study questionIs DNA methylation in mature spermatozoa affected by different culture media? SUMMARY ANSWER: Incubation of mature spermatozoa in hydrogen peroxide (H₂O₂) or palmitate for 24 h did not alter the DNA methylome. WHAT IS KNOWN ALREADY: Children conceived through ART face a higher risk of disorders linked to abnormal DNA methylation. As culture media can modulate DNA methylation in somatic cells, abnormal DNA methylation after ART may be caused by factors present in culture media during sperm incubation. STUDY DESIGN SIZE DURATION: Sperm from four donors underwent targeted shotgun proteomics analysis, focusing on protein size ranges that include epigenetic modifying enzymes. Additionally, sperm samples from 15 men were incubated for 24 h in either standard media, media with H₂O₂, or media containing palmitate, two media known to induce acute DNA methylation changes in somatic cells. DNA methylation profiles of these incubated sperm samples were then compared to those of pre-incubation sperm using Reduced Representation Bisulfite Sequencing (RRBS). PARTICIPANTS/MATERIALS SETTING

methodsSperm samples were obtained from 15 healthy Caucasian men aged 20-40 years. Participants using prescription medications daily or with a history of testicular disease were excluded. Motile sperm were isolated through the swim-up technique. Protein analysis was conducted via in-gel digestion followed by mass spectrometry. DNA methylation was evaluated using RRBS and validated for select genomic regions with targeted bisulfite pyrosequencing. MAIN RESULTS AND THE ROLE OF CHANCE: Proteomic analysis returned 1930 proteins, including 144 newly identified proteins. Consistent with previous studies, we confirmed the presence of DNA methyltransferase 1 (DNMT1), while LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: RRBS analysis covers only a portion of the genome, so DNA methylation changes in unexamined regions cannot be excluded, particularly at repeated regions of the genome. Moreover, since this study tested only three culture media conditions, it remains possible that other media formulations could impact DNA methylation. WIDER IMPLICATIONS OF THE

findingsThis study indicates that the DNA methylome of mature human spermatozoa is stable despite the presence of DNA methylation machinery components. These results imply that DNA methylation changes in response to environmental stress are more likely to happen during earlier stages of spermatogenesis. While the various culture media used in ART are unlikely to alter the DNA methylation patterns of individual sperm cells, the possibility that certain sperm with specific methylation signatures are selectively favored cannot be ruled out.

fundingThis work was supported by a Challenge Programme Grant from the Novo Nordisk Foundation (NNF18OC0033754) to the Gametic Epigenetics Consortium against Obesity (GECKO). The Novo Nordisk Foundation Center for Basic Metabolic Research is an independent research center at the University of Copenhagen, partially funded by an unrestricted donation from the Novo Nordisk Foundation (NNF18CC0034900). This work was supported by the French Government (National Research Agency, ANR) through the 'Investments for the Future' programs LABEX SIGNALIFE ANR-11-LABX-0028-01 and IDEX UCAJedi ANR-15-IDEX-01. DISCLOSURES: R.B. is a recipient of research funding granted by the Novo Nordisk Foundation. E.A. is a shareholder of ExSeed Health Ltd.