单一畸形精子症患者精子印记基因DNA甲基化与DNA损伤的初步研究

发布时间：2018-05-05 05:02

  本文选题：单一畸形精子症 + 流产率　； 参考：《第四军医大学》2014年硕士论文

【摘要】：不育症，是人类现今面临的重大健康问题之一，影响了10-15%的育龄夫妇，其中男性因素占了大约50%。在临床上，男性因素不育通常是根据异常的精子参数（如少精、弱精、畸形精子等）来诊断的。精子作为配子之一，关乎着人类的繁衍生息。据报道，70%的男性不育症患者由精液或者精子异常导致。诸多研究显示，与精液的其他参数（浓度、活力、存活率等）相比，精子形态对胚胎受精潜能和临床妊娠结局具有更好的预测价值，这一结论在体内和体外试验中均得到了验证。正是由于精子形态的重要预测价值和目前男性精液质量日益下降的现状，近年来，WHO关于畸形精子症患者的鉴定阈值一直在更新，正常精子形态百分比由1978年的50%，到1992年的30%，到1999年的15%，尤其是2010年颁布的第五版精液分析标准，将这一阈值定为4%，从而引发了国内外生殖学家的广泛关注和争议。但目前的研究，绝大部分是关于精子形态与辅助生殖技术后的周期结局的相关性分析，而鲜有进一步的机制分析。印记基因是指亲本仅一方来源的同源基因表达，而另一方来源的不表达。这种差异表达由DNA序列上印记基因控制区域的共价修饰来调控，主要参与了胚胎的正常生长、发育及小儿的行为发展。而精子DNA损伤情况与胚胎发育、临床妊娠和流产等均密切相关。因此，本研究以单一畸形精子症为研究对象，从与临床妊娠结局相关性、DNA甲基化修饰和DNA完整性三个方面进行研究。 目的： 回顾性分析单一畸形精子症与临床妊娠结局是否存在相关性；检测精子印记基因（父源H19、母源SNRPN以及Line-1）DNA甲基化改变和精子细胞凋亡。 方法： 本研究以唐都医院生殖医学中心2012年4月-2013年5月期间，因单一畸精症原因接受辅助受孕治疗的患者夫妇为研究对象，首先回顾性分析了精子形态与临床妊娠结局的相关性；进一步挑选了40例单一畸形精子症患者精液样本，通过Isolate法离心洗涤纯化精子后提取DNA，采取亚硫酸氢盐测序PCR的方法分析父源印记基因H19、母源印记基因SNRPN以及Line-1的DNA甲基化状态，并且通过流式细胞术检测了其精子凋亡（DNA损伤）情况。 结果： 1、行IVF助孕后，对照组的胚胎正常受精率、卵裂率、优胚率、临床妊娠率、流产率分别为83.09%、94.52%、49.58%、62%、5.14%，单一畸形精子症患者的胚胎正常受精率、卵裂率、优胚率、临床妊娠率、流产率分别为83.64%、95.26%、41.88%、64.41%、6.95%，各项指标均无显著差异（P＞0.05）；行ICSI助孕后，对照组胚胎正常受精率、卵裂率、优胚率、临床妊娠率分别为86.54%、96.80%、43.08%、56%，单一畸形精子症患者的对应指标分别为87.78%、98.13%、41.67%、56.25%，均无统计学差异（P＞0.05），但单一畸形精子症患者的流产率却显著升高，差异具有统计学意义（6.19%Vs19.5%，P＜0.05）。 2、DNA甲基化的结果：①父源印记基因H19：畸形精子组的克隆丢失率为36.05±2.86%，CpG岛丢失率为3.85±0.41%，CTCF6结合位点区域CpG岛丢失率为2.43±0.19%，而对照组则分别为13.41±3.21%，，0.75±0.17%，0.25±0.06%，差异均具有统计学意义（P＜0.01）；②母源印记基因SNRPN：DNA甲基化改变在畸形精子组与对照组相比，无统计学差异（P＞0.05）；③基因Line-1：在畸形精子组和对照组均呈现高度甲基化的状态（对照组80.54±2.9%Vs畸形精子组79.67±3.2%），且两组之间甲基化程度无统计学差异（P＞0.05）。 3、畸形精子组的细胞凋亡程度高于对照组（3.01±0.38Vs0.65±0.19，P＞0.05），且随着畸形率的升高，细胞凋亡有增高的趋势。 结论： 1、精子畸形率与临床辅助助孕方式选择及妊娠结局密切相关。 2、精子畸形率与H19印记控制区域的低甲基化程度呈显著正相关。 3、精子畸形率越高，精子DNA凋亡程度越高，但需大样本进一步确定。
[Abstract]:Infertility is one of the major health problems facing mankind today, affecting the 10-15% couples of childbearing age, in which male factors account for about 50%. in the clinic. Male infertility is usually diagnosed according to abnormal sperm parameters such as oligospermia, weak sperm, abnormal sperm and so on. Sperm is one of the gametes and is related to human reproduction. It is reported that 70% of male infertility are caused by semen or sperm abnormalities. Many studies have shown that sperm morphology has a better predictive value for embryo fertilization potential and clinical pregnancy outcomes than other seminal parameters (concentration, vitality, survival, etc.). This conclusion has been verified in both in vivo and in vitro tests. The important predictive value of sperm morphology and the current decline in the quality of male semen have been updated in WHO in recent years. The percentage of normal sperm morphology has been updated from 50% in 1978 to 30% in 1992, to 15% in 1999, especially in 2010, the fifth version of semen analysis, the threshold of this threshold. It is defined as 4%, which leads to widespread concern and controversy about reproductive scientists at home and abroad. But most of the current studies are about the correlation analysis of the periodic outcome of sperm morphology and assisted reproductive technology, but few mechanism analyses. This differential expression is regulated by covalent modification of the imprinted gene control region on the DNA sequence. It is mainly involved in the normal growth of the embryo and the development of the child's behavior. The DNA damage of the sperm is closely related to the development of the embryo and the clinical pregnancy and abortion. Therefore, this study is based on the single malformed spermatozoa. The correlation between clinical pregnancy outcome, DNA methylation and DNA integrity were studied in three aspects.
Objective:
A retrospective analysis of the correlation between single malformed spermatozoa and clinical pregnancy outcome, and detection of DNA methylation changes and sperm cell apoptosis in sperm imprinting genes (parent source H19, parent SNRPN and Line-1).
Method锛

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