Mouse Model Of Polycystic Ovary Syndrome Research Articles

Effect of nanocurcumin on fertility in murine model of polycystic ovary syndrome.

The precise pathophysiology of polycystic ovary syndrome (PCOS) is not well-founded. In an attempt to fill this gap, the current study was executed to probe the effect of nanocurcumin (NCC) on ovarian tissue, in vitro fertilization (IVF) and pre-implantation embryo development in a mouse model of PCOS. Fifty adult female mice were randomly categorized into five equal groups including non-treated control and PCOS (receiving 0.20 mg estradiol valerate (EV) intra-peritoneally once a day for 21 days) as well as NCC12.50 + PCOS, NCC25 + PCOS and NCC50 + PCOS (receiving respectively 12.50, 25.00 and 50.00 mg kg-1 NCC daily along with EV injection through oral gavages for 21 days) groups. Subsequently, ovarian histo-architecture and total anti-oxidant capacity, and malonaldehyde and catalase levels as well as in vitro fertilizing potential, early embryonic development and serum testosterone concentration were analyzed. Results showed that NCC in a dose-dependent manner improved ovarian cyto-architectural organization and oxidant/anti-oxidant balance along with IVF rate and pre-implantation embryo development in PCOS mice. These findings revealed that NCC at the doses of 25.00 and 50.00 mg kg-1 could alleviate PCOS-linked reproductive disruptions in female mice.

All-trans retinoic acid and fibroblast growth factor-2 enhance the fertility rate and embryo development in polycystic ovary syndrome mouse model.

Polycystic ovary syndrome (PCOS) causes a developmental arrest of antral follicles and disrupts oocyte maturation. Retinoic acid (RA) and Fibroblast Growth Factor-2 (FGF2) are effective in follicle growth, thus their effects on histopathology and in vitro fertility of oocytes were investigated in PCOS-induced mice. Eighty female NMRI mice were randomly divided into 8 groups including 1-Normal mice, 2-PCOS mice without any treatment, 3-Normal mice treated with RA, 4-Normal mice treated with FGF2, 5-PCOS mice treated with RA, 6- PCOS mice treated with FGF2, 7- PCOS mice treated with RA and FGF2, and 8- Normal mice treated with RA and FGF2. Following PCOS induction, the mice were treated with intraperitoneal RA and FGF2 as a treatment. Then ovarian stimulation, for preparing the oocyte and embryo microscopic examinations was performed. After oocyte morphometry, through in vitro fertilization, the embryo formation was assessed. Data was analyzed by one-way ANOVA and Tukey tests. The results showed simultaneous injection of RA and FGF2 into PCOS-induced mice increases antral follicles and corpus luteum, but decreases cystic follicles. Simultaneous injection of these two substances into healthy mice increases the pre-antral follicles and corpus luteum. Simultaneous injection of RA and FGF2 increases the number of embryos in both control and intervention groups. It can be concluded that RA and FGF2 increase the maturity of ovarian follicles, the number of two-celled embryos, and the number of grade-A embryos in mice with PCOS, which is more effective when these two substances are injected simultaneously.

Dried Zingiber officinale Alleviates Dehydroepiandrosterone‐Induced Ovarian Granulosa Cell Apoptosis via the Reactive Oxygen Species‐Mediated Pathway

Polycystic ovary syndrome (PCOS), a prevalent reproductive endocrine disorder, frequently coincides with insulin resistance, lipid dysregulation, and cellular apoptosis. In this study, we aimed to evaluate the therapeutic potential of Dried Zingiber officinale (DZO), renowned for its antibacterial, antiviral, anti‐inflammatory, and antioxidant properties, in the context of PCOS. To this end, we induced a PCOS mouse model through the administration of dehydroepiandrosterone (DHEA) and a high‐fat diet (HFD), followed by DZO treatment to assess its effects on ovarian pathology, insulin resistance, and hormonal imbalances. The anti‐apoptotic effect of DZO on PCOS ovarian granulosa cells was confirmed through network pharmacological analysis, TUNEL staining, FITC‐PI staining, and protein blotting. Notably, DZO treatment significantly alleviated ovarian pathological changes in PCOS mice and normalized hormone levels, including testosterone, estradiol, and progesterone/follicle‐stimulating hormone ratios. Furthermore, our findings confirmed the anti‐apoptotic effect of DZO on PCOS ovarian granulosa cells. Mechanistically, DZO primarily exerted its therapeutic effects in PCOS by inhibiting apoptosis induced by the accumulation of reactive oxygen species (ROS). In conclusion, our study demonstrates the promising therapeutic role of DZO in the management of obese PCOS patients, particularly in reversing ROS‐mediated apoptosis in ovarian granulosa cells.

