本文選題：精子鞭毛多發(fā)形態(tài)異常　切入點：弱精子癥　出處：《蘇州大學》2016年博士論文　論文類型：學位論文

【摘要】：【背景】精子鞭毛多發(fā)形態(tài)異常,曾稱作纖維鞘發(fā)育不良,因精子鞭毛的嚴重畸形和弱精子癥,導致男性不育。卵胞漿內(nèi)單精子注射治療是患者生育的有效方式,但其攜帶的遺傳缺陷可能繼續(xù)傳遞。中國精子鞭毛多發(fā)形態(tài)異常患者的致病基因尚不明確,基因診斷和遺傳咨詢?nèi)狈碚撘罁?jù)�！灸康摹坑^察精子鞭毛多發(fā)形態(tài)異常(multiple morphological abnormalities of the sperm flagella,MMAF)患者精子光鏡和電鏡下的形態(tài)學特點,分析鞭毛畸形與精子活動率的關系。觀察MMAF患者行卵胞漿內(nèi)單精子注射治療的臨床結局。鑒定中國MMAF患者的致病基因。【方法】第一部分:對表現(xiàn)為弱精子癥或完全性不活動精子的患者,通過光鏡下形態(tài)觀察初步診斷MMAF,并對鞭毛形態(tài)細化分類。通過掃描電鏡和透射電鏡進一步觀察病變精子超微結構,明確纖維鞘、線粒體鞘、外周致密纖維及軸絲畸形,計算中心微管缺失率。分析射精中有無活動精子與鞭毛形態(tài)之間的關系。第二部分:對7例中國MMAF患者采取卵胞漿內(nèi)單精子注射輔助生育。卵胞漿內(nèi)單精子注射首選精液中活動精子。如射精中不能找到活動精子則采取低滲腫脹試驗選取活精子;或采用睪丸精子;或卵子凍存后待下次從射精中尋找活動精子。觀察受精率、胚胎質量并隨訪妊娠結局。第三部分:先后采取了Sanger測序和全外顯子組測序的方法篩選MMAF患者的可能致病基因。首先,通過Sanger法對3例患者AKAP3、AKAP4、MNS1和SPEF2基因外顯子測序,對6例患者DNAH1基因可能的熱點突變位點測序。因未明確致病突變,進而通過Hi Seq X-Ten(Illumina)平臺,對19例MMAF患者采取全外顯子組測序,并進行相關生物信息學分析和驗證�！窘Y果】第一部分:通過光鏡下形態(tài)分析診斷MMAF患者28例,其中15例(53.6%)患者未檢測到活動精子。13例(46.4%)患者可檢測到活動精子,其中12例患者精子活動率10%。精子存活率9.0-80.0%。光鏡和掃描電鏡下可見MMAF精子鞭毛缺失、短、卷曲、彎折和不規(guī)則寬度等多種畸形及其組合,MMAF缺陷精子占94.0±5.9%。透射電鏡表現(xiàn)為精子鞭毛纖維鞘、線粒體鞘等多種結構組裝異常,中心微管缺失率41.4-84.6%。動力蛋白臂缺失或存在,內(nèi)、外側動力蛋白臂均缺失的2例患者精液中無活動精子。射精中無活動精子和存在活動精子的兩組患者間,各種鞭毛畸形構成比例的差異無統(tǒng)計學意義。第二部分:卵胞漿內(nèi)單精子注射治療的7例患者,均表現(xiàn)為嚴重弱精子癥,精子活動率0-8.5%。輔助生殖采用的精子,3例為射精中活動精子,1例為通過低滲腫脹試驗挑選的活精子,2例為睪丸精子,1例因無活動精子凍卵待用。本組MMAF患者ICSI受精率為50.0%(44/88),妊娠率26.7%(4/15),其中2例自然流產(chǎn),2例分別分娩1男嬰和1女嬰。第三部分:通過Sanger測序發(fā)現(xiàn)AKAP3、MNS1和SPEF2基因的多個單核苷酸多態(tài)性位點,未發(fā)現(xiàn)致病突變。DNAH1基因的熱點位點未發(fā)現(xiàn)致病突變。通過全外顯子組測序發(fā)現(xiàn),11例患者攜帶DNAH1基因突變純合或復合雜合突變,占57.9%(11/19);在1例患者中發(fā)現(xiàn)CCDC39純合突變。經(jīng)生物信息學分析和Sanger測序驗證確認DNAH1和CCDC39為MMAF的致病基因�！窘Y論】:第一部分:MMAF是嚴重弱精子癥和完全性不活動精子的原因之一,光鏡下表現(xiàn)為鞭毛缺失、短、卷曲、彎折和不規(guī)則寬度等多種畸形。透射電鏡可見以中心微管缺失為主要特征的整個鞭毛組裝異常。內(nèi)、外側動力蛋白臂均缺失可導致完全性不活動精子。第二部分:MMAF患者精子鞭毛嚴重畸形,卵胞漿內(nèi)單精子注射是一種有效的輔助生育手段。本組MMAF患者ICSI受精率和妊娠率低于其他因素的ICSI患者,最終結論有待大樣本觀察。第三部分:全外顯子組測序是鑒定MMAF致病基因的有效方法;中國MMAF患者的致病基因包括DNAH1和CCDC39等,本組病例中DNAH1為主要致病基因。
[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.

【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R698.2

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