Polycystic Ovary Syndrome Model Research Articles

Butyrate-Mediated Modulation of Paraoxonase-1 Alleviates Cardiorenometabolic Abnormalities in a Rat Model of Polycystic Ovarian Syndrome.

Polycystic ovarian syndrome (PCOS) has been associated with cardiovascular risks and comorbid pathologies, particularly cardiorenal disorder. Short-chain fatty acids (SCFAs), especially butyrate, are essential fatty acids that regulate metabolic health and ameliorate granulosa inflammation in PCOS. However, the effect of butyrate on cardiorenal damage associated with PCOS is unknown. This study investigated the impact of SCFA and butyrate on cardiorenal abnormalities in PCOS rat model and the probable involvement of paraoxonase-1 (PON-1). Eight-week-old female Wistar rats were allotted into three groups, n = 5, namely control (CTL), PCOS (LEZ), and LEZ + BUT. Induction of PCOS with letrozole (1mg/kg) lasted for 21days, while treatment with butyrate (200mg/kg) commenced after the induction and lasted for 6weeks uninterruptedly. PCOS rats showed hyperandrogenism, multiple ovarian cysts, disrupted metabolic indices (fasting insulin and homeostatic model of insulin resistance), and increased (p < 0.05) plasma troponin T, urea, and creatinine, as well as increased cardiac/renal stroma cell-derived factor-1/caspase-6, malondialdehyde/nuclear factor-kappaB, transforming growth factor-β1, and renal ϒ-glutamyl transferase, while a significant decrease (p < 0.05) in systemic nitric oxide/endothelial nitric oxide synthase and cardiac/renal hypoxia-inducible factor-1α and nuclear factor erythroid 2-related factor 2, which were accompanied with a decreased level of PON-1. These systemic and cardiorenal derangements were reversed by butyrate administration. The results demonstrate the therapeutic benefits of SCFAs, butyrate, against cardiorenometabolic disorder in a model of PCOS. This beneficial effect is accompanied by an elevated level of PON-1. The present data possibly provides a preclinical relevance for the management of cardiorenal syndrome in PCOS.

Resveratrol improves follicular development in PCOS rats by inhibiting the inflammatory response and pyroptosis of granulosa cells.

Chronic inflammation is a key characteristic of polycystic ovary syndrome (PCOS) and is associated with follicular dysplasia in PCOS. PCOS patients treated with 1000mg resveratrol (RES) daily for 3months showed significant improvement in menstrual cycle regularity compared to the placebo group. This investigation explores potential impact of RES on a rat model of PCOS. Sprague-Dawley (SD) rats were subjected to a 30-day letrozole/high-fat diet interventions for PCOS model establishment, followed by RES intervention (20mg/kg/d) for an additional 30days. RES intervention mitigated obesity, estrous cycle irregularities, and ovulation disorders while decreasing serum testosterone and lipopolysaccharide (LPS) levels in PCOS rats. Concurrently, inflammatory markers (TNF-α, NLPR3, IL-6,) and pyroptosis-related markers (GSDMD, cleaved-Caspase-1, IL-1β, IL-18) were downregulated. Additionally, KGN cells (a human granulosa-like cell line) were treated with LPS and RES for in vitro assays. It was observed that RES (15μM) significantly reduced ROS production and downregulated inflammatory cytokine expression in LPS-intervened KGN cells. Additionally, RES downregulated the expression levels of pyroptosis-related factors (GSDMD and cleaved-Caspase-1) and attenuated IL-18 and IL-1β secretion in LPS-induced KGN cells. Furthermore, RES intervention improved the pyroptosis-associated morphology of KGN cells after LPS treatment. In conclusion, RES may restore follicular development in PCOS rats by inhibiting inflammation and NLRP3/GSDMD/Caspase-1-mediated pyroptosis of ovarian granulosa cells, providing new insights into potential therapeutic approaches for PCOS.

Evaluation of the Effects of Naringenin on the Hypothalamic mRNA Levels of nesfatin-1 and CRH in a Rat Model of PCOS

Background: Polycystic ovary syndrome (PCOS) is a common endocrine disorder and a leading cause of infertility in women. Studies suggest that naringenin may improve ovarian function; however, its molecular mechanism within the hypothalamus-pituitary-gonad (HPG) axis remains unclear. Objectives: This study investigated the role of naringenin on the expression of corticotropin-releasing hormone (CRH) and nesfatin-1 genes in the hypothalamus of PCOS rats. Methods: Twenty rats, each weighing 180 - 200 g, were divided into four groups (n = 5). Polycystic ovary syndrome was induced by administering estradiol valerate (2 mg per rat). The control and PCOS groups received saline, while the other two PCOS groups received intraperitoneal injections of naringenin at doses of 20 and 50 mg/kg for 14 days. Hypothalamic samples were collected to measure gene expression via real-time PCR. Results: The PCOS group showed a significant decrease in CRH and nesfatin-1 gene expression compared to the control group (P ≤ 0.05). Naringenin treatment significantly increased the expression of CRH and nesfatin-1 genes in comparison to the PCOS group (P ≤ 0.05). Conclusions: Naringenin appears to have therapeutic potential in improving ovarian function in PCOS. Its effects are mediated through up-regulation of hypothalamic neuropeptides upstream of GnRH neurons.

