P-604 Underlying molecular mechanisms of Polycystic Ovary Syndrome and Morphology: focusing on the ovary

Can cluster analysis be used to differentiate between normo-ovulatory women with normal ovaries and normo-ovulatory women with polycystic ovarian morphology (PCOM) in a non-subjective manner? Cluster analysis can be used to accurately and non-subjectively differentiate between normo-ovulatory women with normal ovaries and normo-ovulatory women with PCOM. Currently, PCOM is diagnosed using a fixed threshold level, i.e. 12 or more follicles per ovary, and is one of the diagnostic criteria of polycystic ovary syndrome (PCOS). However, PCOM is also encountered in normo-ovulatory women, suggesting that it could just represent a normal variant. On the other hand, recent studies have shown subtle endocrine abnormalities in women with isolated PCOM that resemble those found in women with PCOS. Because of the strong correlation between anti-Müllerian hormone (AMH) and follicle number, a high serum AMH level has been proposed as a surrogate marker for PCOM and could, therefore, be integrated in the diagnostic classifications for PCOS. This was a retrospective observational cohort study. Original cohorts had been recruited for previous studies between 1998 and 2010. Two hundred ninety-seven regularly cycling women and 700 women with PCOS were eligible for inclusion. Cluster analysis was performed in 297 regularly cycling women. After exclusion of 'PCOM' clusters, each 'non-PCOM' cluster (young, n = 118 and old, n = 100) was included in the construction of a receiver operating characteristics curve to test the diagnostic performance of follicle number per ovary (FNPO) and serum AMH in discriminating similarly aged full-blown PCOS patients (n = 411 and 237, respectively) from normal regularly cycling non-PCOM women. The optimal number of clusters was four; age was the most important classifying variable, followed by the FNPO and serum AMH. Two distinct clusters of normo-ovulatory women with PCOM were isolated and differed solely by age, i.e. 'young' and 'old'. Both 'PCOM' clusters had their similarly aged counterpart of 'non-PCOM' clusters. Likewise, two clusters comprised women younger than 30 years, with (n = 28, 'PCOM regularly cycling women') or without (n = 118, 'normal regularly cycling women') features of PCOM (increased FNPO and/or serum AMH). The two other clusters in older women could be labelled 'normal regularly cycling women' or 'PCOM regularly cycling women' (n = 100 and 51, respectively). The prevalence of PCOM was significantly greater in old than in young regularly cycling women controls. In the young population, after exclusion of the 'PCOM regularly cycling women', the diagnostic performance of AMH, expressed by area under the curve (AUC) (AUC = 0.903; CI (0.876-0.930)) to differentiate PCOS women from normal regularly cycling women was similar to that using the FNPO (AUC = 0.915, CI (0.891-0.940)) (P = 0.25), confirming results from earlier studies. In the old population, the diagnostic performance of AMH was greater than that of FNPO (AUCs = 0.948 (0.927-0.970) vs 0.874 (0.836-0.912), respectively, P = 0.00035). Cut-off levels of AMH and antral follicle count distinguishing regularly cycling non-PCOM women from PCOS women were higher in young women than in older women. Data of normal women were obtained from earlier studies, aiming to measure normal endocrine values. Apparently, the strong effect of age in cluster analysis revealed a dichotomy in the age distribution among the cohort of regularly cycling women included. This was involuntary since in none of the original studies, eligibility was limited by age and there was considerable overlap in age ranges of the cohorts. Transvaginal ultrasound was performed using a 6.5-8 mHz probe and our data confirm that this threshold level for FNPO is still valid if using such probe frequencies, although the use of devices with a maximum frequency lower than 8 mHz has become obsolete. Obviously, newer ultrasound scanner using higher transducer frequency will facilitate the detection of more follicles. Our data support the use of AMH as a surrogate for ultrasound to define PCOM, which is one of the three items of the Rotterdam classification. They also show that age should be taken into account to define the optimal threshold. The fact that the prevalence of PCOM was increased in the older regularly cycling women, may be due to 'attenuated' PCOS, a phenomenon that has been described in ageing women with PCOS. These women might have had anovulatory cycles in the past and have become ovulatory with increasing age, and were, therefore, eligible for this study. However, since most women included at older age have had spontaneous pregnancies in the past, PCOM at older age may be associated with a subclinical form of PCOS, which may also be present in young regularly cycling women. No funding was received for this study. J.S.E.L. has received grants and support from Ferring, MSD, Organon, Merck-Serono, Schering Plough and Serono during recruitment and analysis of data for this study. S.L.F., A.D. and D.D. do not have any conflict of interest.

