REPRODUCTION © 2017 Society for Reproduction and Fertility DOI: 10.1530/REP-17-0092 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org RESEARCH High-throughput mRNA sequencing of stromal cells from endometriomas and endometrium Kadri Rekker 1,2 , Merli Saare 1,2 , Elo Eriste 2 , Tõnis Tasa 3,4 , Viktorija Kukuškina 4,5 , Anne Mari Roost 2 , Kristi Anderson 2 , Külli Samuel 2 , Helle Karro 1,6 , Andres Salumets 1,2,7,8 and Maire Peters 1,2 1 Department of Obstetrics and Gynecology, Institute of Clinical Medicine, University of Tartu, Tartu, Estonia, 2 Competence Centre on Health Technologies, Tartu, Estonia, 3 Institute of Computer Science, University of Tartu, Tartu, Estonia, 4 Estonian Genome Center, University of Tartu, Tartu, Estonia, 5 Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia, 6 Tartu University Hospital’s Women’s Clinic, Tartu, Estonia, 7 Department of Biomedicine, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia and 8 Department of Obstetrics and Gynecology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland Correspondence should be addressed to K Rekker; Email: kadri.rekker@gmail.com Abstract The aetiology of endometriosis is still unclear and to fnd 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 profles 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 in vitro experiments to evaluate the effect of endometriosis patients’ peritoneal fuid (PF) on complement system gene expression levels, but no signifcant impact of PF on C3, CD55 and CFH 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. Reproduction (2017) 154 93–100 Introduction Endometrial tissue outside the uterine cavity can form lesions on ovaries and other peritoneal organs, resulting in endometriosis. As the molecular aetiology of endometriosis is still unclear, it is important to detect gene expression alterations in endometriotic lesions and eutopic endometrium of endometriosis patients. Three major types of endometriotic lesions are known, peritoneal lesions, ovarian endometriomas and deep infltrating lesions (Nisolle & Donnez 1997), that are distinct clinical entities with unique histopathogeneses accompanied by specifc expression profles, depending on the anatomical location of lesions (Meola et al. 2010, Ballester et al. 2012, Filippi et al. 2016). Therefore, it seems reasonable to study endometriomas and lesions from other locations separately. Several transcriptomic studies have been conducted to fnd molecular alterations in endometrial cells lining the inner surface of endometriomas (reviewed in Sanchez et al. 2015). However, despite hundreds to thousands of genes and specifc pathways that have been revealed in these studies, the results remain inconclusive and no common molecular markers or pathways linked to endometriosis have been described. The most probable reason for these discordances could be the tissue heterogeneity of whole-lesion biopsies, as in addition to endometrial cells the endometrioma wall includes various proportions of ovarian stroma, follicles and fbrous tissue that prevent the detection of gene expression alterations characteristic to ectopic endometrial cells (reviewed in Sanchez et al. 2014). There are only a few studies utilizing pure cell populations for exploring gene expression in endometriotic lesions. The frst study applied laser capture microdissection to gather epithelial cells from eu- and ectopic endometrial tissues (Wu et al. 2006), while another study analysed cultured immortalized stromal cells from ovarian endometriosis Downloaded from Bioscientifica.com at 02/26/2019 01:06:18PM via free access