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Women's Health

Women's Health - Poster Session C & D

Z45 - Sex and Duration-Based Differences in the Murine Gut Microbiome with Antibiotics Treatment

Friday, October 10, 2025
10:00 AM - 11:00 AM PDT
Location: Exhibit Hall F, G & H

    Presenting Author(s)

  • Anna M. Kiley

    Undergraduate Student
    Loyola University Chicago
    Clinton, South Carolina, United States

    • Co-Author(s)

  • Cameron Villarreal

    Graduate Student
    Purdue University
    Lafayette, Indiana, United States

    • Last Author(s)

  • Deva D. Chan, Ph.D.

    Associate Professor
    Purdue University
    West Lafayette, Indiana, United States

    • Primary Investigator(s)

  • Deva D. Chan, Ph.D.

    Associate Professor
    Purdue University
    West Lafayette, Indiana, United States


Introduction: : The gut microbiome has been increasingly recognized for its impact on various human diseases, including inflammatory and metabolic disorders [1], prompting the need for better understanding the role of microbiome manipulation. Among the various systems that have been linked to microbiome disruption [2,3], musculoskeletal diseases affect one in two adults in the United States and include injuries, arthritis, and back pain [4]. Furthermore, women are disproportionately impacted: accounting for 80% of osteoporosis patients, 60% of osteoarthritis patients, and 64.5% of bone fractures [5,6,7]. Disruption of the gut microbiome has been linked to changes in bone formation and resorption, regulation of bone mass and metabolism, and altered progression of osteoarthritis and osteoporosis [8,9,10]. These effects may occur through regulation of nutrition, immune responses, and translocation of bacterial products [8]. Inflammatory bowel diseases, mediated by the gut microbiome [11], also show sex-based differences, with women experiencing worsened disease outcomes and higher psychological stress compared to men [12]. One possible explanation lies in sex-based variations in gut microbiome phyla and their interactions with sex hormones [13,14]. While recent literature supports the gut-bone axis [8,15,16], systemic sex-based differences in bone and gut patterns complicate efforts to generalize this connection. Better understanding how efforts to manipulate the microbiome differ by sex could lead to more affordable, patient-specific therapeutic strategies for musculoskeletal disorders. The objective of our study was to evaluate whether sex affects microbial phylum abundance changes in response to an antibiotic cocktail previously shown to affect bone quality in mice [17,18].

Materials and

Methods: : Under institutional approval, we used a commonly used gut microbiome disruption model in male and female C57Bl/6J mice [17,18]. Starting at 5 weeks of age, mice either received no treatment (control) or were administered 1 g/L ampicillin and 0.5 g/L neomycin dissolved in drinking water provided ad libitum. At humane euthanasia after 3, 11, or 19 weeks of treatment, cecum contents were collected (n = 6+ per sex, per treatment group, per treatment duration). These antibiotic treatment durations corresponded to 8, 16, and 24 weeks of age. At each time point 16S rRNA sequencing (Zymo) was used to quantify microbial community relative abundance. We focused analysis on the 6 most abundant phyla: Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Tenericutes, and Verrucomicrobia, which are conserved in the human microbiome [19]. For each treatment duration, we applied a two-way ANOVA (R-Studio) to assess the effects of sex and treatment. Main and interaction effects were considered statistically significant for p < 0.05. Relative abundances of each bacterial phylum and ANOVA tables were visualized using MATLAB to allow for comparison of significant trends across the treatment duration.

Results, Conclusions, and Discussions:: Phylum relative abundance differed by sex and duration of antibiotic treatment (Figure 1), with sex-based differences peaking at 11 weeks of treatment. Notably, this time point coincides with skeletal maturity [20], so we focus below on this duration. Tenericutes had significant effects based off sex, treatment, and their interaction (p=0.01,0.002, < 0.001), increasing in antibiotic-treated females but decreasing in antibiotic-treated males. Bacteroidetes abundance decreased from treatment (p=0.003) and showed that females have a significantly lower abundance than males (p=0.01). Firmicutes also displayed a significant sex effect (p < 0.001), with females having higher abundance. Verrucomicrobia increased in treated groups (p < 0.001).

