【作者】 陆宁霞；

【导师】 王心如；

【作者基本信息】 南京医科大学 ， 卫生毒理学， 2007， 硕士

【摘要】 全世界育龄夫妇中约有10-15％的人被不孕不育所困扰。在所有的不孕不育中，由男方因素造成的约占50％，其中一个重要的原因是男方生精障碍，主要表现为无精或严重少精，约占不育男性的10％。许多环境、行为和遗传因素都会影响男性生殖。近年来研究发现，精子生成的过程受到诸多基因的调控。这些基因的缺失、突变或表达异常都可能导致精子生成障碍，形成少精子症或无精子症，导致男性不育。人类基因组计划的进展为阐明男性不育的基因机制及精确了解基因对睾丸功能的影响提供了机会。基因多态性尤其是单核苷酸多态性(Single Nucleotide Polymorphisms，SNPs)是人类最常见的遗传变异。最近，越来越多的研究表明，男性不育特别是精子生成障碍与某些基因多态性具有相关性。由此看来，一些精子发生相关基因的SNPs与男性不育可能存在关联性。本课题采用病例-对照研究设计，并应用限制性片段长度多态性分析法(Polymorphism chain reaction-Restriction fragment length polymorphism，PCR-RFLP)，研究CYP1A1和p53基因的多个多态性单独或联合作用与男性不育的相关性。第一部分CYP1A1基因多态性与男性不育的相关性研究许多化学物质会导致男性生育能力下降，已证实多环芳烃(Polycyc1ic aromatic hydrocarbons，PAHS)具有生殖毒性。细胞色素P4501A1(CYP1A1)可催化多环芳烃等化合物进入体内的Ⅰ相反应，使其与DNA形成加合物。本研究结合聚合酶链反应技术的应用来探讨CYP1A1基因多态性与男性不育的相关性。采用病例-对照研究方法，选择已经确诊的192例特发性无精和严重少精的男性不育患者和143例精子密度正常的不育患者为病例，226名有正常生育史的男性为对照。通过PCR-RFLP方法检测CYP1A1*2A和CYP1A1*2C两位点的基因型。计算不同基因型人群中男性不育患病的相对危险度(OR)及其95％可信区间(95％CI)。研究发现，CYP1A1*2C位点三种基因型频率在无精和严重少精不育病例组中分别为59.4％(AA)、31.8％(AG)和8.8％(GG)，对照组中分别为46.9％(AA)、45.1％(AG)和8.0％(GG)，存在显著性差异(P=0.019)。在无精和严重少精不育病例与正常对照比较的研究中，Logistic回归分析显示：(1)与CYP1A1*2C AA基因型比较，AG基因型携带者患男性不育的风险显著降低(OR=0.56，95％CI=0.36-0.86，P=0.005)；当合并CYP1A1*2C AG和GG基因型时，AG和GG基因型携带者患男性不育的风险同样显著降低(OR=0.60，95％CI=0.40-0.91，P=0.011)；CYP1A1*2A各基因型在病例组和对照组中的分布无显著性差异。(2)与C-A单倍型比较，T-G单倍型(OR=0.38，95％CI=0.16-0.87)，C-G单倍型(OR=0.44，95％CI=0.27-0.73)和T-A单倍型(OR=0.51，95％CI=0.32-0.79)均降低了个体患男性不育的风险。研究还发现，在精子密度正常的男性不育病例中，CYP1A1*2C AG基因型携带者精子密度、精子总数和精子活率均显著高于CYP1A1*2C AA基因型携带者(P=0.049，P=0.016和P=0.049)。我们得出初步结论，CYP1A1多态性可能与中国汉族人群的男性不育具有一定的相关性，但须进一步进行功能研究并在不同人群中进行验证。第二部分p53基因多态性与男性不育的关联性研究尽管一些遗传因素已被证实会与男性不育具有相关性，但仍有许多遗传因素如基因的变异与男性不育的关系未阐明。p53基因参与了雄性小鼠和大鼠的减数分裂过程，提示该基因可能在精子生成过程中发挥重要作用。本课题采用病例-对照研究方法，选择已经确诊的198例特发性无精和严重少精的男性不育患者为病例，233名有正常生育史的男性为对照。通过PCR-PFLP的方法检测p53 C0don72和p53IVS7+72C＞T两位点的基因型。计算不同基因型人群中男性不育患病的相对危险度(OR)及其95％可信区间(95％CI)。结果表明，p53COdon72和p53 IVS7+72C＞T两位点基因型和等位基因型的频率分布在病例组和对照组中均无显著性差异；两位点的单倍型在病例组和对照组中的频率分布也无显著性差异。我们得出初步结论，P53 codon72和p53 IVS7+72C＞T多态性可能与男性不育无明显相关。更多还原