Nicotinamide Mononucleotide Improves Endometrial Homeostasis in a Rat Model of Polycystic Ovary Syndrome by Decreasing Insulin Resistance and Regulating the Glylytic Pathway.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder that can lead to insulin resistance (IR) and dysregulation of glucose metabolism, resulting in an imbalance in the endometrial environment, which is unfavorable for embryo implantation of PCOS. This study aims to investigate whether nicotinamide mononucleotide (NMN) improves the stability of the endometrium in a rat model of PCOS and identifies whether it is related to reduce IR and increase glycolysis levels and its potential signaling pathway. Female Sprague-Dawley (SD) rats are fed letrozole and a high-fat diet (HFD) to form the PCOS model, then the model rats are treated with or without NMN. It randomly divided into control, PCOS, and PCOS-NMN groups according to the feeding and treating method. Compared with the PCOS group, the regular estrous cycles are restored, the serum androgen (p<0.01) and fasting insulin levels (p<0.05) are reduced, and endometrial morphology (p<0.05) is improved in NMN-PCOS group. Furthermore, NMN inhibits endometrial cell apoptosis, improves endometrial decidualization transition, reduces IR, restores the expression of glycolysis rate-limiting enzymes, and activates the PI3K/AKT pathway in the uterus. These results suggest that NMN enhances endometrial tissue homeostasis by decreasing uterine IR and regulating the glycolysis through the PI3K/AKT pathway.

Mesenchymal Stromal Cell Exosome-Induced Vascular Regeneration in a PCOS Mouse Model.

The efficacy of Bone Marrow Mesenchymal Stem Cell-derived Exosomes (BMSCs-Exo) in addressing the complexities of Polycystic Ovary Syndrome (PCOS) has been explored in a controlled experimental study using a DHEA-induced PCOS model in 6-8-week-old female NMRI mice. This research undertook an in vivo approach with fifteen female murine subjects to investigate the potential of BMSCs-Exo in promoting vascular regeneration and alleviating the adverse effects associated with PCOS. Through a strategic intervention, the study aimed to modulate the pathophysiological markers of oxidative stress and inflammation that are hallmark features of PCOS. Remarkably, the administration of BMSCs-Exo led to decreased CD31 expression in ovarian tissues, suggesting reduced angiogenesis and endothelial activation. Moreover, a significant reduction in pro-inflammatory cytokines and oxidative stress markers was noted, aligning closely with the metrics observed in the control group. These findings illuminate a promising therapeutic avenue utilizing BMSCs-Exo to recalibrate angiogenic, inflammatory, and oxidative stress responses in PCOS. This research not only contributes to the current understanding of PCOS management but also opens new doors for innovative clinical treatments.

Antioxidant activity of self-nanoemulsifying drug delivery system (SNEDDS) of Curcuma longa in polycystic ovary syndrome rat model

Purpose: To investigate the effect of SNEDDS containing Curcuma longa extract on hormonal and histological improvement of polycystic ovary syndrome (PCOS) rat model. Method: A total of 36 female Wistar rats, aged between 8 and 10 weeks, were randomly divided into 6 groups, including control (normal rats), PCOS (untreated PCOS rats), PCOS + M (treated with 20 mg/kg bw/day metformin), PCOS + SC25, PCOS + SC50, and PCOS + SC100 (treated with Curcuma longa extract at 25, 50, and 100 mg/kg/day with SNEDDS, respectively). This study used a posttest-only control group design. The rats were euthanized on day 15, and blood samples were taken to examine malondialdehyde (MDA) and anti-mullerian hormone (AMH) levels. Ovarian tissue was prepared on a histological slide, and various follicles were observed. Data was analyzed using analysis of variance (ANOVA) and Kruskal-Wallis tests. Results: Curcuma longa extract, at 50 and 100 mg/kg, incorporated in SNEDDS resulted in a significant reduction in the corpus luteum number and width of the granulosa layer (p &lt; 0.05). Also, Curcuma longa significantly reduced malondialdehyde (MDA), anti-mullerian hormone (AMH), preantral follicles, and follicular cysts (p &lt; 0.05). Conclusion: Curcuma longa extract in SNEDDS reduces MDA and AMH levels and improves the histology of the ovaries in PCOS model rats. There is a need to conduct further studies of C. longa extract or isolates in SNEDDS using human cell lines of PCOS model.

7869 Conditional deletion of androgen receptors from kisspeptin neurons only partially rescues the reproductive phenotype of a prenatal androgen-induced mouse model of polycystic ovary syndrome

