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
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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。
PART1
THE EFFECTS OF SMOKING ON SEMEN PARAMETERS AND SPERM DNA DAMAGE-A CLINICAL STUDY
Objective: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.
PART2
ACCESSING SPERM DNA METHYLATION VARIANCE BY SMOKING WITH HIGH-DENSITY DNA ARRAYS
Objective: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.
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。
PART1
THE EFFECTS OF SMOKING ON SEMEN PARAMETERS AND SPERM DNA DAMAGE-A CLINICAL STUDY
Objective: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.
PART2
ACCESSING SPERM DNA METHYLATION VARIANCE BY SMOKING WITH HIGH-DENSITY DNA ARRAYS
Objective: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.
引文
1. Georgiou, I., Pardalidis N., Giannakis D., et al. In vitro spermatogenesis as a method to bypass pre-meiotic or post-meiotic barriers blocking the spermatogenetic process:genetic and epigenetic implications in assisted reproductive technology[J]. Andrologia,2007; 39(5):159-176.
2. Steilmann, C., Cavalcanti M. C., Bergmann M., et al. Aberrant mRNA expression of chromatin remodelling factors in round spermatid maturation arrest compared with normal human spermatogenesis[J]. Mol Hum Reprod, 2010; 16(10):726-733.
3. Chohan, K. R. and Badawy S. Z. Cigarette smoking impairs sperm bioenergetics[J]. Int Braz J Urol,2010; 36(1):60-65.
4. Young, S. S., Eskenazi B., Marchetti F. M., et al. The association of folate, zinc and antioxidant intake with sperm aneuploidy in healthy non-smoking men[J]. Hum Reprod,2008;23(5):1014-1022.
5. Taha, E. A., Ez-Aldin A. M., Sayed S. K., et al. Effect of smoking on sperm vitality, DNA integrity, seminal oxidative stress, zinc in fertile men[J]. Urology,2012; 80(4):822-825.
6. Fariello, R. M., Pariz J. R., Spaine D. M., et al. Effect of smoking on the functional aspects of sperm and seminal plasma protein profiles in patients with varicocele[J]. Hum Reprod,2012;27(11):3140-3149.
7. Viloria, T., Meseguer M., Martinez-Conejero J. A., et al. Cigarette smoking affects specific sperm oxidative defenses but does not cause oxidative DNA damage in infertile men[J]. Fertil Steril,2010;94(2):631-637.
8. Zenzes, M. T. Smoking and reproduction:gene damage to human gametes and embryos[J]. Hum Reprod Update,2000;6(2):122-131.
9. Joo, K. J., Kwon Y. W., Myung S. C., et al. The effects of smoking and alcohol intake on sperm quality:light and transmission electron microscopy findings[J]. J Int Med Res,2012;40(6):2327-2335.
10. Colagar, A. H., Marzony E. T. and Chaichi M. J. Zinc levels in seminal plasma are associated with sperm quality in fertile and infertile men[J]. Nutr Res,2009; 29(2):82-88.
11. Maldera, J. A., Vasen G, Ernesto J. I., et al. Evidence for the involvement of zinc in the association of CRISP 1 with rat sperm during epididymal maturation[J]. Biol Reprod,2011; 85(3):503-510.
12. Elshal, M. F., El-Sayed I. H., Elsaied M. A., et al. Sperm head defects and disturbances in spermatozoal chromatin and DNA integrities in idiopathic infertile subjects:association with cigarette smoking[J]. Clin Biochem,2009; 42(7-8):589-594.
13. Egger, G, Liang G, Aparicio A., et al. Epigenetics in human disease and prospects for epigenetic therapy[J]. Nature,2004;429(6990):457-463.
14. Santenard, A. and Torres-Padilla M. E. Epigenetic reprogramming in mammalian reproduction:contribution from histone variants[J]. Epigenetics, 2009; 4(2):80-84.
15. Lukova, M., Todorova A., Todorov T., et al. Different methylation patterns in BWS/SRS cases clarified by MS-MLPA[J]. Mol Biol Rep,2013;40(1): 263-268.
16.梁小薇,卢卫红,陈振文,等过去25年中国有生育力男性精液参数变化的回顾性研究[J].中华男科学杂志,2008,；14(9)：775-778.
17.世界卫生组织编,谷翊群等译,人类精液及精子-宫颈粘液相互作用实验室检验手册(第四版)[M].北京人民卫生出版社.2001.
18. Simon, L., Brunborg G., Stevenson M., et al. Clinical significance of sperm DNA damage in assisted reproduction outcome[J]. Hum Reprod,2010; 25(7): 1594-1608.
19. El Mulla, K. F., Kohn F. M., El Beheiry A. H., et al. The effect of smoking and varicocele on human sperm acrosin activity and acrosome reaction[J]. Hum Reprod,1995; 10(12):3190-3194.
20. Erlich, P. M., Hoffman S. N., Rukstalis M., et al. Nicotinic acetylcholine receptor genes on chromosome 15q25.1 are associated with nicotine and opioid dependence severity[J]. Hum Genet,2010; 128(5):491-499.
21. Keskitalo-Vuokko, K., Hallfors J., Broms U., et al. Chromosome 20 shows linkage with DSM-Ⅳ nicotine dependence in Finnish adult smokers[J]. Nicotine Tob Res,2012;14(2):153-160.
22. 周妮娅,曹佳,崔志鸿,等.吸烟对重庆主城区成年男性精子凋亡及精 液质量的影响[J].中华男科学杂志,2009；15(8)：685-688.
23. 武俊青,李玉艳,高尔生等.吸烟对精液DNA指标影响的研究[J].中国卫生统计,2012；29(1)：50-52.
24. Host, E., Lindenberg S., Ernst E., et al. Sperm morphology and IVF: embryo quality in relation to sperm morphology following the WHO and Kruger's strict criteria[J]. Acta Obstet Gynecol Scand,1999;78(6):526-529.
25. 王益鑫,郑菊芬.男性不育研究新进展[J].中华男科学杂志,2006；12(9)：771-774.
26. Van Voorhis, B. J., Dawson J. D., Stovall D. W., et al. The effects of smoking on ovarian function and fertility during assisted reproduction cycles[J]. Obstet Gynecol,1996;88(5):785-791.
27. Glintborg, D., Mumm H., Hougaard D. M., et al. Smoking is associated with increased adrenal responsiveness, decreased prolactin levels and a more adverse lipid profile in 650 white patients with polycystic ovary syndrome[J]. Gynecol Endocrinol,2012; 28(3):170-174.
28. Colagar, A. H., Jorsaraee G. A. and Marzony E. T. Cigarette smoking and the risk of male infertility [J]. Pak J Biol Sci,2007; 10(21):3870-3874.
29. Linnebur, S. A. Tobacco education:emphasizing impotence as a consequence of smoking[J]. Am J Health Syst Pharm,2006;63(24): 2509-2512.
30. 李义平,王平,王亚文.香烟主流烟雾与侧流烟雾对小鼠致突变作用 的研究[J].中国公共卫生,2001；17(8)：707-708.
31. Arabi, M. and Moshtaghi H. Influence of cigarette smoking on spermatozoa via seminal plasma[J]. Andrologia,2005;37(4):119-124.
32.郭航,张红国,薛百功,等.吸烟、饮酒和桑拿对精子形态的影响[J].中华男科学杂志,2006；12(3)：215-217,221.
33. Saleh, R. A., Agarwal A., Sharma R. K., et al. Effect of cigarette smoking on levels of seminal oxidative stress in infertile men:a prospective study [J]. Fertil Steril,2002; 78(3):491-499.
34. Ozyurt, H., Pekmez H., Parlaktas B. S., et al. Oxidative stress in testicular tissues of rats exposed to cigarette smoke and protective effects of caffeic acid phenethyl ester[J]. Asian J Androl,2006;8(2):189-193.
35. Chia, S. E., Xu B., Ong C. N., et al. Effect of cadmium and cigarette smoking on human semen quality [J]. Int J Fertil Menopausal Stud,1994; 39(5):292-298.
36. Ramlau-Hansen, C. H., Thulstrup A. M., Aggerholm A. S., et al. Is smoking a risk factor for decreased semen quality? A cross-sectional analysis[J].Hum Reprod,2007; 22(1):188-196.
37. Al-Matubsi, H. Y., Kanaan R. A., Hamdan F., et al. Smoking practices in Jordanian people and their impact on semen quality and hormonal levels among adult men[J]. Cent Eur J Public Health,2011;19(1):54-59.
38. Ravnborg, T. L., Jensen T. K., Andersson A. M., et al.Prenatal and adult exposures to smoking are associated with adverse effects on reproductive hormones, semen quality, final height and body mass index[J]. Hum Reprod, 2011; 26(5):1000-1011.
