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Title: Evaluation of biomarkers for a non-invasive diagnosis of endometriosis
Other Titles: Evaluatie van biomarkers voor niet-invasieve diagnose van endometriose
Authors: Vodolazkaia, Alexandra
Issue Date: 15-May-2012
Abstract: 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.
Table of Contents: Table of Contents

Abbreviations

Chapter 1: General introduction and Objective of the PhD project 1
General Introduction 2
1.1. Endometriosis: general background 2
1.1.1. Definition, prevalence, economic impact 2
1.1.2. Aetiology and genetic predisposition 3
1.1.3. Classification of endometriosis 4
1.1.4. Pathogenesis of endometriosis 6
1.2. Diagnosis of endometriosis 9
1.2.1. State of the art 9
1.2.2. Peripheral biomarkers of endometriosis 10
1.2.3. Need for a non-invasive diagnosis 13
1.3. Biobanking approach as a supporting tool in endometriosis research 14
1.4. Multiplex high throughput technologies in endometriosis research 16
1.4.1. Multiplex immunoassay technologies 16
1.4.2. High throughput SNPs assays 19
1.5. Development a biomarker-based non-invasive diagnostic test 20
1.5.1. Phases for the development of a biomarker-based test and application to our study 21
General objectives and specific aims of the project 24
References 25

Chapter 2: Evaluation of a panel of 28 plasma biomarkers for a non-invasive diagnosis of endometriosis 35
2.1. Abstract 36
2.2. Introduction 37
2.3. Materials and Methods 40
2.4. Results 46
2.5. Discussion 52
References 59

Chapter 3: A comparative study of the performance of hsCRP assay vs classical CRP assay in plasma of endometriosis and controls patients 79
3.1. Abstract 80
3.2. Introduction 81
3.3. Materials and Methods 83
3.4. Results 85
3.5. Discussion 88
References 92

Chapter 4: VEGF-pathway in endometriosis: Genetic Variants and Plasma Biomarkers 101
4.1. Abstract 102
4.2. Introduction 104
4.3. Materials and Methods 109
4.4. Results 113
4.5. Discussion 116
References 124




Chapter 5: General Discussion and Further perspectives 141
General Discussion and Further perspectives 142
5.1. Development of a non-invasive diagnostic test for endometriosis 142
5.2. Patient population for a non-invasive diagnostic test of endometriosis 143
5.3. A role of the QUADAS guidelines in endometriosis research 144
5.4. Endometriosis as a heterogeneous disease 145
5.5. Complexity and challenges of endometriosis research 146
5.5.1. Technical variability as a source of complexity in endometriosis research 146
5.5.1.1. Sample matrix: Plasma versus Serum 147
5.5.1.2. Processing of samples 147
5.5.1.3. Analytical variability 148
5.5.2. Biological variability as a source of complexity in endometriosis research 149
5.6. Further perspectives 150
References 152

Scientific Summary 158
Wetenschappelijke Samenvatting 160
Curriculum Vitae 163
Publications 165
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Sexual, Pelvic, Reproductive and Family Studies (-)
Embryo and Stemcells (-)
Section Woman - Miscellaneous (-)
Pregnancy, Foetus and Newborn (-)

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