Evaluation of biomarkers for a non-invasive diagnosis of endometriosis

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 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 165nrpages: 168status: publishe

Salmonella Durban meningitis: case report and genomics study

Abstract Background Bacterial meningitis caused by non-typhoid Salmonella can be a fatal condition which is more common in low and middle-income countries. Case presentation We report the case of a Salmonella meningitis in a Belgian six-month old male infant. The first clinical examination was reassuring, but after a few hours, his general state deteriorated. A blood test and a lumbar puncture were therefore performed. The cerebrospinal fluid analysis was compatible with a bacterial meningitis which was later identified by the NRC (National Reference Center) as Salmonella enterica serovar Durban. Conclusions In this paper, we present the clinical presentation, genomic typing, and probable sources of infection for an unusually rare serovar of Salmonella. Through an extended genomic analysis, we established its relationship to historical cases with links to Guinea

A high sensitivity assay is more accurate than a classical assay for the measurement of plasma CRP levels in endometriosis

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Vascular endothelial growth factor pathway in endometriosis: genetic variants and plasma biomarkers

To study single nucleotide polymorphisms (SNPs) involved in angiogenesis (VEGF, PLGF, VEGFR1, VEGFR2, HIF-1α) and plasma levels of the corresponding proteins (VEGF, PLGF, sVEGFR1, sVEGFR2) in women with and without endometriosis.publisher: Elsevier articletitle: Vascular endothelial growth factor pathway in endometriosis: genetic variants and plasma biomarkers journaltitle: Fertility and Sterility articlelink: http://dx.doi.org/10.1016/j.fertnstert.2015.12.016 content_type: article copyright: Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.status: publishe

Endometriosis is associated with aberrant metabolite profiles in plasma

OBJECTIVE: To identify metabolites that are associated with and predict the presence of endometriosis. DESIGN: Metabolomics study using state-of-the-art mass spectrometry approaches. SETTING: University hospital and universities. PATIENT(S): Twenty-five women with laparoscopically confirmed endometriosis (cases) and 19 women with laparoscopically documented absence of endometriosis (controls). None of the women included in this study had received oral contraception or GnRH agonists for a minimum of 1 month before blood collection. INTERVENTION(S): Plasma collection. MAIN OUTCOME MEASURE(S): Metabolite profiles were generated and interrogated using multiple mass spectrometry methods, that is, high performance liquid chromatography coupled with negative mode electrospray ionization tandem mass spectrometry, UPLC-MS/MS, and ultra performance liquid chromatography-electroSpray ionization-quadrupole time-of-flight (UPLC-ESI-Q-TOF). Metabolite groups investigated included phospholipids, glycerophospholipids, ether-phospholipids, cholesterol-esters, triacylglycerol, sphingolipids, free fatty acids, steroids, eicosanoids, and acylcarnitines. RESULT(S): A panel of acylcarnitines predicted the presence of endometriosis with 88.9% specificity and 81.5% sensitivity in human plasma, with a positive predictive value of 75%. However, due to data limitations the outcome of the receiver operating characteristic curve analysis was not significant. CONCLUSION(S): A diagnostic model based on acylcarnitines has the potential to predict the presence and stage of endometriosi

Endometriosis is associated with aberrant metabolite profiles in plasma

To identify metabolites that are associated with and predict the presence of endometriosis.publisher: Elsevier articletitle: Endometriosis is associated with aberrant metabolite profiles in plasma journaltitle: Fertility and Sterility articlelink: http://dx.doi.org/10.1016/j.fertnstert.2016.12.032 content_type: article copyright: ©2017 American Society for Reproductive Medicine, Published by Elsevier Inc.status: publishe