39. Kunzle, R., Mueller M. D., Hanggi W., et al. Semen quality of male smokers and nonsmokers in infertile couples[J]. Fertil Steril,2003;79(2): 287-291.
40. Vine, M. F., Margolin B. H., Morrison H. I., et al. Cigarette smoking and sperm density:a meta-analysis[J]. Fertil Steril,1994;61(1):35-43.
41.侯翠,王文军,陈静华,等.1050例不孕不育夫妇的精液质量与吸烟、饮酒的关系[J].中国卫生检验杂志,2010；20(6)：1476-1478.
42. Sofikitis, N., Takenaka M., Kanakas N., et al. Effects of cotinine on sperm motility, membrane function, and fertilizing capacity in vitro [J]. Urol Res, 2000; 28(6):370-375.
43. Santos, E. P., Lopez-Costa S., Chenlo P., et al. Impact of spontaneous smoking cessation on sperm quality:case report[J]. Andrologia,2011;43(6): 431-435.
44. Coetzee, K., Kruge T. F. and Lombard C. J. Predictive value of normal sperm morphology:a structured literature review[J]. Hum Reprod Update, 1998; 4(1):73-82.
45. Menkveld, R., Wong W. Y., Lombard C. J., et al. Semen parameters, including WHO and strict criteria morphology, in a fertile and subfertile population:an effort towards standardization of in-vivo thresholds [J]. Hum Reprod,2001; 16(6):1165-1171.
46. De Vos, A., Van De Velde H., Joris H., et al. Influence of individual sperm morphology on fertilization, embryo morphology, and pregnancy outcome of intracytoplasmic sperm injection[J]. Fertil Steril,2003;79(1):42-48.
47. Hauser, R., Yogev L., Botchan A., et al. Intrauterine insemination in male factor subfertility:significance of sperm motility and morphology assessed by strict criteria[J]. Andrologia,2001; 33(1):13-17.
48. Zavos, P. M., Correa J. R., Antypas S., et al. Effects of seminal plasma from cigarette smokers on sperm viability and longevity [J]. Fertil Steril, 1998; 69(3):425-429.
49.杨加周,赵豫凤,苗乃周,等.吸烟对男性不育患者精液质量的影响[J].第四军医大学学报,2005；26(20)：1900-1901.
50. 武俊青,李玉艳,高尔生等.吸烟对精液质量常规指标影响的研究[J].中华流行病学杂志,2012；33(12)：1228-1232.
51. Rubes, J., Lowe X., Moore D.,2nd, et al. Smoking cigarettes is associated with increased sperm disomy in teenage men[J]. Fertil Steril,1998;70(4): 715-723.
52. Oliveira, J. B., Massaro F. C., Baruffi R. L., et al. Correlation between semen analysis by motile sperm organelle morphology examination and sperm DNA damage[J]. Fertil Steril,2010;94(5):1937-1940.
53. Costello, L. C., Franklin R. B. and Feng P. Mitochondrial function, zinc, and intermediary metabolism relationships in normal prostate and prostate cancer[J].Mitochondrion,2005;5(3):143-153.
54. Batra, N., Nehru B. and Bansal M. P. Influence of lead and zinc on rat male reproduction at 'biochemical and histopathological levels'[J].J Appl Toxicol,2001; 21(6):507-512.
55.李付彪,郭淑英,许宗革,等.不育男性精浆锌与精子密度、形态及运动性关系探讨[J].中国现代医学杂志,2008；18(11)：1594-1597.
56. Dissanayake, D., Wijesinghe P., Ratnasooriya W., et al. Relationship between seminal plasma zinc and semen quality in a subfertile population[J]. J Hum Reprod Sci,2010;3(3):124-128.
57. 江莉,李慕军,牛冉冉等.精浆锌含量与精液质量及体外受精结局的相关性[J].山东医药,2012；52(35)：22-24.
58. Oldereid, N. B., Thomassen Y. and Purvis K. Seminal plasma lead, cadmium and zinc in relation to tobacco consumption[J]. Int J Androl,1994; 17(1):24-28.
59. Liu, R. Z., Gao J. C., Zhang H. G, et al. Seminal plasma zinc level may be associated with the effect of cigarette smoking on sperm parameters [J]. J Int Med Res,2010;38(3):923-928.
60. Kumosani, T. A., Elshal M. F., Al-Jonaid A. A., et al. The influence of smoking on semen quality, seminal microelements and Ca2+ -ATPase activity among infertile and fertile men[J]. Clin Biochem,2008; 41(14-15): 1199-1203.
61. Rajpurkar, A., Jiang Y., Dhabuwala C. B., et al. Cigarette smoking induces apoptosis in rat testis[J]. J Environ Pathol Toxicol Oncol,2002; 21(3): 243-248.
62. Beek, J., Nauwynck H., Maes D., et al. Inhibitors of zinc-dependent metalloproteases hinder sperm passage through the cumulus oophorus during porcine fertilization in vitro[J]. Reproduction,2012; 144(6):687-697.
63. Kosower, N. S., Katayose H. and Yanagimachi R. Thiol-disulfide status and acridine orange fluorescence of mammalian sperm nuclei[J]. J Androl, 1992; 13(4):342-348.
64.郭应禄主编.男性不育症[M].人民军医出版社,北京,2003：92.
65. Chohan, K. R., Griffin J. T., Lafromboise M., et al. Comparison of chromatin assays for DNA fragmentation evaluation in human sperm[J]. J Androl,2006; 27(1):53-59.
66. Rowe, L. A., Degtyareva N. and Doetsch P. W. DNA damage-induced reactive oxygen species (ROS) stress response in Saccharomyces cerevisiae[J]. Free Radic Biol Med,2008; 45(8):1167-1177.
67. Cemeli, E. and Anderson D. Mechanistic Investigation of ROS-Induced DNA Damage by Oestrogenic Compounds in Lymphocytes and Sperm Using the Comet Assay [J]. Int J Mol Sci,2011; 12(5):2783-2796.
68. Linschooten, J. O., Laubenthal J., Cemeli E., et al. Incomplete protection of genetic integrity of mature spermatozoa against oxidative stress [J]. Reprod Toxicol,2011; 32(1):106-111.
69. Aitken, R. J., Gordon E., Harkiss D., et al. Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa[J]. Biol Reprod,1998; 59(5):1037-1046.
70. Erenpreiss, J., Elzanaty S. and Giwercman A. Sperm DNA damage in men from infertile couples[J]. Asian J Androl,2008; 10(5):786-790.
71. Bungum, M., Humaidan P., Axmon A., et al. Sperm DNA integrity assessment in prediction of assisted reproduction technology outcome[J]. Hum Reprod,2007; 22(1):174-179.
72.李娜,江莉.精子DNA损伤对体外受精-胚胎移植结局的影响[J].广西医学,2011；33(3)：257-260.
73. Khalili, M. A., Aghaie-Maybodi F., Anvari M., et al. Sperm nuclear DNA in ejaculates of fertile and infertile men:correlation with semen parameters[J]. Urol J,2006;3(3):154-159.
74.黄学锋,金建远,费前进,杨旭.精子DNA损伤：独立的精子质量评价指标[J].温州医学院学报,2010；40(3)：239-242.
1. Miyamoto, T., Tsujimura A., Miyagawa Y., et al. Male infertility and its causes in human[J]. Adv Urol,2012;2012:384520.
2. Gaur, D. S., Talekar M. S. and Pathak V. P. Alcohol intake and cigarette smoking:impact of two major lifestyle factors on male fertility[J]. Indian J Pathol Microbiol,2010;53(1):35-40.
3. Majumdar, S. S. and Bhattacharya I. Genomic and post-genomic leads toward regulation of spermatogenesis[J]. Prog Biophys Mol Biol,2013;Jan 31.[Epub ahead of print]PMID:23375691.
4.张正果.遗传性病因致男性不育的研究进展[J].中国男科学杂志,2010；24(10)：69-72.
5. Nanassy, L. and Carrell D. T. Abnormal methylation of the promoter of CREM is broadly associated with male factor infertility and poor sperm quality but is improved in sperm selected by density gradient centrifugation[J]. Fertil Steril,2011; 95(7):2310-2314.
6. Marques, C. J., Francisco T., Sousa S., et al. Methylation defects of imprinted genes in human testicular spermatozoa[J]. Fertil Steril,2010; 94(2):585-594.
7. Poplinski, A., Tuttelmann F., Kanber D., et al. Idiopathic male infertility is strongly associated with aberrant methylation of MEST and IGF2/H19 ICR1[J]. Int J Androl,2010;33(4):642-649.
