Identification of novel non-invasive biomarkers of urinary chronic pelvic pain syndrome: findings from the Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network

Abstract

Objective: To examine a series of candidate markers for urological chronic pelvic pain syndrome (UCPPS), selected based on their proposed involvement in underlying biological processes so as to provide new insights into pathophysiology and suggest targets for expanded clinical and mechanistic studies.

Methods: Baseline urine samples from Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network study participants with UCPPS (n = 259), positive controls (PCs; chronic pain without pelvic pain, n = 107) and healthy controls (HCs, n = 125) were analysed for the presence of proteins that are suggested in the literature to be associated with UCPPS. Matrix metalloproteinase (MMP)-2, MMP-9, MMP-9/neutrophil gelatinase-associated lipocalin (NGAL) complex (also known as Lipocalin 2), vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGF-R1) and NGAL were assayed and quantitated using mono-specific enzyme-linked immunosorbent assays for each protein. Log-transformed concentration (pg/mL or ng/mL) and concentration normalized to total protein (pg/μg) values were compared among the UCPPS, PC and HC groups within sex using the Student's t-test, with P values adjusted for multiple comparisons. Multivariable logistic regression and receiver-operating characteristic curves assessed the utility of the biomarkers in distinguishing participants with UCPPS and control participants. Associations of protein with symptom severity were assessed by linear regression.

Results: Significantly higher normalized concentrations (pg/μg) of VEGF, VEGF-R1 and MMP-9 in men and VEGF concentration (pg/mL) in women were associated with UCPPS vs HC. These proteins provided only marginal discrimination between UCPPS participants and HCs. In men with UCCPS, pain severity was significantly positively associated with concentrations of MMP-9 and MMP-9/NGAL complex, and urinary severity was significantly positively associated with MMP-9, MMP-9/NGAL complex and VEGF-R1. In women with UCPPS, pain and urinary symptom severity were associated with increased normalized concentrations of MMP-9/NGAL complex, while pain severity alone was associated with increased normalized concentrations of VEGF, and urinary severity alone was associated with increased normalized concentrations of MMP-2. Pain severity in women with UCPPS was significantly positively associated with concentrations of all biomarkers except NGAL, and urinary severity with all concentrations except VEGF-R1.

Conclusion: Altered levels of MMP-9, MMP-9/NGAL complex and VEGF-R1 in men, and all biomarkers in women, were associated with clinical symptoms of UCPPS. None of the evaluated candidate markers usefully discriminated UCPPS patients from controls. Elevated VEGF, MMP-9 and VEGF-R1 levels in men and VEGF levels in women may provide potential new insights into the pathophysiology of UCPPS.

Keywords: Lipocalin 2 (also known as NGAL); Multidisciplinary Approach to the Study of Chronic Pelvic Pain; matrix metalloproteinase; neutrophil gelatinase associated lipocalin; vascular endothelial growth factor; vascular endothelial growth factor receptor 1.

Figure 1

Log biomarker concentrations (ng/mL or pg/mL, subfigure 1A) or normalized concentrations (pg/µg) are presented for male (blue) and female (red) participants by patient cohort (Healthy Control, Positive Control, or UCPPS). Candidate proteins that differed significantly by cohort within sex are indicated by ‘*’. a Biomarker Concentration (ng/mL or pg/mL) by Cohort and Sex b Biomarker Concentration Normalized to Total Protein (pg/µg) by Cohort and Sex

Figure 1

Log biomarker concentrations (ng/mL or pg/mL, subfigure 1A) or normalized concentrations (pg/µg) are presented for male (blue) and female (red) participants by patient cohort (Healthy Control, Positive Control, or UCPPS). Candidate proteins that differed significantly by cohort within sex are indicated by ‘*’. a Biomarker Concentration (ng/mL or pg/mL) by Cohort and Sex b Biomarker Concentration Normalized to Total Protein (pg/µg) by Cohort and Sex

Figure 2

Receiver Operating Characteristic (ROC) curves are presented for markers that differed significantly between cohorts within sex to demonstrate the capacity for candidate proteins to distinguish Urological Chronic Pelvic Pain Syndrome (UCPPS) participants from Healthy Controls (HC). Satisfactory performance is indicated by an area under the ROC curve (AUC) of 0.70. Subfigure 2A shows the potential of candidate protein VEGF concentration to distinguish between female UCPPS and HC. Results are based on a logistic regression model with UCPPS vs HC as the outcome and VEGF concentration as the predictor, fit among female participants only. Subfigure 2B shows the potential of normalized concentrations of MMP9, VEGF, and VEGF-R1 to distribution between male UCPPS and male HC. Results are based on a multivariable logistic regression model with UCPPS vs. HC as the outcome and normalized MMP9, VEGF, and VEGFR1 as predictors, fit among male participants only. a. ROC Analysis of VEGF (ng/mL) for Discriminating UCPPS versus Healthy Control Females, AUC=0.58 b. ROC Analysis of MMP-9, VEGF, and VEGF-R1 (pg/µg) for Discriminating UCPPS versus Healthy Control Males, AUC=0.68

Figure 3

Pain severity is plotted against MMP9/NGAL log concentration (ng/ml, top) or log normalized concentration (pg/µg, bottom) separately for male (left) and female (right) Urological Chronic Pelvic Pain Syndrome participants (UCPPS, red), Positive Controls (PC, green), and Healthy Controls (HC, blue). Trend lines were determined by locally weighted scatterplot smoothing. Pain Severity and MMP9/NGAL by Cohort and Sex