Sex hormone-responsive human vaginal epithelial organoids: a novel in vitro platform for studying Chlamydia trachomatis infection.

The vaginal epithelium, a hormone-sensitive barrier critical for reproductive health, lacks robust in vitro models due to primary tissue scarcity and existing systems’ limitations. Here, we established the first long-term, genetically stable human vaginal epithelial organoids (VEOs) using a tailored Matrigel-based culture medium that preserves the native stratified squamous architecture. Through transcriptomic and functional profiling, we identified progesterone as a key suppressor of immune defense and keratinization. Modeling Chlamydia trachomatis (CT) infection in VEOs recapitulated infection-induced reactive oxygen species (ROS) overproduction, senescence, and inflammation. Progesterone pretreatment significantly reduced ROS and cellular damage, suggesting hormonal modulation of cellular homeostasis. By integrating air-liquid interface (ALI) culture technique and exploring different proportions of Matrigel and collagen composition, we resolved the polarity limitations of three-dimensional (3D) organoids, enabling physiological modeling of infection-induced barrier dysfunction. This study establishes VEOs as a transformative model for dissecting hormone-microbe crosstalk in reproductive health and for accelerating therapeutic development against vaginal infections. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12964-026-02685-7.