Antibiotics strongly disrupted the gut microbiome over time, with the most sex-specific differences observed at 11 weeks of treatment, corresponding to when mice reach skeletal maturity. This suggests a sex-specific growth link between microbial composition and bone maturation [16]. However, previous studies found microbial composition continues to affect bone structure even after skeletal maturity [16]. Notably, postmenopausal women experience greater decline of bone health [21], supporting the idea that hormonal shifts also play a role. The bacterial shifts we observed may therefore mark the beginning of altered interactions between sex hormones and gut microbiome phyla at skeletal maturity.

In conclusion, antibiotic treatment significantly altered the gut microbiome, with sex-based differences in bacterial phylum abundance that varied in significance depending on the treatment duration. These changes peaked near skeletal maturity, reinforcing the link between gut microbiome and bone health and supporting that microbiome composition may explain sex differences in both intestinal and musculoskeletal disease. Ongoing analysis of trabecular bone parameters in the hindlimb will enable correlation of microbial shifts with musculoskeletal outcomes. The sex-based differences can guide future investigation of how sex hormones influence microbial abundance and, in turn, impact musculoskeletal health. If maintaining a healthy microbiome through supplements, prebiotics, and exercise could target bone and gut disease [22], then our work suggests that sex-based differences in both microbial composition and skeletal biology must be considered in therapeutic efforts to modify the microbiome.

Acknowledgements and/or References (Optional): : 1. Festi, D., Schiumerini, R., Eusebi, L. H., Marasco, G., Taddia, M., et al. (2014). Gut microbiota and metabolic syndrome. World Journal of Gastroenterology, 20(43), 16079–16094. https://doi.org/10.3748/wjg.v20.i43.16079

2. Sharon, G., Sampson, T. R., Geschwind, D. H., & Mazmanian, S. K., et al. (2016). The central nervous system and the gut microbiome. Cell, 167(4), 915–932. https://doi.org/10.1016/j.cell.2016.10.027

3. Lambring, C. B., Siraj, S., Patel, K., Sankpal, U. T., Mathew, S., et al. (2020). Impact of the microbiome on the immune system. Critical Reviews in Immunology, 39(5), 313–328. https://doi.org/10.1615/critrevimmunol.2019033233

4. Kiesel, K., Matsel, K., Bullock, G., Arnold, T., & Plisky, P. (n.d.). Risk factors for musculoskeletal health: A review of the literature and clinical application. International Journal of Sports Physical Therapy. https://doi.org/10.26603/001c.123485

5. Office on Women’s Health. (2025, March 21). Osteoporosis. U.S. Department of Health and Human Services. https://womenshealth.gov/a-z-topics/osteoporosis

6. World Health Organization. (2023). Osteoarthritis. Retrieved July 23, 2025, from https://www.who.int/news-room/fact-sheets/detail/osteoarthritis/

7. Bergh, C., Wennergren, D., Möller, M., & Brisby, H. (2020). Fracture incidence in adults in relation to age and gender: A study of 27,169 fractures in the Swedish Fracture Register in a well-defined catchment area. PLOS ONE, 15(12), e0244291. https://doi.org/10.1371/journal.pone.0244291

8. Hernandez, C. J., Guss, J. D., Luna, M., & Goldring, S. R. (2016). Links between the microbiome and bone. Journal of Bone and Mineral Research, 31(9), 1638–1646. https://doi.org/10.1002/jbmr.2887

9. Ding, K., Hua, F., & Ding, W., et al. (2020). Gut microbiome and osteoporosis. Aging and Disease, 11(2), 438–447. https://doi.org/10.14336/AD.2019.0523

