L26/O-078 Integrated single-cell RNA-sequencing and cytokine profiling reveal a systemic inflammatory and fibrotic landscape across lesions, endometrium, and menstrual blood in adenomyosis

Abstract Study question What are the cellular and molecular landscapes driving adenomyosis progression across the ectopic lesion, eutopic endometrium, and menstrual blood? Summary answer Pathogenic CHI3L1+ fibroblasts, unbalanced immune cells, and dysregulated cytokine networks characterize coordinated inflammatory, oxidative stress adaptation, and fibrosis in adenomyosis. What is known already Adenomyosis (AM) is a common gynecological disorder characterized by the aberrant presence of endometrial glands and/or stroma in the myometrium. The pathophysiology of AM is characterized by a state of persistent chronic inflammation and progressive fibrosis, both in ectopic lesions and the eutopic endometrium. Women with AM suffer from menorrhagia, dysmenorrhea, and infertility. Extensive literature and comprehensive reviews have established that various cell populations undergo significant phenotypic and functional alterations in AM. Diverse cytokines and growth factors also act as critical mediators in this process. Study design, size, duration This study employed a laboratory-based experimental design using single-cell RNA sequencing (scRNA-seq) and cytokine microarray. The in vitro experiment included a total of 145 clinical samples, conducted over 24 weeks, with data collection taking place between July 2025 and December 2025. Participants/materials, setting, methods Samples were collected from 80 women with adenomyosis and 65 women without adenomyosis. Among them, scRNA-seq was performed on lesion and normal myometrium (3 patients, 3 controls), and menstrual blood (3 patients, 3 controls). Cytokine arrays were analyzed on matched myometrium-endometrium pairs (3 patients, 3 controls) and menstrual blood (4 patients, 4 controls). The other 113 samples were for validation. Single-cell data of the eutopic endometrium were from a published database (GSA-Human: HRA005777). Main results and the role of chance The most significant alterations within lesions were fibrosis and inflammation. ScRNA-seq identified a distinct CHI3L1+ fibroblast subtype, alongside increased numbers of exhausted CD8+ T cells. A variety of cytokines were overexpressed in AM, including IP-10, IL-2, IL-5, IL-4, IL-16, TGFb1, IFN-γ, bFGF, FGF-4, and GNDF (adjusted P < 0.05). These findings were indicative of a coordinated response involving inflammation, fibrosis, neurogenesis, and excessive vascular regeneration within the lesion. Within the eutopic endometrium, macrophages and stromal cells exhibited a stress-adaptive phenotype (elevated HSPs) and pro-fibrotic signatures (elevated MRC1, CD163, SERPINE1, CCN2, THBS1, and COL4A1), representing their involvement in fibrosis and chemotaxis. In vitro validation demonstrated increased numbers of CD206-positive macrophages and enhanced mean vascular density within the AM endometrium. Endometrial cytokines displayed increased CCL8, CCL18, CCL19, HCC-1, and FGF-4, but reduced bFGF. Menstrual blood stromal cells from patients with adenomyosis exhibited a robust pro-inflammatory and immune-stressed state (elevated S100A8/A9, IL-18, CXCL8). Subsequent cytokine array revealed upregulation of IL-2, IL-4, and GM-CSF in AM patients. Notably, bFGF was consistently downregulated in both eutopic and menstrual endometrium. As a crucial molecule in tissue repair and remodeling of mature blood vessels, its diminished expression may explain the clinical phenomenon of prolonged menstruation in patients with AM. Limitations, reasons for caution First, the samples of scRNA-seq and cytokine originate from different eligible individuals and do not have one-to-one correspondences. Moreover, patients included in this study had either endogenous or diffuse-type adenomyosis, indicating the endometrium was affected by lesions. The present investigation did not concentrate on exogenous or localized adenomyosis. Wider implications of the findings This multi-tissue atlas reveals potential therapeutic targets for adenomyosis. In addition, the expression patterns of bFGF in the endometrium warrant further investigation, as they may provide evidence of mechanisms underlying recurrent implantation failure in AM women. Additionally, analyzing menstrual blood is a promising, noninvasive diagnostic tool for clinical practice. Trial registration number No