Using organoids to decipher endometrial regeneration in Asherman syndrome patients

Abstract

Asherman syndrome (AS) is an acquired endometrial pathology characterized by the presence of intrauterine adhesions. Endometrial injury within the stratum basalis frequently triggers the formation of fibrotic tissues, resulting in functional impairment as normal endometrial tissue becomes substituted by non-functional fibrotic tissue and adhesions within the uterine wall. The European Medicines Agency classifies AS, which has a prevalence of 4.4 cases per 10,000 people, as a rare iatrogenic condition that can cause pelvic/abdominal pain, alterations in menstruation, and, ultimately, infertility. Hysteroscopy represents the gold standard for AS diagnosis and treatment (through adhesion removal), which can restore fertility in certain patients but often encounters restricted long-term clinical outcomes. Poor outcomes are primarily due to the tendency for intrauterine adhesion recurrence, which may result in recurrent pregnancy loss, placental abnormalities, preterm birth, and lower birth weight. Alternative endometrial regeneration-promoting therapies based on stem cells have emerged in response to the limited number of treatment options. Some therapies have reached the clinical trial phase in AS patients, including an approach from our group that employs autologous CD133+ bone marrow-derived stem cells (BMDSCs) to enhance endometrial thickness and improve reproductive function. This doctoral thesis aims to investigate the cellular environment of the AS-affected endometrium in human patients by obtaining endometrial cell transcriptomic profiles and analyzing the potential therapeutic effect of our autologous cell therapy in vitro and in vivo, combining single-cell sequencing techniques and a three-dimensional organoid culture model. We performed single-cell RNA sequencing (scRNA-seq) analyses on nine AS patients and six healthy donors (to which we added ten previously processed samples), covering the secretory phase and the window of implantation. Single-cell transcriptomics helped us to classify eighteen cell identities, which included epithelial, endothelial, stromal, immune, perivascular, and smooth muscle cells. These cell populations displayed significant differences when comparing AS and healthy controls, such as significant reductions in ciliated and glandular secretory epithelial subpopulations or the disappearance of a potentially stem-cell-containing SOX9+ cell population in the AS endometrium. Furthermore, the scRNA-seq analysis identified a disease-associated population – the AS-epithelium - exclusively present in AS samples. Consequently, we targeted the detection of this cell population using RNAscope via characteristic SLPI gene expression as a marker for AS pathology. We also encountered apparent differences in the transcriptomic profiles of cell populations between AS and healthy controls, including the downregulation of genes associated with secretory and receptive functions and the upregulation of genes related to proliferation and extracellular matrix (ECM) synthesis in the AS-affected endometrium. Likewise, we discovered impaired cell-to-cell communication (CCC) analysis associated with AS, featuring enriched pathways (e.g., COLLAGEN, FGF, or ICAM) that supported AS's pro-fibrotic and pro-inflammatory nature. Overall, this approach enabled the establishment of an endometrial "atlas" with cell population and gene expression changes associated with AS, which will serve as a reference for future studies. To address the therapeutic impact of autologous CD133+ BMDSC therapy, we analyzed endometrial biopsies from AS patients after administration via scRNA-seq. This approach demonstrated the reversal of particular disease-associated alterations observed in AS patients, such as an increase in epithelial populations and a significant decrease in the AS-epithelium (supporting the analysis of the AS-epithelium as a diagnostic marker) but without recovering SOX9+ cells, suggesting that endometrial regeneration would not occur through the activity of this cell population. Differential expression analysis after CD133+ BMDSC administration revealed the upregulated expression of angiogenic genes (IGFBP2 and KLF2) and genes related to secretory function and ECM stabilization (TPM1), indicating a reversal of the pathological profiles and the development of new blood vessels. CCC analysis also highlighted restored CCC between the epithelium and stroma and reduced signaling pathway activity related to fibrosis (ITGB2 and FGF2) and inflammation (ICAM and LIGHT). In the hope of addressing the inherent limitations of endometrial biopsies, we developed a three-dimensional epithelial organoid culture model derived from biopsies of AS patients. Beyond demonstrating the successful development of AS organoids, we assessed their ability to mimic specific disease patterns, such as restricted growth or reduced proliferation. Overall, cells of the AS epithelial organoids resembled the cell population patterns seen previously in endometrial biopsies. While we did not detect the AS-epithelium, AS epithelial organoids derived from CD133+ BMDSC-treated patient biopsies exhibited a healthy-like profile, highlighting the potential of this model for future research. Altogether, our findings provide a comprehensive overview of the cellular state of the AS-affected endometrium, establish a reference atlas, and describe the therapeutic effect of autologous CD133+ BMDSC therapy, which results in marked improvements in disease pathology. Developing a novel epithelial organoid culture model for the AS endometrium also opens new possibilities for exploring novel treatments or developing combination therapies