【作者】 陆春城；

【导师】 王心如；

【作者基本信息】 南京医科大学 ， 卫生毒理学， 2010， 博士

【摘要】 已婚育龄夫妇中不育症的发病率高达9-15%。引起不育的病因很多,其中由男方因素引起的约占50%。研究表明,半个多世纪以来,人类精液质量显著下降,精子生成障碍已成为男性不育最常见的病因。而在精子生成障碍的患者中有大约30%的患者是由于遗传学的异常引起的。自1976年Tiepolo等发现Y染色体“无精子因子”(Azoospermia Factor, AZF)以来,Y染色体微缺失成为导致男性生精障碍的重要遗传因素。Y染色体AZF区分布于Y染色体长臂的三个不同区域,称为AZFa、AZFb及AZFc。综合数据显示,在精子生成障碍的病人中,约10℅存在这种缺失。其中,约80%表现为AZFc缺失。近年来研究发现,AZFc区存在各种染色体遗传结构的变化,包括染色体片段倒位、缺失及重复等。AZFc区缺失类型包括AZFc区全缺失及部分缺失(gr/gr缺失、b2/b3缺失等)。其中,AZFc区全缺失是精子生成障碍最常见的致病因素之一;而AZFc区部分缺失在精子生成中的作用目前还没有明确的结论。先前研究证实,AZFc区部分缺失对精子发生的影响存在群体差异性,欧洲人群中已证明的b2/b3缺失单倍群N更充分的验证了这点。Y染色体遗传背景对缺失分布的显著影响提示,在进行Y染色体相关疾病研究时,对样本进行Y染色体分型,检测病例和对照的匹配至关重要。本研究中,我们对中国汉族人群中7个AZFc全缺失家系、756例不育病例(包括220例无精症、199例少精症及337例不育但精子生成正常病人)及391例健康对照进行了AZFc区缺失筛查、DAZ基因拷贝定量分析和Y染色体单倍群分型工作。结果发现在中国汉族人群中部分缺失的发生率明显高于其他人群,且b2/b3部分缺失及DAZ3/4+CDY1a部分缺失亚型与精子生成障碍显著相关。对7例AZFc全缺失家系缺失情况检测后发现,一例完全缺失病人的父亲为部分缺失,说明此例完全缺失是由部分缺失发展而来。结果表明,AZFc全缺失除了常规的直接缺失外,还存在第二种缺失方式-分步缺失。同时,我们还发现在部分缺失高发的单倍群中,完全缺失的比例也显著增高,即AZFc部分缺失会增加全缺失发生的风险。在随后的研究中,我们发现不同部分缺失(gr/gr和b2/b3)发展成为完全缺失的易感性存在差异,b2/b3部分缺失相对gr/gr部分缺失更容易发展成为完全缺失。另外,AZFc区存在基因重复的现象(既可以发生单纯重复,也可以发生缺失后重复),这些事件的发生都伴随相关基因拷贝数的变化,且研究证实,过高的DAZ基因拷贝可能不利于精子的发生。通过研究AZFc区各种染色体结构变异及其所致基因拷贝数的变化对精子发生过程的影响,有助于阐明AZFc区不同基因及其不同拷贝在精子发生过程中的作用;同时,通过对部分缺失及完全缺失关系的研究,将使我们从一个全新的视角审视完全缺失发生的可能机制。更多还原

【Abstract】 It is estimated that about 9~15% of couples suffer from infertility and roughly half of these cases are due to the man’s problem. Related studies have demonstrated that the sperm quality declined remarkably in half a century. The spermatogenic failure has already become the most common cause for male infertility, from which about 30% are genetic abnormalities caused.Since the concept of Azoospermia Factor (AZF) was proposed by Tiepolo et al. in 1976, Y chromosome microdeletion has become one of the most important genetic factors for spermatogenic failure. The AZF, which located in three different regions of the Y chromosome, termed AZFa, AZFb and AZFc are recurrently deleted in about 10% of cases of abnormospermia. Amony which, the AZFc deletions form the majority of these deletions (80%).Recently, many structural variations, including deletion, inversion, duplication, and etc., have been reported in AZFc. The AZFc deletion patterns include AZFc complete deletion and partial deletion. The complete AZFc deletion is one of the most common causes of spermatogenic failure, while the roles of partial AZFc deletions (gr/gr deletion and b2/b3 deletion) in spermatogenesis are controversial.Several studies have demonstrated that the influence of AZFc partial deletion to spermatogenesis varied across population. Namely the genetic background of Y chromosome affect the distributions of AZFc partial deletion. Therefore, Y haplogrouping is highly recommended for studies on Y-linked disease, such as spermatogenic failure.In this study, in order to investigate the roles of these AZFc structural variations in spermatogenic failure, we performed deletion typing, quantitative analysis of DAZ gene copies, and Y chromosome haplogrouping in seven pedigrees of complete AZFc deletion carriers, 736 infertile and 391 healthy men in Chinese. We found that both the b2/b3 partial deletion and the DAZ3/4+CDY1a deletion pattern were associated with spermatogenic failure.After the detection of deletion patterns of seven pedigrees, we observed that a complete AZFc deletion was derived from partial deletion, suggesting that complete deletions of AZFc can be preceded with partial deletions. We also found that the frequency of complete AZFc deletion in haplogroups (with high frequency of partial deletions) was significantly higher than that in the other haplogroups (with low frequency of partial deletions). Namely, the partial AZFc deletions can increase the risk of complete AZFc deletion. In our following study, we found the predisposition differences of partial deletions (gr/gr and b2/b3) to complete AZFc deletion. Our results suggest that the b2/b3 partial deletion was associated with a higher risk of complete AZFc deletion compared with the gr/gr partial deletion. The relationship between partial AZFc deletions and complete AZFc deletion deserves further examination, especially those with a relatively high frequency of b2/b3 and gr/gr partial deletions.In addition, the duplications of DAZ gene (not only pure partial duplication, but also duplication followed by partial deletion) have also be found . In the process of duplication, the copy numbers of key gene have changed as well. Our results suggested that abnormally high copy numbers of DAZ gene are likely to increase the risk of spermatogenic failure.更多还原