本文選題：精子形態(tài) + 體外受精　； 參考：《吉林大學》2014年碩士論文

【摘要】：研究目的： 1.研究精子形態(tài)參數(shù)(正常形態(tài)精子百分率、畸形精子指數(shù)、頂體指數(shù)、頂體完整率)在體外受精方式選擇中的作用，為合理選擇體外受精方式，提高體外受精率，避免體外受精失敗提供一定幫助。 2.建立精子形態(tài)各項參數(shù)預測IVF妊娠結(jié)局的截點值，以各截點值分組，研究精子形態(tài)參數(shù)對IVF結(jié)局的影響。 3.研究特殊異常精子形態(tài)患者生育異常的機制，為此類患者的臨床診治提供依據(jù)。 研究對象： 1.2011年5月-2013年10月來吉林大學第一醫(yī)院生殖中心首次行體外受精助孕治療的265對不孕夫婦，均為女方輸卵管因素。 2.選取特殊異常精子形態(tài)3例：95%無頭精子癥1例(病例1)、98%無頭精子癥1例(病例2)、100%圓頭精子癥1例(病例3)；對照組：20%無頭精子癥1例、15%無頭精子癥1例。 研究方法： 1.采用計算機輔助精液分析系統(tǒng)進行精液分析。常規(guī)方法制備精子形態(tài)涂片，采用Diff-Quik方法進行染色。參照WHO第五版精子形態(tài)分析標準進行形態(tài)學判讀。計算正常形態(tài)精子百分率和畸形精子指數(shù)。在同一張形態(tài)涂片上參照Menkveld方法進行精子頂體形態(tài)分析，計算頂體指數(shù)和頂體完整率。 2.采用Epon812環(huán)氧樹脂包埋精子細胞團，光鏡定位后超薄切片，透射電鏡觀察精子超微結(jié)構(gòu)；常規(guī)石蠟包埋睪丸曲細精管、切片、HE染色，對睪丸生精功能狀態(tài)進行病理檢測；采用精子染色質(zhì)擴散試驗合并精子熒光原位雜交技術(shù)分析精子核DNA完整性和精子染色體非整倍體性。熒光顯微鏡下分析500個精子的DNA損傷情況及18/X/Y號染色體情況，計算精子核DNA損傷指數(shù)及精子非整倍體率。 3.采用SPSS17.0統(tǒng)計學軟件進行分析。計量資料采用兩獨立樣本t檢驗；計數(shù)資料采用四格表卡方檢驗。應(yīng)用MedCalc軟件進行受試者工作特征曲線(Thereceiver operating characteristics,ROC)分析。P0.05差異有統(tǒng)計學意義。研究結(jié)果：1.精子形態(tài)參數(shù)在體外受精方式選擇中的作用 (1)根據(jù)體外受精方式不同，分為IVF組197例、補救ICSI組68例。比較2組間精子形態(tài)參數(shù)，結(jié)果顯示IVF組正常形態(tài)精子百分率、頂體指數(shù)、頂體完整率均高于補救ICSI組，比較有統(tǒng)計學差異(P0.05)； IVF組畸形精子指數(shù)與補救ICSI組比較無統(tǒng)計學差異(P0.05)。 (2)采用ROC曲線分析265例樣本精子形態(tài)各項參數(shù)對受精方式的評價能力和參考值，結(jié)果顯示頂體完整率ROC曲線下面積最大，為0.717。對精子形態(tài)各項參數(shù)進行特異性和敏感性分析，其中頂體指數(shù)特異性最好。正常形態(tài)精子百分率、頂體指數(shù)、頂體完整率在體外受精方式選擇中的截點值分別為1.44%、15.35%、28.57%。 (3)根據(jù)受精方式不同，采用二元邏輯回歸分析精子形態(tài)參數(shù)與受精方式相關(guān)性。結(jié)果顯示，頂體完整率與受精方式具有獨立相關(guān)關(guān)系（OR=1.038，P0.05）。2.精子形態(tài)參數(shù)對IVF結(jié)局的影響 (1)采用ROC曲線分析197例樣本精子形態(tài)各項參數(shù)對IVF妊娠結(jié)局的評價能力和參考值，結(jié)果顯示頂體完整率ROC曲線下面積最大，為0.553。正常形態(tài)精子百分率、畸形精子指數(shù)、頂體指數(shù)、頂體完整率在IVF妊娠結(jié)局評價中的截點值分別為3.92%、1.37、26.34%和30.92%。 (2)根據(jù)精子形態(tài)各項參數(shù)預測IVF妊娠結(jié)局的截點值，將精液標本分別按正常形態(tài)精子百分率、畸形精子指數(shù)、頂體指數(shù)和頂體完整率進行分組，研究精子形態(tài)參數(shù)對IVF結(jié)局的影響，結(jié)果顯示正常形態(tài)精子百分率、頂體指數(shù)和頂體完整率的不同組別間臨床妊娠率均有統(tǒng)計學差異(P0.05)。