【作者】 ELFATEH GASSMALLA FADLALLA AHMED；

【导师】 刘睿智；

【作者基本信息】 吉林大学 ， 细胞生物学， 2013， 博士

【摘要】 不孕不育是指适龄夫妇婚后1年以上未采取任何避孕措施有规律性生活而仍未受孕。20世纪初期,大多数人认为不孕不育由女性因素所致。而今,事实证明,在不孕不育病例中约一半与男性有关。导致不孕不育因素较为复杂,从遗传性因素到性交障碍均可以影响男性的生育能力。其中,遗传因素约占不育的15-30%。遗传因素影响男性的生育能力,包括染色体核型异常和Y染色体微缺失。遗传所致不育男性临床表型多样,有些男性射出精液中完全没有精子(无精子症),还有一些男性射出精液中仅有少量精子。为什么遗传所致的不育男性会有不同的临床表型?精液质量异常和遗传因素之间存在什么样的关系?为了回答这些问题,我们对中国东北的1384例不育男性进行了遗传因素分析。这些患者按精液常规结果分为693例无精子症患者和691例精液质量差的患者。此外,50例年龄在24-37岁之间的正常生育男性作为对照。我们通过G显带技术进行染色体核型分析,通过多重PCR技术进行Y染色体微缺失检测,通过电化学免疫荧光方法检测激素水平。最终,我们对临床和实验室结果进行分析来研究精液质量异常和遗传因素之间的关系。本研究经吉林大学白求恩第一医院伦理委员会同意。结果：1.无精子症患者相关结果1.1中国东北无精子症患者中,核型异常比例为23.52%(163/693)。其中,Klinefelter综合征(KS)最为常见(17.75%),其次为染色体多态性(3.32%)和易位(0.72%)1.2在无精子症患者中,KS和易位携带者的睾丸体积明显小于对照组,而多态和倒位患者睾丸体积和对照组相比没有明显差异。1.3在无精子症患者中,多态和KS组精浆抑制素B水平明显低于对照组,易位组果糖水平明显高于对照组。KS患者和其他异常核型患者相比精浆生化水平无明显差异。1.4相比于对照组,多态和KS患者FSH、LH和E2水平升高,而易位和倒位患者其PRL、FSH、LH和E2水平变化不大。值得注意的是,所有染色体异常患者其T和T/LH值均较对照组明显降低。1.5无精子症患者Y染色体微缺失发病率为7.94%(55/693)。缺失最易发生的趋势为AZFc区。最易缺失的位点为SY152、SY255和SY254。家系研究显示Y染色体微缺失大部分为新发突变,仅有少数病例遗传自父亲。1.6和对照组相比,AZFc区缺失患者的睾丸体积减小,果糖和α糖苷酶水平升高。此外,AZFc、AZFb+c和AZFb区缺失患者抑制素B水平明显低于对照组。1.7和对照组相比,AZFc、AZFb和AZFb+c区缺失患者FSH高：AZFc、 AZFb区缺失患者PRL高；AZFc区缺失患者LH水平高,而AZFc、AZFb和AZFb+c区缺失患者T和T/LH水平低。2.精液质量差患者相关结果2.1中国东北精液质量差患者中,核型异常比例为12.01%(83/691)。其中,KS最为常见(4.49%),其次为染色体多态性(3.76%)和易位(3.18%)。2.2KS和易位携带者的睾丸体积明显小于对照组,而多态患者睾丸体积和对照组相比没有明显差异。2.3和对照组相比,KS患者的FSH和LH水平显著升高,而在多态患者中,仅FSH水平显著升高。此外,所有精液质量差的异常核型患者T和T/LH值均低于对照组。2.4精液质量差的非嵌合KS患者表现为少精子症和隐匿精子症。与对照组相比,这些患者睾丸体积小、FSH和LH水平升高、T和T/LH值降低。2.5精液质量差患者中,Y染色体缺失率为5.50%。其中,AZFc区缺失率最高。最易缺失的位点仍为SY152、SY255和SY254。家系研究显示所有的Y染色体微缺失都是新生突变而来。2.6与对照组和Y染色体未缺失组相比,精液质量差患者的Y染色体微缺失患者睾丸体积小,T/LH值低,FSH和LH水平高。3.无精子症患者与精液质量差患者比较3.1无精子症患者的核型异常比率显著高于精液质量差的患者,而二者Y染色体微缺失发生率无明显差异。3.2无精子症患者(包括遗传因素和非遗传因素所致无精)的年龄和睾丸体积均比精液质量异常患者显著减小。3.3遗传因素所致无精子症患者的α-糖苷酶水平比精液质量差患者低,而抑制素B和果糖水平无明显差异。3.4和精液质量差患者相比,遗传和非遗传因素所致的无精子症患者FSH和LH水平均高,而PRL、T和E2水平无明显差异。3.5和非遗传学因素所致无精子患者相比,遗传因素所致无精子患者其FSH和LH水平显著升高；T和T/LH水平显著降低,而PRL、T和E2水平无明显差异。3.6精液质量差患者中,非遗传因素患者的年龄,睾丸体积,精浆生化,抑制素B和激素水平与遗传因素来源的患者相比均无明显差异。结论：1.中国东北地区无精子患者致病遗传学因素主要为克氏症和Y染色体微缺失。相比其他种类核型异常患者,克氏症患者的睾丸更小,FSH和LH水平更高,T水平更低。Y染色体微缺失的无精子症患者激素异常与克氏症患者相似。2.中国东北精液质量差患者遗传学致病因素较为复杂,其中Y染色体微缺失是致病最主要病因。绝大部分的Y染色体缺失患者都为AZFc区缺失,且这种缺失多是新发突变而来。3.和Y染色体微缺失相比,核型异常更容易导致无精子症发生。激素结果提示遗传因素导致的生殖激素水平的变化和生精功能障碍关系密切。更多还原

【Abstract】 Infertility refers to couples who have not become pregnant after at least1year of unprotected intercourse. Over many years in early20th century the majority if not all of human societies believed that infertility always is a woman’s problem. But nowadays there is a clear fact that male factors contributed about half of infertility cases. Regarding the possible causes of infertility, wide range of disorders ranging from genetic disorders to coital problems can influence male fertility. Among these genetic aberrations were considered as one of the most frequent and interesting causes, that contributed about30%causes of infertile male, involving both chromosomal disorders and single gene mutations.Genetic disorders that influence male fertility including two major groups as follow:chromosomal abnormalities which characterized by abnormal karyotype, including sex chromosome abnormalities, and autosomal chromosomal abnormalities. The second type of genetic disorders is referred to Y chromosome microdeletions which characterized by azoospermia factor gene deletions. The clinical phenotype of infertile men with genetic aberrations is of wide variety, in some cases infertile men attend the reproductive clinic with completely absent of sperm in the ejaculated semen (azoospermia), other cases showed small amount of sperm in their ejaculated semen. Why infertile men with genetic aberrations have specific clinical phenotype? What’s the relationship between abnormal semen quality and genetic