| ObjectiveIn recent years,fertility and developmental toxicity of environmental pollutants on the human body are receiving increasing attention.Polycyclic aromatic hydrocarbons(PAHs)are widespread pollutants in the environment.The impact of these compounds on embryonic development has attracted the attention of scholars from various countries.Previous studies have shown that PAHs are closely related to the occurrence of adverse birth outcomes.However,the mechanism of teratogenicity associated with PAHs is currently unclear.Genetic imprinting is a unique epigenetic regulation that is closely related to embryonic development.Although the most studies have been examining the effect of maternal exposure on fetal development,recent studies showed that PAHs-exposed male animals resulted in the abnormal structure and function of sperm and induced abnormal DNA methylation.However,there has been no report on the effect of paternal PAHs exposure on the methylation of sperm imprinted genes.Therefore,this study aimed to explore the association between male non-occupational PAHs exposure and methylation level of sperm DNA imprinting genes.This data will provide a theoretical basis for the study of the etiology of adverse birth outcomes through paternal exposure to PAHs,and provide a new insight for the prevention of birth defects.MethodsA total of 219 men were enrolled in this study from two Clinics of Health Reproductive Center in Shanxi Province during March-April,2015 and March,2016.The participants visited for the purpose either pre-pregnant check or family infertility examination.Upon enrollment,they were asked to complete a questionnaire including age,occupation,education,family history of birth defects or heredopathia;social-demographic factors,lifestyle,and dietary preference.Urine samples were collected and the level of hydroxy metabolites(OH-PAHs)concentrations was determined by ultra high performance liquid chromatography-tandem mass spectrometry(LC-MS/MS).Semen was collected on site by masturbation without the use of lubricants into a sterile polypropylene collection container.The sperm DNA was extracted.After sulfite treatment,the methylation level was determined in the regions of H19,PEG3 and MEG3 by pyrosequencing.The data were analyzed using SPSS for Windows 22.0 for statistical analyses.The statistical methods included normality test,two independent sample nonparametric tests,Spearman rank correlation,and multiple linear regressions.ResultsDemographic characteristics of the study participants : A total of 219 men were enrolled in this study.The range of age is between 24 ~ 53 years old,with 50% of the participants being between 30 to 35 years old.The educational level of 50% of participants(n = 158)is high school(including secondary specialized schools)or above.There were130 participants with three years of smoking experience.Only a few people(n=39)like to smoke food(more than once a week).Therefore,environmental PAH exposure is likely through the inhalation.There is no significant difference was found in 7 kind of urinary PAHs hydroxyl metabolites(OH-PAHs)with various demographic groups(P > 0.05).1.The distribution of Urine hydroxyl metabolites(OH-PAHs)among different demographic groups:In the current study,we have examined OH-PAHs in urine samples.After normalized by urine gravity to correct the influence derived from urinary concentration,the median level(25%-75%)of OH-PAHs is 2-OHN(0,0-0.428),1-OHN(0,0-0.016),3-OHF(0.004,0-0.039),2-OHF(0.023,0-0.069),2-OHPH(0.129,0-0.319),1-OHPH(0.015,0-0.052),and 1-OHP(0.010,0-0.058),respectively.2.The levels of sperm DNA methylation of imprinting genes: Three imprinting genes,namely H19,PEG3 and MEG3 were selected and measured in this study.The average level of methylation are as follow,H19: 86.74 %(95% CI: 86.05,87.42);PEG3:0.5569%(95% CI: 0.2855,0.8283);and MEG3: 2.267 %(95% CI: 1.685,2.850).3.The correlation analysis between the level of urinary OH-PAHs and the average level of sperm DNA imprinting genes methylation.To examine the relationship between OH-PAHs exposure and methylation level of imprinted gene,the correlation analysis was carried out using 7 OH-PAHs and the average methylation level of each gene.As it can be seen that 1-OHPH and H19 and PEG3 methylation levels were positively correlated with the correlation coefficients of 0.189 and 0.909,respectively.Furthermore,Spearman rank correlation was used to analyze the correlation between the OH-PAHs and the methylation level of the imprinted genes.The results shows that 1-OHPH was positively correlated with the methylation levels of H19(coefficient,0.189)and PEG3(coefficient,0.909),respectively.Furthermore,in order to investigate the correlation between OH-PAHs and specific CpG sites of each gene,we also examined the correlation between each OH-PAHs and individual CpG.the result indicated that 1-OHPH was positively correlated with CpG1of the imprinted gene PEG3 and CpG2 of the imprinted gene MEG3,with correlation coefficients of 0.207 and 0.201,respectively;P<0.05;1-OHP and imprinted gene PEG3CpG1 was positively correlated,and the correlation coefficient was 0.156,P<0.05.4.Multiple linear regression analysis of factors influencing methylation level of sperm DNA imprinted genes.We performed a multiple linear regression analysis(Stepwise)on the mean level of the percentage of methylation of the imprinted genes.The potential confounders,such as age,education,drinking,smoking,smoking food intake were selected as an independent variable in the analysis with the seven OH-PAHs respectively.The results showed that the factors affecting the methylation level of the imprinted gene H19 include urinary 1-OHP and 1-OHPH levels,suggesting that 1-OHP and 1-OHPH levels are factors that affect the methylation level of the imprinted gene H19.The frequency of smoked food intake was associated with the methylation level of the imprinted gene PEG3,with a regression coefficient of 0.154.Conclusions1.A certain level of 7 urinary PAHs hydroxyl metabolites was detected in these participants.And there is no significant difference was found in 7 urinary OH-PAHs among different demographic groups.2.Mean methylation levels of urinary 1-OHPH positively correlated with the methylation levels of H19(4th and 6th CpG),PEG3(1st CpG)and MEG3(2st CpG).In addition,there was a positive correlation between 1-OHP and the first CpG of the imprinted gene PEG3,and the first CpG methylation level of PEG3 increased with the increase of 1-OHP level in urine.3.Factors that affect the methylation level of the imprinted gene H19 include urinary1-OHP and 1-OHPH levels,which illustrated that 1-OHP and 1-OHPH levels are independent factors that affect the level of methylation of the imprinted gene H19.The frequency of smoked food intake is a factor that affects the methylation level of the imprinted gene PEG3. |
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