精子鞭毛多发形态异常的形态学研究和致病基因鉴定

发布时间：2018-03-03 18:16

本文选题：精子鞭毛多发形态异常　切入点：弱精子症　出处：《苏州大学》2016年博士论文　论文类型：学位论文

【摘要】：【背景】精子鞭毛多发形态异常,曾称作纤维鞘发育不良,因精子鞭毛的严重畸形和弱精子症,导致男性不育。卵胞浆内单精子注射治疗是患者生育的有效方式,但其携带的遗传缺陷可能继续传递。中国精子鞭毛多发形态异常患者的致病基因尚不明确,基因诊断和遗传咨询缺乏理论依据。【目的】观察精子鞭毛多发形态异常(multiple morphological abnormalities of the sperm flagella,MMAF)患者精子光镜和电镜下的形态学特点,分析鞭毛畸形与精子活动率的关系。观察MMAF患者行卵胞浆内单精子注射治疗的临床结局。鉴定中国MMAF患者的致病基因。【方法】第一部分:对表现为弱精子症或完全性不活动精子的患者,通过光镜下形态观察初步诊断MMAF,并对鞭毛形态细化分类。通过扫描电镜和透射电镜进一步观察病变精子超微结构,明确纤维鞘、线粒体鞘、外周致密纤维及轴丝畸形,计算中心微管缺失率。分析射精中有无活动精子与鞭毛形态之间的关系。第二部分:对7例中国MMAF患者采取卵胞浆内单精子注射辅助生育。卵胞浆内单精子注射首选精液中活动精子。如射精中不能找到活动精子则采取低渗肿胀试验选取活精子;或采用睾丸精子;或卵子冻存后待下次从射精中寻找活动精子。观察受精率、胚胎质量并随访妊娠结局。第三部分:先后采取了Sanger测序和全外显子组测序的方法筛选MMAF患者的可能致病基因。首先,通过Sanger法对3例患者AKAP3、AKAP4、MNS1和SPEF2基因外显子测序,对6例患者DNAH1基因可能的热点突变位点测序。因未明确致病突变,进而通过Hi Seq X-Ten(Illumina)平台,对19例MMAF患者采取全外显子组测序,并进行相关生物信息学分析和验证。【结果】第一部分:通过光镜下形态分析诊断MMAF患者28例,其中15例(53.6%)患者未检测到活动精子。13例(46.4%)患者可检测到活动精子,其中12例患者精子活动率10%。精子存活率9.0-80.0%。光镜和扫描电镜下可见MMAF精子鞭毛缺失、短、卷曲、弯折和不规则宽度等多种畸形及其组合,MMAF缺陷精子占94.0±5.9%。透射电镜表现为精子鞭毛纤维鞘、线粒体鞘等多种结构组装异常,中心微管缺失率41.4-84.6%。动力蛋白臂缺失或存在,内、外侧动力蛋白臂均缺失的2例患者精液中无活动精子。射精中无活动精子和存在活动精子的两组患者间,各种鞭毛畸形构成比例的差异无统计学意义。第二部分:卵胞浆内单精子注射治疗的7例患者,均表现为严重弱精子症,精子活动率0-8.5%。辅助生殖采用的精子,3例为射精中活动精子,1例为通过低渗肿胀试验挑选的活精子,2例为睾丸精子,1例因无活动精子冻卵待用。本组MMAF患者ICSI受精率为50.0%(44/88),妊娠率26.7%(4/15),其中2例自然流产,2例分别分娩1男婴和1女婴。第三部分:通过Sanger测序发现AKAP3、MNS1和SPEF2基因的多个单核苷酸多态性位点,未发现致病突变。DNAH1基因的热点位点未发现致病突变。通过全外显子组测序发现,11例患者携带DNAH1基因突变纯合或复合杂合突变,占57.9%(11/19);在1例患者中发现CCDC39纯合突变。经生物信息学分析和Sanger测序验证确认DNAH1和CCDC39为MMAF的致病基因。【结论】:第一部分:MMAF是严重弱精子症和完全性不活动精子的原因之一,光镜下表现为鞭毛缺失、短、卷曲、弯折和不规则宽度等多种畸形。透射电镜可见以中心微管缺失为主要特征的整个鞭毛组装异常。内、外侧动力蛋白臂均缺失可导致完全性不活动精子。第二部分:MMAF患者精子鞭毛严重畸形,卵胞浆内单精子注射是一种有效的辅助生育手段。本组MMAF患者ICSI受精率和妊娠率低于其他因素的ICSI患者,最终结论有待大样本观察。第三部分:全外显子组测序是鉴定MMAF致病基因的有效方法;中国MMAF患者的致病基因包括DNAH1和CCDC39等,本组病例中DNAH1为主要致病基因。
[Abstract]:[background] sperm flagella multiple morphological abnormalities, called fibrous sheath dysplasia, due to severe deformity of sperm flagella and asthenospermia, leading to male infertility. Intracytoplasmic sperm injection is effective in treating patients of family, but they carry genetic defects may continue to pass. The pathogenic gene Chinese multiple sperm flagella send abnormal patients is not clear, gene diagnosis and genetic counseling for the lack of theoretical basis. [Objective] to observe the abnormal morphology of multiple sperm flagella (multiple morphological abnormalities of the sperm flagella, MMAF) sperm morphological characteristics of light microscope and electron microscope were the analysis of flagellar deformity and sperm motility. The relationship between the clinical outcome of treatment MMAF underwent intracytoplasmic sperm injection. The pathogenic gene identification Chinese MMAF patients. [method] the first part: the performance of asthenospermia or completely inactive The sperm of patients, through morphological observation under light microscope and the initial diagnosis of MMAF, detailed classification. Through the form of flagellar scanning electron microscopy and transmission electron microscopy to observe the ultrastructure of sperm lesions, clear fibrous sheath, mitochondrial sheath, outer dense fibers and the axoneme deformed, micro tube Computing Center loss rate. The relationship between non motile sperm and the flagellum morphology was analyzed. The second part: take the ejaculation of intracytoplasmic sperm injection in assisted reproduction in 7 cases of MMAF patients. Chinese activities of intracytoplasmic sperm injection of sperm. If the preferred ejaculation cannot find sperm take live sperm hypotonic swelling test selection; or by testicular sperm or eggs; after cryopreservation of sperm from the ejaculate until the next search activity. To