Circulating microRNAs in endometriosis.

Endometriosis is defined as the presence of endometrial tissue outside the uterus and is the leading cause of disability in reproductive‐age women. The pathogenesis of endometriosis remains unclear and no blood test is available for early diagnosis of the disease. MicroRNAs (miRNAs) are endogenous small ribonucleic acids (RNAs) that have important gene‐regulatory roles via posttranscriptional suppression of target genes. The biological importance of miRNAs, initially demonstrated in cancer, has more recently been confirmed in other diseases. In light of the sustained presence of miRNAs in the circulation and given the emerging evidence on aberrant miRNA expression in endometriotic tissue, we hypothesized that endometriosis is associated with unique plasma miRNA signatures that have diagnostic potential and possibly contribute to disease pathogenesis. In order to test this hypothesis, we established a procedure to measure plasma miRNAs and created a tissue bank of prospectively collected blood and eutopic endometrium samples. Next, by using qRT‐PCR‐based arrays we screened the plasma of a small set of women (n = 16) with or without endometriosis for miRNA content at 3 different phases of the menstrual cycle. We demonstrated that plasma miRNAs do not fluctuate across the cycle and identified 12 plasma miRNAs that are differentially abundant in endometriosis. In silico functional analyses revealed that these miRNAs and their predicted targets have functional relevance in endometriosis, being involved in molecular pathways known to be associated with the disease. Using a microarray methodology, we profiled miRNAs in eutopic endometrium from women with and without endometriosis. We demonstrated no correlation between dysregulated miRNAs in endometrium and plasma, suggesting that the differentially abundant circulating miRNAs are not released from the endometrium. Mir‐551a and mir‐148a* were significantly dysregulated in the endometrium from women with endometriosis, and thus are putative diagnostic markers and therapeutic targets. We also identified differences in miRNA expression between endometriosis‐free women with and without pelvic pain, suggesting that pelvic pain might independently modify the endometrial miRNA profile. Finally, we assessed the value of plasma miRNAs as molecular markers for endometriosis in a prospective diagnostic study in a larger cohort (n = 68) of symptomatic women by using singleplex qRT‐PCR. The diagnostic accuracy of circulating miRNAs for patients with endometriosis was assessed with a predictive algorithm incorporating miRNA expression levels and clinical parameters. A model that included mir‐155, mir‐574‐3p, mir‐133a and mir‐30c, history of infertility and previous miscarriages demonstrated an accuracy of 84.1% with 93.5% sensitivity, 58.8% specificity and AUC = 0.831. This thesis presents potential novel biomarkers for early detection of endometriosis, laying the ground work for future efforts to develop blood‐based biomarkers for this disease. An accurate non‐invasive test would reduce the need for a surgical diagnosis, would be more accessible to women, and is likely to lead to an earlier diagnosis and treatment of endometriosis. Our results need to be confirmed in larger independent patient groups in different populations. In addition, this work raises the possibility that plasma miRNAs may provoke some of the adverse health epiphenomenon associated with endometriosis, which potentially could be altered by therapeutic manipulations of endometriosis‐associated plasma miRNAs. Future studies and broader miRNA profiling may elucidate a relationship between miRNAs, endometriotic disease and its severity.Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 201