Urine Mercury Levels Correlate With Sperm DNA Methylation Of Imprinting Genes

ObjectiveMercury(Hg)is a well-recognized environmental pollutant known by its toxicity of development and neurotoxicity.In the body,Hg not only crosses placental barrier to directly damage the early embryonic development,but also readily crosses the blood-testis barrier to inhibit spermatogenesis and decrease sperm capacitation.Although some studies have revealed an association of paternal Hg exposure with adverse pregnant outcome,but the underlying mechanism has not been well understood.Imprinting is a specific epigenetic phenomenon,playing a key role on fetal and postnatal growth and development.Recently,increasing literature with respect to environmental influences on a bunch of imprinted genes suggests that DNA methylation of imprinting genes are sensitive to teratogenic agents,such as alcohol,tobacco,and endocrine disruptor bisphenol A(BPA).However,there is no research about paternal exposure to Hg and DNA methylation of imprinting genes in human sperm.Epidemiological investigation and molecular biology laboratory analysis were employed to investigate the non-occupational paternal Hg exposure levels and examine the association between paternal Hg exposures and the alteration of DNA methylation of imprinting genes H19,Meg3 and Peg3 in human sperm to provide the theory basis for the primary prevention of birth defects through exploring the possible paternal epigenetic mechanisms.Methods1.Epidemiology investigationA total of 618 men were recruited from reproductive Medicine Clinic of Maternal and Child Care Service Center and Andrology Clinic of Shanxi Academy of Medical Sciences during April 2015 and March-April 2016.The participants including those either pre-pregnant check or premarital check-up were asked to complete a questionnaireincluding age,education,individuals or family history of birth defects and of inherited diseases,smoking and drinking and dietary preference.2.Biological samples collecting and measuring2.1 Paternal midstream urine was collected for measuring the mercury concentration by using a fully-automatic double-channel hydride generation atomic fluorescence spectrometer AFS-9700.2.2 Genomic DNA was extracted from sperm by using modified guanidinium thiocyanate method and bisulfite pyrosequencing was employed to examine the DNA methylation of imprinting genes H19,Peg3 and Meg3 of sperm DNA.3.Statistical analysesEstablish the database used Epi Data3.1 with the method of parallel double input data.Statistical description and analysis the data used SPSS19.0.Nonparametric test was used to compare the median levels of Hg between two groups or multiple social-demographic groups,respectively.Spearman's rank correlation analysis was used to determine the correlation between DNA methylation status of 3 imprinting genes and urinary Hg concentrations,and multivariate regression models for further confirming the relationship between internal urinary Hg and methylation levels of the imprinting genes in sperm DNA under controlling potential confounders.Results1.Non-occupational paternal mercury exposure levels of the respondentsIn the present study,although the participants are non-occupational Hg exposure,the median concentration of urinary mercury in these male samples were 9.14 ?g/l(IQR: 5.56-12.52 ?g/l;range = 0.16-71.35 ?g/l).Moreover,a total of 42.7% of these participants are beyond normal level for non-occupational exposure according to the criterion of Hg poisoning(urinary Hg concentration ? 10 ?g/l).The results from nonparametric test indicate no significant difference in median concentrations of Hg among different demographic groups.2.The relationship between urinary mercury levels and DNA methylation of imprinting genes H19,Peg3 and Meg3 in sperm2.1 Spearman's rank correlation analysis indicated that the average DNA methylationlevels of H19 were negatively correlated with urinary Hg concentrations.However,there was no such a correlation found in genes of Peg3 and Meg3 in sperm DNA.2.2 After adjusting age,smoking,drinking,intake of aquatic products and education by multivariate regression analysis,the results have indicated a significant negatively correlated between urinary Hg concentrations and the DNA methylation levels of H19,indicating that an increasing concentration of fathers urinary Hg is an individual relationship with demethylation of H19 in sperm DNA.Conclusions1.Non-occupational paternal mercury exposure levels are at higher levels;2.Mercury non-occupational environmental exposure in men associated with demethylation imprinting gene H19 in sperm DNA,implicating the susceptibility of the sperm DNA epigenetic modification for environmental insults.