【Abstract】 Approximately 10% couples of childbearing ages suffer from some kinds of infertility, and about half of these cases are due to male factors. Previous reports from clinics confirmed that a significant proportion of male infertility is idiopathic infertility, typified by non-obstructive azoospermia or sever oligozoospermia. Many environmental, behavioral and genetic factors affect male infertility. Many genes have been suggested to be involved in spermetogenesis and it is possible that mutations in these genes are also related to human infertility.Single nucleotide polymorphisms are the most common genetic varients. Recently, several studies have focused on the association of polymorphisms with male infertility, especially with spermatogenetic failure. Thus, some gene involved in spermetogenesis may be associated with male infertility. A case-control study was conducted by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to study the association of polymorphisms in CYP1A1 gene and p53 gene with male infertility. PartⅠStudy on polymorphisms in CYP1A1 gene and male infertilityCytocrome P4501A1 (CYP1A1) is a key enzyme in phaseⅠbioactivation of polycyclic aromatic hydrocarbons (PAHs) which have potential reproductive toxicity. The aim of this study was to investigate the association of the CYP1A1 polymorphisms with male infertility in a Han-Chinese population. We genotyped two polymorphisms, CYP1A1*2A and CYP1A1*2C, using PCR-RFLP assay in a hospital-based case-control study including 192 infertile patients with non-obstructive azoospermia or severe oligozoospermia, 143 normospermic infertile men and 226 fertile controls. We found that the genotype distribution of CYP1A1*2C was significantly different between the patients with non-obstructive azoospermia or severe oligozoospermia and controls (P=0.019). Analysis showed that CYP1A1*2C AG genotype was associated with a significantly decreased risk of male infertility with azoospermia or severe oligozoospermia [odds ratio (OR)= 0.56, 95% confidence interval (95% CI)=0.36-0.86, P=0.005] compared with the AA genotype. A statistically significantly decreased risk of male infertility was found to be associated with the CYP1A1*2C AG genotype plus GG genotype compared with CYP1A1*2C AA genotype (OR=0.60, 95% CI=0.40-0.91, P=0.011). No significant association was detected between CYP1A1*2A polymorphism and male infertility. Haplotypic analysis showed a significantly decreased risk of male infertility with azoospermia or severe oligozoospermia associated with the T-G haplotype compared with the C-A haplotype (OR=0.38, 95% CI=0.16-0.87), indicating a synergic effect of those two polymorphisms. Normospermic infertile patients with CYP1A1*2C AG genotype was found to be associated with higher sperm concentration, the total sperm count and motility when compared to those with AA genotype (P=0.049, P=0.016 and P=0.049). Our results suggest that the CYP1A1 polymorphisms may contribute to the pathogenesis of male infertility in Han-Chinese population.PartⅡStudy on polymorphisms in p53 gene and male infertilityAlthough various genetic factors have been demonstrated in human male infertility, many genetic causes involving gene variants for the idiopathic male infertility have not yet been elucidated. The involvement of p53 gene in the meiosis of the male rat and mice suggested that p53 plays a critical role in spermatogenesis. To examine whether the codon72 polymorphism and IVS7+72C＞T polymorphism of the human p53 gene are associated with spermatogenetic failure in Han-Chinese population. A case-control study was conducted with 198 idiopathic infertile patients with non-obstructive azoospermia or severe oligozoospermia and 233 fertile controls. We genotyped the two polymorphisms, codon72 and IVST+72C＞T, using PCR-RFLP assay. The polymorphisms were identified in both infertile patients and fertile controls. We found no significant difference in the allelic and genotypic distributions of the two polymorphisms between the patients and controls. Further analysis showed that there was no statistically significant difference in the haplotype distributions between the patients and controls. The results of this study suggest that the codon72 and IVS7+72C＞T polymorphisms of the p53 gene are unlikely to contribute to the pathogenesis of idiopathic male infertility with spermatogenetic failure.更多还原