Evaluatie van biomarkers voor niet-invasieve diagnose van endometriose

Scientific SummaryEndometriosis is a benign, chronic gynaecological disorder associated with pelvic pain and infertility. At present, the only way to conclusively diagnose endometriosis is laparoscopic inspection, preferably with histological confirmation. A lack of a reliable non-invasive diagnostic test for endometriosis contributes to the diagnostic delay between 6-11 years. Development of a reliable non-invasive test of endometriosis has been identified as one of the top research priorities. In the present thesis (Chapter 2) we evaluated 28 potential plasma biomarkers of endometriosis based on monocentral biobanking approach by using single and multiplex immunoassay technologies; uni- and multivariate statistical approaches and the QUADAS guidelines (in respect to the menstrual cycle phases, different stages of endometriosis and control groups). We developed and validated a non-invasive diagnostic test (based on a panel of 4 biomarkers (Annexin V, VEGF, CA-125, sICAM-1 or glycodelin)) which enabled the diagnosis of endometriosis undetectable by ultrasound with sensitivity of 81-90% and specificity of 63-81% in independent training and test data sets. We confirmed that a panel of biomarkers can improve the sensitivity and specificity of diagnostic test compared with the diagnostic performance of any single biomarker. Indeed, our panel of 4 biomarkers had a better diagnostic performance than any single biomarker in our study. Additional 2 methodological studies (Chapter 2 and Chapter 3) have been performed to ensure the accuracy of the measured plasma biomarkers. We analytically validated the use of the glycodelin ELISA kit (Bioserv Diagnostics, Rostock, Germany) in plasma (Chapter 2) and confirmed that this assay is accurate for EDTA plasma. In Chapter 3 we compared the diagnostic performance of the hsCRP assay and the classical CRP assay to detect low grade inflammation in plasma of women with endometriosis and confirmed that the hsCRP assay was superior to the classical CRP assay for the detection of low CRP levels (indicating subclinical inflammation in plasma of endometriosis patients) and for the diagnosis of moderate-severe endometriosis. In Chapter 4 we investigated the association between development of endometriosis and genetic variants in the VEGF-pathway (VEGF, PLGF, VEGFR1, VEGFR2, HIF-1 alpha) genes in a large population of Caucasian women. We also evaluated a role of plasma biomarkers of VEGF-pathway as non-invasive biomarkers of endometriosis and an association between genetic variants in genes of VEGF family and plasma levels of corresponding proteins as the source of biological variability (Chapter 4). We demonstrated for the first time that, in Caucasian women, endometriosis is associated with single gene polymorphisms in PLGF rs2268614, HIF-1 alpha rs11549465, VEGFR1 rs9582036 and VEGFR2 rs2305948. Genetic variants in HIF-1 alpha (rs11549465) and PLGF (rs2268613) genes significantly affect VEGF and PLGF plasma levels, respectively. We observed elevated VEGF plasma levels (especially in minimal-mild endometriosis) in women with endometriosis compared to women with laparoscopically excluded endometriosis (Chapter 4). Thus, VEGF was included in the panel of the evaluated plasma biomarkers (Chapter 2). Our findings revealed differences in the genetic and protein expression levels in different stages of endometriosis, suggesting different genetic pathways in the pathogenesis of endometriosis depending on type and severity of the disease (Chapter 2-4). Therefore, future studies (based on evidence-based biobanking approach) are needed that will focus on new system biology approaches, which will allow the better understanding of the pathogenesis of development different phenotypes of endometriosis which is needed for the identification of new non-invasive biomarkers of endometriosis and for the development of a higher sensitivity test to be implemented to the clinical settings.