P-283 Spatial transcriptomics identifies dysregulated pathways in adenomyosis proposing potential re-purposed lesion-specific treatments

Abstract

Abstract Study question How does the transcriptome of adenomyosis lesions compare to the matched eutopic endometrium, and can they be targeted with lesion-specific treatments? Summary answer Adenomyosis lesions are transcriptionally distinct from matched eutopic endometrium, exhibiting upregulated cell motility pathways, increased cilia, and altered hormonal regulation, providing targets for lesion-specific treatments. What is known already Adenomyosis affects 10% of women of reproductive age and is caused by the presence of ectopic endometrium within the myometrium, causing heavy periods, chronic pain, and subfertility. The only definitive treatment is hysterectomy and there are no current fertility-sparing treatments available. Spatial transcriptomics allows interrogation of the transcriptome with spatial context, overcoming the limitations of bulk and sc-RNA sequencing technology. In the endometrium, spatial transcriptomics has providing insight into how cellular functions differ depending on their location and communication with neighbouring cells, however it has not yet been used to investigate adenomyosis. Study design, size, duration A retrospective observational study of ten women undergoing hysterectomy for heavy, painful periods, with a histological diagnosis of adenomyosis. All women were in the secretory phase of menstrual cycle and had not taken exogenous hormones for the preceding three months. Paired full-thickness endometrial and myometrial biopsies containing adenomyosis lesions were obtained from each woman. The presence of adenomyosis lesions was confirmed by the presence of endometrial glands and stroma >2.5mm from the endo-myometrial junction. Participants/materials, setting, methods Whole transcriptome profiling of the following regions of interest was performed: luminal epithelium, functionalis, basalis, and adenomyosis lesions, and immunofluorescent morphology markers were employed to identify epithelial, stromal, endothelial, and immune cells within each region. A generalised linear-mixed model was designed and unsupervised clustering, differential gene expression comparison, gene set enrichment analysis (GO and Reactome), ligand-receptor signalling, and in silico drug screening was performed. Statistical analyses were adjusted for a false discovery rate of (<0.05). Main results and the role of chance Adenomyosis lesions were more similar to endometrial basalis than functionalis, with 102 significantly DEG’s between adenomyosis lesions and endometrial basalis, and 282 significantly DEG’s between adenomyosis lesions and endometrial functionalis (adjusted p < 0.05). Adenomyosis lesion epithelium, stroma, and immune cells consistently revealed significantly downregulated DEG’s involved with cellular proliferation and apoptosis (FOS, JUNB, DUSP1) compared to the endometrium (adjusted p = 0.002). Pathway analysis revealed that adenomyosis lesion epithelium was positively enriched for terms relating to ciliation compared to the endometrium (adjusted p < 0.0001), and immunostaining confirmed an increased number of ciliated cells in adenomyosis lesions compared with eutopic endometrium in the same samples (n = 10, p < 0.01). Androgen receptor signalling was negatively enriched in the stroma of adenomyosis lesions compared to endometrial functionalis (adjusted P < 0.0001). Ligand-receptor signalling analysis revealed prominent crosstalk between endothelial-epithelial, and immune-epithelial cells in adenomyosis lesions, and a loss of stromal-epithelial cross-talk. TNFSF10 ligand from endothelial and immune cells showed high regulatory potential of gene receptors on epithelial cells known to be involved in cellular response to stress, regulation of growth, and apoptosis (DUSP1, DUSP5, EGR1, FOS, IER3, JUNB, PPP1R15A, SGK1). In-silico drug repurposing analysis revealed key compounds which may target adenomyosis lesions and spare the eutopic endometrium. Limitations, reasons for caution This is an observational study with a small sample size of ten which is a limitation, although is larger than the sample size of most published transcriptional studies of this type. Further validation work is needed to confirm the exploratory transcriptional findings outlined. Wider implications of the findings Fertility sparing, effective, lesion-specific treatments are urgently needed for women with adenomyosis. This study provides region and cell-type specific comparative transcriptional data between adenomyosis lesions and eutopic endometrium, identifying lesion-specific dysregulated pathways, insight into their biological relevance, and potential treatment targets to be trialled in future pre-clinical studies. Trial registration number Not applicable