Significant alteration of protein profiles in a mouse model of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is an endocrine disorder, affecting women of child-bearing age, and the incidence rate is growing and assuming epidemic proportions. The etiology of PCOS remains unknown and there is no cure. Some animal models for PCOS have been established which have enhanced our understanding of the underlying mechanisms, but omics data for revealing PCOS pathogenesis and for drug discovery are still lacking. In the present study, proteomics analysis was used to construct a protein profile of the ovaries in a PCOS mouse model. The result showed a clear difference in protein profile between the PCOS and control group, with 495 upregulated proteins and 404 downregulated proteins in the PCOS group. The GO term and KEGG pathway analyses of differentially expressed proteins mainly showed involvement in lipid metabolism, oxidative stress, and immune response, which are consistent with pathological characteristics of PCOS in terms of abnormal metabolism, endocrine disorders, chronic inflammation and imbalance between oxidant and antioxidant levels. Also, we found that inflammatory responses were activated in the PCOS ovarium, while lipid biosynthetic process peroxisome, and bile secretion were inhibited. In addition, we found some alteration in unexpected pathways, such as glyoxylate and dicarboxylate metabolism, which should be investigated. The present study makes an important contribution to the current lack of PCOS ovarian proteomic data and provides an important reference for research and development of effective drugs and treatments for PCOS.

#324 : Gasdermin D-Mediated Pyroptosis Underlies Dehydroepiandrosterone-Induced Polycystic Ovary Syndrome

Background and Aims: Intestinal microflora disturbance is one of the important etiologies of polycystic ovary syndrome (PCOS). The purpose of this study was to investigate the mechanism of macrophage pyroptosis induced by intestinal microbiota disturbance in PCOS. Method: The PCOS mouse model was established using dehydroepiandrosterone, and was studied by 16S rDNA sequencing, western blot, ELISA, GSDMD gene knockout/oral pyroptotic inhibitors, single cell transcriptomics and translatomics analysis. Results: PCOS mice showed abnormal estrous cycle, elevated serum testosterone and polycystic ovaries. Fecal 16S rDNA sequencing showed that Muribaculacea (26.76%), and Lactobacillus (40.98%) were the most abundant microbe in the intestinal tracts of control mice and PCOS mice, respectively. Difference analysis showed a significant decrease in the abundance of intestinal probiotics Akkermansia and a significant increase in Gram-negative bacteria (Desulfovibrio and Burkholderia) in PCOS mice. Intestinal microbial disturbance in PCOS mice was associated with increased serum lipopolysaccharide (LPS). LPS intraperitoneal injection can induce pyroptosis of ovarian macrophages, which also exists in the ovaries of PCOS mice. The cleavage of GSDMD protein is the final key step in pyroptosis of macrophages. GSDMD knockout ameliorated PCOS in mice. Oral administration of pyroptotic inhibitors disulfiram and metformin can increase the abundance of intestinal Akkermansia, inhibit pyroptosis of ovarian macrophages and ameliorated PCOS in PCOS group mice. Mechanically, transcriptome and translatome sequencing of primary granulosa cells showed that pyroptosis of macrophages disrupted estrogen production and promoted granulosa cell apoptosis. Conclusion: This study revealed that macrophage pyroptosis induced by intestinal flora disorder can lead to the occurrence of PCOS through granulosa cell apoptosis and estrogen synthesis dysfunction. Macrophage pyroptotic inhibitors may be a potential therapeutic strategy for PCOS.

