study questionIs exome sequencing (ES) an efficient approach for simultaneous analysis of causative single gene defects and copy number variants (CNV) in unexplained premature ovarian insufficiency (POI)? SUMMARY ANSWER: Among 51 idiopathic POI cases, a conservative molecular diagnostic yield of 12% was achieved with equal contribution of pathogenic or likely pathogenic (P/LP) monogenic causes and pathogenic microdeletions. WHAT IS KNOWN ALREADY: POI is a clinically and genetically heterogeneous condition, yet most cases remain idiopathic. Although numerous monogenic causes and CNVs have been implicated in POI, standardized clinical guidelines for comprehensive genetic analysis are still lacking. STUDY DESIGN SIZE DURATION: This observational study of 51 unexplained POI cases implemented an PARTICIPANTS/MATERIALS SETTING

methodsPatients were recruited and phenotyped at two tertiary care women's health and endocrinology centers in Estonia. Bioinformatic processing of ES data and assessment of rare causal variants in POI were performed using an in-house analysis pipeline including automated filtering and manual assessment for pathogenicity, followed by experimental validation via Sanger sequencing (monogenic findings) and chromosomal microarray analysis (CNVs). MAIN RESULTS AND THE ROLE OF CHANCE: Six of 51 unexplained POI cases had confident molecular findings, including three cases with P/LP monogenic variants and three carrying large pathogenic CNVs. Overall molecular diagnostic yield was estimated to be 12% (6 of 51). There was a statistically significant overrepresentation of likely causal genetic findings in primary amenorrhea (5 of 9 cases, 56%) compared to secondary amenorrhea/oligomenorrhea (1 of 42 cases, 2%) ( LARGE SCALE DATA: All variants linked identified in this study have been submitted to the NCBI ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/) and FerGI (https://www.eshre.eu/Specialty-groups/Special-Interest-Groups/Reproductive-Genetics/FeRGI) databases. LIMITATIONS REASONS FOR CAUTION: All recruited participants were of white European ancestry and living in Estonia. Thus, the results might not apply to other ethnic groups. This study was conducted in a relatively small and well-selected cohort. Validation in larger and more diverse cohorts is needed to further assess the utility of ES in solving idiopathic POI cases. WIDER IMPLICATIONS OF THE

findingsThe study's findings support the efficient use of ES as a comprehensive,

fundingThis study was funded by the Estonian Research Council grants PRG1021 and PRG3041 (to M.L.). DISCLOSURES: The authors declare no conflicts of interest.