精子形态与体外受精结局的相关性研究

发布时间：2018-06-24 16:00

本文选题：精子形态 + 体外受精　；参考：《吉林大学》2014年硕士论文

【摘要】：研究目的： 1.研究精子形态参数(正常形态精子百分率、畸形精子指数、顶体指数、顶体完整率)在体外受精方式选择中的作用，为合理选择体外受精方式，提高体外受精率，避免体外受精失败提供一定帮助。 2.建立精子形态各项参数预测IVF妊娠结局的截点值，以各截点值分组，研究精子形态参数对IVF结局的影响。 3.研究特殊异常精子形态患者生育异常的机制，为此类患者的临床诊治提供依据。研究对象： 1.2011年5月-2013年10月来吉林大学第一医院生殖中心首次行体外受精助孕治疗的265对不孕夫妇，均为女方输卵管因素。 2.选取特殊异常精子形态3例：95%无头精子症1例(病例1)、98%无头精子症1例(病例2)、100%圆头精子症1例(病例3)；对照组：20%无头精子症1例、15%无头精子症1例。研究方法： 1.采用计算机辅助精液分析系统进行精液分析。常规方法制备精子形态涂片，采用Diff-Quik方法进行染色。参照WHO第五版精子形态分析标准进行形态学判读。计算正常形态精子百分率和畸形精子指数。在同一张形态涂片上参照Menkveld方法进行精子顶体形态分析，计算顶体指数和顶体完整率。 2.采用Epon812环氧树脂包埋精子细胞团，光镜定位后超薄切片，透射电镜观察精子超微结构；常规石蜡包埋睾丸曲细精管、切片、HE染色，对睾丸生精功能状态进行病理检测；采用精子染色质扩散试验合并精子荧光原位杂交技术分析精子核DNA完整性和精子染色体非整倍体性。荧光显微镜下分析500个精子的DNA损伤情况及18/X/Y号染色体情况，计算精子核DNA损伤指数及精子非整倍体率。 3.采用SPSS17.0统计学软件进行分析。计量资料采用两独立样本t检验；计数资料采用四格表卡方检验。应用MedCalc软件进行受试者工作特征曲线(Thereceiver operating characteristics,ROC)分析。P0.05差异有统计学意义。研究结果：1.精子形态参数在体外受精方式选择中的作用 (1)根据体外受精方式不同，分为IVF组197例、补救ICSI组68例。比较2组间精子形态参数，结果显示IVF组正常形态精子百分率、顶体指数、顶体完整率均高于补救ICSI组，比较有统计学差异(P0.05)； IVF组畸形精子指数与补救ICSI组比较无统计学差异(P0.05)。 (2)采用ROC曲线分析265例样本精子形态各项参数对受精方式的评价能力和参考值，结果显示顶体完整率ROC曲线下面积最大，为0.717。对精子形态各项参数进行特异性和敏感性分析，其中顶体指数特异性最好。正常形态精子百分率、顶体指数、顶体完整率在体外受精方式选择中的截点值分别为1.44%、15.35%、28.57%。 (3)根据受精方式不同，采用二元逻辑回归分析精子形态参数与受精方式相关性。结果显示，顶体完整率与受精方式具有独立相关关系（OR=1.038，P0.05）。2.精子形态参数对IVF结局的影响 (1)采用ROC曲线分析197例样本精子形态各项参数对IVF妊娠结局的评价能力和参考值，结果显示顶体完整率ROC曲线下面积最大，为0.553。正常形态精子百分率、畸形精子指数、顶体指数、顶体完整率在IVF妊娠结局评价中的截点值分别为3.92%、1.37、26.34%和30.92%。 (2)根据精子形态各项参数预测IVF妊娠结局的截点值，将精液标本分别按正常形态精子百分率、畸形精子指数、顶体指数和顶体完整率进行分组，研究精子形态参数对IVF结局的影响，结果显示正常形态精子百分率、顶体指数和顶体完整率的不同组别间临床妊娠率均有统计学差异(P0.05)。顶体指数组别间2PN受精率有统计学差异(P0.05)。3.特殊异常精子形态患者生育异常机制探讨病例1精液常规：液化时间正常，粘稠度适中，量1.5ml，pH7.9，浓度83.55×106/ml，（a+b）级4.19%。Diff-Quik染色示95%为无头精子。衣原体、支原体、淋球菌、TORCH、精浆生化、生殖激素、外周血染色体、AZF检查均正常。精子染色体非整倍体率为0.6%。精子核DNA损伤指数为84.4%。女方于取卵当日获卵10枚。MⅡ期卵母细胞4枚，行ICSI后获得4枚2PN受精卵，均卵裂。移植2枚7Ⅱ级优质胚胎，但移植后未孕。病例2精液常规：液化时间正常，粘稠度适中，量3.1ml，pH7.5，浓度26.14×106/ml，（a+b）级13%，存活率60%。Diff-Quik染色示98%为无头精子。解脲支原体培养阳性。泌乳素(Prolactin,PRL)水平偏高。精浆抗精子抗体、沙眼衣原体、外周血染色体、AZF检查均正常。睾丸穿刺涂片：精原细胞占20%，初级精母细胞占20%，次级精母细胞占2%，精子细胞占25%，，支持细胞占30%，精子占3%，每高倍视野可见1-2个精子。精子染色体非整倍体率为0.8%。精子核DNA损伤指数为95%。超微结构：标本头部、颈部及中段、尾部均可见到程度不同的结构异常：全片大多数精子仅见尾部；顶体发育不良，缺失，顶体膜结构不完整；核内有空泡；线粒体大小不均，排列紊乱；部分精子尾部包绕多个轴丝，且轴丝结构不完整。睾丸病理：大部分曲细精管硬化，细胞成分消失，只见界膜。界膜增厚明显，无精子生成。个别曲细精管相对正常。睾丸间质纤维化，可见炎细胞渗出。夫妻双方均不同意行辅助生殖助孕。病例3精液常规：液化时间60min，稍稠，量1.2ml，pH7.8，浓度35.99×106/ml，（a+b）级0.00%，存活率38%。Diff-Quik染色示100%为圆头精子。生殖激素、外周血染色体、无精子因子(Azoospermia factor,AZF)检查均正常。精子染色体非整倍体率为0.2%。女方于取卵当日获卵21枚，MⅡ期卵母细胞20枚。行ICSI后获得2枚2PN受精卵，均卵裂，移植1枚5Ⅱ级的非优质胚胎。移植后14d血HCG418U/L，移植后34d见宫内单活胎，其内可见胚芽及心管搏动。2013年6月诞生1名健康男婴。对照组20%无头精子症患者的精子染色体非整倍体率为0.4%，精子核DNA损伤指数为34%。15%无头精子症患者的精子染色体非整倍体率为0.2%，精子核DNA损伤指数为27%。结论： 1.正常形态精子百分率、顶体指数和顶体完整率均可作为体外受精方式选择的重要依据，且顶体形态参数特异性优于正常形态精子百分率。 2.正常形态精子百分率、顶体指数、顶体完整率均可作为预测IVF妊娠结局的有效参考指标。 3.精子超微结构异常和DNA损伤程度高可能是精子形态异常患者生育异常的原因，即使此类患者行ICSI后能够成功助孕，其后代远期安全性仍有待长期随访。
[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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.8

【参考文献】