Exploratory Multi-Omics Analysis of Gut Microbiota and Fecal Metabolites in Relation to Serum S-Equol Levels in Older Adults with Osteoporosis from a Tropical Community: A Pilot Study

Wenting Cao¹Rui Li¹Hanxiang Zhang¹Tianxia Zhang¹Hongxin Pan¹Wen Sun²Lingqi Wang³Jiashu Ke¹Jindong Ding Petersen^1,4,5Ping Zhang^1*

• ¹Key Laboratory of Tropical Translational Medicine of Ministry of Education, School of Public Health, Hainan Academy of Medical Sciences, Hainan Medical University, Haikou, China
• ²Department of Nuclear Medicine, First Affiliated Hospital of Hainan Medical University, Haikou, China
• ³Department of Laboratory Diagnosis, Fifth Affiliated Hospital of Harbin Medical University, Daqing, China
• ⁴Department of Public Health, University of Copenhagen, Copenhagen, Denmark
• ⁵Department of Public Health, University of Southern Denmark, Odense, Denmark

Background: Osteoporosis (OP) is a multifactorial skeletal disorder influenced by host metabolism, inflammation, and gut microbiota-derived metabolites such as S-equol. However, the interplay between intestinal microbiota, S-equol production, and host metabolic profiles in OP remains incompletely understood. Objective: To conduct a preliminary multi‑omics investigation integrating metagenomic and metabolomic analyses to identify gut microbiota and metabolite biomarkers associated with serum S‑equol levels in older adults with OP. Methods: A cross-sectional study was conducted in 39 community-dwelling adults aged ≥50 years in Haikou, China. Participants were grouped into OP and control groups based on lumbar spine T-scores, using a cut-off value of ≤−2.5 to define osteoporosis. Serum biomarkers (S-equol, inflammatory cytokines, oxidative stress indicators) were assessed by ELISA. Fecal samples underwent metagenomic sequencing and untargeted metabolomics. LEfSe, Spearman correlation, machine learning, and KEGG enrichment were used to explore microbiota-metabolite-bone health axes. Results: Serum S-equol levels were significantly lower in the OP group compared to controls (3561 ± 304 vs. 3855 ± 469 pg/mL, P = 0.026), whereas most inflammatory markers were comparable between groups, apart from a modest increase in IL-1β in OP. Metagenomic analysis revealed a lower relative abundances of key SCFA-producing taxa in OP (e.g., Faecalibacterium prausnitzii, Roseburia hominis, Bacteroides uniformis). Metabolomic profiling identified distinct alterations in amino acid and tryptophan pathways, with KEGG analysis highlighting disruptions in glycerophospholipid, glycine-serine-threonine, and choline metabolism. Discriminative metabolites (e.g., Gln-Val-Ile-Asp, 5-oxooctanoic acid) showed diagnostic potential (AUC > 0.75). S-equol levels positively correlated with these beneficial microbes and with amino acid-related metabolites (e.g., D-tryptophan, 3-indoleacrylic acid, N-methylglutamate). Network and heatmap analyses illustrated differences in microbial-metabolite association patterns between groups. Conclusion: In conclusion, low levels of serum S-equol in older adults with osteoporosis were associated with distinct changes in gut microbiota composition and fecal metabolic profiles in this pilot study.