Nanocomposites based on nanoceria regulate the immune microenvironment for the treatment of polycystic ovary syndrome

The immune system is closely associated with the pathogenesis of polycystic ovary syndrome (PCOS). Macrophages are one of the important immune cell types in the ovarian proinflammatory microenvironment, and ameliorate the inflammatory status mainly through M2 phenotype polarization during PCOS. Current therapeutic approaches lack efficacy and immunomodulatory capacity, and a new therapeutic method is needed to prevent inflammation and alleviate PCOS. Here, octahedral nanoceria nanoparticles with powerful antioxidative ability were bonded to the anti-inflammatory drug resveratrol (CeO2@RSV), which demonstrates a crucial strategy that involves anti-inflammatory and antioxidative efficacy, thereby facilitating the proliferation of granulosa cells during PCOS. Notably, our nanoparticles were demonstrated to possess potent therapeutic efficacy via anti-inflammatory activities and effectively alleviated endocrine dysfunction, inflammation and ovarian injury in a dehydroepiandrosterone (DHEA)-induced PCOS mouse model. Collectively, this study revealed the tremendous potential of the newly developed nanoparticles in ameliorating the proinflammatory microenvironment and promoting the function of granulosa cells, representing the first attempt to treat PCOS by using CeO2@RSV nanoparticles and providing new insights in combating clinical PCOS.

Hyperandrogenism drives ovarian inflammation and pyroptosis: A possible pathogenesis of PCOS follicular dysplasia

Hyperandrogenemia and persistent chronic inflammation, two main striking features of polycystic ovary syndrome (PCOS), have been proven involved in follicular dysgenesis in PCOS. However, the association between hyperandrogenism and inflammation activation in PCOS is not fully understood. Excess testosterone（T） induces inflammation and pyroptosis activation in a mouse model of PCOS, leading to ovarian dysfunction and fibrosis. Excessive endoplasmic reticulum (ER) stress is present in ovarian granulosa cells (GCs), testosterone-induced PCOS mouse and cellular models. This study found higher levels of interleukin (IL)-1β, IL-8, IL-17, and IL-18 in the follicular fluid of PCOS patients with hyperandrogenemia undergoing IVF treatment. In addition, pyroptosis in GCs was demonstrated, which was significantly elevated in PCOS patients. To clarify the association of hyperandrogenism, inflammation, and pyroptosis activation in PCOS, dehydroepiandrosterone(DHEA)-treated mouse PCOS model and T-treated KGN cell line were explored for PCOS mechanism. Markers of inflammatory activation and pyroptosis were significantly increased after DHEA treatment in mice and T treatment in KGN cells. In addition, ER stress sensor proteins were increased simultaneously. However, suppression of inflammation by genipin(GP) led to decreased pyroptosis in KGN cells but no variation in ER stress sensor proteins. In contrast, when treated with tauroursodeoxycholic acid(TUDCA) to attenuate ER stress, the markers of inflammatory factors were significantly reduced, accompanied by a reduction in pyroptosis. Our results suggest that persistent hyperandrogenemia of PCOS promotes local inflammatory activation of the ovary, and the imbalanced inflammatory microenvironment leads to pyroptosis of GCs, which is mediated by ER stress activation.

Dehydroepiandrosterone-induced polycystic ovary syndrome mouse model requires continous treatments to maintain reproductive phenotypes

BackgroundPolycystic ovary syndrome (PCOS) is the most common endocrinopathy associated with infertility and metabolic disorder in women of reproductive age. Animal models have been developed and used as tools to unravel the pathogenesis of PCOS, among which most postnatal models employ continuing experimental manipulations. However, the persistence and stability of these animals after modeling is unknown. Dehydroepiandrosterone (DHEA)-induced PCOS mouse model is commonly used in PCOS studies. Thus the aim of the present study was to investigate the reproductive features of DHEA-induced PCOS mice fed a normal chow or an high-fat diet (HFD) with treatment withdrawal or consecutive treatments after PCOS mouse models were established.MethodsPrepubertal C57BL/6 J mice (age 25 days) were injected (s.c.) daily with DHEA on a normal chow or a 60% HFD for 20 consecutive days to induce PCOS mouse models. Mice injected with the vehicle sesame oil were used as controls. After 20 days, mice were divided into 2 groups, namely “Continue dosing group” and “Stop dosing group”. The animals were consecutively treated with DHEA or DHEA + HFD, or housed without any treatment for 2 or 4 weeks. Estrous cycles were evaluated during this period. At the end of the experiment, serum testosterone (T) levels were measured and the morphology of ovaries was evaluated.ResultsThe mice in Continue dosing groups maintained reproductive phenotypes of PCOS mouse models. In contrast, 2 or 4 weeks after PCOS models were established, the mice with treatment withdrawal in Stop dosing groups exhibited normal serum testosterone levels, regular estrous cycle, and relatively normal ovarian morphology. In addition, even with consecutive treatments, there was no marked difference in body weight between DHEA mice on the normal chow or an HFD in Continue dosing groups and the control animals 3 weeks after modeling.ConclusionsAfter PCOS mice were induced with DHEA or DHEA + HFD, the mice still need consecutive treatments to maintain reproductive phenotypes to be regarded as PCOS mice that meet the diagnostic criteria of PCOS defined by the 2003 Rotterdam criteria.