8. Navarro-Costa, P., Nogueira P., Carvalho M., et al. Incorrect DNA methylation of the DAZL promoter CpG island associates with defective human sperm[J]. Hum Reprod,2010;25(10):2647-2654.
9. Conen, D., Everett B. M., Kurth T., et al. Smoking, smoking cessation, [corrected] and risk for symptomatic peripheral artery disease in women:a cohort study[J]. Ann Intern Med,2011;154(11):719-726.
10. Rea, T. D., Heckbert S. R., Kaplan R. C., et al. Smoking status and risk for recurrent coronary events after myocardial infarction[J]. Ann Intern Med, 2002; 137(6):494-500.
11. Braber, S., Henricks P. A., Nijkamp F. P., et al. Inflammatory changes in the airways of mice caused by cigarette smoke exposure are only partially reversed after smoking cessation[J]. Respir Res,2010; 11:99.
12.Bouloukaki, I., Tsiligianni I. G., Tsoumakidou M., et al. Sputum and nasal lavage lung-specific biomarkers before and after smoking cessation[J]. BMC Pulm Med,2011; 11:35.
13. Louhelainen, N., Stark H., Mazur W., et al. Elevation of sputum matrix metalloproteinase-9 persists up to 6 months after smoking cessation:a research study [J]. BMC Pulm Med,2010; 10:13.
14. Luo, J., Margolis K. L., Wactawski-Wende J., et al. Association of active and passive smoking with risk of breast cancer among postmenopausal women:a prospective cohort study [J]. BMJ,2011;342:d1016.
15. Vineis, P., Alavanja M., Buffler P., et al. Tobacco and cancer:recent epidemiological evidence[J]. J Natl Cancer Inst,2004; 96(2):99-106.
16. Petros, W. P., Younis I. R., Ford J. N., et al. Effects of Tobacco Smoking and Nicotine on Cancer Treatment[J]. Pharmacotherapy,2012; 32(10):920-31.
17. D'Addario, C., Francesco A. D., Pucci M., et al. Epigenetic mechanisms and endocannabinoid signalling[J]. FEBS J,2013; Jan 11.[Epub ahead of print]PMID:23305292.
18. Breitling, L. P., Yang R., Korn B., et al. Tobacco-smoking-related differential DNA methylation:27K discovery and replication[J]. Am J Hum Genet,2011; 88(4):450-457.
19. Wan, E. S., Qiu W., Baccarelli A., et al. Cigarette smoking behaviors and time since quitting are associated with differential DNA methylation across the human genome[J]. Hum Mol Genet,2012; 21(13):3073-3082.
20. Pop, C. and Salvesen G S. Human caspases:activation, specificity, and regulation[J]. J Biol Chem,2009;284(33):21777-21781.
21. Soung, Y. H., Lee J. W., Kim S. Y, et al. Somatic mutations of CASP3 gene in human cancers[J]. Hum Genet,2004;115(2):112-115.
22.黄健清,梁红玲,张绪超,等.CASP3与Cleaved-CASP3在肺癌中的表达及意义[J].实用医学杂志,2012；28(8)：1247-1250.
23.倪勤,刘冰,金明娟,等.CASP3基因多态及单体型分布与乳腺癌危险 性的关联研究[J].浙江大学学报(医学版),2011；40(3)：259-264.
24.邱广蓉,刘培燕,姜红垄,等.CASP3基因R101H多态性与法洛四联症的相关性[J].实用儿科临床杂志,2012；27(11)：856-858.
25. Lan, Q., Zheng T., Chanock S., et al. Genetic variants in caspase genes and susceptibility to non-Hodgkin lymphoma[J]. Carcinogenesis,2007; 28(4): 823-827.
26. Wang, D. H., Hu J. R., Wang L. Y, et al. The apoptotic function analysis of p53, Apafl, Caspase3 and Caspase7 during the spermatogenesis of the Chinese fire-bellied newt Cynops orientalis[J]. PLoS One,2012;7(6): e39920.
27. Almeida, C., Correia S., Rocha E., et al. Caspase signalling pathways in human spermatogenesis [J]. J Assist Reprod Genet,2013, Jan 29. [Epub ahead of print]PMID:23359247.
28. Cabrillana, M. E., Monclus M. A., Saez Lancellotti T. E., et al. Characterization of flagellar cysteine-rich sperm proteins involved in motility, by the combination of cellular fractionation, fluorescence detection, and mass spectrometry analysis[J]. Cytoskeleton (Hoboken),2011; 68(9): 491-500.
29. 王勇.精子鞭毛相关蛋白在人精子中的表达特征及其临床意义[D].汕头大学,2009.
30. Chen, J., Wang Y., Xu X., et al. Differential expression of ODF1 in human ejaculated spermatozoa and its clinical significance[J]. Zhonghua Nan Ke Xue,2009; 15(10):891-894.
31. Yang, K., Meinhardt A., Zhang B., et al. The small heat shock protein ODF1/HSPB10 is essential for tight linkage of sperm head to tail and male fertility in mice[J]. Mol Cell Biol,2012; 32(1):216-225.
32. Portela, A. and Esteller M. Epigenetic modifications and human disease[J]. Nat Biotechnol,2010; 28(10):1057-1068.
33. Attwood, J. T., Yung R. L. and Richardson B. C. DNA methylation and the regulation of gene transcription[J]. Cell Mol Life Sci,2002;59(2):241-257.
1. Egger, G., Liang G., Aparicio A., et al. Epigenetics in human disease and prospects for epigenetic therapy[J]. Nature,2004;429(6990):457-463.
2. Hales, B. F., Grenier L., Lalancette C., et al. Epigenetic programming:from gametes to blastocyst[J]. Birth Defects Res A Clin Mol Teratol,2011; 91(8): 652-665.
3. Katari, S., Turan N., Bibikova M., et al. DNA methylation and gene expression differences in children conceived in vitro or in vivo[J]. Hum Mol Genet,2009; 18(20):3769-3778.
4. Vanyushin, B. F. A view of an elemental naturalist at the DNA world (base composition, sequences, methylation)[J]. Biochemistry (Mosc),2007; 72(12): 1289-1298.
5. Hammoud, S. S., Purwar J., Pflueger C., et al. Alterations in sperm DNA methylation patterns at imprinted loci in two classes of infertility [J]. Fertil Steril,2010; 94(5):1728-1733.
6. Bird, A. DNA methylation patterns and epigenetic memory[J]. Genes Dev, 2002; 16(1):6-21.
7. Okano, M., Bell D. W., Haber D. A., et al. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development[J]. Cell,1999; 99(3):247-257.
8. Goll, M. G. and Bestor T. H. Eukaryotic cytosine methyltransferases[J]. Annu Rev Biochem,2005; 74:481-514.
9. Marandel, L., Labbe C., Bobe J., et al. nanog 5'-upstream sequence, DNA methylation, and expression in gametes and early embryo reveal striking differences between teleosts and mammals[J]. Gene,2012; 492(1):130-137.
10. Kreth, S., Thon N., Eigenbrod S., et al. O-methylguanine-DNA methyltransferase (MGMT) mRNA expression predicts outcome in malignant glioma independent of MGMT promoter methylationfJ]. PLoS One,2011; 6(2):e17156.
11. Kaneko, R., Kawaguchi M., Toyama T., et al. Expression levels of Protocadherin-alpha transcripts are decreased by nonsense-mediated mRNA decay with frameshift mutations and by high DNA methylation in their promoter regions[J]. Gene,2009;430(1-2):86-94.
12. Gokul, G, Gautami B., Malathi S., et al. DNA methylation profile at the DNMT3L promoter:a potential biomarker for cervical cancer[J]. Epigenetics,2007; 2(2):80-85.
13. Gupta, S., Pathak R. U. and Kanungo M. S. DNA methylation induced changes in chromatin conformation of the promoter of the vitellogenin II gene of Japanese quail during aging[J]. Gene,2006; 377:159-168.
14. Ozdemir, O., Bulut H. E., Korkmaz M., et al. Increased cell proliferation and R.Msp1 fragmentation induced by 5-aza-2'-deoxycytidine in rat testes related to the gene imprinting mechanism[J]. Exp Toxicol Pathol,2000; 52(4):317-322.
15. Santenard, A. and Torres-Padilla M. E. Epigenetic reprogramming in mammalian reproduction:contribution from histone variants[J]. Epigenetics, 2009; 4(2):80-84.
16. Lukova, M., Todorova A., Todorov T., et al. Different methylation patterns in BWS/SRS cases clarified by MS-MLPA[J]. Mol Biol Rep,2013;40(1): 263-268.