頂體指數(shù)組別間2PN受精率有統(tǒng)計學差異(P0.05)。3.特殊異常精子形態(tài)患者生育異常機制探討 病例1精液常規(guī)：液化時間正常，粘稠度適中，量1.5ml，pH7.9，濃度83.55×106/ml，（a+b）級4.19%。Diff-Quik染色示95%為無頭精子。衣原體、支原體、淋球菌、TORCH、精漿生化、生殖激素、外周血染色體、AZF檢查均正常。精子染色體非整倍體率為0.6%。精子核DNA損傷指數(shù)為84.4%。 女方于取卵當日獲卵10枚。MⅡ期卵母細胞4枚，行ICSI后獲得4枚2PN受精卵，均卵裂。移植2枚7Ⅱ級優(yōu)質(zhì)胚胎，但移植后未孕。 病例2精液常規(guī)：液化時間正常，粘稠度適中，量3.1ml，pH7.5，濃度26.14×106/ml，（a+b）級13%，存活率60%。Diff-Quik染色示98%為無頭精子。解脲支原體培養(yǎng)陽性。泌乳素(Prolactin,PRL)水平偏高。精漿抗精子抗體、沙眼衣原體、外周血染色體、AZF檢查均正常。睪丸穿刺涂片：精原細胞占20%，初級精母細胞占20%，次級精母細胞占2%，精子細胞占25%，，支持細胞占30%，精子占3%，每高倍視野可見1-2個精子。精子染色體非整倍體率為0.8%。精子核DNA損傷指數(shù)為95%。 超微結(jié)構(gòu)：標本頭部、頸部及中段、尾部均可見到程度不同的結(jié)構(gòu)異常：全片大多數(shù)精子僅見尾部；頂體發(fā)育不良，缺失，頂體膜結(jié)構(gòu)不完整；核內(nèi)有空泡；線粒體大小不均，排列紊亂；部分精子尾部包繞多個軸絲，且軸絲結(jié)構(gòu)不完整。 睪丸病理：大部分曲細精管硬化，細胞成分消失，只見界膜。界膜增厚明顯，無精子生成。個別曲細精管相對正常。睪丸間質(zhì)纖維化，可見炎細胞滲出。 夫妻雙方均不同意行輔助生殖助孕。 病例3精液常規(guī)：液化時間60min，稍稠，量1.2ml，pH7.8，濃度35.99×106/ml，（a+b）級0.00%，存活率38%。Diff-Quik染色示100%為圓頭精子。生殖激素、外周血染色體、無精子因子(Azoospermia factor,AZF)檢查均正常。精子染色體非整倍體率為0.2%。 女方于取卵當日獲卵21枚，MⅡ期卵母細胞20枚。行ICSI后獲得2枚2PN受精卵，均卵裂，移植1枚5Ⅱ級的非優(yōu)質(zhì)胚胎。移植后14d血HCG418U/L，移植后34d見宮內(nèi)單活胎，其內(nèi)可見胚芽及心管搏動。2013年6月誕生1名健康男嬰。 對照組20%無頭精子癥患者的精子染色體非整倍體率為0.4%，精子核DNA損傷指數(shù)為34%。15%無頭精子癥患者的精子染色體非整倍體率為0.2%，精子核DNA損傷指數(shù)為27%。 結(jié)論： 1.正常形態(tài)精子百分率、頂體指數(shù)和頂體完整率均可作為體外受精方式選擇的重要依據(jù)，且頂體形態(tài)參數(shù)特異性優(yōu)于正常形態(tài)精子百分率。 2.正常形態(tài)精子百分率、頂體指數(shù)、頂體完整率均可作為預測IVF妊娠結(jié)局的有效參考指標。 3.精子超微結(jié)構(gòu)異常和DNA損傷程度高可能是精子形態(tài)異�；颊呱惓５脑颍词勾祟惢颊咝蠭CSI后能夠成功助孕，其后代遠期安全性仍有待長期隨訪。
[Abstract]:The purpose of the study is:
1. study the role of sperm morphological parameters (percentage of normal sperm, abnormal sperm index, acrosome index, acrosome integrity) in the selection of in vitro fertilization, which can help to select in vitro fertilization mode, improve the rate of in vitro fertilization and avoid the failure of in vitro fertilization.
