Spatial and single-cell transcriptomics landscape of adenomyosis

Adenomyosis is a prevalent gynaecological disorder affecting women of reproductive age, yet the underlying etiology and cellular-molecular mechanisms driving its pathogenesis remain poorly understood. Gonadotropin-releasing hormone agonists (GnRHa) are commonly used to improve clinical pregnancy rates in affected patients; however, their effects on specific cellular niches within the diseased uterus have not been fully elucidated.

This study aimed to profile adenomyosis at both the single-cell and spatial transcriptomic levels to examine various cell populations and systematically evaluate the effects of GnRHa treatment.

To characterize in depth the cellular and molecular landscape of adenomyosis, we employed single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (Geo-seq) to profile 15 participants, including 11 patients with adenomyosis (three untreated and eight treated with GnRHa) and four control participants. For validation using immunofluorescence and immunohistochemistry, we included an additional two control subjects and 11 patients with adenomyosis. Furthermore, publicly available scRNA-seq datasets from samples collected during the proliferative phase of the menstrual cycle were incorporated for comparison.

Our analysis revealed that ectopic endometrial glands in adenomyosis were enriched with ciliated epithelial cells, a feature that persisted following GnRHa treatment, suggesting a potential role of these cells in disease pathogenesis. Immune-inflammatory signatures, including elevated CD4⁺ T cells and LYVE1⁺ macrophages, were prominent in untreated adenomyosis, contributing to a pro-angiogenic microenvironment. This inflammatory and angiogenic activity was partially mitigated by GnRHa therapy. Notably, mast cells were found to be concentrated in the junctional zone, implicating this region as a potential site of disease initiation.

Our findings provide evidence for the invagination theory, highlighting the involvement of ciliated epithelial cells and immune-angiogenic crosstalk in adenomyosis pathogenesis. Moreover, this study demonstrates that GnRHa exerts therapeutic effects through the normalization of immune cell composition and restoration of epithelial-stromal interaction, offering novel mechanistic insights into its mode of action.