Inflammatory and Molecular Mechanisms of Adenomyosis Associated Pain: Insights from Multiple Analytic Approaches

Adenomyosis, a common gynecologic condition of the uterus, affects women with diverse symptoms including pain. Traditionally evaluated pathologically, advanced imaging modality has led to more diagnoses including asymptomatic women. This noninvasive diagnosis has raised questions about disease progression and symptom development. This study aimed to investigate the cellular and molecular patterns associated with adenomyosis and adenomyosis-related pain, focusing on inflammatory processes. We employed an integrative, retrospective bioinformatic design combining publicly available bulk RNA-sequencing data from adenomyotic and control endometrium and myometrium with single-cell RNA-sequencing data from adenomyosis patients stratified by the presence of pain.

We performed enrichment analysis, gene expression profile analysis with core inflammatory genes, protein-protein network analysis, and cell-cell communication analysis.

Activation levels of core inflammation-associated modules revealed elevated inflammatory activity in endometrial tissues compared to myometrial tissues in adenomyosis. Single-cell RNA-seq identified significant upregulation of inflammation modules in cell types such as endothelial, mesenchymal, and NK/T cells, particularly in patients experiencing pain. NK/T cells disclosed co-upregulation in non-canonical inflammatory pathways, implicating them in pain-associated immunity. Also, combinatorial analyses through enrichment analysis and core inflammatory-associated gene expression pattern analysis emphasized key inflammatory genes, including CGAS, TNF, and IL1B, along with pain-specific alterations in DNAJB9, GSTA1, CXCL8, and CXCL3 across cell subpopulations. Additionally, protein-protein interaction (PPI) network analysis identified multipotent stem cells and NK/T cells as key drivers of nociceptive processes, mediated by chemokines such as CCL11 and CXCL13. Moreover, cell-cell communication analysis exhibited pain-associated disruptions in IL-1, estradiol, and 2-AG pathways.

These findings underscore the significance of inflammation, mitochondrial immunity, and specific chemokine pathways in the pathophysiology of adenomyosis and its pain. Because the single-cell analyses were based on a small cohort (n = 2 per group), these results should be regarded as hypothesis-generating and require validation in larger, well-characterized cohorts. Understanding these mechanisms could nonetheless help guide the development of targeted therapies to alleviate symptoms and enhance outcomes.