10. Ramires, L. C., Santos, G. S., Ramires, R. P., da Fonseca, L. F., Jeyaraman, M., et al. (2022). The association between gut microbiota and osteoarthritis: Does the disease begin in the gut? International Journal of Molecular Sciences, 23(3), 1494. https://doi.org/10.3390/ijms23031494

11. Glassner, K. L., Abraham, B. P., & Quigley, E. M. M. (2020). The microbiome and inflammatory bowel disease. Journal of Allergy and Clinical Immunology, 145(1), 16–27. https://doi.org/10.1016/j.jaci.2019.11.003

12. Greuter, T., Manser, C., Pittet, V., Vavricka, S. R., & Biedermann, L. (2020). Gender differences in inflammatory bowel disease. Digestion, 101(Suppl 1), 98–104. https://doi.org/10.1159/000504701

13. d’Afflitto, M., Upadhyaya, A., Green, A., & Peiris, M. (2022). Association between sex hormone levels and gut microbiota composition and diversity: A systematic review. Journal of Clinical Gastroenterology, 56(5), 384–392. https://doi.org/10.1097/MCG.0000000000001676

14. Tian, X., & Zhang, B. (2025). The association between sex hormones and bone mineral density in US females. Scientific Reports, 15, Article 5546. https://doi.org/10.1038/s41598-025-89985-z

15. Tu, Y., Yang, R., Xu, X., & Zhou, X. (2021). The microbiota-gut-bone axis and bone health. Journal of Leukocyte Biology, 110(3), 525–537. https://doi.org/10.1002/JLB.3MR0321-755R

16. Liu, C., Cyphert, E. L., Stephen, S. J., Wang, B., Morales, A. L., Nixon, J. C., Natsoulas, N. R., Garcia, M., Carmona, P. B., Vill, A. C., Donnelly, E., Brito, I. L., Vashishth, D., & Hernández, C. J. (2024, October 29). Microbiome‑induced increases and decreases in bone matrix strength can be initiated after skeletal maturity. Journal of Bone and Mineral Research, 39(11), 1621–1632. https://doi.org/10.1093/jbmr/zjae157

17. Guss, J. D., Taylor, E., Rouse, Z., Roubert, S., Higgins, C. H., Thomas, C. H., Baker, S. P., Vashishth, D., Donnelly, E., Shea, M. K., Booth, S. L., & Hernandez, C. J. (2019). The microbial metagenome and bone tissue composition in mice with microbiome-induced reductions in bone strength. Bone, 127, 146–154. https://doi.org/10.1016/j.bone.2019.06.010

18. Thabit, A. K., Fatani, D. F., Bamakhrama, M. S., Barnawi, O. A., Basudan, L. O., & Alhejaili, S. F. (2019). Antibiotic penetration into bone and joints: An updated review. International Journal of Infectious Diseases, 81, 128–136. https://doi.org/10.1016/j.ijid.2019.02.005

19. AboNahas, H.H. et al. (2022). Trust Your Gut: The Human Gut Microbiome in Health and Disease. In: Sayyed, R.Z., Khan, M. (eds) Microbiome-Gut-Brain Axis. Springer, Singapore. https://doi.org/10.1007/978-981-16-1626-6_3

20. Somerville, J.M., Aspden, R.M., Armour, K.E. et al. Growth of C57Bl/6 Mice and the Material and Mechanical Properties of Cortical Bone from the Tibia. Calcif Tissue Int 74, 469–475 (2004). https://doi.org/10.1007/s00223-003-0101-x

21. Ji, M.-X., & Yu, Q. (2015). Primary osteoporosis in postmenopausal women. Chronic Diseases and Translational Medicine, 1(1), 9–13. https://doi.org/10.1016/j.cdtm.2015.02.006

22. McCabe, L., Britton, R. A., & Parameswaran, N. (2015). Prebiotic and probiotic regulation of bone health: Role of the intestine and its microbiome. Current Osteoporosis Reports, 13(6), 363–371. https://doi.org/10.1007/s11914-015-0292-x