Antibiotic therapy with metronidazole reduces endometriosis disease progression in mice: a potential role for gut microbiota

Abstract

Study question: Does altering gut microbiota with antibiotic treatment have any impact on endometriosis progression?

Summary answer: Antibiotic therapy reduces endometriosis progression in mice, possibly by reducing specific gut bacteria.

What is known already: Endometriosis, a chronic condition causing abdominal pain and infertility, afflicts up to 10% of women between the ages of 25 and 40, ~5 million women in the USA. Current treatment strategies, including hormone therapy and surgery, have significant side effects and do not prevent recurrences. We have little understanding of why some women develop endometriosis and others do not.

Study design, size, duration: Mice were treated with broad-spectrum antibiotics or metronidazole, subjected to surgically-induced endometriosis and assayed after 21 days.

Participants/materials, setting, methods: The volumes and weights of endometriotic lesions and histological signatures were analysed. Proliferation and inflammation in lesions were assessed by counting cells that were positive for the proliferation marker Ki-67 and the macrophage marker Iba1, respectively. Differences in faecal bacterial composition were assessed in mice with and without endometriosis, and faecal microbiota transfer studies were performed.

Main results and the role of chance: In mice treated with broad-spectrum antibiotics (vancomycin, neomycin, metronidazole and ampicillin), endometriotic lesions were significantly smaller (~ 5-fold; P < 0.01) with fewer proliferating cells (P < 0.001) than those in mice treated with vehicle. Additionally, inflammatory responses, as measured by the macrophage marker Iba1 in lesions and IL-1β, TNF-α, IL-6 and TGF-β1 in peritoneal fluid, were significantly reduced in mice treated with broad-spectrum antibiotics (P < 0.05). In mice treated with metronidazole only, but not in those treated with neomycin, ectopic lesions were significantly (P < 0.001) smaller in volume than those from vehicle-treated mice. Finally, oral gavage of faeces from mice with endometriosis restored the endometriotic lesion growth and inflammation (P < 0.05 and P < 0.01, respectively) in metronidazole-treated mice.

Large-scale data: N/A.

Limitations, reasons for caution: These findings are from a mouse model of surgically-induced endometriosis. Further studies are needed to determine the mechanism by which gut bacteria promote inflammation, identify bacterial genera or species that promote disease progression and assess the translatability of these findings to humans.

Wider implications of the findings: Our findings suggest that gut bacteria promote endometriosis progression in mice. This finding if translated to humans, could aid in the development of improved diagnostic tools and personalised treatment strategies.

Study funding and competing interest(s): This work was funded, in part, by: a National Institutes of Health (NIH)/ National Institute of Child Health and Human Development (NICHD) grant (R00HD080742) to RK; Washington University School of Medicine start-up funds to RK; an Endometriosis Foundation of America Research Award to R.K.; and an NIH/NICHD grant (R01HD091218) to IUM. The authors report no conflict of interest.

Keywords: endometriosis; gut bacteria; inflammation; metronidazole; microbiome.

© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Figures

Figure 1

Treatment with broad-spectrum antibiotics prevents early endometriotic lesion growth and progression. (A and E) Schematic of experimental timeline and procedures. (B–D and F–H) Representative gross images (B and F), volumes (C and G), and masses (D and H) of ectopic endometriotic lesions from the indicated treatment groups 21 days after surgical induction of endometriosis. Data are presented as mean ± SE(n = 5) *P < 0.05, **P < 0.01.

Figure 2

Treatment with broad-spectrum antibiotics reduces endometriotic lesion proliferation and inflammation. (A) Schematic of experimental timeline and procedures. (B–D) Representative gross images (B), volumes (C) and masses (D) of ectopic endometriotic lesions from the indicated treatment groups 21 days after surgical induction of endometriosis. Data are presented as mean ± SE; endo-vehicle (n = 15) and endo-VNMA (n = 14). (E) Representative Hematoxylin and Eosin-stained cross-section images of the eutopic uteri and ectopic lesions from the indicated treatment groups. The scale bar (0.5 μm) applies to all images; n = 5. (F–G) Representative cross-sectional images (left) of the eutopic uteri and ectopic lesions stained for Ki-67 (F) and Iba1 (G); respective graphs on the right show positively stained cells counted in at least five different areas in ectopic lesions and plotted as percent positive cells relative to total cells. The scale bar (0.5 μm) applies to all images; n = 5. ‘E’, ‘G’ and ‘S’ denote epithelia, glands and stroma, respectively. (H) ELISA-based quantification of IL-1β, TNF-α, IL-6, IL-10 and TGF-β1 levels in peritoneal fluid from the indicated treatment groups. Data are presented as mean ± SE (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, non-significant.

Figure 3

Bacteroidesare enriched in faeces from mice with endometriotic lesions. (A) Heat map representation of relative abundances of the phyla in faecal samples from non-endo (n = 5), endo-vehicle (n = 5) and endo-VNMA (n = 4) mice. (B) Stacked bar plots of the phyla belonging to the 10 most abundant OTUs. (C) Heat map depiction of the relative abundances of the genera in each faecal sample. (D) The genera belonging to the 10 most abundant OTUs across each group are shown as stacked bar plots.

Figure 4

Metronidazole treatment reduces endometriotic lesion growth. (A–C) Representative gross images (A), volumes (B) and masses (C) of ectopic endometriotic lesions from the indicated treatment groups; (n = 5). (D) Representative Hematoxylin and Eosin-stained cross-section images of ectopic lesions from the indicated treatment groups; n = 5. Scale bars represent 200 μm (upper panel) or 500 μm (lower panel). (E) Quantification of IL-1β concentration in peritoneal fluid from the indicated treatment groups. (F) Representative cross-sectional images of ectopic lesions stained for Iba1; graph on the right shows the number of positively stained cells counted in at least five different areas in ectopic lesions and plotted as percent positive cells relative to total cells. ‘E’, ‘G’ and ‘S’ denote epithelia, glands and stroma, respectively. Data are presented as mean ± SE; (n = 5). *P < 0.05, ***P < 0.001, ****P < 0.0001, and ns, non-significant.

Figure 5

Oral gavage of faeces from endometriotic mice promotes endometriotic lesion growth in antibiotic-treated mice. (A) Schematic of experimental timeline and procedures. (B–D) Representative gross images (B), volumes (C) and masses (D) of ectopic endometriotic lesions from the indicated treatment groups 28 days after surgical induction of endometriosis; ‘V’, ‘M’ and ‘ME’ denote endo-vehicle, endo-metronidazole + non-endo faeces, and endo-metronidazole + endo faeces, respectively; n = 4–5. (E) Representative Hematoxylin and Eosin-stained cross-section images of ectopic lesions from the indicated treatment groups; n = 4–5. Scale bars represent 200 μm (upper panel) and 500 μm (lower panel). (F) Representative cross-sectional images of ectopic lesions stained for Iba1. (G) Quantification of Iba1-positive cells counted in at least five different areas in ectopic lesions and plotted as percent positive cells relative to total cells (left panel) and quantification of IL-1β concentration in peritoneal fluid from the indicated treatment groups (right panel). Data are presented as mean ± SE (n = 4–5). ‘E’, ‘G’ and ‘S’ denote epithelia, glands and stroma, respectively. *P < 0.05, ***P < 0.001, and ns, non-significant.