Is intrinsic dysregulation of granulosa cells (GC) and consequent increases in the per-follicle production of anti-Müllerian hormone (AMH), correlated with the phenotypic presentation of women with polycystic ovaries? Involvement of intrinsic GC dysregulation in oligo-anovulation associated with polycystic ovary syndrome (PCOS) is likely because among women with PCOS, those with oligo-amenorrhea have higher per-follicle AMH production than those who ovulate normally, irrespective of their androgen and/or metabolic status. Women with PCOS have higher serum AMH level than non-PCOS women due to an increased follicle number and excessive AMH production per follicle, the latter reflecting a putative GC dysfunction that may vary between PCOS phenotypes. This is a retrospective analysis of data collected from 1021 women undergoing infertility evaluation from March 2011 to October 2013. The study included women with polycystic ovarian morphology (PCOM) who met the Rotterdam criteria for PCOS (n = 272), women with PCOM only (n = 168) and controls (n = 581). We used serum AMH to antral follicle count (AFC) ratio (AMH/AFC) as a marker of per-follicle AMH production and checked whether this ratio was associated with the PCOS phenotype and to the menstrual, androgen and metabolic status in women with PCOS, women with PCOM only and in controls. AMH/AFC was significantly higher in oligo-amenorrheic women with PCOS than in eumenorrheic women with PCOS or PCOM (P < 0.001) but also in the latter group compared with controls (P < 0.001) regardless of androgen status. Stepwise discriminant analysis yielded a significant score for the menstrual status with a discriminant power of 26.5% (P < 0.001). This score included AFC, AMH/AFC, waist circumference and LH with partial R(2) of 0.172, 0.042, 0.024 and 0.023, respectively. The AMH to AFC ratio as a surrogate marker for average AMH may be subject to error because follicles below the sensitivity limit of the ultrasonography used may also contribute to serum AMH concentration and secondly, AFC can be subjective. The higher AMH/AFC in women with PCOM only than in controls suggests that isolated PCOM may represent a PCOS-like phenotype in which an inherent dysfunction of GC exists but is too mild to affect the ovulatory process. No funding was obtained for this study. There are no conflicts of interest to be declared. Non-applicable.

Steroid metabolome profiling of follicular fluid in normo- and hyperandrogenic women with polycystic ovary syndrome

PCOS is a common endocrine disorder in women of reproductive age, with granulosa cells playing a key role in its development. This study aims to identify shared differentially expressed genes (DEG) in granulosa cells and blood from PCOS patients, offering potential biomarkers and therapeutic targets. Transcriptomic data were obtained from the Gene Expression Omnibus (GEO) database: GSE95728 (granulosa cells; 7 PCOS, 7 controls) and two blood-based datasets, GSE85932 and GSE54248 (12 PCOS, 12 controls). In silico tools were employed to identify DEG, hub genes, and protein-protein interactions and to explore predicted drug targets. DEG analysis revealed that Vanin-2 (VNN2) and Interleukin-1 receptor type 2 (IL1R2) were consistently dysregulated in PCOS patients. Dysferlin (DYSF), Carbonic Anhydrase 4 (CA4), and Solute Carrier Family 2 Member 14 (SLC2A14) were differentially expressed between the blood datasets, while Aquaporin 9 (AQP9), C-X-C Motif Chemokine Receptor 1 (CXCR1), and Annexin A3 (ANXA3) were dysregulated in GSE95728 (granulosa cells) and GSE54248 (blood). Functional enrichment highlighted immune and metabolic pathways. Protein-protein interaction analysis identified AQP9 as a hub gene. Drug prediction analysis suggested that IL1R2 could be targeted by anakinra, while VNN2 was predicted to interact with pantothenic acid. The dysregulation of VNN2 and IL1R2 across tissue types suggests their involvement in immune-inflammatory processes in PCOS. The expression of AQP9 and other metabolism-related genes indicates possible immune-metabolic crosstalk, aligning with known PCOS pathophysiology. VNN2 and IL1R2 show potential as biomarkers or therapeutic targets in PCOS. Further validation is needed to confirm their clinical significance and roles.