2. establish cut-off values of various parameters of sperm morphology to predict IVF pregnancy outcome, and study the effects of sperm morphological parameters on IVF outcome by grouping the cut-off points.
3. to study the mechanism of abnormal fertility in patients with special abnormal sperm morphology, so as to provide evidence for clinical diagnosis and treatment.
Research object:
In May, -2013, October, the 265 infertile couples who received first in vitro fertilization and assisted reproductive treatment in No.1 Hospital of Jilin University reproductive center were all oviduct factors of the 1.2011 women.
2. the special abnormal sperm morphology was selected in 3 cases: 1 cases of head spermatozoa (1 cases), 98% azoospermia (2), 1 cases of round head spermatozoa (3), 95% cases of azoospermia in the control group, and 95% cases of spermatozoospermia.
Research methods:
1. the sperm morphology smear was prepared by the computer assisted semen analysis system. The sperm morphologic smear was prepared by the conventional method. The Diff-Quik method was used to stain the sperm. According to the morphologic criteria of the fifth version of the sperm, the percentage of normal sperm and the abnormal sperm index were calculated. The Menkveld method was used on the same form smear. The acrosome morphology was analyzed and acrosome index and acrosome integrity were calculated.
2. the sperm cell group was embedded with Epon812 epoxy resin, the ultrathin sections were sectioning after the light microscope, and the ultrastructure of sperm was observed by transmission electron microscope. The normal paraffin embedded testicular seminiferous tubules, slices, and HE staining were used to detect the function of spermatogenesis, and the sperm chromatin diffusion test combined with sperm fluorescence in situ hybridization was used to analyze sperm. The integrity of nuclear DNA and the aneuploidy of sperm chromosomes. Under the fluorescence microscope, the DNA damage and the 18/X/Y chromosome of 500 sperm were analyzed, and the DNA damage index and the aneuploidy rate of sperm were calculated.
3. SPSS17.0 statistical software was used for analysis. Two independent sample t test was used for measurement data; counting data was tested by four grid card square test. MedCalc software was used to carry out Thereceiver operating characteristics, ROC to analyze.P0.05 differences in statistical significance. Research results: 1. sperm morphologic parameters in The role of the selection of in vitro fertilization
(1) according to the different methods of in vitro fertilization, 197 cases were divided into IVF group, and 68 cases of ICSI group were remedial. The morphological parameters of sperm were compared between the 2 groups. The results showed that the percentage of normal morphology and sperm, acrosome index and acrosome integrity of group IVF were higher than that of the remedial group (P0.05), and there was no statistical difference between the IVF group and the remedial ICSI group. Difference (P0.05).
(2) the ROC curve was used to analyze the evaluation ability and reference value of the parameters of sperm morphology in 265 samples. The results showed that the acreage was the largest under the ROC curve of the acrosome integrity, which was the specificity and sensitivity analysis of the parameters of the sperm morphology, in which the acrosome index specificity was best. The percentage of normal sperm and the acrosome index were the best. The cut-off value of acrosome integrity rate in IVF selection was 1.44%, 15.35%, 28.57%.
(3) according to the different fertilization methods, the correlation between sperm morphological parameters and fertilization mode was analyzed by two element logic regression analysis. The results showed that the effect of the acrosome integrity and fertilization mode (OR=1.038, P0.05).2. sperm morphology parameters on the outcome of IVF
(1) the ROC curve was used to analyze the evaluation ability and reference value of the parameters of sperm morphology in 197 samples on the outcome of IVF pregnancy. The results showed that the acreage was the largest under the ROC curve of the acrosome integrity rate, the percentage of normal 0.553. sperm, the abnormal sperm index, acrosome index and the acrosome integrity rate in the evaluation of IVF pregnancy outcome were 3.92%, 1.3, respectively. 7,26.34% and 30.92%.
