Menstruation involves cyclical shedding and scarless regeneration of the human endometrium, yet the cellular states and interactions that coordinate regeneration remain incompletely defined. Investigation of the perimenstrual window has been particularly challenging because this approximately five-day period is difficult to access experimentally and has been difficult to reproduce in vitro. The study by Nikolakopoulou and colleagues addresses this limitation by focusing on the epithelial compartment and using tissue-derived human endometrial organoids (EOs), a reproducible and hormone-responsive model that enables investigation of epithelial dynamics without the confounding factors associated with co-culture systems.
The researchers aimed to establish an in vitro menstrual cycle (IVMC) protocol capable of reproducing the cyclical epithelial changes associated with hormonal differentiation, menstrual breakdown, and regeneration. The protocol was designed to capture repeated cycles rather than a single round of hormonal stimulation, allowing examination of epithelial transitions during the otherwise inaccessible perimenstrual period. Established EOs were subjected to hormonal differentiation with estrogen and progesterone, followed by hormone withdrawal, mechanical breakdown, (by disrupting Matrigel droplets to mimic extracellular matrix degradation in vivo), and regeneration. This experimental framework was intended to provide a controlled system for investigating how distinct epithelial states emerge and transition during regeneration.
To validate the IVMC system, the researchers benchmarked organoid responses against human endometrial samples spanning the menstrual window using histological, transcriptomic, and multiplex secreted-protein analyses.
Furthermore, transcriptomic profiling—including bulk RNA sequencing and single-cell RNA sequencing (scRNA-seq) was combined with functional validation assays, such as in situ hybridization, quantitative PCR, Western blotting, Luminex analysis, CRISPR-mediated WNT7A knockout, and wound-healing assays with human endothelial cells, to examine candidate mechanisms and epithelial-endothelial interactions.
The IVMC protocol reproduced key epithelial dynamics observed during repeated menstrual cycles. A regeneration-associated luminal epithelial state became most abundant 24 hours after breakdown, representing approximately 40% of cells at that time point before decreasing during subsequent estrogen-differentiation phases. This state was characterized by a wound-healing transcriptional program and by expression of WNT7A, which emerged as a defining marker of the regenerative window. WNT7A transcripts showed a transient peak in expression at 24 hours post-breakdown—a pattern consistently observed across continuous menstrual cycles— while genetic deletion of WNT7A impaired long-term organoid survival. The study further identified strong predicted interactions between regeneration-associated epithelial cells and endothelial populations. Following breakdown, organoids secreted IL-8 and other factors including FGFb and GROβ, while conditioned medium from post-breakdown organoids significantly increased endothelial wound-closure velocity in both HUVECs and primary human uterine microvascular endothelial cells.
Together, these findings establish an organoid-based in vitro framework that captures cyclical epithelial breakdown and regeneration and enables controlled analysis of cellular transitions during the perimenstrual window. The identification of a transient WNT7A-expressing luminal epithelial population and its association with long-term organoid maintenance supports a functional role for this cell state in regeneration. The observed epithelial-endothelial signaling further indicates that the regenerating epithelium functions as a signaling hub during tissue recovery. According to the authors, the IVMC system provides a framework for dissecting the regenerative program of the human endometrium and for investigating the cellular mechanisms underlying this process, ultimately extending its relevance beyond reproduction to fundamental processes of tissue renewal and wound healing.
Reference:Nikolakopoulou, K., Ybañez, W., Klaeylé, L., Frugoli, L., Cindrova-Davies, T., Hotz, H.-R., Soneson, C., & Turco, M. Y. (2026). An in vitro menstrual cycle using organoids captures epithelial cell transitions during menstruation and regeneration of the human endometrium. Cell Stem Cell, 33(5), 747-762. https://doi.org/10.1016/j.stem.2026.04.005.
Author: Yağmur Aktepe
Editor: Elinsu Ak
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