On The Impact Of Smoking On Male Semen Parameters In Clinical Research And Smoking Male Sperm Genome Methylation Mutation Analysis

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.