(2) according to the parameters of sperm morphology to predict the cut-off point of IVF pregnancy outcome, the sperm specimens were grouped according to the percentage of normal sperm, abnormal sperm index, acrosome index and acrosome integrity, and the effects of sperm morphological parameters on the IVF outcome were studied. The results showed that the percentage of normal sperm, the acrosome index and the acrosome integrity rate. The clinical pregnancy rates of different groups were statistically different (P0.05). The rate of 2PN fertilization between the acrosome index groups was statistically different (P0.05) the mechanism of abnormal fertility in patients with.3. special abnormal sperm morphology
Case 1 semen routine: normal liquefaction time, moderate viscosity, 1.5ml, pH7.9, concentration of 83.55 x 106/ml, and (a+b) 4.19%.Diff-Quik staining 95% for head free sperm. Chlamydia, mycoplasma, gonococcal, TORCH, seminal plasma biochemistry, reproductive hormones, peripheral blood chromosomes, AZF examination are normal. The sperm chromosome aneuploidy rate is 0.6%. sperm nucleus DNA damage The index is 84.4%.
On the day of ovum collection, the female obtained 10 eggs from.M phase II, 4 ICSI after obtaining 4 2PN fertilized eggs, all of which were cleavage. 2 7 grade II embryos were transplanted, but no pregnancy after transplantation.
Case 2 semen routine: normal liquefaction time, moderate viscosity, 3.1ml, pH7.5, 26.14 x 106/ml, (a+b) 13%, 60%.Diff-Quik staining 98% for head sperm. Mycoplasma urealyticum culture positive. Prolactin (Prolactin, PRL) level is high. Seminal plasma antigol antibody, Chlamydia trachomatis, peripheral blood chromosomes, AZF examination are normal. The pellet puncture smear: spermatogonial cells accounted for 20%, primary spermatocyte accounted for 20%, secondary spermatocyte accounted for 2%, spermatocyte accounted for 25%, supporting cells accounted for 30%, sperm accounted for 3%, and 1-2 sperm were seen in every high field of vision. The sperm chromosome aneuploidy rate of 0.8%. sperm nucleus DNA damage index was 95%.
Ultrastructure: in the head, neck and middle section of the specimen, the tail can be seen in the tail of different structural abnormalities: most of the sperm only see the tail; the apical body is dysplasia, missing, the acrosome membrane structure is incomplete; there are vacuoles in the nucleus; the size of mitochondria is uneven, and the tail of some spermatozoa is wrapped around a number of axles, and the shaft silk structure is incomplete.
Testicular pathology: most of the seminiferous tubules sclerosis, the cell components disappear, only the boundary membrane. The thickening of the boundary membrane is obvious, no spermatogenesis. The individual seminiferous tubules are relatively normal. The interstitial fibrosis of the testis and the exudation of the inflammatory cells.
Both husband and wife do not agree to assist reproductive pregnancy.
Case 3 semen routine: liquefaction time 60min, slightly thickened, 1.2ml, pH7.8, concentration of 35.99 x 106/ml, (a+b) 0%, 38%.Diff-Quik staining showed that 100% were round head sperm. Reproductive hormones, peripheral blood chromosomes, Azoospermia factor, AZF were all normal. The chromosome aneuploidy rate of sperm was 0.2%..
The women got 21 eggs on the day of taking eggs, 20 M II oocytes. After ICSI, 2 2PN fertilized eggs were obtained and 1 5 II grade non quality embryos were transplanted. After transplantation, 14d blood was HCG418U/L, and the single live fetus was found in the uterus after transplantation. The embryo and cardiac tube pulsation was found in 1 healthy male babies in June.
In the control group, the sperm chromosome aneuploidy rate of 20% azoospermia patients was 0.4%, the sperm nuclear DNA damage index was 0.2% of the sperm chromosome aneuploidy in 34%.15% anspermatozoa, and the DNA damage index of the sperm nucleus was 27%.
Conclusion:
1. the percentage of normal sperm, the acrosome index and the acrosome integrity can be used as an important basis for the selection of in vitro fertilization, and the speciation of the acrosome morphological parameters is better than the percentage of normal sperm.
2. normal morphology sperm percentage, acrosome index and acrosome integrity rate can be used as effective reference indicators for predicting the outcome of IVF pregnancy.
3. the abnormal ultrastructure of sperm and the high degree of DNA damage may be the cause of abnormal fertility in patients with abnormal sperm morphology. Even if such patients can succeed in helping pregnancy after ICSI, the long-term safety of their offspring is still to be followed up for a long time.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R714.8

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