17. van Montfoort, A. P., Hanssen L. L., de Sutter P., et al. Assisted reproduction treatment and epigenetic inheritance[J]. Hum Reprod Update, 2012; 18(2):171-197.
18. Kallen, B., Finnstrom O., Nygren K. G., et al. In vitro fertilization (IVF) in Sweden:risk for congenital malformations after different IVF methods[J]. Birth Defects Res A Clin Mol Teratol,2005; 73(3):162-169.
19. Tanasijevic, B., Dai B., Ezashi T., et al. Progressive accumulation of epigenetic heterogeneity during human ES cell culture[J]. Epigenetics,2009; 4(5):330-338.
20. Wagner, W. Implications of long-term culture for mesenchymal stem cells: genetic defects or epigenetic regulation?[J].Stem Cell Res Ther,2012;3(6): 54.
21. Hansen, M., Milne E., de Klerk N., et al. ART, birth defects and subfertility--what should prospective patients be told?[J]. J Assist Reprod Genet,2011; 28(12):1229-1230.
22. Favaedi, R., Shahhoseini M. and Akhoond M. R. Comparative epigenetic analysis of Oct4 regulatory region in RA-induced differentiated NT2 cells under adherent and non-adherent culture conditions[J]. Mol Cell Biochem, 2012; 363(1-2):129-134.
23. Minor, A., Chow V. and Ma S. Aberrant DNA methylation at imprinted genes in testicular sperm retrieved from men with obstructive azoospermia and undergoing vasectomy reversal[J]. Reproduction,2011; 141(6): 749-757.
24. Huh, J. H., Bauer M. J., Hsieh T. F., et al. Cellular programming of plant gene imprinting[J]. Cell,2008;132(5):735-744.
25. Katz, D. J., Edwards T. M., Reinke V., et al. A C. elegans LSD1 demethylase contributes to germline immortality by reprogramming epigenetic memory[J]. Cell,2009;137(2):308-320.
26. Okada, Y., Scott G, Ray M. K., et al. Histone demethylase JHDM2A is critical for Tnpl and Prml transcription and spermatogenesis[J]. Nature, 2007; 450(7166):119-123.
27. Isidori, A. M., Pozza C., Gianfrilli D., et al. Medical treatment to improve sperm quality[J]. Reprod Biomed Online,2006; 12(6):704-714.
28. Matzuk, M. M. and Lamb D. J. The biology of infertility:research advances and clinical challenges[J]. Nat Med,2008;14(11):1197-1213.
29. Aoki, V. W., Christensen G. L., Atkins J. F., et al. Identification of novel polymorphisms in the nuclear protein genes and their relationship with human sperm protamine deficiency and severe male infertility [J]. Fertil Steril,2006; 86(5):1416-1422.
30. Hammoud, S., Emery B. R., Dunn D., et al. Sequence alterations in the YBX2 gene are associated with male factor infertility [J]. Fertil Steril,2009; 91(4):1090-1095.
31. Aston, K. I. and Carrell D. T. Genome-wide study of single-nucleotide polymorphisms associated with azoospermia and severe oligozoospermia[J]. J Androl,2009; 30(6):711-725.
32. Steel, A. J. and Sutcliffe A. Long-term health implications for children conceived by IVF/ICSI[J]. Hum Fertil (Camb),2009;12(1):21-27.
33. Reefhuis, J., Honein M. A., Schieve L. A., et al. Assisted reproductive technology and major structural birth defects in the United States [J]. Hum Reprod,2009; 24(2):360-366.
34. Yan, J., Huang G, Sun Y., et al. Birth defects after assisted reproductive technologies in China:analysis of 15,405 offspring in seven centers (2004 to 2008)[J]. Fertil Steril,2011; 95(1):458-460.
35. Kalra, S. K. and Molinaro T. A. The association of in vitro fertilization and perinatal morbidity[J]. Semin Reprod Med,2008; 26(5):423-435.
36. Odom, L. N. and Segars J. Imprinting disorders and assisted reproductive technology[J]. Curr Opin Endocrinol Diabetes Obes,2010; 17(6):517-522.
37. Manipalviratn, S., DeCherney A. and Segars J. Imprinting disorders and assisted reproductive technology [J]. Fertil Steril,2009; 91(2):305-315.
38. Dasoula, A., Georgiou I., Kontogianni E., et al. Methylation status of the SNRPN and HUMARA genes in testicular biopsy samples[J]. Fertil Steril, 2007; 87(4):805-809.
39. Poplinski, A., Tuttelmann F., Kanber D., et al. Idiopathic male infertility is strongly associated with aberrant methylation of MEST and IGF2/H19 ICR1[J]. Int J Androl,2010;33(4):642-649.
40. Dada, R., Kumar M., Jesudasan R., et al. Epigenetics and its role in male infertility [J]. J Assist Reprod Genet,2012;29(3):213-223.
41. Kobayashi, H., Hiura H., John R. M., et al. DNA methylation errors at imprinted loci after assisted conception originate in the parental sperm[J]. Eur J Hum Genet,2009;17(12):1582-1591.
42. Carrell, D. T. Epigenetics of the male gamete[J]. Fertil Steril,2012; 97(2): 267-274.
43. Seisenberger, S., Andrews S., Krueger F., et al. The dynamics of genome-wide DNA methylation reprogramming in mouse primordial germ cells[J]. Mol Cell,2012;48(6):849-862.
44. Hyldig, S. M., Ostrup O., Vejlsted M., et al. Changes of DNA methylation level and spatial arrangement of primordial germ cells in embryonic day 15 to embryonic day 28 pig embryos[J]. Biol Reprod,2011; 84(6):1087-1093.
45. Jang, H. J., Lee M. O., Kim S., et al. Biallelic expression of the L-arginine:glycine amidinotransferase gene with different methylation status between male and female primordial germ cells in chickens[J]. Poult Sci, 2013; 92(3):760-769.
46. Pathak, S., D'Souza R., Ankolkar M., et al. Potential role of estrogen in regulation of the insulin-like growth factor2-H19 locus in the rat testis[J]. Mol Cell Endocrinol,2010; 314(1):110-117.
47. Hammoud, S. S., Cairns B. R. and Carrell D. T. Analysis of gene-specific and genome-wide sperm DNA methylation[J]. Methods Mol Biol,2013; 927: 451-458.
48. Pacheco, S. E., Houseman E. A., Christensen B. C., et al. Integrative DNA methylation and gene expression analyses identify DNA packaging and epigenetic regulatory genes associated with low motility sperm[J]. PLoS One,2011; 6(6):e20280.
49. Marques, C. J., Francisco T., Sousa S., et al. Methylation defects of imprinted genes in human testicular spermatozoa[J]. Fertil Steril,2010; 94(2):585-594.
50. Nanassy, L. and Carrell D. T. Analysis of the methylation pattern of six gene promoters in sperm of men with abnormal protamination[J]. Asian J Androl,2011;13(2):342-346.
51. Nanassy, L. and Carrell D. T. Paternal effects on early embryogenesis[J]. J Exp Clin Assist Reprod,2008; 5:2.
52. Carrell, D. T. Elucidating the genetics of male infertility:understanding transcriptional and translational regulatory networks involved in spermatogenesis[J]. Int J Androl,2008; 31(5):455-456.
53. Yamashita, S., Hosoya K., Gyobu K., et al. Development of a novel output value for quantitative assessment in methylated DNA immunoprecipitation-CpG island microarray analysis[J]. DNA Res,2009; 16(5):275-286.
54. Kagami, M., Matsuoka K., Nagai T., et al. Paternal uniparental disomy 14 and related disorders:placental gene expression analyses and histological examinations [J]. Epigenetics,2012; 7(10):1142-1150.
55. Mitalipov, S., Clepper L., Sritanaudomchai H., et al. Methylation status of imprinting centers for H19/IGF2 and SNURF/SNRPN in primate embryonic stem cells[J]. Stem Cells,2007;25(3):581-588.
56. Wang, M., Zhang X., Kang L., et al. Molecular characterization of porcine NECD, SNRPN and UBE3A genes and imprinting status in the skeletal muscle of neonate pigs[J]. Mol Biol Rep,2012; 39(10):9415-9422.
57. Peng, H. H., Chang S. D., Chao A. S., et al. DNA methylation patterns of imprinting centers for H19, SNRPN, and KCNQ1OT1 in single-cell clones of human amniotic fluid mesenchymal stem cell[J]. Taiwan J Obstet Gynecol, 2012; 51(3):342-349.
58. Inawaka, K., Kawabe M., Takahashi S., et al. Maternal exposure to anti-androgenic compounds, vinclozolin, flutamide and procymidone, has no effects on spermatogenesis and DNA methylation in male rats of subsequent generations[J]. Toxicol Appl Pharmacol,2009;237(2):178-187.
