Cannabis use in the United States is increasing, with highest consumption among men at their peak reproductive years. We previously demonstrated widespread changes in sperm DNA methylation with cannabis exposure in humans and rats, including genes important in neurodevelopment. Here, we use an in vitro human spermatogenesis model to recapitulate chronic cannabis use and assess DNA methylation at imprinted and autism spectrum disorder (ASD) candidate genes in spermatogonial stem cell (SSC)- and spermatid-like cells. Methylation at maternally imprinted genes SGCE and GRB10 was significantly altered in SSC- and spermatid-like cells, respectively, while PEG3 was significantly differentially methylated in spermatid-like cells. Two of ten randomly selected ASD candidate genes, HCN1 and NR4A2, had significantly altered methylation with cannabis exposure in SSC-like cells. These results support our findings in human cohorts and provide a new tool with which to gain mechanistic insights into the association between paternal cannabis use and risk of ASD in offspring.

Semen parameters, including sperm counts, have rapidly declined in men across the globe over the last five decades. Although this decline remains unexplained, lifestyle factors may affect male fertility. Recently, several studies highlighted a potential link between non-steroidal anti-inflammatory drug (NSAID) usage, such as naproxen and ibuprofen, and declining male fertility. However, the mechanisms by which these common analgesics affect male fertility, including their effects on the blood–testis barrier (BTB), remain poorly characterized. Utilizing an in vitro rhesus macaque non-human primate (NHP) BTB model, we demonstrate that serum levels of naproxen and ibuprofen alter the function of BTB. Following short-term naproxen and ibuprofen treatment of NHP primary Sertoli cells, we show that these NSAIDs increase the transepithelial electrical resistance, indicating an overall strengthening of the Sertoli cell junctions. Furthermore, naproxen and ibuprofen treatment alter the expression of genes involved in maintaining the BTB. Specifically, the genes that were significantly expressed in response to ibuprofen exposure were enriched for human phenotypic abnormalities linked to male factor infertility. Together, these results suggest that short-term naproxen and ibuprofen treatment disrupt the function of the BTB by altering the integrity of the Sertoli cell junctions, proposing a potential role of NSAIDs in male factor infertility.