## 2 Materials and Methods  
### Patients and Control Groups  
The participants in the study were 53 adolescent girls, with a mean age of 15.8 (11.9-17.9) years, all followed at one and the same unit and included in subsequent manner according to their clinic visits. They were divided into three groups. The PCOS group consisted of 34 girls, fulfilling the criteria of the International Evidence-based Guideline for PCOS in adolescents--irregular menstrual cycle (<21 or >45 days) and clinical (hirsutism, acne, seborrhea) and/or biochemical HA (10). Other causes of HA were excluded. The healthy control group included 11 BMI- and age-matched girls with regular menstrual cycles and without clinical or biochemical HA. The hyperandrogenic control group (HAC) consisted of 8 girls with clinical and/or biochemical HA but without men strual disturbances, thus not fulfilling the criteria for PCOS but considered as being at risk for developing PCOS, according to the International Evidence-based Guideline [1]. The exclusion criteria for all participants were age above 18 years, less than 2 years post-menarche, other disorders with OA and/or HA (Cushing syndrome, androgen-producing tumors, non-classical or classical congenital adrenal hyperplasia, hyperprolactinemia, hypopro /hyperthyroidism), use of contraceptive pills or other drugs affecting the hormonal levels in the past 3 months. All participants underwent a complete clinical examination and auxological assessment with measurement of height, weight and waist circumference (WC) using a standard technique. BMI was calculated with the formula Weight (kg)/Height\({}^{2}\) (m). The presence of clinical HA and OA was assessed using a questionnaire and physical examination. A modified version of the Ferriman-Gallwey scale (FGS) with semi-subjective analysis of hair growth in nine androgen-dependent zones was used for assessment of hirsutism, with a score of \(\geq\)8 indicating hirsutism. All participants underwent metabolic and hormonal measurements using commercial laboratory kits and assay specifications according to the manufacturer's recommendations. The hormonal analyses were performed during the early follicular phase of the menstrual cycle in girls with regular menstruation and, when possible, in girls with PCOS. The free androgen index (FAI) was calculated with the formula FAI = (T (mmol/L)/SHBG (mmol/L)) \(\times\) 100, with FAI > 5 indicating androgen excess [16]. Biochemical HA was defined as testosterone levels above 1.4 nmol/L and/or FAI above 5. For calculation of HOMA-IR (Homeostatic Model Assessment for Insulin Resistance) the formula HOMA-IR = (fasting blood glucose (mmol/l) \(\times\) fasting insulin (mmol/L))/22.5 was used, with HOMA-IR > 2.5 indicating insulin resistance [17]. Plasma samples were collected from all participants and stored at \(-\)80 \({}^{\circ}\)C.  
We decided to investigate 6 miRNAs--miR-451a, miR-15a-5p, miR-320-5p, miR-28-5p, miR-103a-5p and miR-194-5p--most of which have not yet been studied in PCOS and, apart from miR-451a, miR194-5p and miR-15a-5p, have not been studied in adolescent girls. MiR-451a was found to be significantly down-regulated in PCOS girls, compared to healthy controls, with 100% sensitivity and 100% specificity for diagnosing PCOS [13]. MiR-103a-3p and miR-28-3p were also found significantly down-regulated in patients with PCOS [18]. Mir-320 is reported to be a potential biomarker for disorders of the lipid and glucose metabolism (type 2 diabetes mellitus, hyperlipidemia, atherosclerosis, fatty liver disease), which are often related to PCOS, and is found to be up-regulated in these conditions but down-regulated in PCOS [19; 20; 21]. MiR-194 was found to be overexpressed in women with PCOS and impaired glucose metabolism and correlated with HOMA-IR [22]. It was also found to be increased in granulosa cells of PCOS women, where it induced apoptosis [23]. We decided to investigate if we would find similar changes in the plasma levels of other members of miR-103, miR-28, miR-320 and miR-194 families.