Close relationship between polycystic ovary syndrome (PCOS) and bisphenol A (BPA) has drawn much attention in recent years, while the underlying mechanisms are poorly understood. In our study, we aim to detect BPA concentration in the follicular fluid and investigate its effect on estradiol synthesis in human granulosa cells from PCOS and non-PCOS patients. Follicular fluid and granulosa cells were collected from women who underwent controlled ovarian stimulation for in vitro fertilization or intracytoplasmic sperm injection. BPA concentration in the follicular fluid from PCOS patients (440.50 ± 63.70 pg/ml) was significantly higher than that from non-PCOS patients (338.00 ± 57.88 pg/ml). Expression of aromatase and estradiol synthesis in cultured granulosa cells was examined after treatment with BPA from 0.01 to 1 μM for 24 h. Expression of aromatase and estradiol synthesis was downregulated by BPA in a dose-dependent manner in PCOS, but no effect was observed in granulosa cells from non-PCOS patients. These findings provide evidence that increased BPA concentration in the follicular fluid of PCOS patients may play an important role in its pathogenesis by attenuating the expression of aromatase in granulosa cells.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder characterized by menstrual irregularities, androgen excess, and polycystic ovarian morphology, but its pathogenesis remains largely unknown. This review focuses on how androgen excess influences the molecular basis of energy metabolism, mitochondrial function, and mitophagy in granulosa cells and oocytes, summarizes our current understanding of the pathogenesis of PCOS, and discuss perspectives on future research directions. A search of PubMed and Google Scholar databases were used to identify relevant studies for this narrative literature review. Female offspring born of pregnant animals exposed to androgens recapitulates the PCOS phenotype. Abnormal mitochondrial morphology, altered expression of genes related to glycolysis, mitochondrial biogenesis, fission/fusion dynamics, and mitophagy have been identified in PCOS patients and androgenic animal models. Androgen excess causes uncoupling of the electron transport chain and depletion of the cellular adenosine 5'-triphosphate pool, indicating further impairment of mitochondrial function. A shift toward mitochondrial fission restores mitochondrial quality control mechanisms. However, prolonged mitochondrial fission disrupts autophagy/mitophagy induction due to loss of compensatory reserve for mitochondrial biogenesis. Disruption of compensatory mechanisms that mediate the quality control switch from mitophagy to apoptosis may cause a disease phenotype. Furthermore, genetic predisposition, altered expression of genes related to glycolysis and oxidative phosphorylation, or a combination of these factors may also contribute to the development of PCOS. In conclusion, fetuses exposed to a hyperandrogenemic intrauterine environment may cause the PCOS phenotype possibly through disruption of the compensatory regulation of the mitophagy-apoptosis axis.

Polycystic ovary syndrome (PCOS) represents the most common endocrine abnormality in women of reproductive age. The cause of PCOS remains largely unknown, but studies suggest an intrinsic ovarian abnormality. The objective of the study was to test our hypothesis that differences in granulosa cell proliferation and apoptosis may underlie abnormalities that affect follicular development. Granulosa cells were prepared from follicular fluid aspirated from 4- to 8-mm follicles of unstimulated ovaries during routine laparoscopy or laparotomy from women with anovulatory PCOS and those with regular ovulatory cycles. The study was conducted at a university hospital. Fourteen women with anovulatory PCOS and nine women with regular ovulatory cycles participated in the study. Immunocytochemistry on granulosa cells to investigate apoptotic and proliferation rates, together with real-time RT-PCR to analyze gene expression profiles of apoptotic regulators, was measured. Significantly lower apoptotic rates were found in granulosa cells from patients with PCOS, compared with women with regular ovulatory cycles (P=0.004). Lower apoptotic rates were associated with decreased levels of the apoptotic effector caspase-3 (P=0.001) and increased levels of the anti-apoptotic survival factor cellular inhibitor of apoptosis proteins-2 in the PCOS group that were coupled to higher proliferation rates (P=0.032). Gene expression profiling confirmed the immunocytochemical findings. Our findings indicate that there are significant differences in the rate of cell death and proliferation in granulosa cell populations in PCOS patients. These are associated with decreased expression of apoptotic effectors and increased expression of a cell survival factor. These results provide new insights that may be useful in developing specific therapeutic intervention strategies in PCOS.

