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

Background

Endometritis is a bacterial infection that leads to the disruption of the endometrial epithelial barrier, causing a range of adverse effects. With the growth of assisted reproductive technologies, gaining a better understanding of endometritis has become increasingly important, especially in developing models that assess how pathogenic bacteria impact the endometrial barrier and its receptivity [1–2]. In vitro models have become essential for exploring the mechanisms underlying endometritis and the interactions between the host and pathogens [3]. Cell-based systems provide a controlled environment that closely replicates human endometrial cells and physiological conditions [4]. These models allow for the examination of immune responses, pathogen evasion strategies, and the role of virulence factors in disease progression [5]. Additionally, in vitro platforms are crucial for evaluating the effectiveness and toxicity of therapeutic compounds in the early stages of drug development, reducing the dependency on expensive animal models. However, despite the growing interest in advanced culture systems for studying endometrial infections, there is still a lack of comprehensive reviews on these methodologies.

Research on endometrial models has evolved from the use of in vitro cell lines to animal models. In vitro 2D culture systems limit essential cell–cell interactions required to replicate the in vivo microenvironment [6–8]. Additionally, the use of non-human primates in research poses challenges, including high costs, specialized resources, and ethical concerns. Significant anatomical and physiological differences between human and mouse reproductive systems necessitate careful consideration of interspecies variations in research [9]. Recent advancements in the development of self-organizing three-dimensional organoids have addressed these limitations [10–11]. Following enzymatic digestion, endometrial tissue forms hollow, spherical organoids in specialized media, preserving genetic integrity during cell division and differentiation. These organoids can also recover secretory functions post-cryopreservation [12–13]. Furthermore, the epithelial cells of endometrial organoids (EOs) show positive responses to estrogen and progesterone, opening new avenues for research into endometrial-related disorders [5,14]. Traditional 2D endometrial cell cultures have limited lifespans and rapidly lose their phenotypic and hormonal responsiveness. In contrast, organoids preserve these characteristics and demonstrate remarkable expansion potential, overcoming the senescence typically observed in primary cell cultures [15–16]. Endometrial organoids hold promise for uncovering key intracellular signaling pathways and providing insights into the overall morphological features of tissues, thus offering a deeper understanding of human endometrial physiology and pathology at the cellular level [17–18]. When endometrial organoids are cultured in Matrigel, their apical surface faces inward, mimicking the native structure of the endometrium. In this polarized state, the epithelial barrier function is likely intact, with tight cell junctions and minimal antigen exposure, resulting in lower susceptibility to pathogen infection. However, when Matrigel is removed and the organoids are transferred to low-adhesion plates, the polarity is reversed, and the epithelial surface is exposed outward. This change increases the likelihood of pathogen interaction, as the exposed cell surface may facilitate pathogen attachment. The outward-facing polarity may also lead to looser cell junctions or expose specific receptors, further promoting pathogen adhesion and infection.

In endometritis, pathogens first encounter the surface of the endometrial epithelium before initiating infection. The organoids we have constructed resemble the deeper glandular structures of the endometrium, where the epithelial surface is located at the center of the glands. In humans, under the influence of hormones, these glands gradually move upward and extend, eventually forming the endometrial epithelium, with the epithelial surface becoming exposed. Therefore, we reverse the polarity of the organoids to replicate this process, ensuring that the epithelial surface is on the exterior, which is critical for subsequent experimental procedures, such as pathogen infection studies. This polarity reversal allows for a more accurate simulation of how pathogens interact with the surface epithelium during infection, improving the relevance and reliability of our model. Our novel endometrial organoid infection model addresses these limitations by optimizing bacterial exposure parameters, enhancing infection consistency, and maintaining organoid viability. This improved model closely mimics the in vivo endometrial microenvironment, providing a robust platform for investigating host–pathogen interactions, inflammatory responses, and potential therapeutic interventions. Additionally, this protocol may be adapted for studying other bacterial pathogens or exploring the impact of various environmental factors on endometrial health.