Gut Microbiome and Breast Cancer-Related Endogenous Hormone Risk Factors in US Women: Findings from the Nurses' Health Study II

Abstract Background: Endogenous estrogens are well-established risk factors for breast cancer. A subset of gut microbes, collectively termed the estrobolome, plays a fundamental role in estrogen metabolism and reabsorption. However, existing studies examining associations between the gut microbiome and circulating estrogen levels in humans have been few and small in scale, and no prior work has established an estrobolome linked to circulating estrogen levels using shotgun metagenomic sequencing. Methods: This study included 141 non-hormone users from a microbiome sub-study nested within the Nurses’ Health Study II. Stool samples were collected using a home-based self-collection protocol, followed by blood collection approximately 3-4 months later. Circulating estrone (E1), estradiol (E2), and testosterone levels were assayed at the Mayo Clinic Laboratory by liquid chromatography-tandem mass spectrometry. Shotgun metagenomic sequencing was conducted using the 100 nt Illumina HiSeq platform. Taxonomic and functional profiling were performed using the bioBakery 4.0 workflow. Microbial α-diversity (Inverse Simpson index) and β-diversity (Bray-Curtis dissimilarity) were calculated. LASSO regression was applied to identify microbial features predictive of hormone levels, and age and BMI-adjusted generalized linear models were used for association analyses with multiple comparisons correction. Results: Taxonomic and functional profiling identified 1,860 species and 512 MetaCyc pathways. Microbial α-diversity was inversely correlated with E1 (R = -0.16, p = 0.07) and E2 (R = -0.16, p = 0.06), and positively correlated with testosterone (R = 0.12, p = 0.16). In age-adjusted models, α-diversity was significantly inversely associated with E2 (β = -0.39; 95% CI: -0.70, -0.07), though the association attenuated after further adjustment for BMI at stool collection (β = -0.16; 95% CI: -0.47, 0.15). PERMANOVA analyses revealed significant associations between taxonomic variation and E1 (R2 = 0.28%), E2 (R2 = 0.34%), and testosterone (R2 = 0.16%) (all p < 0.005). LASSO feature selection identified 23 species predictive of E1 and 16 predictive of E2, with the majority belonging to the genera Bacteroides, Clostridium, Eubacterium, and Alistipes. E2 was further associated with microbial pathways related to glycogen degradation and queuosine biosynthesis. Conclusion: This study represents the first and largest shotgun metagenomic investigation of gut microbiome composition in relation to circulating sex hormones in an epidemiological cohort. Reduced microbial diversity and higher abundance of estrobolome species with β-glucuronidase activity (e.g., Bacteroides fragilis) may be associated with elevated circulating estrogen levels, suggesting that structural variation in gut microbial communities contributes to inter-individual differences in estrogen metabolism. Citation Format: Tengteng Wang, Tong Cheng, Walter C. Willett, Curtis Huttenhower, Yang-Yu Liu, A. Heather Eliassen. Gut microbiome and breast cancer-related endogenous hormone risk factors in US women: findings from the Nurses’ Health Study II [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2328.