This study aims to investigate gut microbiota and metabolomes of fecal, cecum, serum, and ovarian tissues in a DHEA-induced rat polycystic ovary syndrome (PCOS) model and explore the gut-ovary axis in PCOS pathogenesis. Rats were treated with DHEA or sesame oil to create PCOS and Control groups. The PCOS model was confirmed by ovarian morphology, estrous cycle, and serum hormone levels (FSH, LH, and E2). Stool, serum, cecum, and ovarian tissues underwent untargeted metabolomic analysis, and gut microbiota were assessed using 16S rRNA sequencing. Differentially expressed metabolites (DEMs) were analyzed using KEGG function and pathway enrichment analyses. Pearson's correlation analysis between DEMs and gut microbiota was performed. RT-PCR and Western blotting analyses determined intestinal integrity markers. Western blotting validated key pathways. DHEA treatment induced PCOS in rats as indicated by hormonal imbalances, ovarian histopathological changes, and estrous cycle disruption. PCOS rats exhibited increased alpha diversity and beta-diversity in gut microbiota and gut microbial community compositions, indicating dysbiosis. PCOS group fecal, cecal, serum, and ovarian metabolic phenotypes differed significantly from those of the control group. KEGG function and pathway enrichment analysis of DEMs indicated that all PCOS group sample types exhibited steroid hormone and lipid metabolism disturbances. DEMs in fecal, cecal, serum, and ovarian samples were closely correlated with differential gut microbiota and serum hormones relevant to PCOS. Expression levels of CLDN3 and Occludin that are involved in intestinal integrity were altered, and protein expression levels of Cyp4b1 and 3B-HSD that are involved in ovarian steroid hormone metabolism were disordered. DHEA-induced PCOS rats exhibited significant microbiota and metabolomes disruptions, particularly in the steroid, lipid, and amino acid pathways. Key metabolites (17α-Hydroxyprogesterone, Ethyltestosterone, and Pregnenolone) were closely linked to PCOS-related hormones and altered microbiota such as Bacillus and Akkermansia. These findings suggest the potential involvement of the gut-ovary axis in PCOS development.
