In preprints: shedding light - chemogenetic induction of menstruation in mice.

In summary, the preprint by Çevrim and colleagues ( Çevrim et al., 2025 preprint) introduces X-Mens as a physiologically relevant and repeatable mouse model of menstruation, representing a significant advancement over previous approaches. By closely mimicking key hallmarks of human menstruation – cyclical shedding, scarless regeneration and hormone-dependent decidualization – X-Mens represents a substantial improvement over previous models, with potential for continued refinement to better replicate human menstruation. The integration of spatial transcriptomics and cross-species comparisons delivers new insights into endometrial compartmentalization and fibroblast differentiation, expanding our understanding of uterine architecture and regeneration. An important gap still remains in understanding the distinctions between bleeding during menstruation and miscarriage. Only recently, Burns and colleagues ( Burns et al., 2025 preprint) demonstrated that the ‘classic’ decidualization markers – IGFBP1, PRL, FOXO1 – are strongly specific to first-trimester decidua. These markers peak in first-trimester tissue, whereas their expression is lowest in the early secretory phase and rises gradually through mid- and late-secretory phases, yet never reaches first-trimester levels. Morphologically, first-trimester stromal cells display rounded, epithelioid features with prominent nuclei – hallmarks of decidualization – while these characteristics are absent in mid-secretory tissue. These findings underscore fundamental molecular and structural differences between mid-secretory decidualization and pregnancy-associated decidualization. Moving forward, a key priority must be to identify clear markers that distinguish menstrual, pregnancy-independent decidualization from post-implantation decidualization. To refine mouse models of menstruation – whether using DREADDs or oil-induced protocols – it will be essential to confirm that stromal cells truly mimic secretory-phase decidual stromal cells rather than pregnancy-associated stromal cells. Additionally, demonstrating that bleeding can be induced via oral contraceptive withdrawal, rather than mifepristone (which triggers miscarriage-like bleeding), will be essential for validating these models as physiologically relevant to menstruation.

In a recent preprint, Çevrim and colleagues ( Çevrim et al., 2025 preprint) developed X-Mens, a chemogenetic mouse system that reproduces the core anatomical and functional characteristics of menstruation. The system employs engineered G-protein-coupled receptors known as DREADDs (Designer Receptors Exclusively Activated by Designer Drugs), expressed in the endometrium. Unlike native receptors, DREADDs are inert to endogenous ligands but can be selectively activated by synthetic agonists such as clozapine-N-oxide (CNO), enabling precise, cell type–specific control of intracellular signaling pathways like Ca 2+ and cAMP. By activating these pathways in a progesterone-primed endometrium, the model induces premenstrual decidualization followed by hormone-dependent tissue breakdown. Upon CNO administration, mice exhibited vaginal bleeding ~7.5 days after pseudopregnancy onset, coinciding with progesterone decline, as is also observed in the oil-induced decidualization model. Bleeding persisted for about 4 days – closely resembling the duration of human menstruation. This phenotype was accompanied by strong expression of the decidual marker Prl8a2 and uterine enlargement, confirming successful decidualization. Histological analysis revealed extensive vascular remodeling and detachment of the decidualized layer, consistent with tissue shedding observed in human menstruation. However, histology alone does not clearly define the boundaries of the functionalis and basalis layers; additional characterization of glandular morphology within these zones is required to confirm the presence of two distinct layers. Notably, menstrual effluent contained viable endometrial fragments capable of forming organoids in culture, indicating that tissue shedding involves live cells rather than necrotic debris. Thus, the ability to achieve sustained bleeding and confirming the viability of shed endometrial tissue using organoid culture highlights the fidelity of this system in mimicking key aspects of human menstruation.

To interrogate the spatial and transcriptional dynamics of menstruation, Çevrim and colleagues ( Çevrim et al., 2025 preprint) combined slide-tags spatial transcriptomics, single-nucleus RNA-sequencing and immunofluorescence imaging of full-thickness endometrial sections from X-Mens mice across key stages of the cycle, alongside human samples from the secretory and menstrual phases. This multimodal approach provided insights into functionalis–basalis organization, with concentric decidual clusters forming a radial maturation gradient that compresses underlying fibroblasts and creates cleavage planes for tissue detachment. These findings are consistent with previous single-cell ( Burns et al., 2025 preprint; Marečková et al., 2024 ) and single-nucleus ( Marečková et al., 2024 ) transcriptomic studies that identified distinct stromal cell subsets across the menstrual/early proliferative and decidual stromal cell subsets during early, mid and late secretory phases and characterized their unique gene-expression signatures. Additionally, Çevrim and colleagues ( Çevrim et al., 2025 preprint) identified dynamic fibroblast states, including proliferative and induced subtypes. With the advent of single-cell transcriptomics, it has become increasingly evident that fibroblasts are a heterogeneous population involved in endometrial repair ( Kirkwood et al., 2022 ; Wang et al., 2020 ; Winkler et al., 2024 ). Cross-species integration demonstrated strong conservation of decidualization programs, with 31% overlap in pseudotime-associated genes between human and X-Mens samples, including key regulators such as Cebpb , Foxo1 , and Wnt5a . These findings position X-Mens as a physiologically relevant menstruation model that can be used to study spatial organization and gene regulatory networks involved in shedding and repair.

A key strength of the X-Mens system is its ability to support repeated cycles of menstruation without compromising uterine integrity or function. Following the initial induction, endometrial morphology returned to its pre-induction state within days, characterized by restoration of a continuous luminal epithelium, normalization of gland distribution and absence of decidual cells. This regenerative capacity enabled the protocol to be repeated up to five times in the same animal, with each cycle exhibiting robust decidualization and bleeding, demonstrating that the endometrium retains its responsiveness over successive inductions. Importantly, post-menstrual uteri were competent for implantation and pregnancy, confirming functional recovery of the regenerated tissue. Variability in bleeding onset and duration was observed across cycles, paralleling inter-cycle variation in humans. This variability was linked to differences in the timing of progesterone decline during pseudopregnancy; administration of mifepristone synchronized luteolysis and markedly reduced variation, with all animals initiating bleeding within one day of treatment. Together, these findings establish X-Mens as a reproducible and hormonally tunable model that captures both the cyclical nature of menstruation and the regenerative resilience of the endometrium.