59. Ouko, L. A., Shantikumar K., Knezovich J., et al. Effect of alcohol consumption on CpG methylation in the differentially methylated regions of H19 and IG-DMR in male gametes:implications for fetal alcohol spectrum disorders[J]. Alcohol Clin Exp Res,2009;33(9):1615-1627.
60. WHO. WHO laboratory manual for the examination and processing of human semen[M].5th ed. Geneva:World Health Organization,2010.
2. Steilmann, C., Cavalcanti M. C., Bergmann M., et al. Aberrant mRNA expression of chromatin remodelling factors in round spermatid maturation arrest compared with normal human spermatogenesis[J]. Mol Hum Reprod, 2010; 16(10):726-733.
3. Chohan, K. R. and Badawy S. Z. Cigarette smoking impairs sperm bioenergetics[J]. Int Braz J Urol,2010; 36(1):60-65.
4. Young, S. S., Eskenazi B., Marchetti F. M., et al. The association of folate, zinc and antioxidant intake with sperm aneuploidy in healthy non-smoking men[J]. Hum Reprod,2008;23(5):1014-1022.
5. Taha, E. A., Ez-Aldin A. M., Sayed S. K., et al. Effect of smoking on sperm vitality, DNA integrity, seminal oxidative stress, zinc in fertile men[J]. Urology,2012; 80(4):822-825.
6. Fariello, R. M., Pariz J. R., Spaine D. M., et al. Effect of smoking on the functional aspects of sperm and seminal plasma protein profiles in patients with varicocele[J]. Hum Reprod,2012;27(11):3140-3149.
7. Viloria, T., Meseguer M., Martinez-Conejero J. A., et al. Cigarette smoking affects specific sperm oxidative defenses but does not cause oxidative DNA damage in infertile men[J]. Fertil Steril,2010;94(2):631-637.
8. Zenzes, M. T. Smoking and reproduction:gene damage to human gametes and embryos[J]. Hum Reprod Update,2000;6(2):122-131.
9. Joo, K. J., Kwon Y. W., Myung S. C., et al. The effects of smoking and alcohol intake on sperm quality:light and transmission electron microscopy findings[J]. J Int Med Res,2012;40(6):2327-2335.
10. Colagar, A. H., Marzony E. T. and Chaichi M. J. Zinc levels in seminal plasma are associated with sperm quality in fertile and infertile men[J]. Nutr Res,2009; 29(2):82-88.
11. Maldera, J. A., Vasen G, Ernesto J. I., et al. Evidence for the involvement of zinc in the association of CRISP 1 with rat sperm during epididymal maturation[J]. Biol Reprod,2011; 85(3):503-510.
12. Elshal, M. F., El-Sayed I. H., Elsaied M. A., et al. Sperm head defects and disturbances in spermatozoal chromatin and DNA integrities in idiopathic infertile subjects:association with cigarette smoking[J]. Clin Biochem,2009; 42(7-8):589-594.
13. Egger, G, Liang G, Aparicio A., et al. Epigenetics in human disease and prospects for epigenetic therapy[J]. Nature,2004;429(6990):457-463.
14. Santenard, A. and Torres-Padilla M. E. Epigenetic reprogramming in mammalian reproduction:contribution from histone variants[J]. Epigenetics, 2009; 4(2):80-84.
15. Lukova, M., Todorova A., Todorov T., et al. Different methylation patterns in BWS/SRS cases clarified by MS-MLPA[J]. Mol Biol Rep,2013;40(1): 263-268.
16.梁小薇,卢卫红,陈振文,等过去25年中国有生育力男性精液参数变化的回顾性研究[J].中华男科学杂志,2008,；14(9)：775-778.
17.世界卫生组织编,谷翊群等译,人类精液及精子-宫颈粘液相互作用实验室检验手册(第四版)[M].北京人民卫生出版社.2001.
18. Simon, L., Brunborg G., Stevenson M., et al. Clinical significance of sperm DNA damage in assisted reproduction outcome[J]. Hum Reprod,2010; 25(7): 1594-1608.
19. El Mulla, K. F., Kohn F. M., El Beheiry A. H., et al. The effect of smoking and varicocele on human sperm acrosin activity and acrosome reaction[J]. Hum Reprod,1995; 10(12):3190-3194.
20. Erlich, P. M., Hoffman S. N., Rukstalis M., et al. Nicotinic acetylcholine receptor genes on chromosome 15q25.1 are associated with nicotine and opioid dependence severity[J]. Hum Genet,2010; 128(5):491-499.
21. Keskitalo-Vuokko, K., Hallfors J., Broms U., et al. Chromosome 20 shows linkage with DSM-Ⅳ nicotine dependence in Finnish adult smokers[J]. Nicotine Tob Res,2012;14(2):153-160.
22. 周妮娅,曹佳,崔志鸿,等.吸烟对重庆主城区成年男性精子凋亡及精 液质量的影响[J].中华男科学杂志,2009；15(8)：685-688.
23. 武俊青,李玉艳,高尔生等.吸烟对精液DNA指标影响的研究[J].中国卫生统计,2012；29(1)：50-52.
24. Host, E., Lindenberg S., Ernst E., et al. Sperm morphology and IVF: embryo quality in relation to sperm morphology following the WHO and Kruger's strict criteria[J]. Acta Obstet Gynecol Scand,1999;78(6):526-529.
25. 王益鑫,郑菊芬.男性不育研究新进展[J].中华男科学杂志,2006；12(9)：771-774.
26. Van Voorhis, B. J., Dawson J. D., Stovall D. W., et al. The effects of smoking on ovarian function and fertility during assisted reproduction cycles[J]. Obstet Gynecol,1996;88(5):785-791.
27. Glintborg, D., Mumm H., Hougaard D. M., et al. Smoking is associated with increased adrenal responsiveness, decreased prolactin levels and a more adverse lipid profile in 650 white patients with polycystic ovary syndrome[J]. Gynecol Endocrinol,2012; 28(3):170-174.
28. Colagar, A. H., Jorsaraee G. A. and Marzony E. T. Cigarette smoking and the risk of male infertility [J]. Pak J Biol Sci,2007; 10(21):3870-3874.
29. Linnebur, S. A. Tobacco education:emphasizing impotence as a consequence of smoking[J]. Am J Health Syst Pharm,2006;63(24): 2509-2512.
30. 李义平,王平,王亚文.香烟主流烟雾与侧流烟雾对小鼠致突变作用 的研究[J].中国公共卫生,2001；17(8)：707-708.
31. Arabi, M. and Moshtaghi H. Influence of cigarette smoking on spermatozoa via seminal plasma[J]. Andrologia,2005;37(4):119-124.
32.郭航,张红国,薛百功,等.吸烟、饮酒和桑拿对精子形态的影响[J].中华男科学杂志,2006；12(3)：215-217,221.
33. Saleh, R. A., Agarwal A., Sharma R. K., et al. Effect of cigarette smoking on levels of seminal oxidative stress in infertile men:a prospective study [J]. Fertil Steril,2002; 78(3):491-499.
34. Ozyurt, H., Pekmez H., Parlaktas B. S., et al. Oxidative stress in testicular tissues of rats exposed to cigarette smoke and protective effects of caffeic acid phenethyl ester[J]. Asian J Androl,2006;8(2):189-193.
35. Chia, S. E., Xu B., Ong C. N., et al. Effect of cadmium and cigarette smoking on human semen quality [J]. Int J Fertil Menopausal Stud,1994; 39(5):292-298.
36. Ramlau-Hansen, C. H., Thulstrup A. M., Aggerholm A. S., et al. Is smoking a risk factor for decreased semen quality? A cross-sectional analysis[J].Hum Reprod,2007; 22(1):188-196.
37. Al-Matubsi, H. Y., Kanaan R. A., Hamdan F., et al. Smoking practices in Jordanian people and their impact on semen quality and hormonal levels among adult men[J]. Cent Eur J Public Health,2011;19(1):54-59.
38. Ravnborg, T. L., Jensen T. K., Andersson A. M., et al.Prenatal and adult exposures to smoking are associated with adverse effects on reproductive hormones, semen quality, final height and body mass index[J]. Hum Reprod, 2011; 26(5):1000-1011.
39. Kunzle, R., Mueller M. D., Hanggi W., et al. Semen quality of male smokers and nonsmokers in infertile couples[J]. Fertil Steril,2003;79(2): 287-291.
40. Vine, M. F., Margolin B. H., Morrison H. I., et al. Cigarette smoking and sperm density:a meta-analysis[J]. Fertil Steril,1994;61(1):35-43.