Exposure to 6PPD-Q induces dysfunctions of ovarian granulosa cells: Its potential role in PCOS.

Increased total and phosphorylated glycogen synthase kinase-3 (GSK-3) in the adipocytes of patients with the polycystic ovary syndrome (PCOS)

Is there a relationship between serum uric acid and fructose levels in polycystic ovary syndrome (PCOS)? Elevated serum uric acid levels in women with PCOS positively correlate with serum fructose levels, and elevated serum fructose levels are an independent risk factor for hyperuricemia in women with PCOS. Our previous study suggested a link between elevated serum fructose levels and PCOS. Fructose is unique as it generates uric acid during metabolism, and high uric acid levels are associated with metabolic disorders and an increased risk of anovulation. However, the relationship between serum uric acid and fructose levels in women with PCOS remains unclear. In a case-control study of 774 women (482 controls and 292 patients with PCOS) between May and October 2020 at the Shengjing Hospital of China Medical University, the relationship between uric acid and fructose levels in women with PCOS was examined. Participants were divided into subgroups based on various factors, including BMI, insulin resistance, dyslipidemia, metabolic syndrome, and hyperuricemia. Serum uric acid concentrations were measured using enzymatic assays, and serum fructose levels were determined using a fluorescent enzyme immunoassay. Dietary fructose data were collected through a validated food-frequency questionnaire of 81 food items. We applied restricted cubic splines to a flexibly model and visualized the linear/nonlinear relationships between serum uric acid and fructose levels in PCOS. Multivariate logistic analysis was executed to assess the association between serum fructose levels and hyperuricemia in PCOS. Human granulosa cell and oocyte mRNA profile sequencing data were downloaded for mapping uric acid and fructose metabolism genes in PCOS. Further downstream analyses, including Gene Ontology, Kyoto Encyclopedia of Genes and Genomes analysis, and protein-protein interactions were then carried out on the differentially expressed genes (DEGs). The correlation between uric acid and fructose metabolism genes was calculated using the Pearson correlation coefficient. The GeneCards database was used to identify DEGs related to uric acid and fructose metabolism in PCOS, and then several DEGs were confirmed by quantitative real-time PCR. Both serum fructose and uric acid levels were significantly increased in women with PCOS compared with the control women (P < 0.001), and there was no statistically significant difference in dietary fructose intake between PCOS and controls, regardless of metabolic status. There was a positive linear correlation between serum uric acid and fructose levels in women with PCOS (Poverall < 0.001, Pnon-linear = 0.30). In contrast, no correlation was found in control women (Poverall = 0.712, Pnon-linear = 0.43). Additionally, a non-linear association was observed in the obese subgroup of patients with PCOS (Poverall < 0.001, Pnon-linear = 0.02). Serum uric acid levels were linearly and positively associated with serum fructose levels in patients with PCOS with insulin resistance, dyslipidemia, and metabolic syndrome. Furthermore, even after adjusting for confounding factors, elevated serum fructose levels were an independent risk factor for hyperuricemia in patients with PCOS (P = 0.001; OR, 1.380; 95% CI, 1.207-1.577). There were 28 uric acid and 25 fructose metabolism genes which showed a significant correlation in PCOS. Seven upregulated genes (CAT, CRP, CCL2, TNF, MMP9, GCG, and APOB) related to uric acid and fructose metabolism in PCOS ovarian granulosa cells were ultimately successfully validated using quantitative real-time PCR. Due to limited conditions, more possible covariates (such as smoking and ethnicity) were not included, and the underlying molecular mechanism between fructose and uric acid levels in women with PCOS remains to be further investigated. The results of this study and our previous research indicate that the high uric acid status of PCOS may be mediated by fructose metabolism disorders, highlighting the importance of analyzing fructose metabolism, and especially its metabolic byproduct uric acid, during the clinical diagnosis of PCOS. These results suggest the adverse effects of high uric acid in PCOS, and the importance of taking early interventions regarding uric acid levels to reduce the occurrence and development of further clinical signs, such as metabolic disorders in women with PCOS. This work was supported by: the National Natural Science Foundation of China (No. 82371647, No. 82071607, and No. 32100691); LiaoNing Revitalization Talents Program (No. XLYC1907071); Fok Ying Tung Education Foundation (No. 151039); and Outstanding Scientific Fund of Shengjing Hospital (No. 202003). No competing interests were declared. N/A.

