STUDY QUESTION: Can plasma miRNAs be used for the non-invasive diagnosis of endometriosis in infertile women? SUMMARY ANSWER: miRNA-based diagnostic models for endometriosis failed the test of independent validation. WHAT IS KNOWN ALREADY: Circulating miRNAs have been described to be differentially expressed in patients with endometriosis compared with women without endometriosis, suggesting that they could be used for the non-invasive diagnosis of endometriosis. However, these studies have shown limited consistency or conflicting results, and no miRNA-based diagnostic test has been validated in an independent patient cohort. STUDY DESIGN, SIZE, DURATION: We performed genome-wide miRNA expression profiling by small RNA sequencing to identify a set of plasma miRNAs with discriminative potential between patients with and without endometriosis. Expression of this set of miRNAs was confirmed by RT-qPCR. Diagnostic models were built using multivariate logistic regression with stepwise feature selection. In a final step, the models were tested for validation in an independent patient cohort. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Plasma of all patients was available in the biobank of the Leuven Endometriosis Centre of Excellence. Biomarker discovery and model development were performed in a discovery cohort of 120 patients (controls = 38, endometriosis = 82), and models were tested for validation in an independent cohort of 90 patients (controls = 30, endometriosis = 60). RNA was extracted with the miRNeasy Plasma Kit. Genome-wide miRNA expression analysis was done by small RNA sequencing using the NEBNext small RNA library prep kit and the NextSeq 500 System. cDNA synthesis and qPCR were performed using the Qiagen miScript technology. MAIN RESULTS AND THE ROLE OF CHANCE: We identified a set of 42 miRNAs with discriminative power between patients with and without endometriosis based on genome-wide miRNA expression profiling. Expression of 41 miRNAs was confirmed by RT-qPCR, and 3 diagnostic models were built. Only the model for minimal–mild endometriosis (Model 2: hsa-miR-125b-5p, hsa-miR-28-5p and hsa-miR-29a-3p) had diagnostic power above chance performance in the independent validation (AUC = 60%) with an acceptable sensitivity (78%) but poor specificity (37%). LIMITATIONS, REASONS FOR CAUTION: The diagnostic models were built and tested for validation in two patient cohorts from a single tertiary endometriosis centre. Further validation tests in large cohorts with patients from multiple endometriosis centres are needed. WIDER IMPLICATION OF THE FINDINGS: Our study supports a possible biological link between certain miRNAs and endometriosis, but the potential of these miRNAs as clinically useful biomarkers is questionable in women with infertility. Large studies in well-described patient cohorts, with rigorous methodology for miRNA expression analysis, sufficient statistical power and an independent validation step, are necessary to answer the question of whether miRNAs can be used as diagnostics markers for endometriosis. STUDY FUNDING/COMPETING INTEREST(S): The project was funded by a grant from the Research Foundation - Flanders (FWO). A.V., D.F.O. and D.P. are PhD fellows from the FWO. T.D. is vice president and Head of Global Medical Affairs Fertility, Research and Development, Merck KGaA, Darmstadt, Germany. He is also a professor in Reproductive Medicine and Biology at the Department of Development and Regeneration, Group Biomedical Sciences, KU Leuven (University of Leuven), Belgium and an adjunct professor at the Department of Obstetrics and Gynecology in the University of Yale, New Haven, USA. Neither his corporate role nor his academic roles represent a conflict of interest with respect to the work done by him for this study. The other co-authors have no conflict of interest. Trial registration number: Not applicable.

