High-throughput mRNA sequencing of stromal cells from endometriomas and endometrium

Abstract

The aetiology of endometriosis is still unclear and to find mechanisms behind the disease development, it is important to study each cell type from endometrium and ectopic lesions independently. The objective of this study was to uncover complete mRNA profiles in uncultured stromal cells from paired samples of endometriomas and eutopic endometrium. High-throughput mRNA sequencing revealed over 1300 dysregulated genes in stromal cells from ectopic lesions, including several novel genes in the context of endometriosis. Functional annotation analysis of differentially expressed genes highlighted pathways related to cell adhesion, extracellular matrix-receptor interaction and complement and coagulation cascade. Most importantly, we found a simultaneous upregulation of complement system components and inhibitors, indicating major imbalances in complement regulation in ectopic stromal cells. We also performed experiments to evaluate the effect of endometriosis patients' peritoneal fluid (PF) on complement system gene expression levels, but no significant impact of PF on, and levels was observed. In conclusion, the use of isolated stromal cells enables to determine gene expression levels without the background interference of other cell types. In the future, a new standard design studying all cell types from endometriotic lesions separately should be applied to reveal novel mechanisms behind endometriosis pathogenesis.

Chemosensitivity and chemoresistance in endometriosis – differences for ectopic versus eutopic cells

“…Endometrial tissue samples and peritoneal endometriotic lesions were collected from 11 endometriosis patients (see Table 2) undergoing laparoscopy at the Tartu University Hospital Women's Clinic. Tissue samples were immediately placed into the cryopreservation medium and processed as described previously (Rekker et al, 2017). At least one endometriotic lesion sample from each patient was placed into formalin and the diagnosis was confirmed by histopathological examination of specimens.…”

A relational database to identify differentially expressed genes in the endometrium and endometriosis lesions

“…Comparing the gene expression profiles of disease tissue to that of a normal healthy tissue is a powerful approach to understand the underlying cellular events in the etiology of any disease 8 . Accordingly, gene expression changes associated with endometriosis have also been analyzed in previous studies using various microarray platforms by comparing the endometriosis lesions with eutopic endometrial tissues 9 – 13 , or by comparing the endometrium of the patients to that of healthy controls 14 – 16 . All these studies have offered some essential understanding into the transcriptional differences related to endometriosis, however, only a limited number of samples were included due to various constraints, with samples size ranging from between 6 and 25.…”

DNA methylation alterations—potential cause of endometriosis pathogenesis or a reflection of tissue heterogeneity?†

“…Second, the uncultured single cells or cell populations from lesion biopsies and endometrium can be isolated using fluorescently labelled antibodies against cell surface markers in combination with fluorescence activated cell sorting (FACS). FACS methodology has previously been used to isolate specific cell types from endometrium of healthy women [71] and from endometrium of women suffering from endometriosis [72,73] for gene expression studies.…”

“…We have shown the usefulness of this methodology for isolation of CD10-positive stromal cells from endometrioma biopsies for transcriptome study [72] and the amount of cells obtained by FACS should also be suitable for DNA methylation analysis. Currently, the main limitation of this methodology is the absence of specific antibodies to discriminate endometrial epithelial cells from lesion biopsy.…”