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Environmental insults during germline epigenetic reprogramming may affect DNAme, presenting a potential mechanism for transmission of environmental exposures across multiple generations.<\/jats:p><\/jats:sec><jats:sec><jats:title>Objectives<\/jats:title><jats:p>We investigated how germ cell DNAme is impacted by lifetime exposures to diets containing either low or high, clinically relevant, levels of the methyl donor folic acid and whether resulting DNAme alterations were inherited in germ cells of male offspring of subsequent generations.<\/jats:p><\/jats:sec><jats:sec><jats:title>Materials and methods<\/jats:title><jats:p>Female mice were placed on a control (FCD), 7\u2010fold folic acid deficient (7FD) or\u00a010\u2010\u00a0to\u00a020\u2010fold supplemented (10FS and 20FS) diet before and during pregnancy. Resulting F1 litters were weaned on the respective diets. F2 and F3 males received control diets. Genome\u2010wide DNAme at cytosines (within CpG sites) was assessed in F1 spermatogonia, and in F1, F2 and F3 sperm.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>In F1 germ cells, a greater number of differentially methylated cytosines (DMCs) were observed in spermatogonia as compared with F1 sperm for all folic acid diets. DMCs were lower in number in F2 versus F1 sperm, while an unexpected increase was found in F3 sperm. DMCs were predominantly hypomethylated, with genes in neurodevelopmental pathways commonly affected in F1, F2 and F3 male germ cells. While no DMCs were found to be significantly inherited inter\u2010 or transgenerationally, we observed over\u2010representation of repetitive elements, particularly young long\u00a0interspersed\u00a0nuclear\u00a0elements\u00a0(LINEs).<\/jats:p><\/jats:sec><jats:sec><jats:title>Discussion and conclusion<\/jats:title><jats:p>These results suggest that the prenatal window is the time most susceptible to folate\u2010induced alterations in sperm DNAme in male germ cells. Altered methylation of specific sites in F1 germ cells was not present in later generations. However, the presence of DNAme perturbations in the sperm of males of the F2 and F3 generations suggests that epigenetic inheritance mechanisms other than DNAme may have been impacted by the folate diet exposure of F1 germ cells.<\/jats:p><\/jats:sec>","DOI":"10.1111\/andr.13399","type":"journal-article","created":{"date-parts":[[2023,1,26]],"date-time":"2023-01-26T03:54:43Z","timestamp":1674705283000},"page":"927-942","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":15,"title":["Transgenerational impact of grand\u2010paternal lifetime exposures to both folic acid deficiency and supplementation on genome\u2010wide DNA methylation in male germ cells"],"prefix":"10.1111","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6860-8384","authenticated-orcid":false,"given":"Donovan","family":"Chan","sequence":"first","affiliation":[{"name":"Research Institute of the McGill University Health Centre  Montreal QC Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lundi","family":"Ly","sequence":"additional","affiliation":[{"name":"Research Institute of the McGill University Health Centre  Montreal QC Canada"},{"name":"Department of Human Genetics McGill University  Montreal QC Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Edgar Mart\u00ednez Duncker","family":"Rebolledo","sequence":"additional","affiliation":[{"name":"Research Institute of the McGill University Health Centre  Montreal QC Canada"},{"name":"Department of Human Genetics McGill University  Montreal QC Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jos\u00e9e","family":"Martel","sequence":"additional","affiliation":[{"name":"Research Institute of the McGill University Health Centre  Montreal QC Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Myl\u00e8ne","family":"Landry","sequence":"additional","affiliation":[{"name":"Research Institute of the McGill University Health Centre  Montreal QC Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marie\u2010Pier","family":"Scott\u2010Boyer","sequence":"additional","affiliation":[{"name":"Centre de Recherche du CHU de Qu\u00e9bec\u2010Universit\u00e9 Laval  Qu\u00e9bec QC Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Arnaud","family":"Droit","sequence":"additional","affiliation":[{"name":"Centre de Recherche du CHU de Qu\u00e9bec\u2010Universit\u00e9 Laval  Qu\u00e9bec QC Canada"},{"name":"D\u00e9partement de M\u00e9decine Mol\u00e9culaire Universit\u00e9 Laval  Qu\u00e9bec QC Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4830-042X","authenticated-orcid":false,"given":"Jacquetta M.","family":"Trasler","sequence":"additional","affiliation":[{"name":"Research Institute of the McGill University Health Centre  Montreal QC Canada"},{"name":"Department of Human Genetics McGill University  Montreal QC Canada"},{"name":"Departments of Pediatrics and Pharmacology &amp; 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