Clinical and single-cell integration identifies a TCF21-associated stromal death–inflammation signature in endometriosis

Background Stromal heterogeneity, regulated-cell-death-associated stress, and chronic inflammation contribute to endometriosis, but their relationships within specific stromal populations remain incompletely understood. This study integrated clinical characterization with single-cell and spatial transcriptomics to investigate a TCF21-associated stromal death–inflammation signature. Methods A retrospective cohort of 150 patients with histopathologically confirmed endometriosis was characterized clinically. Public single-cell datasets (GSE179640 and GSE213216) were analyzed to identify stromal populations, reconstruct transcriptional trajectories, evaluate death-associated and inflammatory programs, and infer intercellular communication. Spatial transcriptomic analysis assessed TCF21-associated expression patterns in endometriotic lesions. Multiplex immunofluorescence, RT-qPCR, TCF21 silencing, EdU incorporation, Transwell assays, and three-dimensional spheroid culture were used for experimental evaluation. Results Single-cell analysis identified 13 major cell classes and a fib-TCF21-associated stromal state characterized by TCF21, NEXN, IFI44L, WNT2B, and SPRR2F. Cell-death-associated and inflammatory transcriptional programs varied across stromal states, with fib-TCF21 exhibiting selective rather than uniformly elevated program activity. Independent single-cell analysis identified a related but anatomically heterogeneous stromal expression pattern. Spatial transcriptomics demonstrated localized TCF21 expression and heterogeneous distributions of stromal, stress-associated, and inflammatory programs. Tissue immunofluorescence supported stromal-associated TCF21 expression, while lesion-derived ihESCs exhibited higher basal TCF21 expression than the eutopic stromal-cell model. TCF21 silencing was associated with reduced EdU incorporation, Transwell membrane traversal, and three-dimensional spheroid growth. Conclusion Integration of clinical, single-cell, spatial, and experimental findings identifies a TCF21-associated stromal transcriptional signature linked to heterogeneous death-related stress and inflammatory programs in endometriosis. These findings support further investigation of TCF21-associated stromal biology and its potential relevance to molecularly targeted interventions.