Dysbiosis in pregnant mice induced by transfer of human vaginal microbiota followed by reversal of pathological changes in the uterus and placenta via progesterone treatment

Abstract

Objective: The vaginal microbiota dysbiosis induces inflammation in the uterus that triggers tissue damage and is associated with preterm birth. Progesterone is used to prevent labor in pregnant women at risk of preterm birth. However, the mechanism of action of progesterone still needs to be clarified. We aimed to show the immunomodulatory effect of progesterone on the inflammation of uterine tissue triggered by dysbiotic vaginal microbiota in a pregnant mouse model.

Methods: Healthy (n = 6) and dysbiotic (n = 7) vaginal microbiota samples isolated from pregnant women were transferred to control (n = 10) and dysbiotic (n = 14) pregnant mouse groups. The dysbiotic microbiota transferred group was treated with 1 mg progesterone (n = 7). Flow cytometry and immunohistochemistry analyses were used to evaluate inflammatory processes. Vaginal microbiota samples were analyzed by 16 S rRNA sequencing.

Results: Vaginal exposure to dysbiotic microbiota resulted in macrophage accumulation in the uterus and cellular damage in the placenta. Even though TNF and IL-6 elevations were not significant after dysbiotic microbiota transplantation, progesterone treatment decreased TNF and IL-6 expressions from 49.085 to 31.274% (p = 0.0313) and 29.279-21.216% (p = 0.0167), respectively. Besides, the macrophage density in the uterus was reduced, and less cellular damage in the placenta was observed.

Conclusion: Analyzing the vaginal microbiota before or during pregnancy may support the decision for initiation of progesterone therapy. Our results also guide the development of new strategies for preventing preterm birth.

Keywords: Animal model; Dysbiosis; Inflammation; Pregnancy; Preterm birth; Progesterone; Vaginal microbiota.

Fig. 1

A Compositions of vaginal microbiota transplanted into mice. Control group: Lactobacillus crispatus dominated CST I type microbiota, Dysbiosis group; Gardnerella vaginalis or Atopobium vaginae dominated CST IV type microbiota. B The relative abundance of the bacterial species was detected in the mice vagina on 18.5 days of pregnancy in CST I and CST IV groups

Fig. 2

Comparison of dysbiotic microbiota transferred mice vaginal microbiota species proportion before and after progesterone treatment. C: Healthy vaginal microbiota (CST-I) transferred mice, C + P: Healthy vaginal microbiota (CST-I) transferred mice after progesterone treatment, D: Dysbiotic vaginal microbiota (CST-IV) transferred mice, D + P: Dysbiotic vaginal microbiota (CST-IV) transferred mice after progesterone treatment

Fig. 3

Lymphocyte cells isolated from the uterine tissue (a). Effect of human dysbiotic vaginal microbiota and progesterone treatment on the production of CD3/CD4 + immune cells in uterine tissues (b), CD25/CD69 + cells (c), tumor necrosis factor-alpha (TNF) (d), pro-inflammatory cytokines interleukin-6 (IL-6) (e), interleukin-1β (IL-1β) (f). C (Control Group); healthy vaginal microbiota transplanted mice (n = 6), D (Dysbiosis group); vaginal dysbiosis microbiota transplanted mice (n = 7), (C + P); Control group with progesterone treatment; mice receiving 1 mg/ml progesterone (n = 4), (D + P); Dysbiosis group with progesterone treatment; mice receiving 1 mg/ml progesterone (n = 7)

Fig. 4

H&E-stained formalin-fixed mouse placental section (A) scoring of the placental cellular damage examination C; Control group n = 3, C + P; Progesterone treated control group n = 2, D; Dysbiosis group n = 7, D + P; Progesterone treated dysbiosis group n = 5 (B). Cellular damage score; 0: absent/barely, 1: occasional/mild, 2: moderate/high

Fig. 5

Evaluation of macrophage infiltration with IBA-1 antibody in mouse uterus sections. Hematoxylin-eosin sections (left panel) demonstrated dysbiotic mice harboring increased macrophage infiltration compared to control mice (with and without progesterone). Under progesterone treatment, dysbiotic mice had fewer macrophages. The findings were also seen with anti-IBA-1 staining (right panel). Macrophage accumulation was marked in the red circle area