The Establishment of 3D Polarity-Reversed Organoids From Human Endometrial Tissue as a Model for Infection-Induced Endometritis

Abstract

Endometritis is a prevalent gynecological condition, often resulting from bacterial infections, which poses significant risks to women's reproductive health, including recurrent pregnancy loss, spontaneous abortion, and intrauterine adhesions. While conventional in vitro models have provided valuable insights into the pathogenesis of bacterial-induced endometritis, they often fail to replicate the complex cellular architecture and microenvironment of the endometrium due to species-specific differences and variations in the menstrual cycle. In this study, we present a novel organoid-based culture system that establishes a bacterial-induced endometritis model using endometrial organoids derived from primary epithelial cells. This protocol involves culturing endometrial organoids in a Matrigel-based three-dimensional matrix, followed by infection with Escherichia coli at a defined multiplicity of infection (MOI). The model effectively recapitulates key pathological features of bacterial-induced endometritis, including disruption of the epithelial barrier, release of inflammatory cytokines, and cellular damage. By preserving epithelial polarity, this approach offers enhanced physiological relevance, improves host-pathogen interaction studies, and provides a robust platform for evaluating potential therapeutic interventions. Key features • Establishes apical-out endometrial organoids to model pathogen-induced endometritis via natural infection routes. • Utilizes primary human endometrial epithelial cells to preserve cellular diversity and mimic the native endometrial microenvironment. • Provides a versatile platform for investigating host-pathogen interactions and evaluating potential therapeutic interventions in bacterial-induced endometritis. • Developed apical-out endometrial organoids to better mimic tissue structure and enhance pathogen infection for host-pathogen interaction studies.

Keywords: Endometrial epithelial cells; Endometrial organoid; Endometrial organoid infection model; Endometritis; Infection model.

Figure 1.. Morphology of endometrial organoids during tissue isolation, digestion, and culture.

(A) Representative images of endometrial tissue morphology after PBS washing. (B) Brightfield images of endometrial organoids at Days 1, 3, 5, and 7 of culture. Scale bar: 100 μm. (C) Brightfield images of endometrial organoids at passages P1, P3, P5, and P7 following passaging. Scale bar: 100 μm.

Figure 2.. Formation process of apical-out endometrial organoids.

(A) Sequential process of apical-out transformation in endometrial organoids: (a) before apical-out, (b) 1.5 h after apical-out induction, (c) 1 day, (d) 2 days, (e) 4 days, and (f) 6 days post-induction. Scale bar: 100 μm. (B) Immunofluorescence staining of MUC1 and ZO-1 in endometrial organoids before and after apical-out transformation. (a) MUC1 staining of endometrial organoids; (b) MUC1 staining of polarity-reversed endometrial organoids; (c) ZO-1 staining of endometrial organoids; (d) ZO-1 staining of polarity-reversed endometrial organoids. The arrows indicate red fluorescence signals of MUC1 and ZO-1. Scale bar: 20 μm.

Figure 3.. Escherichia coli infection in endometrial organoids.

(A) Brightfield images of endometrial organoids at 1 h and 24 h post-infection with E. coli. Scale bar: 200 μm. (B) Expression levels of inflammatory factors compared between endometrial organoids infected with E. coli and uninfected controls. The results are shown as the mean ± SD and are representative of three independent experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.