objective:Male infertility means that a couple couldn’t give birth because of male without the use of any contraceptive measures more than 1 year, set by the World Health Organization(WHO). In recent years, some couples of worldwide are affected by reduced fertility because of environmental, psychological, social and other factors.The causes of male infertility could be testicular hypoplasia, varicocele, endocrine or immune diseases, taking hormone drugs or cancer drugs, environmental toxicants and genetic factors and so on. One of the important reasons is dyszoospermia, mainly for azoospermia or severe oligospermia. More and more researches have shown that spermatogenesis is regulated by many genes in recent years, gene mutation or abnormal expression may change the male reproductive ability, resulting oligoasthenospermia or azoospermia, leading to male infertility.A large number of studies have verified that there is a great relationship between the microdeletion of distal AZF region from Y chromosome and spermobstacles. Therefore, in order to analyze the association between spermiogenesis-related gene polymorphism and male infertility, we must exclude the influence of the microdeletion of distal AZF region from Y chromosome.Steroid hormones have a crucial role in male reproduction and spermatogenesis.The synthesis and metabolism of estrogen is a very complex process, which requires the participation of cytochrome P450(CYP17, CYP11, CYP19), hydroxyl steroid(HSD17), and sulfur transferase(SULTs) and so on.CYP17A1 plays a major role in the steroidogenic pathway, which has both 17alpha-hydroxylase and 17,20-lyase activities. The mutations of CYP17 gene results in impaired synthesis of androgen and estrogen. Feigelson et al. reported variants of rs743572 locus of CYP17A1 are related to the synthesis of sex hormone in female,there is higher level of progesterone and estradiol in TC/CC genotype carriers. In2013, Jung-Hoon Park explored the contribution of variants of CYP17A1 gene to male infertility in South Korean population, their research revealed that there was no association between polymorphism of rs743572 locus and male infertility, while variants of rs17115149 locus of CYP17A1 gene were associated with greater risk for male infertility.Cytochrome P450 aromatase is a key enzyme in the synthesis of endogenous estrogen, which catalyzes the irreversible conversion of androgen to estrogen.Cytochrome P450 aromatase is the product of CYP19 gene, which located on chromosome 15. Cytochrome P450 aromatase regulates oestrogen production and androgen bioavailability. Leandros Lazaros’ study has shown an association of CYP19(TAAA)n polymorphism with sperm quality in Greek Caucasian population.At present, there is no relevant research report in domestic. So we tested theassociations between variants of estrogen-related genes and male infertility in Henan Han Chinese population.Our study first excluded the men with the microdeletion of distal AZF region from Y chromosome by using multiplex polymerase chain reaction(Mutiplex PCR)in case group. Then we used the Sna Pshot minisequencing to exam the polymorphisms of rs743572 and rs17115149 loci of CYP17A1 gene, the CYP19 genotyping was detected by PCR and polyacrylamide gel electrophoresis. We made the association study between the three loci polymorphisms in Henan Han population and male fertility, in order to provide a relevant theoretical basis for the clinical detection and treatment of male infertility.Study population:1.Case group261 infertility patients(mean age of 29.3±6.0) in the Outpatient Laboratory Department of the First Affiliated Hospital of Zhengzhou University were enrolled from March 2014 to October 2014. Case group was based on WHO criteria. They were diagnosed as azoospermia or oligozoospermia while had 3 routine semen analysis with sperm concentration <20×106spermatozoa/ml. Patients suffering kidney diseases, varicocel, orchitis, abnormalities, endocrine diseases, genital diseases or other illnesses were excluded from the study.2.Control group230 fertile men(mean age of 30.2±6.3)who had at least one child and never requiring assisted reproduction technology were included as control group.All of the research objects were Henan Han population and there is no genetic relationship between the objects of study. Subjects were all agreed by themselves.MethodsWhole genomic DNA was extracted using the standard phenol-chloroform method. For CYP17A1 gene, genotyping was performed by using the Sna Pshot minisequencing while in CYP19 gene the experimental method was polyacrylamide gel electrophoresis page followed by silver staining. The PCR products of different genotypes were selected randomly and verified by sequencing.Statistical Analysis:Hard-Weinberg equilibrium(HWE) testing was carried out by using SHEsis software in CYP17A1 gene, and using HWE 1.20 software in CYP19 gene. All statistical tests were performed with SPSS version 17.0. The Student’s t-test was used to test for differences in age between case and control group. The chi-square test or Fisher’s exact test was used to compare the genotype and allele distributions of the polymorphisms between case and control groups. We estimated the association between risk factors and male infertility by calculating the odds ratio(OR) value and95% confidence interval(CI). The adjusted p value for significance was set at 0.05.Results:1.The distribution frequency of rs743572, rs17115149 loci in CYP17A1 gene and STR loci in CYP19 gene in the case and control groups was consistent with Hardy-Weinberg equilibrium(The P value were 0.846 and 0.154, 0.852 and 0.888,0.774 and 0.557, respectively)2. The distribution of allele and genotype frequencies of rs743572 locus in CYP17A1 gene between the case and control groups shows a significant difference(P<0.05). The A allele and AA genotype were revealed to be associated with an increased risk of male infertility in Henan Han population(P=0.01, OR=1.44,95%CI:1.11-1.86; P=0.00, OR=2.25, 95%CI:1.30-3.89). There was no difference in Methods:allele and genotype frequencies distribution between the case group and control group of rs17115149 locus.3. Through haplotype analysis of rs743572 and rs17115149 loci in CYP17A1 gene, we found that there was a significant difference between the case and control groups(P<0.05) in the frequencies of A-G and G-G haplotypes. A-G haplotype was associated with an increased risk for male infertility( P=0.01, OR=1.44,95%CI:1.11-1.86), while G-G haplotype was connected with a decreased risk for male infertility(P=0.03,OR=0.75,95%CI:0.58-0.97).4. The population was divided into two groups by using the(TAAA)9 allele as a cutoff point, and the results showed that there was a significant difference between short alleles(alleles with ≦9) and long alleles(alleles with >9) frequencies(P<0.05).Risk analysis indicated that short alleles were positively correlated with the susceptibility of male infertility in Henan Han population( P=0.00, OR=1.43,95%CI:1.10-1.85).Conclusions:1.CYP17A1 rs743572 A allele and AA genotypes were potential risk factors for male infertility in Henan Han population.2.The A-G haplotype of CYP17A1 gene may be a genetic risk factor of male infertility while G-G haplotype was a genetic protective factor of male infertility in Henan Han population.3.Short CYP19 alleles may be associated with an increased risk of male infertility in Henan Han population. |
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