Abstract Disclosure: A.G. Novak: None. A.V. Petriv: None. D.B. Lohr: None. K.M. Dillon: None. N.T. Neifert: None. A.M. Moore: None. Polycystic ovary syndrome (PCOS) is a prevalent cause of infertility in people with ovaries of reproductive age. Although the etiology of PCOS remains unclear, research in the clinic and with animal models suggests that hyperandrogenism (the presence of elevated circulating androgens) plays a significant role in the development of reproductive dysfunction, including irregular menstrual/estrous cycles and altered ovarian morphology. Although the site at which androgens disrupt the reproductive axis is unclear, neurons expressing kisspeptin are crucial regulators of the hypothalamic-pituitary-gonadal axis and express androgen receptors. Therefore, we hypothesize that androgen signaling at kisspeptin cells is a critical mediator of reproductive dysfunction in PCOS. To test this, we utilized Cre-lox technology to conditionally delete androgen receptors from kisspeptin cells (KARKO) in a commonly used mouse model of PCOS induced by using prenatal dihydrotestosterone treatment (prenatal androgen-treated (PNA) mice). To assess estrous cyclicity, vaginal cytology was examined daily for three weeks beginning at 50 days of age. Prenatal vehicle-treated (PNV) wild-type (WT) and PNV KARKO mice (n=6/group) exhibited regular estrous cycles with no significant differences in the percentage of time spent in proestrus, estrus, or metestrus/diestrus, indicating androgen receptors are not required to maintain fertility. As expected, PNA WT mice (n=6) rarely entered proestrus, spent significantly less time in estrus (p&amp;lt;0.05), and spent significantly more time in metestrus and diestrus (p&amp;lt;0.001) compared to the PNV groups (n=6/group, two-way ANOVA), replicating the previously reported acyclic phenotype of PNA mice. No significant differences in the percentage of time spent in cycle stages were detected between PNA WT and PNA KARKO (n=5) mice in this analysis, suggesting androgen signaling at kisspeptin cells is not required to induce acyclicity in PNA mice. Despite this acyclic phenotype, preliminary data using ovarian sections (5μm thickness) stained with hematoxylin and eosin revealed that the percentage of ovarian area composed of corpora lutea was significantly elevated in PNA KARKO mice (n=3) compared to PNA WT mice (n=4) (p&amp;lt;0.05, one-way ANOVA). Further, in this analysis, the area composed of corpora lutea in PNA KARKO mice was not significantly different from PNV WT and KARKO controls (n=6/group), suggesting PNA KARKO mice exhibit a comparable ovulation rate to fertile controls. Together, these data show that androgen signaling at kisspeptin cells partially contributes to the development of anovulation in PNA mice. However, additional mechanisms likely contribute to developing the full PCOS-like reproductive phenotype. Presentation: 6/1/2024

8686 scRNA-seq of pituitary from letrozole-induced PCOS mouse model reveals abnormal prolactin secretion

Abstract Disclosure: G. De Robles: None. N. Ujagar: None. Z. Del Mundo: None. D. Nicholas: None. Polycystic ovary syndrome (PCOS) has a complex pathophysiology and is the most common endocrine disorder among reproductive-aged women. Ovarian hormonal dynamics are regulated via the secretion of the gonadotropin-releasing hormone (GnRH), which stimulates the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary gland. High serum levels of LH in PCOS patients indicate an increase in GnRH levels, but the mechanisms within the anterior pituitary at a cellular level are unclear. We performed single-cell mRNA sequencing (scRNA-seq) on the pituitaries of the letrozole-induced (LET) mouse model of PCOS to profile the functional genomics of endocrine cell populations relative to serum hormone levels. The population percentage of lactotropes significantly decreased; in contrast, two gonadotrope populations significantly increased in the LET mice compared to the placebo-treated mice. Consistent with these changes, an increasing trend of serum LH levels and a decreasing trend of serum prolactin (PRL) levels were observed in the LET mice, while no change occurred in serum growth hormone levels. A gene set enrichment analysis (GSEA) on the differentially expressed genes (DEGs) from the lactotropes revealed an association with negative regulation of protein kinase activity. These DEGs included Hspb1, Park7, Pcp4, and Ppia, which all share the function of mediating stress-induced apoptosis. A GSEA on the DEGs between the two gonadotrope clusters in the LET mice also confirmed a variation in biological pathways linked to ribosomal subunits, transcription regulation, and the regulation of steroid metabolic processes. Based on the scRNA-seq results of the anterior pituitaries of LET mice, alterations in the proportions of hormone-secreting cells may be a response to hyperandrogenism, in which functionally diverse gonadotropes could contribute to heterogeneity in reproductive phenotypes. Changes in the lactotrope population and PRL serum levels also support further research on the lactation hormone and its role in PCOS. PRL is involved in metabolic homeostasis and reproduction, and understanding its dysregulations may provide additional insight into the pathophysiology of PCOS. Presentation: 6/3/2024

