Abstract Study question Would we able to identify specific microRNA which is specific in premature ovarian insufficiency? Summary answer Our study provides compelling evidence suggesting that microRNA-4516 would be a diagnostic marker in Korean premature ovarian insufficiency women. What is known already Premature ovarian insufficiency is diagnosed in women before the age of 40 years old, when there is no menstruation for at least for six months with hypergonadotropic hypogonadism in serum evaluation. It is a disease which ovarian functions are irreversible, but women visit outpatient clinic only when their ovarian functions are already diminished. Exosomes are functional vehicles which transport complex proteins, lipids and nucleic acids between cells, containing numerous microRNAs and it may include disease-specific miRNA signature that can be a valuable diagnostic tool. Study design, size, duration This was a prospective study including patients diagnosed with premature ovarian insufficiency(POI)/Turner syndrome and control individuals who visited outpatient clinic in Department of Gynecology in Gil Hospital, Gachon University from January 2019 to August 2021. We divided the patients into two groups depending on the time period of enrollment; Sequencing cohort and Validation cohort. In sequencing cohort, total 19 patients (7/7/5) were enrolled, and in validation cohort, total 46 patients (15/11/20) were enrolled. Participants/materials, setting, methods The patients in POI group were participants diagnosed with POI but they not Turner syndrome. After collecting first morning urine samples, we extracted exosomes and RNA sequencing was done based on microRNA library. We did real-time PCR using a selected hsa-miR-4516 for confirmation. Then we did confirmation study using POI mouse model (Cyclophosphamide + Busulfan). Main results and the role of chance Expression of hsa-miR-4516 detected via qRT-PCR analysis of RNA from the validation cohort exactly mapped the RNA sequencing results of the sequencing cohort. Moreover, it was significantly upregulated in patients with POI and Turner syndrome compared to the control group. The TaqMan PCR assay confirmed the upregulation of hsa-miR-4516 expression in patients with POI and Turner syndrome. To evaluate miR-4516 as a biomarker of POI, we established a chemotherapy-induced POI mouse model by injecting the mice with CTX and BUS. Next, we assessed the expression of mmumiR-4516 in the ovaries of the chemotherapy-induced POI mouse model; mmu-miR-4516 expression was significantly increased in the ovaries but not in the uteri in the POI mouse model compared to the control. These results suggest that upregulation of mmumiR-4516 expression is associated with pathological changes in the ovaries. Limitations, reasons for caution We also observed inconsistencies between the sequencing and qRT-PCR results. The cause of POI is highly diverse, which can lead to discrepancies in exosomal miRNA expression between the cohorts. The cohort is small and limited compared to the validation cohort, and this may have caused inconsistencies between the cohorts. Wider implications of the findings We suggest that hsa-miR-4516 could be developed as a biomarker of POI within an easy and convenient diagnostic test using non-invasive sampling methods. Trial registration number -