Abstract

5-neurosteroids such as allopregnanolone and isopregnanolone play critical roles in neurological health and mood regulation, yet current therapeutic production faces significant limitations. We demonstrate that specific gut microbes represent a previously unrecognized source of bioavailable 5-neurosteroids that reach the central nervous system via the gut-brain axis. Through integrated metabolomic and genomic analyses of progesterone-amended fecal cultures, we identified Holdemania as a major producer of isopregnanolone via microbial steroid 5-reductase (BaiJ type 2) and 3{beta}-hydroxysteroid dehydrogenase/reductase. Phylogenetic analysis revealed that BaiJ-like sequences cluster predominantly within Firmicutes, with Holdemania species forming a distinct clade. In female C57BL/6 mice administered progesterone and H. filiformis, 5-neurosteroids including isopregnanolone predominated in gut tissues while allopregnanolone was the major hepatic neurosteroid. Critically, using stable isotope tracing with [3,4-13C2]progesterone, we detected 13C-labeled isopregnanolone in brain tissue, providing direct evidence for gut-to-brain transport of microbiota-derived neurosteroids. High-fat diet significantly enhanced brain 5-neurosteroid accumulation. Global meta-analysis reveals reduced Holdemania abundance in PCOS patients (n = 346) compared to healthy women (n = 321). These findings identify gut microbiota as pharmacologically relevant neurosteroid producers and position H. filiformis as a promising probiotic candidate for enhancing endogenous neurosteroid production to treat mood disorders and other neuropsychiatric conditions.