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Male fertility has been declining worldwide especially in countries with high levels of endocrine disrupting chemicals (EDCs). Per- and polyfluorinated alkyl Substances (PFAS) have been classified as EDCs and have been linked to adverse male reproductive health. The mechanisms of these associations and their implications on offspring health remain unknown. The aims of the current study were to assess the effect of PFAS mixtures on the sperm methylome and transcriptional changes in offspring metabolic tissues (i.e., liver and fat). C57BL/6 male mice were exposed to a mixture of PFAS (PFOS, PFOA, PFNA, PFHxS, Genx; 20 µg/L each) for 18-weeks or water as a control. Genome-wide methylation was assessed on F0 epidydimal sperm using reduced representation bisulfite sequencing (RRBS) and Illumina mouse methylation array, while gene expression was assessed by bulk RNA sequencing in 8-week-old offspring derived from unexposed females. PFAS mixtures resulted in 2,861 (RRBS) and 83 (Illumina) sperm DMRs (q < 0.05). Functional enrichment revealed that PFAS-induced sperm DMRs were associated with behavior and developmental pathways in RRBS, while Illumina DMRs were related to lipid metabolism and cell signaling. Additionally, PFAS mixtures resulted in 40 and 53 differentially expressed genes (DEGs) in the liver and fat of males, and 9 and 31 DEGs in females, respectively. Functional enrichment of DEGs revealed alterations in cholesterol metabolism and mitotic cell cycle regulation in the liver and myeloid leukocyte migration in fat of male offspring, while in female offspring, erythrocyte development and carbohydrate catabolism were affected in fat. Our results demonstrate that exposure to a mixture of legacy and newly emerging PFAS chemicals in adult male mice result in aberrant sperm methylation and altered gene expression of offspring liver and fat in a sex-specific manner. These data indicate that preconception PFAS exposure in males can be transmitted to affect phenotype in the next generation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Preconception environmental conditions have been demonstrated to shape sperm epigenetics and subsequently offspring health and development. Our previous findings in humans showed that urinary anti-androgenic phthalate metabolites in males were associated with altered sperm methylation and blastocyst-stage embryo development. To corroborate this, we examined the effect of preconception exposure to di(2-ethylhexyl) phthalate (DEHP) on genome-wide DNA methylation and gene expression profiles in mice. Eight-week old C57BL/6J male mice were exposed to either a vehicle control, low, or high dose of DEHP (2.5 and 25 mg/kg/weight, respectively) for 67 days (~2 spermatogenic cycles) and were subsequently mated with unexposed females. Reduced representation bisulfite sequencing (RRBS) of epididymal sperm was performed and gastrulation stage embryos were collected for RRBS and transcriptome analyses in both embryonic and extra-embryonic lineages. Male preconception DEHP exposure resulted in 704 differentially methylated regions (DMRs; q-value < 0.05; ≥10% methylation change) in sperm, 1,716 DMRs in embryonic, and 3,181 DMRs in extra-embryonic tissue. Of these, 29 DMRs overlapped between sperm and F1 tissues, half of which showed concordant methylation changes between F0 and F1 generations. F1 transcriptomes at E7.5 were also altered by male preconception DEHP exposure including developmental gene families such as Hox, Gata, and Sox. Additionally, gene ontology analyses of DMRs and differentially expressed genes showed enrichment of multiple developmental processes including embryonic development, pattern specification and morphogenesis. These data indicate that spermatogenesis in adult may represent a sensitive window in which exposure to DEHP alters the sperm methylome as well as DNA methylation and gene expression in the developing embryo., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)