Androgen assessment is a key element for diagnosing polycystic ovary syndrome (PCOS), and defining a “normal” level of circulating androgens is critical for epidemiological studies. We determined the upper normal limits (UNLs) for androgens in a population-based group of premenopausal “healthy control” women, overall and by ethnicity (Caucasian and Asian), in the cross-sectional Eastern Siberia PCOS Epidemiology and Phenotype (ESPEP) Study (СlinicalTrials.gov ID: NCT05194384) conducted in 2016–2019. Overall, we identified a “healthy control” group consisting of 143 healthy premenopausal women without menstrual dysfunction, hirsutism, polycystic ovaries, or medical disorders. We analyzed serum total testosterone (TT) by using liquid chromatography with tandem mass spectrometry (LC-MS/MS), and DHEAS, sex-hormone-binding globulin (SHBG), TSH, prolactin, and 17-hydroxyprogesterone (17OHP) were assessed with an enzyme-linked immunosorbent assay (ELISA). The UNLs for the entire population for the TT, free androgen index (FAI), and DHEAS were determined as the 98th percentiles in healthy controls as follows: 67.3 (95% confidence interval (CI): 48.1, 76.5) ng/dl, 5.4 (3.5,14.0), and 355 (289, 371) μg/dl, respectively. The study results demonstrated that the UNLs for TT and FAI varied by ethnicity, whereas the DHEAS UNLs were comparable in the ethnicities studied.

Abnormalities in gut microbiota diversity are considered important mechanisms in metabolic disorders in polycystic ovarian syndrome (PCOS). However, the data on the association of these disorders with the PCOS phenotype remain controversial. The objectives of this study were to estimate the alpha diversity of the gut microbiota of healthy women and PCOS patients depending on phenotype. The study participants (184 premenopausal women: 63 with PCOS, 121 without PCOS) were recruited during the annual employment assessment in the Irkutsk Region and the Buryat Republic (Russia) in 2016–2019. For PCOS diagnosis, we used the Rotterdam (2003) criteria and definitions of PCOS phenotypes. Five indexes of alpha diversity (ASV, Shannon, Simpson, Chao, and ACE) were estimated for the gut microbiota in all participants using amplicon metasequencing. As a result, two out of five alpha diversity indexes showed a statistical difference between the non-PCOS and PCOS groups. We did not find a significant difference in the alpha diversity of gut microbiota in the subgroups of women with hyperandrogenic PCOS phenotypes vs non-androgenic phenotype D and the group of women with the presence of only one of the PCOS criteria. Nevertheless, “classic” PCOS phenotypes demonstrated the most significant decrease in alpha diversity compared with healthy women without any signs of PCOS.

