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Advanced paternal age at fertilization has been suggested to be a risk factor for neurodevelopmental, psychiatric and other disorders in offspring. One emerging hypothesis suggests that altered offspring phenotype is linked with age-related accumulation of epigenetic changes in the sperm of fathers. Given that paternal age is increasing in the developed world, understanding aging-related epigenetic changes in sperm is needed as well as environmental factors that modify such changes. In this study, we characterize age-dependent changes in sperm DNA methylation profiles between young pubertal (postnatal day (PNDs) 65) and mature (PND120) Wistar rats. We also analyze these changes in rats exposed perinatally to 0.2 mg/kg of ubiquitous environmental xenobiotic 2,2’,4,4’-tetrabromodiphenyl ether (BDE-47). Reduced representation bisulfite sequencing (RRBS) libraries were prepared from caudal epididymal sperm DNA and differentially methylated regions (DMRs; ≥ 10x coverage depth, ≥ 3 CpGs per cluster, ≥ 5% methylation change, q < 0.05) were identified via MethPipe package. In control animals, 5,319 age-dependent DMRs were identified, with 99.3% DMRs hypermethylated in mature animals compared to young pubertal rats. These age-related DMRs were enriched for functional categories essential for embryonic development, such as pattern specification, forebrain and sensory organ development, Hippo and Wnt pathways. Age-related changes in sncRNA, reported in different study, target similar list of genes and biological categories.In BDE-47 exposed rats, sperm DNA methylation was higher in young pubertal and lower in mature animals when compared to controls, which resulted in a significant attenuation in the number of age-dependent DMRs (N = 189) identified in the exposed group. In conclusion, our results indicate that the natural aging process has profound effects on sperm methylation levels and this effect may be modified by environmental exposures. Moreover, our results further support the role of epigenetic mechanisms as a likely link betwen paternal age and offspring health and development.

Study question Are preconception phthalate and phthalate replacements associated with sperm differentially methylated regions (DMRs) among men undergoing IVF? Summary answer Ten phthalate metabolites were associated with 131 sperm DMRs that were enriched in genes related to growth and development, cell movement and cytoskeleton structure. What is known already Several phthalate compounds and their metabolites are known endocrine disrupting compounds and are pervasive environmental contaminants. Rodent studies report that prenatal phthalate exposures induce sperm DMRs, but the influence of preconception phthalate exposure on sperm DNA methylation in humans is unknown. Study design, size, duration An exploratory cross-sectional study with 48 male participants from the Sperm Environmental Epigenetics and Development Study (SEEDS). Participants/materials, setting, methods The first 48 couples provided a spot urine sample on the same day as semen sample procurement. Sperm DNA methylation was assessed with the HumanMethylation 450 K array. Seventeen urinary phthalate and 1,2-Cyclohexane dicarboxylic acid diisononyl ester (DINCH) metabolite concentrations were measured from spot urine samples. The A-clust algorithm was employed to identify co-regulated regions. DMRs associated with urinary metabolite concentrations were identified via linear models, corrected for false discovery rate (FDR). Main results and role of chance Adjusting for age, BMI, and current smoking, 131 DMRs were associated with at least one urinary metabolite. Most sperm DMRs were associated with anti-androgenic metabolites, including mono(2-ethylhexyl) phthalate (MEHP, n = 83), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP, n = 16), mono-n-butyl phthalate (MBP, n = 22) and cyclohexane-1,2-dicarboxylic acid-monocarboxy isooctyl (MCOCH, n = 7). The DMRs were enriched in lincRNAs as well as in regions near coding regions. Functional analyses of DMRs revealed enrichment of genes related to growth and development as well as cellular function and maintenance. Finally, 13% of sperm DMRs were inversely associated with high quality blastocyst-stage embryos after IVF. Limitations, reasons for caution Our modest sample size only included 48 males and additional larger studies are necessary to confirm our observed results. Non-differential misclassification of exposure is also a concern given the single spot urine collection. Wider implications of the findings To our knowledge, this is the first study to report that preconception urinary phthalate metabolite concentrations are associated with sperm DNA methylation in humans. These results suggest that paternal adult environmental conditions may influence epigenetic reprogramming during spermatogenesis, and in turn, influence early-life development. Study funding/competing interest(s) This work was supported by grant K22-ES023085 from the National Institute of Environmental Health Sciences. The authors declare no competing interests.