The follicular fluid (FF) of human ovaries contains insulin-like growth factor binding proteins (IGFBPs) which may regulate the bioavailability of the IGFs. Previously we showed discrete changes in IGFBP concentrations in FF which correlated with the physiological state of the follicle. The purpose of the present study was to test the hypothesis that IGFBPs in FF may be increased in polycystic ovarian disease (PCO). FF was aspirated from PCO follicles and size matched healthy and atretic follicles from normal ovaries of naturally cycling women. The IGFBPs in FF samples were studied by ligand blot analysis with 125I-IGF-II and by immunoblot analysis using specific antisera to five human IGFBPs. Of six IGFBP bands in PCO FF, three were identified as IGFBP-2, IGFBP-3, and IGFBP-4. Bands (29K and 31K) were not identified by any of the five IGFBP antisera. The total IGF binding capacity was increased in PCO FF relative to normal healthy or atretic FF. IGFBP-3 was the predominant BP present in FF from PCO follicles as well as normal healthy and atretic follicles with no significant difference in any of the groups of follicles studied. IGFBP-2, IGFBP-4, and the 29K BP were present in smaller amounts, but there were significantly higher levels of each of these BPs in PCO follicles than in normal healthy follicles. Only IGFBP-4 was elevated in PCO follicles relative to normal atretic follicles indicating that the pattern of IGFBP expression in PCO FF was very similar to the pattern observed in atretic follicles. To determine the source of the IGFBPs in FF, granulosa or theca cells were cultured (up to 6 days) in serum-free medium. Ligand blot analysis of the conditioned medium revealed basal secretion of IGFBP-3 by both theca and granulosa cells. FSH inhibited granulosa cell IGFBP-3 production but increased the 29K BP in the medium. Transforming growth factor-beta stimulated basal IGFBP-3 secretion and reversed the FSH effects. Human CG (100 ng/mL) inhibited theca cell IGFBP-3 production but did not stimulate any other IGFBP. The results of our studies indicate that IGFBP-2, IGFBP-4, and the 29K BP are significantly increased in PCO FF and that gonadotropins and TGF-beta regulate the production of IGFBPs by human theca and granulosa cells.

Polycystic ovarian syndrome (PCOS) is the main cause of infertility in women. It is frequently associated with reduced progesterone production by human luteinised granulosa cells (hlGCs). However, the molecular mechanisms involved in these steroidogenesis alterations in PCOS patients are unclear. In a dihydrotestosterone-induced PCOS mouse model, steroid production is maintained in the setting of chemokine-like receptor 1 (Cmklr1) knockout. Thus, chemerin and chemerin receptors in terms of expression and progesterone regulation could be different in control and PCOS hlGCs. We first confirmed that progesterone levels in both plasma (P < 0.0001) and follicular fluid (FF) (P < 0.0001) were significantly reduced in PCOS normal weight women compared to control women. These data were associated with a lower STAR mRNA expression in both in vivo (P < 0.0001) and in vitro (P < 0.0001) hlGCs from PCOS women. Secondly, chemerin FF levels (P < 0.0001) and RARRES2 (P < 0.05) and CMKLR1 (P < 0.0001) mRNA levels in GCs were higher in PCOS normal weight patients. Thirdly, treatment of hlGCs with a specific nanobody (the VHH CA4910) targeting the human receptor for CMKLR1 leading to its inactivation abolished chemerin-induced progesterone inhibition, suggesting the involvement of CMKLR1 in this process. Furthermore, the inhibition of progesterone secretion induced by chemerin was two-fold higher in PCOS hlGCs (P < 0.05). Moreover, the VHH CA4910 reinstated a normal progesterone secretion with lower concentrations in PCOS hlGCs, suggesting a different chemerin sensitivity between PCOS and control hlGCs. Thus, chemerin, through CMKLR1, could be involved in the steroidogenesis alterations in PCOS hlGCs.

