Trimethylamine-N-oxide disrupts spermatogenesis by inducing mitochondrial oxidative stress injury through Hippo signaling

Background: The gut-testis axis is increasingly recognized as a regulator of male reproductive health; however, the key microbial contributors, metabolites, and underlying mechanisms remain unclear.

Methods: We performed fecal metagenomic Sequencing in 107 participants to identify microbial taxa associated with abnormal semen parameters. Serum trimethylamine-N-oxide (TMAO) levels were measured and correlated with semen quality. In mouse models, including fecal microbiota transplantation, dietary choline supplementation, mono-colonization, and direct TMAO administration, we assessed sperm morphology, testicular androgen synthesis, and testicular histology. Testicular transcriptomics, in vitro Leydig cell assays, and mitochondrial function analyses were conducted to investigate the effects of TMAO on Hippo signaling, Oxidative Phosphorylation, mitochondrial membrane damage, and steroidogenesis.

Results: Choline-to-trimethylamine converting bacteria, including Phocaeicola massiliensis, Veillonella spp., and Klebsiella pneumoniae, were enriched in men with abnormal semen parameters. Circulating TMAO levels were inversely associated with semen volume, total sperm count, and motile sperm count. In mouse models, elevated TMAO induced testicular dysfunction characterized by impaired sperm morphology, reduced testicular androgen synthesis, and histological abnormalities. Consistently, gene set enrichment analysis (GSEA) of testicular transcriptomes revealed significant suppression of mitochondrial translation, membrane integrity, Oxidative Phosphorylation, and adenosine triphosphate (ATP) metabolism. TMAO also suppressed steroidogenesis by reducing the expression of steroidogenic acute regulatory protein (StAR). Mechanistic studies in TM3 Leydig cells further demonstrated that TMAO, by promoting YAP phosphorylation, disrupted mitochondrial structure and morphology, decreased mitochondrial membrane potential, increased mitochondrial Reactive Oxygen Species (ROS) levels, impaired ATP synthesis, and promoted mitochondrial fragmentation with upregulation of the mitochondrial fission molecule (Fis1).

Conclusions: Our findings demonstrate that TMAO activates Hippo signaling to induce mitochondrial dysfunction and suppress testosterone synthesis, thereby impairing spermatogenesis. These results highlight TMAO biosynthesis and its downstream signaling as potential therapeutic targets for improving male fertility.

Gut microbiota; Leydig cells; Mitochondria; Spermatogenesis; TMAO.