disorders? To answer these questions, we investigated1384infertile men from Northeast of China. These patients were divided into693azoospermic patients and691patients with poor semen quality by semen analysis. Other50normal fertile men aged24-37years old were included as control. Karyotype analysis was done by using G-banding method and Y chromosome microdeletion was detected by using polymerase chain reaction assay PCR. Moreover, hormone level was tested by eleterocheiluminescence immunoassays. All the clinical and lab results were analyzed to illuminate the relationship between abnormal semen quality and genetic disorders. This study was approved by the ethics committee of the First Bethune Hospital of Jilin University.Results:1. Associated results of azoospermic men1.1Abnormal karyotype among infertile azoospermic patients in Northeast China was detected at a rate of23.52%(163/693). Of these, Klinefelter syndrome (KS) is the most frequent (17.75%of the azoospermic patients), followed by Chromosome polymorphism (3.32%), and Translocation (0.72%).1.2The volumes testis of Klinefelter syndrome and Translocation carriers were significantly smaller than those of control fertile males in azoospermic group, respectively, while no significant difference was noted in those of Polymorphic, and inversion group when compared with control.1.3The levels of inhibin B of seminal plasmal of Polymorphic and Klinefelter syndrome azoospermic patients were significantly lower than those of control fertile males, respectively. Moreover, the level of Fructose in Translocation group was found significantly higher than those of control fertile males. No significant difference was noted in different biochemical of seminal plasma of other azoospermic abnormal karyotype patients compared with those of azoospermic of Klinefelter syndrome carners.1.4FSH, LH and E2hormones levels of Polymorphic and Klinefelter syndrome azoospermic patients were significantly higher than those of control, respectively, while no significant difference of PRL, FSH, LH and E2levels was found between Translocation/Inversion and control group. Interesting, T hormone and T/LH ratio of all chromosomal abnormalities among azoospermic patients were significantly lower than those of control.1.5The incidence of Y chromosome microdeletion was found at a rate of7.94%(55/693) among azoospermic patients. AZFc is most deleted region, and the most frequent deletion pattern is deletion of sY152, sY255and sY254.Additionally, most of deletions were de novo mutations, but some of cases were inherited from their fathers.1.6The testicular volumes of AZFc azoospermic deletions patient were significantly smaller, and the levels of Fructose and a-glucosidase of AZFc deletions were significantly higher than those of control fertile males. Moreover, the levels of Inhibin B of patients with AZFc, AZFb+c, and AZFb deletions were significantly lower than those of control, respectively.1.7FSH levels of patients with AZFc, AZFb+c, and AZFb deletions, LH level of patients with AZFc deletions and PRL level of patients with AZFc and AZFb deletions were significantly higher, while T and T/LH ratio of patients with AZFc, AZFb+c, and AZFb deletions were significantly lower than those of control.2. Associated results of patients with poor semen quality2.1Abnormal karyotype among patients with poor semen quality in Northeast China was detected at a rate of12.01%(83/691). Of these, Klinefelter syndrome is the most frequent (4.49%of the patients with poor semen quality), followed by Chromosome polymorphism (3.76%), and Translocation (3.18%).2.2The volumes testis of Klinefelter syndrome and Translocation carriers were significantly smaller than those of control fertile males in poor semen quality groups, respectively, while