吸烟对男性精液参数影响的临床研究与吸烟男性精子基因组甲基化变异分析

【作者】 江莉；

【导师】 李慕军；

【作者基本信息】 广西医科大学 ， 儿科学（专业学位）， 2013， 博士

【摘要】 第一部分吸烟对男性精液参数及精子DNA损伤影响的临床研究目的：探讨吸烟对男性精子密度、活率、活力,精子形态、精浆锌浓度和精子脱氧核糖核酸(DNA)损伤的影响,分析吸烟与临床精液参数的相关性。方法：将1036例广西地区男性分为吸烟与不吸烟组,对吸烟者按日吸烟支数和吸烟年限分组,分析了吸烟对男性精子密度、活率、活力参数的影响,其中375例同时运用Diff-Quick精子形态分析法进行严格精子形态学分析；150例精浆锌定量检测试剂盒(5-Br-PAPS法)行精浆锌含量检测；80例精子染色质扩散(SCD)法进行精子DNA完整性检测,将不吸烟组与吸烟组分别与各精液参数变化进行分析,探讨吸烟对男性精液质量的影响。结果：1、吸烟组精子密度与不吸烟组比较无统计学差异(P>0.05)；吸烟组精子活率、a级、a+b级活力均下降,差异有统计学意义(P<0.05)。其中按日吸烟支数分组：轻度吸烟组活率、a级、a+b级精子率无明显改变(P>0.05)；中度吸烟组活率无明显改变,而a级、a+b级精子率下降(P<0.05)；重度吸烟组的精液活率、a级、a+b级精子率均降低(P<0.05)。按吸烟年限分组：短烟龄组、中烟龄组与不吸烟组各指标相比无统计学差异(P>0.05),长烟龄组男性精液的密度、活率、a级、a+b级精子百分率均下降(P<0.05)。吸烟组中精液密度、a级、a+b级活力正常的男性所占比例均低于不吸烟组(P<0.01),差异有明显统计学意义。2、吸烟组的正常精子率、头部、体部、尾部异常率和不吸烟组相比无统计学差异(P>0.05),轻度、中度吸烟组正常精子率、头部、体部、尾部异常率和不吸烟组相比无统计学差异(P>0.05),短烟龄组、中烟龄组正常精子率、头部、体部、尾部异常率和不吸烟组相比无统计学差异(P>0.05),而在重度吸烟组和长烟龄组头部精子异常率均明显高于不吸烟组(P<0.05),差异有统计学意义。3、吸烟者与不吸烟者相比精浆锌浓度显著降低(P<0.05)；不吸烟组精浆锌含量正常率(68.6%)高于吸烟组中锌的正常率(51.3%)(P<0.05)；吸烟组精浆锌含量异常对比该组正常含量：精子密度下降,a级、a+b级活力均降低(P<0.01),差异有统计学意义。4、吸烟组精子DNA断裂指数(DFI)高于不吸烟组,精子DNA碎片增多(P<0.05),差异有统计学意义。结论：男性将烟草中的有害物质吸入后导致精子活率、活力降低、精子头部畸形率增加；随着吸烟年限的增加,精子密度也随之下降；吸烟使精液中的精浆锌水平下降,抗氧化能力降低,精浆锌含量低下影响精子密度、活力；吸烟增加精子DNA碎片的产生。精子质量随着每天吸烟支数和吸烟年限增加的影响而呈下降趋势,从而影响男性生育力。第二部分基于高密度芯片的吸烟男性精子基因组甲基化变异分析目的：探讨吸烟对男性精子基因组启动子甲基化状态的影响,了解吸烟和部分基因启动子区CpG岛甲基化程度的相关性,从甲基化变异分析角度探讨吸烟对男性精子基因组的表观遗传影响。