41.侯翠,王文军,陈静华,等.1050例不孕不育夫妇的精液质量与吸烟、饮酒的关系[J].中国卫生检验杂志,2010；20(6)：1476-1478.
42. Sofikitis, N., Takenaka M., Kanakas N., et al. Effects of cotinine on sperm motility, membrane function, and fertilizing capacity in vitro [J]. Urol Res, 2000; 28(6):370-375.
43. Santos, E. P., Lopez-Costa S., Chenlo P., et al. Impact of spontaneous smoking cessation on sperm quality:case report[J]. Andrologia,2011;43(6): 431-435.
44. Coetzee, K., Kruge T. F. and Lombard C. J. Predictive value of normal sperm morphology:a structured literature review[J]. Hum Reprod Update, 1998; 4(1):73-82.
45. Menkveld, R., Wong W. Y., Lombard C. J., et al. Semen parameters, including WHO and strict criteria morphology, in a fertile and subfertile population:an effort towards standardization of in-vivo thresholds [J]. Hum Reprod,2001; 16(6):1165-1171.
46. De Vos, A., Van De Velde H., Joris H., et al. Influence of individual sperm morphology on fertilization, embryo morphology, and pregnancy outcome of intracytoplasmic sperm injection[J]. Fertil Steril,2003;79(1):42-48.
47. Hauser, R., Yogev L., Botchan A., et al. Intrauterine insemination in male factor subfertility:significance of sperm motility and morphology assessed by strict criteria[J]. Andrologia,2001; 33(1):13-17.
48. Zavos, P. M., Correa J. R., Antypas S., et al. Effects of seminal plasma from cigarette smokers on sperm viability and longevity [J]. Fertil Steril, 1998; 69(3):425-429.
49.杨加周,赵豫凤,苗乃周,等.吸烟对男性不育患者精液质量的影响[J].第四军医大学学报,2005；26(20)：1900-1901.
50. 武俊青,李玉艳,高尔生等.吸烟对精液质量常规指标影响的研究[J].中华流行病学杂志,2012；33(12)：1228-1232.
51. Rubes, J., Lowe X., Moore D.,2nd, et al. Smoking cigarettes is associated with increased sperm disomy in teenage men[J]. Fertil Steril,1998;70(4): 715-723.
52. Oliveira, J. B., Massaro F. C., Baruffi R. L., et al. Correlation between semen analysis by motile sperm organelle morphology examination and sperm DNA damage[J]. Fertil Steril,2010;94(5):1937-1940.
53. Costello, L. C., Franklin R. B. and Feng P. Mitochondrial function, zinc, and intermediary metabolism relationships in normal prostate and prostate cancer[J].Mitochondrion,2005;5(3):143-153.
54. Batra, N., Nehru B. and Bansal M. P. Influence of lead and zinc on rat male reproduction at 'biochemical and histopathological levels'[J].J Appl Toxicol,2001; 21(6):507-512.
55.李付彪,郭淑英,许宗革,等.不育男性精浆锌与精子密度、形态及运动性关系探讨[J].中国现代医学杂志,2008；18(11)：1594-1597.
56. Dissanayake, D., Wijesinghe P., Ratnasooriya W., et al. Relationship between seminal plasma zinc and semen quality in a subfertile population[J]. J Hum Reprod Sci,2010;3(3):124-128.
57. 江莉,李慕军,牛冉冉等.精浆锌含量与精液质量及体外受精结局的相关性[J].山东医药,2012；52(35)：22-24.
58. Oldereid, N. B., Thomassen Y. and Purvis K. Seminal plasma lead, cadmium and zinc in relation to tobacco consumption[J]. Int J Androl,1994; 17(1):24-28.
59. Liu, R. Z., Gao J. C., Zhang H. G, et al. Seminal plasma zinc level may be associated with the effect of cigarette smoking on sperm parameters [J]. J Int Med Res,2010;38(3):923-928.
60. Kumosani, T. A., Elshal M. F., Al-Jonaid A. A., et al. The influence of smoking on semen quality, seminal microelements and Ca2+ -ATPase activity among infertile and fertile men[J]. Clin Biochem,2008; 41(14-15): 1199-1203.
61. Rajpurkar, A., Jiang Y., Dhabuwala C. B., et al. Cigarette smoking induces apoptosis in rat testis[J]. J Environ Pathol Toxicol Oncol,2002; 21(3): 243-248.
62. Beek, J., Nauwynck H., Maes D., et al. Inhibitors of zinc-dependent metalloproteases hinder sperm passage through the cumulus oophorus during porcine fertilization in vitro[J]. Reproduction,2012; 144(6):687-697.
63. Kosower, N. S., Katayose H. and Yanagimachi R. Thiol-disulfide status and acridine orange fluorescence of mammalian sperm nuclei[J]. J Androl, 1992; 13(4):342-348.
64.郭应禄主编.男性不育症[M].人民军医出版社,北京,2003：92.
65. Chohan, K. R., Griffin J. T., Lafromboise M., et al. Comparison of chromatin assays for DNA fragmentation evaluation in human sperm[J]. J Androl,2006; 27(1):53-59.
66. Rowe, L. A., Degtyareva N. and Doetsch P. W. DNA damage-induced reactive oxygen species (ROS) stress response in Saccharomyces cerevisiae[J]. Free Radic Biol Med,2008; 45(8):1167-1177.
67. Cemeli, E. and Anderson D. Mechanistic Investigation of ROS-Induced DNA Damage by Oestrogenic Compounds in Lymphocytes and Sperm Using the Comet Assay [J]. Int J Mol Sci,2011; 12(5):2783-2796.
68. Linschooten, J. O., Laubenthal J., Cemeli E., et al. Incomplete protection of genetic integrity of mature spermatozoa against oxidative stress [J]. Reprod Toxicol,2011; 32(1):106-111.
69. Aitken, R. J., Gordon E., Harkiss D., et al. Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa[J]. Biol Reprod,1998; 59(5):1037-1046.
70. Erenpreiss, J., Elzanaty S. and Giwercman A. Sperm DNA damage in men from infertile couples[J]. Asian J Androl,2008; 10(5):786-790.
71. Bungum, M., Humaidan P., Axmon A., et al. Sperm DNA integrity assessment in prediction of assisted reproduction technology outcome[J]. Hum Reprod,2007; 22(1):174-179.
72.李娜,江莉.精子DNA损伤对体外受精-胚胎移植结局的影响[J].广西医学,2011；33(3)：257-260.
73. Khalili, M. A., Aghaie-Maybodi F., Anvari M., et al. Sperm nuclear DNA in ejaculates of fertile and infertile men:correlation with semen parameters[J]. Urol J,2006;3(3):154-159.
74.黄学锋,金建远,费前进,杨旭.精子DNA损伤：独立的精子质量评价指标[J].温州医学院学报,2010；40(3)：239-242.
1. Miyamoto, T., Tsujimura A., Miyagawa Y., et al. Male infertility and its causes in human[J]. Adv Urol,2012;2012:384520.
2. Gaur, D. S., Talekar M. S. and Pathak V. P. Alcohol intake and cigarette smoking:impact of two major lifestyle factors on male fertility[J]. Indian J Pathol Microbiol,2010;53(1):35-40.
3. Majumdar, S. S. and Bhattacharya I. Genomic and post-genomic leads toward regulation of spermatogenesis[J]. Prog Biophys Mol Biol,2013;Jan 31.[Epub ahead of print]PMID:23375691.
4.张正果.遗传性病因致男性不育的研究进展[J].中国男科学杂志,2010；24(10)：69-72.
5. Nanassy, L. and Carrell D. T. Abnormal methylation of the promoter of CREM is broadly associated with male factor infertility and poor sperm quality but is improved in sperm selected by density gradient centrifugation[J]. Fertil Steril,2011; 95(7):2310-2314.
6. Marques, C. J., Francisco T., Sousa S., et al. Methylation defects of imprinted genes in human testicular spermatozoa[J]. Fertil Steril,2010; 94(2):585-594.
7. Poplinski, A., Tuttelmann F., Kanber D., et al. Idiopathic male infertility is strongly associated with aberrant methylation of MEST and IGF2/H19 ICR1[J]. Int J Androl,2010;33(4):642-649.
8. Navarro-Costa, P., Nogueira P., Carvalho M., et al. Incorrect DNA methylation of the DAZL promoter CpG island associates with defective human sperm[J]. Hum Reprod,2010;25(10):2647-2654.
9. Conen, D., Everett B. M., Kurth T., et al. Smoking, smoking cessation, [corrected] and risk for symptomatic peripheral artery disease in women:a cohort study[J]. Ann Intern Med,2011;154(11):719-726.
