Modeling the window of implantation: insights from endometrial biopsy and menstrual blood-derived organoids and endometrial stromal cells

STUDY QUESTION Can menstrual blood-derived organoids (MB-organoids) and human endometrial stromal cells (MB-ESCs) serve as a physiologically relevant, non-invasive model for studying endometrial function and hormonal response? SUMMARY ANSWER MB-organoids and ESCs recapitulate key structural and functional features of the endometrium, responding to hormonal stimulation in a manner comparable to biopsy-derived models, supporting their use in reproductive research. WHAT IS KNOWN ALREADY Endometrial organoids derived from biopsy samples have provided valuable insights into endometrial physiology and implantation. However, their reliance on invasive tissue sampling limits their clinical and research applications. Menstrual blood contains viable endometrial cells, yet its potential for generating functional three-dimensional (3D) models remains underexplored. STUDY DESIGN, SIZE, DURATION This cross-sectional, in vitro cell culture study established and characterized 3D-organoids and ESCs derived from menstrual blood, assessing their structural and functional properties as well as their response to hormonal stimulation over a culture period of several weeks. The work was carried out between October 2023 and December 2024, in two European University hospitals. PARTICIPANTS/MATERIALS, SETTING, METHODS Menstrual blood samples were collected from healthy fertile donors (n = 6). Isolated endometrial cells were cultured using a three-layer gradient system to generate MB-organoids or selected for deriving MB-ESCs. MB-organoids were characterized based on morphological features, including periodic acid-Schiff (PAS) staining for glycogen deposition, scanning electron microscopy (SEM) for pinopode analysis, and immunofluorescence for epithelial (CK8/18) and stromal (vimentin) markers. ESCs were assessed for decidualization by measuring IGFBP-1 and ZBTB16 expression after hormonal stimulation, with mifepristone used to terminate progesterone signaling. MAIN RESULTS AND THE ROLE OF CHANCE MB-organoids demonstrated structural and functional characteristics similar to biopsy-derived endometrial organoids, including glycogen accumulation and pinopode formation, indicative of endometrial receptivity. Immunofluorescence confirmed the presence of both epithelial and stromal populations as well as glycodelin A production. MB-ESCs underwent decidualization in response to hormonal stimulation, with IGFBP-1 and ZBTB16 upregulation, which was suppressed by mifepristone, confirming their functional relevance. LIMITATIONS, REASONS FOR CAUTION This in vitro culture system models key endometrial features but lacks the complexity of in vivo conditions. While menstrual blood derived organoids and ESCs respond to hormonal cues, donor variability and the absence of immune and vascular components limit their physiological relevance. Larger studies and more advanced co-culture systems are needed to improve reproducibility and better replicate the dynamic endometrial environment. WIDER IMPLICATIONS OF THE FINDINGS Menstrual blood provides a non-invasive, accessible source for generating functional endometrial models. MB-organoids and MB-ESCs offer promising applications in reproductive medicine, including drug screening, disease modeling, and personalized therapies for endometrial disorders. STUDY FUNDING/COMPETING INTEREST(S) This work is supported by the Italian Ministry of University and Research - NextGenerationEU PNRR «THE» (Tuscany Health Ecosystem), Spoke 6 - Precision Medicine & Personalized Healthcare ECS_00000017, the Estonian Research Council grant no. PRG1076, Swedish Research Council grant no. 2024-02530, Novo Nordisk Foundation grant no. NNF24OC0092384, and Horizon Europe grant NESTOR, grant no. 101120075. The University of Siena Open Access funding partially supported the APC fees. The authors declare no competing interests.