8421 The Effect of Dietary Glycotoxins on Insulin Resistance in Polycystic Ovary Syndrome

Abstract Disclosure: A. Weidner: None. K. Vann: None. J. Zhang: None. A. Roy: None. O. Astapova: None. Polycystic ovary syndrome (PCOS) is a chronic systemic disorder that affects both metabolism and reproduction. PCOS is characterized by hyperandrogenism and insulin resistance, which are closely intertwined in this disease pathophysiology. PCOS affects an estimated 10% of people with ovaries and increases risk of endometrial cancer, type 2 diabetes, and major cardiovascular events. Furthermore, treatment options are generally poorly tolerated. Despite its prevalence, severity, and enormous public health burden, the etiology of PCOS is not well-understood. Insulin resistance likely plays a central role in PCOS pathogenesis, and often leads to hyperinsulinemia as the pancreas attempts to compensate for insulin’s decreased efficacy in the body. Moreover, the Western diet is becoming increasingly rich in advanced glycation end products (AGEs): non-enzymatically modified biomolecules that have recently been found to have a harmful effect on insulin sensitivity and are associated with diabetes, aging, and oxidative stress. Serum AGE levels have been reported to be higher in both lean and obese PCOS patients, regardless of PCOS subtype. We have also found higher levels of AGE precursors in the serum of our chronic dihydrotestosterone-induced mouse model of PCOS. Using this mouse model, we have shown that ovarian granulosa cells (GCs) remain insulin-sensitive despite systemic insulin resistance and hyperinsulinemia, manifested by impaired glucose tolerance and increased HOMA-IR. To assess whether GCs respond to insulin through alternative receptor pathways, we knocked down insulin receptor in human granulosa-derived KGN cells and found that insulin-stimulated AKT phosphorylation was only minimally reduced, suggesting that insulin may signal in large part through the insulin-like growth factor 1 receptor (IGF1R) in these cells. Therefore, we believe that compensatory hyperinsulinemia plays a key role in chronic GC hyperstimulation, possibly by rerouting the insulin signal through IGF1R to overcome the resistance to insulin receptor activation, suggesting a mechanism for AGE toxicity in PCOS. We hypothesize that in PCOS, this results in impaired GC function and exacerbation of ovarian dysfunction. We will use our PCOS mouse model to study the effects of a reduced-AGE diet in ameliorating the reproductive and metabolic phenotype of an androgenized PCOS mouse. In preliminary experiments, we have found that a low-AGE diet results in lower fasting glucose in C57BL/6J mice after only 4 weeks. We expect that reproductive symptoms, such as estrous cyclicity, and metabolic symptoms, such as GTT and circulating insulin, will improve in our PCOS mouse model when mice are fed a low-AGE diet throughout development and adulthood. This project has significant clinical implications, as reducing dietary AGEs could provide an effective, tolerable, non-drug therapy option for patients with PCOS. Presentation: 6/3/2024

7872 Adiponectin Enhances Hepatic Insulin Sensitivity in a Rat Model of Polycystic Ovary Syndrome: Role of PI3K/Akt/mTORC2 Pathway

Abstract Disclosure: A. Abdelhameed: None. S. Rezq: None. R.M. Vinson: None. J. Flaherty: None. B. Kim: None. D.G. Romero: None. L.L. Yanes Cardozo: None. Background: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in reproductive-age women and is associated with a high prevalence of obesity and insulin resistance (IR). Phosphoinositide-3-kinase (PI3K)/Akt/ the mammalian target of rapamycin complex 2 (mTORC2) pathway is one major insulin signaling pathway and its impairment is involved in multiple metabolic disorders by inducing IR. Phosphorylation of protein kinase B (Akt) at (Ser)-473 by mTORC2 leads to its full activation and subsequently enhances insulin sensitivity. Adiponectin is a peptide secreted by adipocytes that increases insulin sensitivity. PEG-BHD1028 is a novel potent peptide adiponectin receptor agonist that enhances insulin sensitivity in different models of diabetes. However, its potential to attenuate androgen-mediated hepatic IR by modulating PI3K/Akt/mTORC2 pathway remains unknown in PCOS. Hypothesis: We tested the hypothesis that PEG-BHD1028 attenuates hepatic IR via modulating PI3K/Akt/mTORC2 pathway in a rat model of PCOS. Methods: To induce PCOS, four-week-old female Sprague Dawley rats were implanted with dihydrotestosterone (DHT) silastic tubes (7.5mg) or placebo (PBO) for 90 days (n=4-8/group). During the final three weeks of DHT treatment, rats received PEG-BHD1028 (25μg/kg/day) subcutaneously. Body weight (BW) by gravimetry, fat and lean mass were measured by Echo-MRI. Fasting plasma insulin was measured by ELISA while fasting plasma glucose was measured with a clinical chemistry analyzer. HOMA-IR, an index of systemic IR, was calculated. Protein levels of insulin signaling markers including IRS1, p-IRS1 (Ser1101), PI3 kinase p110, Akt, p-Akt (Ser473), mTOR, p-mTOR (Ser2481) and mTORC2 regulatory protein Rictor were quantified in the liver by Western-blot. Results: DHT significantly (p&amp;lt;0.05) increased BW, fat mass and lean mass by 1.4-, 2.2-, 1.4- folds, respectively compared to PBO group. DHT significantly (p&amp;lt;0.05) increased HOMA-IR by 3.8- fold, compared to PBO group. DHT downregulated hepatic p-Akt, Akt and mTOR protein expression by 20% to 40% while upregulating IRS-1 by 1.6- fold with no impact on PI3 kinase, p-mTOR, Rictor and p-IRS1 compared to the PBO group. PEG-BHD1028 significantly decreased BW, fat mass and lean mass while attenuating IR (34.7% reduction in HOMA-IR) in PCOS rats. Interestingly, PEG-BHD1028 significantly (p&amp;lt;0.05) increased PI3K, active Akt (pAKT), mTOR and Rictor levels by 1.6-, 2.2-, 1.5-, 1.8- folds, respectively with no effect on the other insulin signaling markers compared to vehicle-treated group. Conclusion: Our findings suggest that PEG-BHD1028 is a novel and effective therapeutic agent to attenuate hepatic IR in PCOS via activating PI3K/Akt/mTORC2 pathway. Significance: Targeting adiponectin signaling could be a novel and effective therapeutic approach to mitigate metabolic dysfunction in females with excess androgens. Presentation: 6/2/2024