10. Rea, T. D., Heckbert S. R., Kaplan R. C., et al. Smoking status and risk for recurrent coronary events after myocardial infarction[J]. Ann Intern Med, 2002; 137(6):494-500.
11. Braber, S., Henricks P. A., Nijkamp F. P., et al. Inflammatory changes in the airways of mice caused by cigarette smoke exposure are only partially reversed after smoking cessation[J]. Respir Res,2010; 11:99.
12.Bouloukaki, I., Tsiligianni I. G., Tsoumakidou M., et al. Sputum and nasal lavage lung-specific biomarkers before and after smoking cessation[J]. BMC Pulm Med,2011; 11:35.
13. Louhelainen, N., Stark H., Mazur W., et al. Elevation of sputum matrix metalloproteinase-9 persists up to 6 months after smoking cessation:a research study [J]. BMC Pulm Med,2010; 10:13.
14. Luo, J., Margolis K. L., Wactawski-Wende J., et al. Association of active and passive smoking with risk of breast cancer among postmenopausal women:a prospective cohort study [J]. BMJ,2011;342:d1016.
15. Vineis, P., Alavanja M., Buffler P., et al. Tobacco and cancer:recent epidemiological evidence[J]. J Natl Cancer Inst,2004; 96(2):99-106.
16. Petros, W. P., Younis I. R., Ford J. N., et al. Effects of Tobacco Smoking and Nicotine on Cancer Treatment[J]. Pharmacotherapy,2012; 32(10):920-31.
17. D'Addario, C., Francesco A. D., Pucci M., et al. Epigenetic mechanisms and endocannabinoid signalling[J]. FEBS J,2013; Jan 11.[Epub ahead of print]PMID:23305292.
18. Breitling, L. P., Yang R., Korn B., et al. Tobacco-smoking-related differential DNA methylation:27K discovery and replication[J]. Am J Hum Genet,2011; 88(4):450-457.
19. Wan, E. S., Qiu W., Baccarelli A., et al. Cigarette smoking behaviors and time since quitting are associated with differential DNA methylation across the human genome[J]. Hum Mol Genet,2012; 21(13):3073-3082.
20. Pop, C. and Salvesen G S. Human caspases:activation, specificity, and regulation[J]. J Biol Chem,2009;284(33):21777-21781.
21. Soung, Y. H., Lee J. W., Kim S. Y, et al. Somatic mutations of CASP3 gene in human cancers[J]. Hum Genet,2004;115(2):112-115.
22.黄健清,梁红玲,张绪超,等.CASP3与Cleaved-CASP3在肺癌中的表达及意义[J].实用医学杂志,2012；28(8)：1247-1250.
23.倪勤,刘冰,金明娟,等.CASP3基因多态及单体型分布与乳腺癌危险 性的关联研究[J].浙江大学学报(医学版),2011；40(3)：259-264.
24.邱广蓉,刘培燕,姜红垄,等.CASP3基因R101H多态性与法洛四联症的相关性[J].实用儿科临床杂志,2012；27(11)：856-858.
25. Lan, Q., Zheng T., Chanock S., et al. Genetic variants in caspase genes and susceptibility to non-Hodgkin lymphoma[J]. Carcinogenesis,2007; 28(4): 823-827.
26. Wang, D. H., Hu J. R., Wang L. Y, et al. The apoptotic function analysis of p53, Apafl, Caspase3 and Caspase7 during the spermatogenesis of the Chinese fire-bellied newt Cynops orientalis[J]. PLoS One,2012;7(6): e39920.
27. Almeida, C., Correia S., Rocha E., et al. Caspase signalling pathways in human spermatogenesis [J]. J Assist Reprod Genet,2013, Jan 29. [Epub ahead of print]PMID:23359247.
28. Cabrillana, M. E., Monclus M. A., Saez Lancellotti T. E., et al. Characterization of flagellar cysteine-rich sperm proteins involved in motility, by the combination of cellular fractionation, fluorescence detection, and mass spectrometry analysis[J]. Cytoskeleton (Hoboken),2011; 68(9): 491-500.
29. 王勇.精子鞭毛相关蛋白在人精子中的表达特征及其临床意义[D].汕头大学,2009.
30. Chen, J., Wang Y., Xu X., et al. Differential expression of ODF1 in human ejaculated spermatozoa and its clinical significance[J]. Zhonghua Nan Ke Xue,2009; 15(10):891-894.
31. Yang, K., Meinhardt A., Zhang B., et al. The small heat shock protein ODF1/HSPB10 is essential for tight linkage of sperm head to tail and male fertility in mice[J]. Mol Cell Biol,2012; 32(1):216-225.
32. Portela, A. and Esteller M. Epigenetic modifications and human disease[J]. Nat Biotechnol,2010; 28(10):1057-1068.
33. Attwood, J. T., Yung R. L. and Richardson B. C. DNA methylation and the regulation of gene transcription[J]. Cell Mol Life Sci,2002;59(2):241-257.
1. Egger, G., Liang G., Aparicio A., et al. Epigenetics in human disease and prospects for epigenetic therapy[J]. Nature,2004;429(6990):457-463.
2. Hales, B. F., Grenier L., Lalancette C., et al. Epigenetic programming:from gametes to blastocyst[J]. Birth Defects Res A Clin Mol Teratol,2011; 91(8): 652-665.
3. Katari, S., Turan N., Bibikova M., et al. DNA methylation and gene expression differences in children conceived in vitro or in vivo[J]. Hum Mol Genet,2009; 18(20):3769-3778.
4. Vanyushin, B. F. A view of an elemental naturalist at the DNA world (base composition, sequences, methylation)[J]. Biochemistry (Mosc),2007; 72(12): 1289-1298.
5. Hammoud, S. S., Purwar J., Pflueger C., et al. Alterations in sperm DNA methylation patterns at imprinted loci in two classes of infertility [J]. Fertil Steril,2010; 94(5):1728-1733.
6. Bird, A. DNA methylation patterns and epigenetic memory[J]. Genes Dev, 2002; 16(1):6-21.
7. Okano, M., Bell D. W., Haber D. A., et al. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development[J]. Cell,1999; 99(3):247-257.
8. Goll, M. G. and Bestor T. H. Eukaryotic cytosine methyltransferases[J]. Annu Rev Biochem,2005; 74:481-514.
9. Marandel, L., Labbe C., Bobe J., et al. nanog 5'-upstream sequence, DNA methylation, and expression in gametes and early embryo reveal striking differences between teleosts and mammals[J]. Gene,2012; 492(1):130-137.
10. Kreth, S., Thon N., Eigenbrod S., et al. O-methylguanine-DNA methyltransferase (MGMT) mRNA expression predicts outcome in malignant glioma independent of MGMT promoter methylationfJ]. PLoS One,2011; 6(2):e17156.
11. Kaneko, R., Kawaguchi M., Toyama T., et al. Expression levels of Protocadherin-alpha transcripts are decreased by nonsense-mediated mRNA decay with frameshift mutations and by high DNA methylation in their promoter regions[J]. Gene,2009;430(1-2):86-94.
12. Gokul, G, Gautami B., Malathi S., et al. DNA methylation profile at the DNMT3L promoter:a potential biomarker for cervical cancer[J]. Epigenetics,2007; 2(2):80-85.
13. Gupta, S., Pathak R. U. and Kanungo M. S. DNA methylation induced changes in chromatin conformation of the promoter of the vitellogenin II gene of Japanese quail during aging[J]. Gene,2006; 377:159-168.
14. Ozdemir, O., Bulut H. E., Korkmaz M., et al. Increased cell proliferation and R.Msp1 fragmentation induced by 5-aza-2'-deoxycytidine in rat testes related to the gene imprinting mechanism[J]. Exp Toxicol Pathol,2000; 52(4):317-322.
15. Santenard, A. and Torres-Padilla M. E. Epigenetic reprogramming in mammalian reproduction:contribution from histone variants[J]. Epigenetics, 2009; 4(2):80-84.
16. Lukova, M., Todorova A., Todorov T., et al. Different methylation patterns in BWS/SRS cases clarified by MS-MLPA[J]. Mol Biol Rep,2013;40(1): 263-268.
17. van Montfoort, A. P., Hanssen L. L., de Sutter P., et al. Assisted reproduction treatment and epigenetic inheritance[J]. Hum Reprod Update, 2012; 18(2):171-197.
18. Kallen, B., Finnstrom O., Nygren K. G., et al. In vitro fertilization (IVF) in Sweden:risk for congenital malformations after different IVF methods[J]. Birth Defects Res A Clin Mol Teratol,2005; 73(3):162-169.
19. Tanasijevic, B., Dai B., Ezashi T., et al. Progressive accumulation of epigenetic heterogeneity during human ES cell culture[J]. Epigenetics,2009; 4(5):330-338.
