Gut Bacteroidales and AMH/INH-B ratio predict sperm retrieval: mechanistic insights via SCFA-mediated regulation of blood-testis barrier and steroidogenesis

Objective To establish a non-invasive predictive model for microdissection testicular sperm extraction (micro-TESE) outcomes in FSH-normal non-obstructive azoospermia (NOA) patients by integrating gut microbiota profiling with serum biomarkers. Methods We conducted a retrospective clinical analysis of 58 men and established a busulfan-induced FSH-normal NOA mouse model. Serum hormone levels (FSH, INH-B, AMH, testosterone) were measured by ELISA, and gut microbiota was analyzed via 16S rRNA sequencing. Testicular histology and ultrastructure were assessed by H&E staining and TEM, while protein expression was evaluated by IHC, IF, and Western blot. Receiver operating characteristic (ROC) curve analysis was used to evaluate the predictive efficacy of the serum AMH/INH-B ratio for sperm retrieval outcomes. Results In both patients and model mice, serum INH-B, AMH, and the AMH/INH-B ratio were significantly decreased ( P < 0.01), correlating with severe spermatogenic impairment. Mice exhibited a marked reduction in the abundance of Bacteroidales and Muribaculaceae . Fecal microbiota transplantation (FMT) restored these microbial populations, improved testicular function, and upregulated key proteins involved in proliferation (PCNA, PGK2), blood-testis barrier integrity (ZO-1, Claudin11), and steroidogenesis (StAR, CYP17A1) ( P < 0.05). Mechanistically, FMT increased serum short-chain fatty acid (SCFA) levels, which served as the chemical messengers correlating directly with the recovery of BTB proteins and steroidogenic enzymes. Clinically, the serum AMH/INH-B ratio showed strong predictive efficacy for micro-TESE outcomes, with an area under the ROC curve (AUC) of 0.92 (95% CI: 0.86-0.98), optimal cut-off value of 0.65, sensitivity of 88.2%, and specificity of 85.7%. The gut Bacteroidales abundance (from mouse data) was mechanistically linked to spermatogenic function, suggesting its potential as a future clinical biomarker pending validation. Conclusions Our findings elucidate an SCFA-mediated gut-testis axis, highlighting the therapeutic potential of microbiota modulation and providing a novel tool to guide clinical decision-making, potentially reducing unnecessary surgeries in FSH-normal NOA.Additionally, the serum AMH/INH-B ratio serves as a robust non-invasive biomarker for predicting micro-TESE outcomes in FSH-normal NOA, while gut Bacteroidales abundance may represent a complementary mechanistic target for future clinical investigation.