7939 Impact of genetic background on chronic inflammation in a letrozole-induced mouse model of PCOS

Abstract Disclosure: N. Ujagar: None. G. De Robles: None. L.M. Velez: None. M. Seldin: None. D. Nicholas: None. The etiology behind polycystic ovary syndrome (PCOS) is elusive due to the heterogeneity of its symptoms and varying severity in patients. There are a wide variety of factors associated with PCOS, like hyperinsulinemia, hyperandrogenism, endotoxemia and chronic inflammation that have been implicated as causal factors in the condition. However mechanisms of cause and effect are difficult to establish in human cohorts. Endotoxemia, observed in humans and mouse models of PCOS, is known to trigger chronic inflammation by activating immune cells. Although correlation of chronic inflammation with PCOS has been established, the type of inflammation in PCOS patients has not been defined, in part due to the genetic variance from person to person. Using a systems immunology approach to mimic human genetic diversity, combined with a letrozole-induced PCOS mouse model, we aim to distinguish the types of inflammation present in PCOS. We analyzed the immune cells from lymph nodes and spleens of 21 different strains of PCOS induced and control mice (130 mice in total), and found that based on genetic background the reproductive and metabolic phenotypes differed- similar to how PCOS presents clinically. We were able to categorize each letrozole-induced mouse strain into the sub-phenotypes of PCOS based on the current guidelines for diagnosing PCOS published in 2023 and extensive characterization of each strain. Splenocytes from these control and PCOS mice were activated in vitro to trigger T cell and myeloid cell cytokine secretion, which was measured via luminex. Clustering analysis of these multi-analyte cytokine measurements together with flow cytometry of the immune cells allowed us to identify T-helper 17 (Th17) cells as the prominent immune cell driving inflammation in letrozole-induce mouse model of PCOS independent of genetic background. This systems immunology approach has allowed us to define PCOS associated inflammation and will be the basis for integrating human inflammation in PCOS with mouse models that appropriately recapitulate disease. The discovery of Th17 cells as key inflammatory mediators provide targets for understanding the mechanisms linking chronic inflammation to reproductive and metabolic outcomes in PCOS, and will assist in future development of anti-inflammatory therapies. Presentation: 6/2/2024

7939 Impact of genetic background on chronic inflammation in a letrozole-induced mouse model of PCOS

Abstract Disclosure: N. Ujagar: None. G. De Robles: None. L.M. Velez: None. M. Seldin: None. D. Nicholas: None. The etiology behind polycystic ovary syndrome (PCOS) is elusive due to the heterogeneity of its symptoms and varying severity in patients. There are a wide variety of factors associated with PCOS, like hyperinsulinemia, hyperandrogenism, endotoxemia and chronic inflammation that have been implicated as causal factors in the condition. However mechanisms of cause and effect are difficult to establish in human cohorts. Endotoxemia, observed in humans and mouse models of PCOS, is known to trigger chronic inflammation by activating immune cells. Although correlation of chronic inflammation with PCOS has been established, the type of inflammation in PCOS patients has not been defined, in part due to the genetic variance from person to person. Using a systems immunology approach to mimic human genetic diversity, combined with a letrozole-induced PCOS mouse model, we aim to distinguish the types of inflammation present in PCOS. We analyzed the immune cells from lymph nodes and spleens of 21 different strains of PCOS induced and control mice (130 mice in total), and found that based on genetic background the reproductive and metabolic phenotypes differed- similar to how PCOS presents clinically. We were able to categorize each letrozole-induced mouse strain into the sub-phenotypes of PCOS based on the current guidelines for diagnosing PCOS published in 2023 and extensive characterization of each strain. Splenocytes from these control and PCOS mice were activated in vitro to trigger T cell and myeloid cell cytokine secretion, which was measured via luminex. Clustering analysis of these multi-analyte cytokine measurements together with flow cytometry of the immune cells allowed us to identify T-helper 17 (Th17) cells as the prominent immune cell driving inflammation in letrozole-induce mouse model of PCOS independent of genetic background. This systems immunology approach has allowed us to define PCOS associated inflammation and will be the basis for integrating human inflammation in PCOS with mouse models that appropriately recapitulate disease. The discovery of Th17 cells as key inflammatory mediators provide targets for understanding the mechanisms linking chronic inflammation to reproductive and metabolic outcomes in PCOS, and will assist in future development of anti-inflammatory therapies. Presentation: 6/2/2024