20. Wagner, W. Implications of long-term culture for mesenchymal stem cells: genetic defects or epigenetic regulation?[J].Stem Cell Res Ther,2012;3(6): 54.
21. Hansen, M., Milne E., de Klerk N., et al. ART, birth defects and subfertility--what should prospective patients be told?[J]. J Assist Reprod Genet,2011; 28(12):1229-1230.
22. Favaedi, R., Shahhoseini M. and Akhoond M. R. Comparative epigenetic analysis of Oct4 regulatory region in RA-induced differentiated NT2 cells under adherent and non-adherent culture conditions[J]. Mol Cell Biochem, 2012; 363(1-2):129-134.
23. Minor, A., Chow V. and Ma S. Aberrant DNA methylation at imprinted genes in testicular sperm retrieved from men with obstructive azoospermia and undergoing vasectomy reversal[J]. Reproduction,2011; 141(6): 749-757.
24. Huh, J. H., Bauer M. J., Hsieh T. F., et al. Cellular programming of plant gene imprinting[J]. Cell,2008;132(5):735-744.
25. Katz, D. J., Edwards T. M., Reinke V., et al. A C. elegans LSD1 demethylase contributes to germline immortality by reprogramming epigenetic memory[J]. Cell,2009;137(2):308-320.
26. Okada, Y., Scott G, Ray M. K., et al. Histone demethylase JHDM2A is critical for Tnpl and Prml transcription and spermatogenesis[J]. Nature, 2007; 450(7166):119-123.
27. Isidori, A. M., Pozza C., Gianfrilli D., et al. Medical treatment to improve sperm quality[J]. Reprod Biomed Online,2006; 12(6):704-714.
28. Matzuk, M. M. and Lamb D. J. The biology of infertility:research advances and clinical challenges[J]. Nat Med,2008;14(11):1197-1213.
29. Aoki, V. W., Christensen G. L., Atkins J. F., et al. Identification of novel polymorphisms in the nuclear protein genes and their relationship with human sperm protamine deficiency and severe male infertility [J]. Fertil Steril,2006; 86(5):1416-1422.
30. Hammoud, S., Emery B. R., Dunn D., et al. Sequence alterations in the YBX2 gene are associated with male factor infertility [J]. Fertil Steril,2009; 91(4):1090-1095.
31. Aston, K. I. and Carrell D. T. Genome-wide study of single-nucleotide polymorphisms associated with azoospermia and severe oligozoospermia[J]. J Androl,2009; 30(6):711-725.
32. Steel, A. J. and Sutcliffe A. Long-term health implications for children conceived by IVF/ICSI[J]. Hum Fertil (Camb),2009;12(1):21-27.
33. Reefhuis, J., Honein M. A., Schieve L. A., et al. Assisted reproductive technology and major structural birth defects in the United States [J]. Hum Reprod,2009; 24(2):360-366.
34. Yan, J., Huang G, Sun Y., et al. Birth defects after assisted reproductive technologies in China:analysis of 15,405 offspring in seven centers (2004 to 2008)[J]. Fertil Steril,2011; 95(1):458-460.
35. Kalra, S. K. and Molinaro T. A. The association of in vitro fertilization and perinatal morbidity[J]. Semin Reprod Med,2008; 26(5):423-435.
36. Odom, L. N. and Segars J. Imprinting disorders and assisted reproductive technology[J]. Curr Opin Endocrinol Diabetes Obes,2010; 17(6):517-522.
37. Manipalviratn, S., DeCherney A. and Segars J. Imprinting disorders and assisted reproductive technology [J]. Fertil Steril,2009; 91(2):305-315.
38. Dasoula, A., Georgiou I., Kontogianni E., et al. Methylation status of the SNRPN and HUMARA genes in testicular biopsy samples[J]. Fertil Steril, 2007; 87(4):805-809.
39. Poplinski, A., Tuttelmann F., Kanber D., et al. Idiopathic male infertility is strongly associated with aberrant methylation of MEST and IGF2/H19 ICR1[J]. Int J Androl,2010;33(4):642-649.
40. Dada, R., Kumar M., Jesudasan R., et al. Epigenetics and its role in male infertility [J]. J Assist Reprod Genet,2012;29(3):213-223.
41. Kobayashi, H., Hiura H., John R. M., et al. DNA methylation errors at imprinted loci after assisted conception originate in the parental sperm[J]. Eur J Hum Genet,2009;17(12):1582-1591.
42. Carrell, D. T. Epigenetics of the male gamete[J]. Fertil Steril,2012; 97(2): 267-274.
43. Seisenberger, S., Andrews S., Krueger F., et al. The dynamics of genome-wide DNA methylation reprogramming in mouse primordial germ cells[J]. Mol Cell,2012;48(6):849-862.
44. Hyldig, S. M., Ostrup O., Vejlsted M., et al. Changes of DNA methylation level and spatial arrangement of primordial germ cells in embryonic day 15 to embryonic day 28 pig embryos[J]. Biol Reprod,2011; 84(6):1087-1093.
45. Jang, H. J., Lee M. O., Kim S., et al. Biallelic expression of the L-arginine:glycine amidinotransferase gene with different methylation status between male and female primordial germ cells in chickens[J]. Poult Sci, 2013; 92(3):760-769.
46. Pathak, S., D'Souza R., Ankolkar M., et al. Potential role of estrogen in regulation of the insulin-like growth factor2-H19 locus in the rat testis[J]. Mol Cell Endocrinol,2010; 314(1):110-117.
47. Hammoud, S. S., Cairns B. R. and Carrell D. T. Analysis of gene-specific and genome-wide sperm DNA methylation[J]. Methods Mol Biol,2013; 927: 451-458.
48. Pacheco, S. E., Houseman E. A., Christensen B. C., et al. Integrative DNA methylation and gene expression analyses identify DNA packaging and epigenetic regulatory genes associated with low motility sperm[J]. PLoS One,2011; 6(6):e20280.
49. Marques, C. J., Francisco T., Sousa S., et al. Methylation defects of imprinted genes in human testicular spermatozoa[J]. Fertil Steril,2010; 94(2):585-594.
50. Nanassy, L. and Carrell D. T. Analysis of the methylation pattern of six gene promoters in sperm of men with abnormal protamination[J]. Asian J Androl,2011;13(2):342-346.
51. Nanassy, L. and Carrell D. T. Paternal effects on early embryogenesis[J]. J Exp Clin Assist Reprod,2008; 5:2.
52. Carrell, D. T. Elucidating the genetics of male infertility:understanding transcriptional and translational regulatory networks involved in spermatogenesis[J]. Int J Androl,2008; 31(5):455-456.
53. Yamashita, S., Hosoya K., Gyobu K., et al. Development of a novel output value for quantitative assessment in methylated DNA immunoprecipitation-CpG island microarray analysis[J]. DNA Res,2009; 16(5):275-286.
54. Kagami, M., Matsuoka K., Nagai T., et al. Paternal uniparental disomy 14 and related disorders:placental gene expression analyses and histological examinations [J]. Epigenetics,2012; 7(10):1142-1150.
55. Mitalipov, S., Clepper L., Sritanaudomchai H., et al. Methylation status of imprinting centers for H19/IGF2 and SNURF/SNRPN in primate embryonic stem cells[J]. Stem Cells,2007;25(3):581-588.
56. Wang, M., Zhang X., Kang L., et al. Molecular characterization of porcine NECD, SNRPN and UBE3A genes and imprinting status in the skeletal muscle of neonate pigs[J]. Mol Biol Rep,2012; 39(10):9415-9422.
57. Peng, H. H., Chang S. D., Chao A. S., et al. DNA methylation patterns of imprinting centers for H19, SNRPN, and KCNQ1OT1 in single-cell clones of human amniotic fluid mesenchymal stem cell[J]. Taiwan J Obstet Gynecol, 2012; 51(3):342-349.
58. Inawaka, K., Kawabe M., Takahashi S., et al. Maternal exposure to anti-androgenic compounds, vinclozolin, flutamide and procymidone, has no effects on spermatogenesis and DNA methylation in male rats of subsequent generations[J]. Toxicol Appl Pharmacol,2009;237(2):178-187.
59. Ouko, L. A., Shantikumar K., Knezovich J., et al. Effect of alcohol consumption on CpG methylation in the differentially methylated regions of H19 and IG-DMR in male gametes:implications for fetal alcohol spectrum disorders[J]. Alcohol Clin Exp Res,2009;33(9):1615-1627.
60. WHO. WHO laboratory manual for the examination and processing of human semen[M].5th ed. Geneva:World Health Organization,2010.