方法：收集12例吸烟者与12例对照组非吸烟者(精子活力正常的正常生育男性)男性精液标本,提取精子基因组DNA,进行重亚硫酸盐转化,应用高密度基因芯片技术(Illumina HD450K Infinium Methylation BeadChip)分析吸烟组和非吸烟组男性精液DNA的甲基化差异水平,并对甲基化差异基因进行功能聚类和代谢通路等生物信息学分析。从甲基化水平差异显著的候选基因库中选取与少精子症、弱精子症相关的CASP3(胱氨酸蛋白酶-3)和ODF1(精子尾部外层致密纤维1)基因,采用焦磷酸测序法(Pyrosequencing)验证分析60例轻、中和重度吸烟者与非吸烟者相关基因启动子CpG岛的甲基化状态。结果：通过对甲基化芯片杂交数据统计显示男性吸烟人群和与非吸烟人群精子共有189个基因存在甲基化水平差异(Diff Score值50,P<0.00001),启动子甲基化水平显著上调的基因共121个,包括细胞凋亡相关的CASP3基因、睾丸组织特异性基因：如PLAC1L基因(胎盘特异样基因-1,placenta-specific1-like)等。启动子甲基化水平显著下调的基因共68个,包括与精子发生调节因子GPC1(磷脂酰基醇蛋白聚糖-1,glypican1)。随后,进一步对甲基化显著差异的基因进行了G0聚类分析,结果发现：甲基化显著差异的基因多集中在转录调节、RNA/DNA聚合酶活性、分子蛋白伴侣、细胞骨架、细胞凋亡相关和细胞周期蛋白类相关基因簇。经过对基因的功能的进一步注释,选择了与精子发生直接相关的CASP3和ODF1基因,进行系统的验证。生物信息分析结果显示,两个基因的5’上游序列启动子区均含有1个CpG岛,以CpG岛区序列为模板设计特异性焦磷酸测序引物进行甲基化差异验证。结果显示,CASP3基因启动子呈较低的甲基化状态,ODF1基因启动子呈较高的甲基化状态。CASP3基因启动子区CpG岛的平均甲基化频率在非吸烟组、轻、中和重度吸烟组分别为4.29%、4.56%、5.71%和7.18%,吸烟造成CASP3基因启动子甲基化水平上调,与基因芯片结果一致。ODF1基因启动子启动子区CpG岛的平均甲基化频率在非吸烟组、轻、中和重度吸烟组分别为86.02%、87.06%、91.77%和97.45%。经单因素方差分析结果显示,ODF1基因各组间甲基化水平差异有统计学意义(F=59.588,P<0.05),其中,重度吸烟组甲基化水平最高,与其它各组差异均有统计学意义(P<0.01),吸烟同样造成了ODF1基因启动子甲基化水平上调。结论：通过高密度全基因组甲基化芯片对吸烟与非吸烟男性精子DNA甲基化状态分析,发现吸烟引起了部分基因启动子甲基化水平上调或下调。对甲基化水平显著上调的精子发生直接相关CASP3和ODF1基因进行了扩大群体样本分析,同样显示吸烟造成了CASP3和ODF1基因启动子区甲基化水平显著上调,推测可能会造成基因的表达水平下调,进而造成了吸烟男性精子发生障碍,可能是造成不同类型男性不育症的重要危险因素之一,对今后深入探讨吸烟与男性不育的分子机制奠定了基础。更多还原