no significant difference was noted in those of Polymorphic group compared with control.2.3FSH and LH hormones levels of Klinefelter syndrome patients with poor semen quality were significantly higher than those of control, while in Polymorphism patients, only FSH level were significantly higher than those of control. T hormone and T/LH ratio of all chromosomal abnormalities among poor semen quality patients were significantly lower than those of control.2.4Non-moisaic Klinefelter syndrome men with poor semen quality were all oligozoospermic and cryptozoospermic. Compared with control, all these patients showed smaller testis volume, higher FSH and LH and lower T and T/LH.2.5The incidence of Y chromosome microdeletion was found at a rate of5.50%(38/691) among patients with poor semen quality. AZFc is most deleted region, and the most frequent deletion pattern is also deletion of sY152, sY255and sY254. All the deletions which referred in pedigree study were de novo mutation.2.6Compared with control and without AZF deletion group, poor semen quality patients with AZF deletion showed smaller testis volume, higher FSH and LH and lower T and T/LH.3. Comparison between azoospermic and poor-semen quality patients3.1The incidence of abnormal karyotype among infertile azoospermic patients compared with that of poor semen quality patients is significantly higher, while the incidence of Y chromosome microdeletion was found to be not statistically different from that among poor semen quality patients.3.2Compared with corresponding patients with poor semen quality, the age and testis volumes of all azoospermic group, including group of azoospermia with genetic factor and group of azoospermia without genetic factor, were significantly smaller.3.3Compared with corresponding patients with poor semen quality, the levels of a-glycosidase of azoospermia, and azoospermia with genetic factor patients were significantly lower. However, there was no statistically different was noted in levels of inhibin B and Fructose.3.4Compared with corresponding patients with poor semen quality, the levels of FSH, LH were significantly higher in all azoospermic patients of different etiology. No significant difference was noted in PRL, T and E2levels.3.5Compared with group of azoospermia without genetic factor, the age and testis volumes were smaller, the level of Fructose, a-glucosidase, FSH and LH were higher, and the level of T and T/LH were lower in azoospermic patients with genetic factor. No significant difference was noted on inhibin B, PRL and E2levels.3.6Compared with group of poor-semen without genetic factor, no significant difference was noted about age, testis volumes, biochemicals of seminal plasma, inhibin B and hormone levels in group of poor-semen with genetic factor.Conclusions1. In Northeast of China, important genetic factor of azoospermic patients were Klinefelter syndrome and Y chromosome microdeletion. Klinefelter syndrome patients suffered from smaller testis, higher FSH and LH, and lower T and T/LH than other abnormal karyotype carriers. In addition, the azoospermic patients with Y chromosome microdeletions had similar sex hormones abnormality as KS patients.2. In Northeast of China, genetic factors of patients with poor semen quality were diverse. Y chromosome microdeletion played the most important role for causing poor semen quality. Almost all this kind of patients had gene microdeletion in the AZFc region, and these deletions were derived from de novo mutation.3. Compared with Y chromosome microdeletion, abnormal karyotype tends to cause azoospermia. Genetic disorder may leads to azoospermia by elevated FSH, LH and low T. Moreover, the extent of dyszoospermia was closely association with the value of FSH, LH and T.更多还原