Development and validation of healthy endometrium and endometriosis models for high-throughput phenotypic screening

Introduction: Endometriosis affects 10-15% of women and is characterized by the presence of endometrial-like tissue outside of the uterus, leading to chronic pelvic pain, infertility, and diminished quality of life. Current diagnostic methods, including imaging and laparoscopy, often fail to detect early-stage and microscopic lesions, critical for effective intervention and management. This diagnostic inadequacy, coupled with a limited understanding of the disease's pathophysiology, contributes to delayed treatments and suboptimal patient outcomes. Furthermore, the lack of targeted therapies underscores a pressing need for innovative approaches to identify and evaluate novel therapeutic targets and diagnostic tools.

Materials and

Methods: To address this need, our study focused on the development and application of two distinct three-dimensional (3D) in vitro models designed to emulate the physiological and pathological conditions of the endometrium in both healthy states and in endometriosis. Employing Design of Experiments (DOE) with a central composite face design, we analyzed the effects of six variables across 47 unique hydrogel formulations to study their impact on endometrial thickness, microvessel formation, epithelial invasion, and fibroblast activation. Cultured in IBIDI angiogenesis plates, the models underwent hormonal treatment with estrogen, progesterone, and luteinizing hormone gradients for 72 hours, mimicking the hormonal flux during ovulation that maximizes endometrial lining thickness. Models were were assessed through 85 µm z-stack imaging every three hours and cell dynamics were evaluated using our automated image analysis software.

Results, Conclusions, and Discussions: Results. All of the input variables evaluated (collagen I, collagen IV, fibronectin, laminin, fibroblasts, estrogen, and progesterone) significantly influenced all of the cell responses through first- or second-order interactions (p ≤ 0.05) (Fig. 1B). An adjusted R2 ≥ 0.7 suggested the number of model terms adequately fit the model and a difference between R2 and Q2 values < 0.3 validated that the models worked independently of the data used to develop the model. By tuning the concentrations of these components we successfully replicated the complex layered architecture and cellular interactions characteristic of both healthy and endometriotic endometrium. These models, compatible with high-throughput screening and automatic evaluation, offer promising tools for in-depth mechanistic studies on endometriosis's onset and progression, potentially leading to novel diagnostic and therapeutic strategies.
Conclusion. Our 3D in vitro multilayer multicellular models provided representative screening platforms for both healthy and endometriotic endometrium. These platforms can be used to evaluate thousands of compounds simultaneously, empowering large-scale mechanistic studies to investigate the progression of endometriosis, as well as enable drug discovery and personalized medicine.

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