【Abstract】 PART1THE EFFECTS OF SMOKING ON SEMEN PARAMETERS AND SPERM DNA DAMAGE-A CLINICAL STUDYObjective:To explore the effects of smoking on sperm density, motility, vitality, sperm morphology, seminal plasma zinc concentration and sperm deoxyribonucleic acid (DNA) damage, and analyse the correlation between smoking and clinical semen parameters.Methods:We divided1,036male cases in Guangxi region into smoking and non-smoking group,grouped by daily cigarette consumption and years of smoking. Analysed the impact of smoking on sperm density, motility and vitality parameters, of which375cases using Diff-Quick sperm morphology analysis for strict sperm morphology analysis at the same time;150cases of applying seminal plasma zinc quantitative detection kit (5-Br-PAPS method) for detection of seminal plasma zinc content;80cases utilizing sperm Chromatin Dispersion (SCD) method for sperm DNA integrity test. The changes of semen parameters were analyzed in the non-smoking and smoking groups to explore the impact of smoking on semen quality. Results:1.Sperm density showed no statistically significant difference between smoking group and non-smoking group(P>0.05); sperm motility,(a) and (a+b) level of vitality in smoking group decreased, which was statistically significant (P <0.05). Grouped by daily cigarette consumption:the mild smoking group showed no significantly changes in sperm motility,(a) and (a+b) level of vitality (P>0.05),while the moderate smoking group had the (a) and (a+b) level of sperm vitality decreased (P<0.05) and no significantly change in motility; the heavy smoking group showed the significantly changes in sperm motility n (a) and (a+b) level of vitality(P<0.05). Grouped by years of smoking:the short smoked group, medium smoked group compared with the non-smoking group was no significant difference (P>0.05),while the long smoked group had the sperm density, motility,(a) and (a+b) level of vitality decreased (P<0.05). The proportion of normal density,(a) and (a+b) level of vitality parameters in smoking group were lower than the non-smoking group (P<0.01), which had a statistically significant difference.2. The rate of sperm morphology in normal sperm count and abnormal head, body, tail count was no significant difference between smoking and non-smoking group (P>0.05). When compared with the non-smoking group, the mild smoking group and moderate smoking group showed no significantly changes in the rate of sperm morphology of normal sperm count and abnormal head, body, tail count (P>0.05), and so did the short smoked group, medium smoked group(P>0.05).But the heavy smoking group and long smoked group had the abnormal head rate statistically significant increase (P<0.05).3. The seminal plasma zinc concentration decreased significantly in the smoking group compared with non-smoking group(P<0.05). The normal rate of seminal plasma zinc concentration in the non-smoking group (68.6%) was higher than that of smoking group (51.3%)(P<0.05). The abnormal rate of seminal plasma zinc concentration contrast to the normal content in the same smoking group showed the statistically significant decline in sperm density,(a) and (a+b) level of vitality (P<0.01).4. The sperm DNA fragmentation index (DFI) in smoking group was statistically higher than non-smoking group, with the sperm DNA fragmentation increase(P <0.05).Conclusions:The harmful substances in tobacco inhalated by smokers will cause the sperm motility and vitality decrease, while increase the malformation rate of sperm head. The sperm density decreases with the increase smoking time. Smoking leads to the decline of seminal plasma zinc levels in the semen, which can reduce antioxidant capacity.Low seminal plasma zinc concentration affects sperm density and motility. Smoking increases sperm DNA fragmentation. With the increase in smoking years and daily cigarette consumption, the negative impact of smoking on sperm quality is on the rise, which affects male fertility. PART2ACCESSING SPERM DNA METHYLATION VARIANCE BY SMOKING WITH HIGH-DENSITY DNA ARRAYSObjective:To investigate the effects of smoking on methylation status of sperm DNA and identify relationships between smoking and methylation levels of several gene promoter CpG islands,aslo explore the impact of smoking on sperm genome epigenetic change,exactly methylation change.Methods:Methylation status was examined in24samples,They were divided into2equal groups:healthy fertile nonsmokers whose sperm motility is normal and smokers.All samples were detected methylation status used Illumina HD450k infinium Methylation BeadChip after extracting DNA and bisulfite-convert, for GO category and Metabolic pathways,the standard GO analysis and the standard KEGG analysis were used respectly.Our results were also confirmed by selected oligozoospermia、asthenospermia candidate gene CASP3and ODF1from the chip results using Pyrosequencing among60samples divided into a control group of non-smokers, a light smokers,a moderate smokers group and a heavy smokers group.Results:We detected189genes methylation levels were significantly difference between smokers sprem DNA and nonsmokers sperm DNA(P<0.00001),121 genes were upmethylation in smokers sperm DNA,inclued apoptosis genes,such as CASP3,Testicular tissue-specific gene,such as PLACIL and so on;68genes were downmethylation in smokers sperm DNA,inclued GPC1.GO category analysis the differenlly genes,most genes were belong toTranscriptional regulation、cell apoptosis、cell cyclin and so on.Methylation states of gene-specific promoters (CASP3、ODF1) were quantified by pyrosequencing with special primers.Methylation level of CASP3CPG islands were4.29%,4.56%,5.71%,7.18%among non-smokers,light smokers,moderate smokers and heavy smokers,CASP3DNA methylation was evaluated in relation to smoking,Consistent with arrays results.Methylation level of ODF1CpG islands were86.02%,87.06%,91.77%,97.45%among non-smokers,light smokers,moderate smokers and heavy smokers,the methylation level were Significant difference among groups,smoking also evaluated methylation level of ODF1CpG islands.Conclusions:In this experiment,we indicates that cigarette smoking is associated with sperm DNA methylation,with high-density DNA arrays analysised sperm DNA methylation status between smokers and nonsmokers,we expounded that smoking could change promoter region methylationlevel of several gene.Selected differently gene spermatogenesis related from the array results,CASP3and ODF1,their promoter CpG region methylation level were also evaluated significantly,which maybe down regulated their Mrna expression, Cause spermatogenesis disorder,with adverse effects on male reproduction, Lead to male infertility. Results of our study may help shedding light on the molecular mechanisms of smoking and male infertility for further explore.更多还原