Published by Bioscientifica Ltd. Printed in Great Britain © 2021 European Society of Endocrinology https://eje.bioscientifca.com https://doi.org/10.1530/EJE-21-0552 European Journal of Endocrinology 185:5 637–652 A Romero-Ruiz, B Pineda, D Ovelleiro and others microRNA profling and PCOS diagnosis Molecular diagnosis of polycystic ovary syndrome in obese and non-obese women by targeted plasma miRNA profiling Antonio Romero-Ruiz 1,2,3, *, Beatriz Pineda 1,3, *, David Ovelleiro 4, *, Cecilia Perdices-Lopez 1,2,5 , Encarnación Torres 1,2,3 , María J Vazquez 1,2,3 , Ipek Guler 1 , Álvaro Jiménez 1 , Rafael Pineda 1,2 , Mariasara Persano 1,3 , Cristina Romero-Baldonado 3 , José E Arjona 3 , Juan Lorente 3 , Concepción Muñoz 1,3 , Elier Paz 6 , Fe-Isabel Garcia-Maceira 6 , Álvaro Arjona-Sánchez 1,3 and Manuel Tena-Sempere 1,2,3,5,7 1 Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), 2 Department of Cell Biology, Physiology and Immunology, University of Córdoba, Córdoba, Spain, 3 Hospital Universitario Reina Sofa, Córdoba, Spain, 4 Area of Cellular Biology, Department of Experimental Biology, University of Jaen, Jaen, Spain, 5 CIBER Fisiopatología de la Obesidad y Nutrición, Instituto de Salud Carlos III, Córdoba, Spain, 6 Canvax Biotech, Córdoba, Spain, and 7 Institute of Biomedicine, University of Turku, Turku, Finland *(A Romero-Ruiz, B Pineda and D Ovelleiro contributed equally to this work) Abstract Objective: Polycystic ovary syndrome (PCOS) is diagnosed based on the clinical signs, but its presentation is heterogeneous and potentially confounded by concurrent conditions, such as obesity and insulin resistance. miRNA have recently emerged as putative pathophysiological and diagnostic factors in PCOS. However, no reliable miRNA- based method for molecular diagnosis of PCOS has been reported. The aim of this study was to develop a tool for accurate diagnosis of PCOS by targeted miRNA profling of plasma samples, defned on the basis of unbiased biomarker-fnding analyses and biostatistical tools. Methods: A case–control PCOS cohort was cross-sectionally studied, including 170 women classifed into four groups: non-PCOS/lean, non-PCOS/obese, PCOS/lean, and PCOS/obese women. High-throughput miRNA analyses were performed in plasma, using NanoString technology and a 800 human miRNA panel, followed by targeted quantitative real-timePCR validation. Statistics were applied to defne optimal normalization methods, identify deregulated biomarker miRNAs, and build classifcation algorithms, considering PCOS and obesity as major categories. Results: The geometric mean of circulating hsa-miR-103a-3p, hsa-miR-125a-5p, and hsa-miR-1976, selected among 125 unchanged miRNAs, was defned as optimal reference for internal normalization (named mR3-method). Ten miRNAs were identifed and validated after mR3-normalization as diferentially expressed across the groups. Multinomial least absolute shrinkage and selection operator regression and decision-tree models were built to reliably discriminate PCOS vs non-PCOS, either in obese or non-obese women, using subsets of these miRNAs as performers. Conclusions: We defne herein a robust method for molecular classifcation of PCOS based on unbiased identifcation of miRNA biomarkers and decision-tree protocols. This method allows not only reliable diagnosis of non-obese women with PCOS but also discrimination between PCOS and obesity. Capsule: We defne a novel protocol, based on plasma miRNA profling, for molecular diagnosis of PCOS. This tool not only allows proper discrimination of the condition in non-obese women but also permits distinction between PCOS and obesity, which often display overlapping clinical presentations. Correspondence should be addressed to A Romero-Ruiz or M Tena-Sempere Email antonioromeroruiz@gmail. com or fi1tesem@uco.es Clinical Study European Journal of Endocrinology (2021) 185, 637–652 Downloaded from Bioscientifica.com at 04/12/2023 08:19:45AM via free access