Single-Cell and Spatial Transcriptomic Dissection of Macrophage-Associated Inflammatory Signalling in Endometriosis-Associated Pain: A Hypothesis-Generating Study of Candidate Tissue-Level Genes

Background: Endometriosis is an oestrogen-dependent chronic inflammatory disease with chronic pelvic pain as a predominant and disabling symptom that severely compromises patient quality of life. The molecular mechanisms governing endometriosis-associated pain remain poorly elucidated. Given the lack of individual clinical pain scores in public datasets, this hypothesis-generating study aimed to characterise pain-correlated cell subsets and signalling programmes at single-cell resolution using a literature-based inflammatory gene module, and to identify replicable tissue-level candidate genes across independent cohorts. Methods: A refined single-cell atlas containing 103,434 cells was reconstructed from 31 valid biopsies of 14 patients (GSE179640), with irrelevant organoid samples excluded. Comprehensive analyses were performed, including standardised single-cell preprocessing, Harmony batch integration, Louvain clustering, pseudobulk-based differential analysis, transcription factor activity evaluation, CellChat-based intercellular communication prediction, and nested cross-validated Lasso regression. A 60-gene computational module score was applied to proxy pain phenotypes, with biopsies and patients defined as statistical units. Results: Macrophages dominated the high pain-score cell population (68.8%), while stromal fibroblasts also contributed considerably. High-score macrophages exhibited typical M1-like pro-inflammatory features, activated innate immune and NF-κB pathways, and upregulated the activities of NFKB2, CEBPB and STAT4. Macrophage-derived galectin, MIF, SPP1 and chemokine signalling were prominently enriched. The Lasso model showed limited discriminative power. Only TYROBP and CXCL12 maintained statistical significance after multiple-test correction across two bulk cohorts, whereas spatial co-expression patterns were unstable and section-dependent. Conclusions: Macrophages are the primary cellular population enriched with pain-related transcriptional signatures, though such enrichment is largely driven by the inflammatory components of the gene set. This study provides hypothesis-generating findings. The evaluated pain phenotype is a computational surrogate, and all candidate genes await further experimental and clinical validation.