Perinatal exposures to polybrominated diphenyl ethers permanently reprogram liver metabolism and induce a nonalcoholic fatty liver disease-like phenotype and insulin resistance in rodents. Aim: To test if these changes are associated with altered liver epigenome. Materials & methods: Expression of small RNA and changes in DNA methylation in livers of adult rats were analyzed following perinatal exposure to 2,2′,4,4′-tetrabromodiphenyl ether, the polybrominated diphenyl ether congener most prevalent in human tissues. Results: We identified 33 differentially methylated DNA regions and 15 differentially expressed miRNAs. These changes were enriched for terms related to lipid and carbohydrate metabolism, insulin signaling, Type-2 diabetes and nonalcoholic fatty liver disease. Conclusion: Changes in the liver epigenome are a likely candidate mechanism of long-term maintenance of an aberrant metabolic phenotype.

DNA methylation is a crucial regulatory mechanism of gene expression, affected in many human pathologies. Therefore, it is not surprising that nowadays, in the era of high-throughput methods, a lot of data sets representing DNA methylation in various conditions are available and the amount of such data keeps growing. In this chapter, we discuss those aspects of experiment planning and data analysis, which we consider the most important for reliability and reproducibility of DNA methylation studies: usage of replicates, data quality control at various stages, selection of a statistical model, and incorporation of DNA methylation into the multi-omics analysis.

DNA methylation is a crucial regulatory mechanism of gene expression, affected in many human pathologies. Therefore, it is not surprising that nowadays, in the era of high-throughput methods, a lot of data sets representing DNA methylation in various conditions are available and the amount of such data keeps growing. In this chapter, we discuss those aspects of experiment planning and data analysis, which we consider the most important for reliability and reproducibility of DNA methylation studies: usage of replicates, data quality control at various stages, selection of a statistical model, and incorporation of DNA methylation into the multi-omics analysis.

Background The association of exposure to endocrine disrupting chemicals in the peripubertal period with subsequent sperm DNA methylation is unknown. Objective We examined the association of peripubertal serum 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) concentrations with whole-genome bisulfite sequencing (WGBS) of sperm collected in young adulthood. Methods The Russian Children’s Study is a prospective cohort of 516 boys who were enrolled at 8–9 years of age and provided semen samples at 18–19 years of age. WGBS of sperm was conducted to identify differentially methylated regions (DMR) between highest (n = 4) and lowest (n = 4) peripubertal TCDD groups. Results We found 52 DMRs that distinguished lowest and highest peripubertal serum TCDD concentrations. One of the top scoring networks, “Cellular Assembly and Organization, Cellular Function and Maintenance, Carbohydrate Metabolism”, identified estrogen receptor alpha as its central regulator. Conclusion Findings from our limited sample size suggest that peripubertal environmental exposures are associated with sperm DNA methylation in young adults.

Polybrominated diphenyl ethers (PBDEs) are a group of ubiquitous reproductive toxins. Given that spermatogenesis requires extensive epigenetic changes, we hypothesize that PBDEs impact sperm DNA methylation. Pregnant Wistar rats were exposed perinatally to 0.2mg/kg 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and caudal epididymal sperm were collected from offspring on postnatal days (PNDs) 65 and 120. Libraries were prepared from sperm DNA and sequenced with an average of 18.0 million unique reads per sample. Differential methylated regions (DMRs) were identified via MethPipe package. BDE-47 exposure increased DNA methylation of epididymal sperm on PND 65 in genes, promoters and intergenic regions; however, on PND120 methylation decreased in these genomic elements. We identified 21 and 9 exposure-related DMRs in sperm collected on PND65 and PND120, respectively. Two DMRs overlapped between the two time-points. This is the first study to demonstrate that environmentally-relevant perinatal exposure to PBDE results in long-lasting changes in sperm DNA methylation.