Background There is a lack of data on the prevalence of PCOS and its phenotype in many geographic regions. Siberia is a unique region of the Russian Federation with a multi-raced population living in similar geographic and socio-economic conditions for centuries. Therefore, we considered this population optimal for epidemiological research. Objectives To determine the prevalence of PCOS and the PCOS phenotypes in unselected women in the Eastern Siberia region. Population: We performed the institution-based, cross-sectional Eastern Siberia PCOS Epidemiology & Phenotype (ESPEP) Study during 2016-2019 (СlinicalTrials.gov ID: NCT05194384) and recruited 1148 premenopausal women aged 34.3±6.3 yrs., of which 63.2% were Caucasians, 27.6% Asians, and 9.2% Mixed-race. All subjects provided written informed consent. Exclusion criteria were: current pregnancy or lactation, history of hysterectomy, bilateral oophorectomy, endometrial ablation, uterine artery embolization; and current or previous hormonal medications or insulin-sensitizers intake. The study was approved by the Institutional Ethics Committee of the Scientific Center for Family Health a Human Reproduction (Irkutsk, Russian Federation). Methods include questionnaires, anthropometry, vital signs, gynecological examination, mF-G scoring, pelvic U/S, and blood sampling. For PCOS diagnosis we used the Rotterdam (2003) criteria. Serum samples were analyzed for total testosterone (TT) using LC-MS/MS. DHEAS, SHBG, prolactin, TSH, and 17-OHP were assessed by ELISA. Free Androgen Index (FAI) was calculated (i.e. [TT/SHBG]×100). The upper normal limit (UNL) for the mF-G score was 4, as determined using a 2k-cluster analysis in the total study population. The upper normal limits (UNL) for androgens were determined from the 98th percentiles for these parameters in 143 women, identified as the "super-controls". Pearson Chi-square and Fisher exact one-tailed tests were used to comparing proportions and categorical variables. A p-value of 0.05 was considered statistically significant. Results The total prevalence of PCOS in premenopausal women from Eastern Siberia was estimated as 13.3%, with the following distribution of PCOS phenotypes: 29.1% (A), 9.9% (B), 26.2%(C), and 34.8% (D). There was no significant difference in PCOS prevalence by race: 13.4% in Caucasians, 11.0% in Asians, and 19.8% in Mixed race women (pχ2=0.07). Classic PCOS phenotype A was found in a comparable number of PCOS women (28% in Caucasians, 31.2% in Asians, and 30% in Mixed race); whereas Asian PCOS patients demonstrated the highest proportion of phenotype B (25% vs 5.6% in Caucasians and 5% in mixed-race). The number of hirsute women (with mF-G score >4) was dependent on race and reached 22%, 29%, and 36% among Caucasians, Asians, and mixed-race women, respectively (p χ2=0.001). Conclusions The results of the ESPEP study, conducted in a multi-race unselected population of premenopausal women from Eastern Siberia demonstrated a 13.3% total prevalence of PCOS and race-dependent difference in the clinical manifestation of PCOS. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Testosterone assessment is essential for detecting biochemical hyperandrogenism, one of the important diagnostic criteria of polycystic ovary syndrome (PCOS) both in clinical practice and in epidemiological studies. Currently, tandem liquid chromatography-mass spectrometry (LC-MS/MS) is the most preferred technique to measure testosterone level in women. Its validation is important to reproducibility of androgen tests results for clinical practice and for epidemiological studies of the prevalence PCOS.The aim of the study. To develop and validate a method for determining total testosterone in blood serum using highly efficient LC-MS/MS to assess androgenemia in the epidemiological study of the prevalence of PCOS and its phenotypes in Eastern Siberia (ESPEP STUDY).Materials and methods. We determined a total testosterone level in serum blood using triple quadrupole mass spectrometer LCMS-8060 (Shimadzu, Japan). The protocol of technique was developed using self-prepared purified human testosteronefree serum with a known concentration of analyzed compound. We used the serum samples of women of reproductive age to test the developed method.Results. Optimum chromatographic conditions were obtained with a Kromasil 100-2.5-C18 column (2.1 mm × 100 mm; AkzoNobel, Netherlands), and an isocratic elution mode using a mobile phase consisting of acetonitrile and 0.1 % aqueous solution of formic acid. The total flow rate was 0.35 ml/min. The lower limit of quantification was 5 ng/dl with an average accuracy of 100.2 %. During the approbation of the method in a test population sample of 1138 premenopausal women (mean age – 34.3 ± 6.3 years), the median testosterone concentration was 26.9 ng/dl.Conclusion. It was found that the proposed method for determining testosterone in blood serum has acceptable linearity and reproducibility and meets the requirements for bioanalytical methods under the regulatory documentation. This method can be used for clinical practice and epidemiological study of the prevalence of PCOS.

Aim. To establish cut-off values for the concentrations of complement C3 and ceruloplasmin, diagnostic markers of reduced antral follicle count (AFC) and anti-Müllerian hormone (AMH) which both indicate diminished ovarian reserve, in women of reproductive age.Materials and Methods. Here we enrolled 864 women (18-40 years of age, average age 31.70 ± 5.14 years) who underwent an annual medical examination in 2017–2019 in the Irkutsk Region and the Republic of Buryatia. Reduced AFC was defined as ≤ 5 antral follicles in each ovary at pelvic ultrasound examination whilst reduced AMH was defined as < 1.2 ng/mL. In total, 112 women had reduced ovarian reserve and 752 were included into the control group. In addition to AMH, we also measured serum prolactin, gonadotropins, inhibin B, estradiol, complement C3, and ceruloplasmin using enzyme-linked immunosorbent assay. The cut-off values were determined by plotting a receiver operating characteristic (ROC) curve and calculating the area under the curve (AUC).Results. The cut-off level of complement C3 was 894 (867; 1355.5) mg/mL [AUC: 0.769 (0.635; 0.904)] in women with reduced AFC (≤ 5) and 981.5 (916.5; 1467.5) mg/mL [AUC: 0.62 (0.493; 0.746)] in women with reduced AMH (< 1.2 ng/mL). The cut-off level of ceruloplasmin was 1.745 (1.625; 1.975) mg/mL [AUC: 0.859 (0.759; 0.96)] in women with reduced AFC (≤ 5) and 1.975 (1.665; 2.15) mg/mL, [AUC: 0.662 (0.542; 0.782)] in women with reduced AMH (< 1.2 ng/mL).Conclusion. We have established the cut-off values for the serum complement C3 and ceruloplasmin in women with reduced AFC and AMH, indicators defining diminished ovarian reserve in women of reproductive age.