7310 System-level Analysis in Polycystic Ovary Syndrome on a Panel of Genetically Diverse Strains of Mice

Abstract Disclosure: L.M. Velez: None. C. Johnson: None. I. Tamburrini: None. M. Zhou: None. C. Viesi: None. N. Ujagar: None. D. Ashbrook: None. M. Nelson: None. A. Senior: None. D. James: None. R. Williams: None. D. Nicholas: None. M. Seldin: None. Polycystic ovary syndrome (PCOS) is the most common endocrinopathy in women, with a prevalence of ∼4-20% in women of reproductive age. The diagnosis of the syndrome generally occurs when the patient consults for fertility issues and is only based on a reproductive criterion, which includes (1) hyperandrogenism, (2) oligo-anovulation, and (3) polycystic ovary morphology. However, the overlap of PCOS with cardiometabolic diseases is significant. To put in numbers; up to 75% of PCOS women present some degree of insulin insensitivity, 38-88% present obesity or overweight, 20-50% develop type 2 diabetes by age 40, and PCOS women are at increased risk of cardiovascular disease. Despite these facts, shared reproductive/metabolic mechanisms are largely underexplored. Moreover, studies addressing the genetic architecture of PCOS are missing. Here, we induced a PCOS-like condition in 25 recombinant and classical inbred female strains and matched placebo controls over 6 weeks. Comprehensive in vivo and terminal reproductive/metabolic analyses were performed, as well as ovary and adipose RNA-Seq. These strains varied in PCOS response in a number of key metabolic and reproductive traits, including circulating hormone levels, glucose metabolism, and cardiac function. We applied a linear mixed-effects model to estimate heritability, and genetic (h2), PCOS, and gene-by-PCOS interactions. High h2 was observed for lean and fat mass, glucose, and AUC, whereas PCOS effects were high for the BW change, testosterone, and AUC. Substantial gene-by-PCOS interactions were found for reproductive hormones. Undirected network construction and centrality estimates showed that the reproductive hormones LH and LH/FSH ratio were the strongest central traits connecting metabolic phenotypes. We also showed select strains represent subtypes of human PCOS-metabolism interaction with varied susceptibilities to disease in a PCOS setting. Ovarian RNA-seq analysis of PCOS DEGs showed strong enrichments with human disease settings such as hyperandrogenism, inflammation, and pregnancy hypertension. Similar analyses in GWAT RNA-seq showed enrichments in weight gain, liposarcoma, inflammation, and reproductive diseases were at the top, with adipose genes connecting these diseases and potentially involved with PCOS. In conclusion, we established a PCOS model to study relevant mechanisms intersecting reproduction with metabolism in the context of genetic variation. Presentation: 6/2/2024

Superior metabolic improvement of polycystic ovary syndrome traits after GLP1-based multi-agonist therapy

Polycystic ovary syndrome (PCOS) is a heterogeneous condition, defined by oligo-/anovulation, hyper-androgenism and/or polycystic ovaries. Metabolic complications are common in patients suffering PCOS, including obesity, insulin resistance and type-2 diabetes, which severely compromise the clinical course of affected women. Yet, therapeutic options remain mostly symptomatic and of limited efficacy for the metabolic and reproductive alterations of PCOS. We report here the hormonal, metabolic and gonadal responses to the glucagon-like peptide-1 (GLP1)-based multi-agonists, GLP1/Estrogen (GLP1/E), GLP1/gastric inhibitory peptide (GLP1/GIP) and GLP1/GIP/Glucagon, in two mouse PCOS models, with variable penetrance of metabolic and reproductive traits, and their comparison with metformin. Our data illustrate the superior efficacy of GLP1/E vs. other multi-agonists and metformin in the management of metabolic complications of PCOS; GLP1/E ameliorates also ovarian cyclicity in an ovulatory model of PCOS, without direct estrogenic uterotrophic effects. In keeping with GLP1-mediated brain targeting, quantitative proteomics reveals changes in common and distinct hypothalamic pathways in response to GLP1/E between the two PCOS models, as basis for differential efficiency. Altogether, our data set the basis for the use of GLP1-based multi-agonists, and particularly GLP1/E, in the personalized management of PCOS.

Dihydrotestosterone induces reactive oxygen species accumulation and mitochondrial fission leading to apoptosis of granulosa cells

Dihydrotestosterone (DHT), which has significant androgenic activity，is a major player in follicle development and ovary function in females. However, an excess of androgens may result in increased follicular apoptosis with adverse effects on female fertility. This study aimed to explore the mechanism by which DHT induces apoptosis in human ovarian granulosa cells (GCs). The association between DHT and GC apoptosis was explored by the construction of rat models of polycystic ovary syndrome (PCOS). It was found that serum DHT levels were negatively correlated with thickness of the GC layer in PCOS model rats (R2=0.8342, p＜0.0001), compared with control rats, together with significant increases in cofactors (Fis1: p=0.008; MFF: p=0.044). The GC SVOG cell line was used to clarify the mechanism by which DHT influenced GC apoptosis in in vitro experiments. The results confirmed that apoptosis in SVOG cells was positively associated with the DHT dose. The expression of the autophagy-related proteins LC3A/B (p=0.027) and the proapoptotic protein Bax (p=0.0095) were increased, while that of the anti-apoptotic protein Bcl-2 (p=0.0005) was decreased in the high-dose DHT group. ROS levels were significantly increased (p=0.0237) and the mitochondrial membrane potential ΔΨm was decreased (p=0.0194). Moreover, ultrastructural analysis of the mitochondria indicated significant damage. The results of RT-qPCR and western blotting showed that two fission cofactor-Fis1（p=0.034） and MFF (p=0.039) were significantly increased after treatment with high doses of DHT. Even though the overall expression of Drp1 did not change significantly (p=0.5961), that of activated Phosphor-Drp1(Ser616) was significantly increased (p=0.046), while the expression of Phosphor-Drp1 (Ser637) was markedly reduced (p=0.007) following exposure to high concentrations of DHT. All these effects could be reversed by the Drp1 inhibitor Mdivi-1. These findings indicated the impact of DHT on ROS aggregation and mitochondrial fission, resulting in GC apoptosis. An imbalance in Drp1 phosphorylation may be the key link in DHT-induced excessive mitochondrial fission.

