Human exposure to polycyclic aromatic hydrocarbons (PAHs) results in adverse health implications. However, the specific impact of paternal preconception PAHs exposure has not been fully studied. In this study, a total of 219 men aged 24–53 were recruited and an investigation was conducted using a questionnaire requesting information about age, occupation, education, family history, lifestyle, and dietary preferences. Urine and semen samples were examined for the levels of the hydroxyl metabolites of PAHs (OH-PAHs) using ultra-high-performance liquid chromatography–tandem mass spectrometry and sperm DNA methylation by pyrosequencing. The results from the correlation analysis using seven OH-PAHs and the average methylation levels of the imprinting genes H19, PEG3, and MEG3 indicated that 1-OHPH is positively correlated with H19/PEG3 methylation levels. We further examined the correlation between each OH-PAH and the methylation levels at the individual CpGs. The results showed 1-OHPH is specifically correlated with CpG4 and CpG6 of the imprinted gene H19, CpG1 and CpG2 of PEG3, and CpG2 of MEG3; whereas 1-OHP is positively correlated with PEG3 at CpG1. Multivariate regression model analysis confirmed that 1-OHPH and 1-OHP are independent risk factors for the methylation of H19. These data show that sperm DNA imprinting genes are sensitive to adverse environmental perturbations.

BackgroundMercury (Hg) is a well-recognized environmental pollutant known by its toxicity of development and neurotoxicity, which results in adverse health outcomes. However, the mechanisms underlying the teratogenic effects of Hg are not well understood. Imprinting genes are emerging regulators for fetal development subjecting to environmental pollutants impacts. In this study, we examined the association between preconceptional Hg exposure and the alteration of DNA methylation of imprinting genes H19, Meg3, and Peg3 in human sperm DNA.MethodsA total of 616 men, aged from 22 to 59, were recruited from Reproductive Medicine Clinic of Maternal and Child Care Service Center and the Urologic Surgery Clinic of Shanxi Academy of Medical Sciences during April 2015 and March 2016. Demographic information was collected through questionnaires. Urine was collected and urinary Hg concentrations were measured using a fully-automatic double-channel hydride generation atomic fluorescence spectrometer. Methylation of imprinting genes H19, Meg3 and Peg3 of sperm DNA from 242 participants were examined by bisulfite pyrosequencing. Spearman’s rank and multivariate regression analysis were used for correlation analysis between sperm DNA methylation status of imprinting genes and urinary Hg levels.ResultsThe median concentration of Hg for 616 participants was 9.14μg/l (IQR: 5.56–12.52 μg/l; ranging 0.16–71.35μg/l). A total of 42.7% of the participants are beyond normal level for non-occupational exposure according to the criterion of Hg poisoning (≥10 μg/L). Spearman’s rank analysis indicated a negative correlation between urinary Hg concentrations and average DNA methylation levels of imprinted genes H19 (rs = −0.346, p <0.05), but there was no such a correlation for Peg3 and Meg3. Further, we analyzed the correlation between methylation level at individual CpG site of H19 and urinary Hg level. The results showed a negative correlation between urinary Hg concentrations and three out of seven CpG sites on H19 DMR, namely CpG2 (rs = −0.137, p <0.05), CpG4 (rs = −0.380, p <0.05) and CpG6 (rs = −0.228, p <0.05). After adjusting age, smoking, drinking, intake of aquatic products and education by multivariate regression analysis, the results have confirmed the correlation as mentioned above.ConclusionsMercury non-occupational environmental exposure in reproductive-aged men was associated with altered DNA methylation outcomes at imprinting gene H19 in sperm, implicating the susceptibility of the developing sperm for environmental insults.

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