observe the fertilization rate, embryo quality and pregnancy outcomes were followed up. The third part has adopted the method of Sanger sequencing and whole exome sequencing screening MMAF May the pathogenic gene of patients. First of all, through the Sanger method in 3 cases of patients with AKAP3, AKAP4, MNS1 and SPEF2 gene exon sequencing, of 6 cases of patients with DNAH1 gene mutation sequencing. Because of not clear mutation, and then through the Hi Seq X-Ten (Illumina) platform, 19 cases of MMAF patients to take full exome sequencing and bioinformatics analysis and verification. [results] the first part: analysis of 28 cases of MMAF patients diagnosed by morphology under light microscope, 15 cases (53.6%) were not detected in sperm.13 cases (46.4%) were detected in 12 cases of sperm activity, sperm activity the rate of 10%. sperm survival rate of 9.0-80.0%. under visible light microscope and scanning electron microscope MMAF sperm flagella is missing, short, curly, bent and irregular width and other anomalies and their combination, MMAF defects in sperm accounted for 94 + 5.9%. transmission electron microscope showed the sperm flagellar fibrous sheath, mitochondria The sheath and other abnormal assembly structure, the loss rate of 41.4-84.6%. central microtubule dynein arms, or lack of existence, no activity in 2 patients with sperm outer dynein arms are lack of sperm. Two groups of patients with no activity of sperm and sperm exist in the ejaculate, various malformations constitute no significant proportion of flagella significance. The second part 7 cases of intracytoplasmic sperm injection in the treatment of patients showed severe asthenospermia, rate of sperm 0-8.5%. assisted reproductive by sperm, 3 cases in 1 cases of ejaculation sperm, to live sperm through the hypoosmotic swelling test selected, 2 cases of testicular sperm, 1 cases with no sperm frozen egg aside. The patients with MMAF ICSI fertilization rate was 50% (44/88), the pregnancy rate was 26.7% (4/15), including 2 cases of spontaneous abortion, 2 cases of 1 boys and 1 girls were delivered. The third part: the AKAP3 through Sanger sequencing, MNS1 and SPEF2 Because of the multiple single nucleotide polymorphism, no mutations were found in the hot sites of.DNAH1 gene mutation by whole exome sequencing showed that 11 patients with DNAH1 mutations are homozygous or compound heterozygous mutations, accounting for 57.9% (11/19); in 1 patients the CCDC39 homozygous mutation the bioinformatics analysis and Sanger sequencing confirmed that DNAH1 and CCDC39 as the causative gene of MMAF. [Conclusion]: the first part: MMAF is one of the reasons for serious weak sperm and completely inactive sperm, light microscope showed flagellar deletion, short, curly, bent and irregular width and other malformation transmission electron microscopy showed that microtubules in the center. The main features of the whole lack of flagella anomalies. Within the outer dynein arms are missing can lead to complete non motile sperm. The second part: MMAF patients with severe deformity of sperm flagella, intracytoplasmic sperm injection Shooting is an effective means of assisted reproduction. This group of patients with MMAF ICSI fertilization rate and pregnancy rate were lower than other factors in patients with ICSI, the final conclusion needs large sample observation. The third part: whole exome sequencing is an effective method for identification of pathogenic gene MMAF; pathogenic gene Chinese MMAF patients including DNAH1 and CCDC39. DNAH1 in this group of cases as the main pathogenic gene.

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R698.2

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