Increased monocyte chemoattractant protein-1 levels indicating early vascular damage in lean young PCOS patients

Objective: MicroRNAs (miRNAs) are gene expression regulators. Altered miRNA levels are associated with diabetes, insulin resistance, and inflammation. Insulin resistance and inflammation are both features of Polycystic ovary syndrome (PCOS). The aim of this study was first to assess differences in selected miRNAs (miR-146a, miR-155, miR-320, miR-370, miR-486), involved in insulin sensitivity regulation and inflammation, in women with or without PCOS. Second, to investigate relationships among these miRNAs, insulin, High mobility group box 1 (HMGB1), and IL-6 in follicular fluid (FF), serum 17-beta estradiol (E2), and the number of dominant follicles.Methods: Thirty PCOS and thirty-six non-PCOS women undergoing in vitro fertilization were enrolled. RNA from granulosa cells (GC) and FF was extracted and the specific miRNAs were evaluated using qRT-PCR. HMGB1, insulin, and IL-6 in FF, and serum E2 were assayed using specific kits.Results: MiR-146a, miR-155, miR-486 were upregulated and miR-320 and miR-370 were downregulated in GC from the PCOS patients. In FF, miR-146a, miR-155, and miR-486 showed lower levels in PCOS, whereas miR-320 and miR-370 showed an opposite trend but no significant changes were observed. These miRNAs showed relationships with Body Mass Index (BMI), age, E2, number of dominant follicles, insulin, and HMGB1.Conclusion: In conclusion, the miRNAs analyzed showed changes in PCOS ovaries and had relationships with indices of inflammation and insulin sensitivity within the ovary, providing evidence for new regulatory mechanisms.

Our study aimed to investigate the association of telomerase activity (TA) and telomere length (TL) in granulosa cells (GCs) with IVF outcomes of polycystic ovary syndrome (PCOS) patients, and the effects of oral contraceptive pill (OCP) pretreatment on these two parameters. One hundred sixty-three infertile women were enrolled and divided into a PCOS group (n=65) and a non-PCOS group (n=98). The PCOS group was further divided into an OCP pretreatment group (n=35) and a non-OCP pretreatment group (n=30), a TA <0.070 group (n=34) and a TA ≥0.070 group (n=31), and a TL <1 group (n=41) and a TL ≥1 group (n=24), respectively. No obvious differences were observed in TA between these groups. The TL was 0.971 in PCOS group and 1.118 in non-PCOS group (P=0.005). The patients with TL ≥1 accounted for 36.9% in PCOS group and 54.1% in non-PCOS group (P=0.032). The average duration of infertility for PCOS patients was 5years in TA <0.070 group and 4years in TA ≥0.070 group (P=0.038), and 5years in TL <1 group and 3years in TL ≥1 group (P=0.006), respectively. No obvious differences were observed in IVF outcomes between these groups. No obvious differences were observed in TA, TL, or IVF outcomes between OCP pretreatment group and non-OCP pretreatment group in PCOS patients. Shorter TL was found in PCOS patients. The TA levels did not change significantly in PCOS patients. PCOS patients with a lower TA level and shorter telomeres had an earlier onset of infertility symptoms. No predictive value was found for TA and TL in terms of embryo quality or IVF outcomes in PCOS patients, and no effect OCP pretreatment was observed on either TA and TL.