SPRY4 regulates ERK1/2 phosphorylation to affect oxidative stress and steroidogenesis in polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder in women of childbearing age. The role of Sprouty RTK Signaling Antagonist 4 (SPRY4) in ovarian function in PCOS was investigated herein, focusing on its regulation of ERK1/2 phosphorylation. PCOS models were established in mice using dehydroepiandrosterone (DHEA). The expression levels of SPRY4 in ovarian tissues were analyzed through RT-qPCR and immunohistochemistry. SPRY4 knockdown was achieved via lentivirus, and its effects on endocrine function, ovarian morphology, oxidative stress, and ERK1/2 phosphorylation were evaluated. Afterwards, granulosa cells were isolated and treated with DHEA and ERK2 agonist tert-Butylhydroquinone. The impacts of ERK2 activation on the regulation of SPRY4 knockdown were assessed using ELISA, fluorescent probes, western blotting, and biochemical assays. SPRY4 knockdown normalized the estrous cycle, reduced serum levels of testosterone, anti-Müllerian hormone, and luteinizing hormone/follicle-stimulating hormone ratio, and improved ovarian morphology. Additionally, SPRY4 knockdown alleviated oxidative stress by decreasing reactive oxygen species and malondialdehyde levels while increasing superoxide dismutase activity. It also restored steroidogenic enzyme expression, which were disrupted by DHEA induction. In vitro, SPRY4 knockdown enhanced granulosa cell viability and reduced ERK1/2 phosphorylation, with tert-Butylhydroquinone reversing these effects and restoring oxidative stress and steroidogenesis disruptions. Together, SPRY4 modulates ERK1/2 phosphorylation to influence oxidative stress and steroidogenesis in PCOS. Targeting SPRY4 may provide novel therapeutic avenues for improving ovarian function and managing PCOS.

Patuletin Ameliorates Inflammation and Letrozole-Induced Polycystic Ovarian Syndrome in Rats.

Concerns about inflammation-related issues affecting female reproductive health are growing. Chronic low-grade inflammation in women with polycystic ovarian syndrome (PCOS) affects follicular growth, ovulation, and androgen production. The present investigation aimed to elucidate the efficacy of flavonoid patuletin in ameliorating the letrozole-induced PCOS and associated inflammation in rats. Female Wistar rats (32 days old) were divided into five groups (n = 12): Group I, control; Group II, vehicle control; Group III, letrozole oral (1 mg/kg) for 28 days; Group IV and Group V treatment groups, patuletin i.p. (25 mg/kg) and clomiphene citrate + metformin i.p. (50 mg/kg + 300 mg/kg), respectively. Leterozole-induced PCOS and ovarian inflammation were ameliorated by patuletin, as reflected in the improved histopathology, prevention of cyst formation, significant upregulation of growth factors such as growth differentiation factor 9 (GDF-9) and bone morphogenetic protein-15 (BMP-15) expression, and a decrease in the pro-inflammatory cytokines TNF-α, IL-6, and COX-2. Additionally, the plasma levels of reproductive hormones were restored. Upregulation of FSH-R, PR, and CYP19a1, along with downregulation of ERα, LHR, CYP17a1, CYP11a1 and HSDβ17a1, showed the regulation of gonadotropin receptors and steroid biosynthesis genes in ovarian tissues. Patuletin demonstrated a promising protective approach against the biological model of PCOS by increasing the inflammation in ovarian tissues with consequent regulation of growth factors, enzymes, and hormones, and might be used as adjuvant therapy in the treatment of problems related to female reproductive health.

METTL3 silencing inhibits ferroptosis to suppress ovarian fibrosis in PCOS by upregulating m6A modification of GPX4.

Methyltransferase-like 3 (METTL3) is extensively reported to be involved in organ fibrosis. Ovarian fibrosis is a main characteristic of polycystic ovary syndrome (PCOS). However, the reaction mechanism of METTL3 in PCOS is poorly investigated. This paper was intended to reveal the role and the mechanism of METTL3 in PCOS. Animal and cell models of PCOS were induced by dehydroepiandrosterone (DHEA). H&E staining was performed to detect the pathological alterations in ovary tissues. Masson staining, immunofluorescence, along with western blot measured fibrosis both in vitro and in vivo. To evaluate estrous cycle, vaginal smear was performed. Lipid peroxidation and ferroptosis were evaluated by MDA assay kits, GSH assay kits, immunohistochemistry, Prussian blue staining and western blot. qRT-PCRand western blot were adopted to estimate METTL3 and GPX4 expression. The m6A and hormone secretion levels were respectively assessed by m6A RNA Methylation Quantitative Kit and corresponding kits. The interaction between METTL3 and GPX4 was testified by immunoprecipitation. The fibrosis and ferroptosis were aggravated and m6A and METTL3 expression were increased in ovarian tissues of DHEA-induced PCOS mice. METTL3 silencing alleviated pathological changes, affected hormone secretion level, and repressed fibrosis, lipid peroxidation and ferroptosis in the ovarian tissues of PCOS mice. In vitro, DHEA stimulation increased m6A and METTL3 expression and induced ferroptosis and fibrosis. METTL3 knockdown promoted GPX4 expression in DHEA-induced granulosa cells by m6A modification and restrained DHEA-induced fibrosis, lipid peroxidation and ferroptosis in granulosa cells via elevating GPX4. METTL3 silence inhibited ovarian fibrosis in PCOS, which was mediated through suppressing ferroptosis by upregulating GPX4 in m6A-dependent manner.

