Differential Sperm DNA Methylation Profiles in Healthy Men With High Versus Low Adherence to the Mediterranean Diet in the Led‐Fertyl Study

BACKGROUND: Diet is known to play an essential role not only in sperm quality but also in offspring health, likely mediated through the sperm epigenome. Moreover, one of the main components of the Mediterranean diet (MedDiet) (i.e., nuts) have shown to impact sperm DNA methylation patterns. OBJECTIVES: This study aims to investigate the association between adherence to the MedDiet and sperm DNA methylation patterns in healthy men. MATERIAL/METHODS: This cross-sectional study was conducted on 60 healthy males of reproductive age (18-40 years) randomly selected, based on the MedDiet adherence level (high vs. low adherence), from 200 participants enrolled for the Lifestyle and Environmental Determinants of Seminogram and Other Male Fertility-Related Parameters (Led-Fertyl) study. Detailed lifestyle and dietary information and semen samples were collected. Sperm DNA was extracted and subjected to bisulfite conversion and then hybridized to Infinium MethylationEPIC v2.0 array according to established protocols. Sperm DNA methylation was analyzed using R statistical software, with CpG sites considered differentially methylated when showing an absolute mean methylation difference ≥ 0.10 between high and low adherence groups and an adjusted p-value < 0.05 RESULTS: There were no significant differences in the general and anthropometric characteristics between participants with high adherence and low adherence to MedDiet. Supervised differential methylation analyses found that there were 42 significantly differentially methylated CpGs, with 17 hypermethylated and 25 hypomethylated CpG sites in the participants with high adherence (n = 30) to MedDiet compared to the participants with low adherence (n = 30), after adjustment for age, smoking status, and BMI. The genes involved within differentially methylated sites underwent a gene ontology (GO) analysis, and we observed that 12 GO terms were significantly enriched (adjusted p-value < 0.05), mainly related to cyclic nucleotide signaling, ion channel activity, membrane trafficking/endocytosis, cytoskeletal organization, and transcriptional regulation. DISCUSSION: These novel and preliminary findings point to a possible relationship between diet quality and the sperm epigenome. Further studies should seek to confirm these observations in larger, more diverse groups including older men and individuals. CONCLUSION: Overall, our findings suggest that adherence to the MedDiet, a dietary pattern rich in natural methyl donors and cofactors, may modulate sperm DNA methylation at specific genomic loci.