The aim of this systematic review is to assess the power of circulating miRNAs as biomarkers as a diagnostic tool in endometriosis. In endometriosis-suspected women with uncertain imaging, the only way to confirm or exclude endometriosis with certainty is currently laparoscopy. This creates a need for non-invasive diagnostics. We searched the literature through the PubMed database using the Mesh terms ‘endometriosis’ and ‘miRNAs’. Some, but limited, overlap was found between the 32 articles included, with a total of 20 miRNAs reported as dysregulated in endometriosis in two or more studies. MiR-17-5p was reported as dysregulated in six studies, followed by miR-451a and let-7b-5p in four studies and miR-20a-5p, miR-143-3p, miR-199a-5p and miR-3613-5p in three studies. Furthermore, a possible impact of the menstrual phase on miRNA expression was noted in five studies, while no influence of hormonal intake was observed in any included study. The modest reproducibility between studies may be attributable to biological variability as well as to the lack of universal protocols, resulting in pre- and analytical variability. Despite the identification of several suitable candidate biomarkers among the miRNAs, the need for high-quality studies with larger and well-defined population cohorts and the use of standardized protocols lingers.

Endometriosis is a common gynecological disease that is characterized by the presence of functional endometrial-like lesions in the abdominal cavity. Aside from epithelial cells, these lesions consist of stromal cells that have the capacity to migrate, adhere, proliferate, and induce neuro- and lymphangiogenesis, which allows them to survive at ectopic locations. However, the exact underlying mechanisms that regulate these changes are yet to be elucidated. The common ground of these processes, however, is the second messenger, calcium. In this regard, members of the superfamily of transient receptor potential (TRP) ion channels, which are known to be calcium-permeable and expressed in the endometrium, have emerged as key regulators. Here, we assessed the molecular and functional expression of TRP channels in stromal cells isolated from the eutopic endometrium of endometriosis patients and controls. Using RT-qPCR, high mRNA levels of TRPV2, TRPV4, TRPM4, TRPM7, TRPC1, TRPC3, TRPC4, and TRPC6 were observed in the whole endometrium throughout the menstrual cycle. Additionally, and in line with previous reports of control patients, TRPV2, TRPV4, TRPC1/4, and TRPC6 were present in human endometrial stromal cells (hESC) from endometriosis patients both at the molecular and functional level. Moreover, proliferation and migration assays illustrated that these parameters were not affected in stromal cells from endometriosis patients. Furthermore, comparison between eutopic and ectopic endometrial samples revealed that the RNA expression pattern of TRP channels did not differ significantly. Collectively, although a functional expression of specific ion channels in hESCs was found, their expression did not correlate with endometriosis.

Endometriosis-associated pain has debilitating effects on the quality of life of patients. Despite its high prevalence in reproductive-aged women, the pathophysiology is still unknown, impeding the development of targeted treatment approaches. The prospective ExPAND study proposes the neurosteroids pregnenolone sulphate (PS) and dehydroepiandrosterone sulphate (DHEAS) as potential contributors to endometriosis-associated pain, due to their agonistic action at the pain-related ion channel TRPM3. To this end, endometrium, deep endometriosis lesions, and peritoneal fluid were prospectively collected in four demarcated patient groups, which were characterised based on their pain symptoms, as scored via the WERF-EPHect questionnaire, i.e., (1) control (n = 44), (2) endometriosis patients with no pain symptoms (n = 24), (3) with only severe dysmenorrhea (n = 54), or (4) with both severe dysmenorrhea and non-cyclic pelvic pain (n = 78). Tissue mRNA expression levels of steroidogenic enzymes were investigated and showed significantly increased levels of CYP17A1 in the endometrium of patients with severe pain symptoms compared to control tissue. In addition, liquid chromatography with tandem mass spectrometry (LC-MS/MS) was performed to investigate neurosteroid concentrations in the peritoneal fluid. Both neurosteroids PS and DHEAS were present in the peritoneal fluid at concentrations that are known to stimulate TRPM3 activity in vitro. Finally, using microfluorimetric Ca²⁺ imaging, we demonstrate that both DHEAS and PS stimulate human stem-cell-derived sensory neurons in a TRPM3-dependent manner. Taken together, these data indicate a potential contribution of steroidogenesis and TRPM3 in endometriosis-associated pain.