Endocrine Abstracts

Background and Methods: PCOS is common and associated with significant comorbidity. Yet, its pathogenesis is complex and poorly understood. We have developed new methods for deep phenotyping discovery proteomic profiling of urine to identify disease mechanisms and novel non-invasive biomarkers for PCOS in adolescents. We undertook proteomic analysis (nano 2D-LC-QTOF MS^e) on a subset of 15 samples from a longitudinal PCOS cohort of 40 participants. We compared the urinary proteome of adolescents with PCOS (n = 6), controls (n = 6), and insulin resistance (IR) (n = 3), to identify markers of PCOS as distinct from those of IR. Subsequently, we ran a novel assay of 96 pro/anti-inflammatory associated proteins.

Results: In the discovery proteomic analysis, we identified 3,793 proteins, of which, 314 were significantly and differentially expressed (P < 0.05) in the PCOS cohort in comparison to healthy controls, and 397 in the PCOS vs. IR analysis. Of these, 66 proteins are potential novel biomarkers for PCOS – being differentially expressed in the PCOS cohort in comparison to both controls and IR. Gene ontology and bioinformatic Ingenuity Pathway Analysis (IPA) revealed that many proteins were mediators of complement, coagulation and apoptosis cascades or pro-inflammatory cytokines. Almost half of all significant biological pathways (10/23; 43%) were related to inflammatory/immunological responses and the thrombotic/fibrinolytic systems, and the inflammatory response was the most significant biological process associated with PCOS (P < 0.001). To validate these findings, we used an in-house, multiplexed and targeted proteomic assay of known inflammatory markers. This panel allowed us to identify and quantitate 80 of 96 pro/anti-inflammatory proteins in our cohorts. Multivariate analysis of all inflammatory proteins revealed clear separation between the three cohorts. Univariate analysis revealed four significantly different proteins between PCOS vs. control cohorts, six proteins between PCOS vs. IR cohorts, and one significant protein between all three cohorts.

Conclusions: We have utilised non-invasive matrices to map the proteome in adolescent PCOS and identified 66 potential novel biomarkers, providing greater insight into its molecular underpinnings. Our initial findings suggest inflammation is a major contributory factor in the pathophysiology of PCOS, which we corroborated through targeted assays. Future studies include proteomic analysis of the entire longitudinal cohort.