Abstract P189: Maternal Hyperandrogenemia Programs Hypoandrogenemia and Intrarenal Activation of Histone Deacetylase in Adult Male Offspring

Polycystic ovary syndrome ( PCOS ) is the most common endocrinopathy in young women. In-utero, children of ~80% of PCOS women in the US are exposed to maternal “hyperandrogenemia and obesity”. It remains unclear whether this exposure affects their cardiovascular ( CV ) health later in life. Using a well-characterized rat model of maternal PCOS, the hyperandrogenemic obese ( HAO ) dams, we previously showed that adult male, not female, offspring ( HAO F1 ) have enhanced blood pressure ( BP ) response to chronic angiotensin ( Ang ) II compared to sex-matched control offspring ( CON F1 ), suggesting their increased risk of later CV disease. Sex steroids have the potential to modulate epigenetics. Histone deacetylase ( HDAC ) and histone acetyltransferase ( HAT ) are markers of epigenetic modifications. HDACs have been shown to promote tumor necrosis factor ( TNF )-α expression and play a role in Ang II-induced hypertension. The present study was designed to test the hypothesis that male HAO F1 have intra-renal activation of HDAC and increased TNF-α that could play a role in their Ang II BP response. Methods: At 4 weeks ( wks ) of age, female SD rats were implanted (s.c.) with either 5α-dihydrotestosterone pellets (7.5 mg/90 d; HAO) or placebo pellets (CON) and mated at 9-12 wks. Offspring were obtained and weaned at 3 wks. Serum testosterone ( T; by LC/MS-MS), renal cortical TNF-α (Bioplex) and renal cortical nuclear HDAC and HAT activities (colorimetric assays) were measured in male offspring (1/litter, 16-24 wks, n=5-9). Results: Male HAO F1 had significantly lower serum T, higher renal cortical HDAC activity and TNF-α (430.2±124 vs 120.6±14 ng/dl, 0.97±0.1 vs 4.51±0.96 OD/min/mg protein and 423.7±47 vs 579.8±47 pg/g tissue, in male CON F1 vs male HAO F1 , respectively, p&lt;0.05), and no change in their renal cortical HAT activity (vs male CON F1 ). Conclusion: Male offspring born to HAO dams develop hypoandrogenemia that is associated with intra-renal activation of HDAC that could be mediating their increased intra-renal inflammation (TNF-α expression) and mediating their exaggerated BP response to Ang II. Future studies will determine the causative relationship between hypoandrogenemia, HDAC activation and TNF-a and their role in the exaggerated pressor response to Ang II in those male offspring. Funding: AHA-24DIVSUP1273026 (JA), NIH-NIGMS P20GM121334 (JFR, NMS), R01HL135089 &amp; R01AG075963 (JFR), AHA-22CDA938320 and 2024 Dean Franklin Award (NMS).

O53 RENOMETABOLIC DISORDER IN EXPERIMENTAL RAT MODEL OF PCOS IS REVERSED VIA INHIBITION OF PYRUVATE DEHYDROGENASE KINASE 4

Background and Objective: Chronic Kidney disorders is a global public health problem, including in women with polycystic ovarian syndrome (PCOS), and is characterized by renal fibrosis, nephrotoxicity and glomerulonephritis, which increases the possibility of renal failure and organ transplant. Pyruvate dehydrogenase kinase 4 (PDK4) has been implicated in mitochondria dysfunction, contributing to metabolic dysregulation in different organs, including kidney. Studies have shown the metabolic regulatory impact of probiotics in human and experimental models. Hence, the present study investigated the therapeutic potential of probiotics on renometabolic disorders associated with experimental PCOS model. The study in addition elucidates the probable involvement of PDK4 in PCOS-associated renometabolic disorders. Methods: Eight-week-old nulliparous female Wistar rats were randomly allotted into four groups (n=6). Letrozole (1 mg/kg) was used to induce PCOS for 3 weeks. Thereafter, probiotics (2 x 107 CFU) was administered for 6 weeks, uninterruptedly. Biochemical parameters, histological analysis, immunohistochemistry and gene expression were carried out with appropriate methods. Results Experimental PCOS rats were characterized with elevated circulating testosterone and the presence of multiple ovarian cysts. In addition, rat with PCOS also manifested insulin resistance, increased plasma urea and creatinine levels, increased renal Gamma glutamyltransferase (GGT), MDA, NF-kappa B, TNF-alpha, TGF-beta 1, caspase-6, HDAC2, while a decrease in G6PD, GSH, renal NO and eNOS, when compared with animals in the control rats. These were associated with elevated level of PDK4 in the renal tissue. However, administration of probiotics ameliorates these renal/metabolic abnormalities. Conclusion: Altogether, the results from the present study revealed that probiotics ameliorates renal dysfunction in PCOS via downregulation of PDK4.