Oocyte quality is impaired in a hyperandrogenic PCOS mouse model by increased Foxo1 expression

One of the most important characteristics of patients with polycystic ovary syndrome (PCOS) is excess androgen, which has adverse effects on pregnancy outcomes and increases the risk of offspring developing metabolic disorders. Foxo1 has been shown to play an important role in PCOS, but whether it has an affect on oocyte’s quality in PCOS remains unclear. The current research investigated the effect of excess androgen exposure on mouse oocyte quality, as well as the possible molecular mechanism. Timelapse incubator was used to culture oocytes in vitro and evaluate the maturation process. The level of reactive oxygen species (ROS) and mitochondrial membrane potential were detected by laser confocal microscope. Immunofluorescence staining assays were performed to examine the expression of Foxo1 and γ-H2AX. Relative mRNA level of Foxo1 and Caspase3 were examined by RT-qPCR. Results showed Germinal vesicle breakdown and maturation rates of oocytes from hyperandrogenic PCOS mice were significantly decreased in vitro, while in vitro maturation showed a marked delay from the germinal vesicle breakdown to metaphase II stage in PCOS group. Expression levels of reactive oxygen species, Foxo1, Caspase3, and γ-H2AX were significantly increased, whereas mitochondrial membrane potential was significantly decreased in oocytes from PCOS mice. These results indicate that excess androgen exposure induced oxidative stress, which further induced high expression of Foxo1, resulting in more DNA damage and apoptosis in oocytes. The current findings provide new knowledge for exploring the mechanism of decreased oocyte quality in hyperandrogenic PCOS.

THU641 Single-cell Transcriptomic Analysis Reveal Dysregulated Immune Modulation In The Pituitaries Of Letrozole-induced Mouse Model Of PCOS

Abstract Disclosure: G. De Robles: None. The most common endocrine disorder, affecting both the reproduction and metabolism of females, is polycystic ovary syndrome (PCOS). Characterizations of the heterogeneous disorder include anovulation, biochemical hyperandrogenemia, insulin resistance, and obesity, which can be recapitulated with a letrozole-induced PCOS mouse model. Immune abnormalities have also been reported in PCOS human patients, including pro-inflammatory factor secretion imbalance and endothelial cell dysfunction. The extent to which the letrozole-induced mouse model for PCOS can represent these immune dysregulations remain unclear, specifically its relevance to disrupting hormone-regulating centers, such as the pituitary gland. Using a PCOS mouse model developed via letrozole induction, we performed single-cell mRNA sequencing on the pituitaries to study impairment in hormone activity and immune regulation. We also carried out a multiplexed immunoassay on stimulated splenocytes to profile cytokines under different immune cell activations. We identified B cells, neutrophils, microglia, T memory cells, and interestingly, sub-populations of macrophages in the pituitary. Differential expression analysis revealed affected genes in the total macrophages of PCOS mice, such as the up-regulation of Pf4, Il1a, and Ccl7. Differentially expressed genes, such as Lyz2, Ctss, Apoe, and H2-Aa, were also down-regulated in the T memory cells of PCOS mice. Pituitary cell types also exhibited dysregulated expressions, including lactotropes, corticotropes, gonadotropes, and somatotropes. Differences in cytokine expression from the multiplex immunoassays also allowed the mice to be distinct from those treated with letrozole or not via principal component analysis. Altogether, the letrozole-treated mice presented disrupted immune responses and hormonal imbalances in their transcription and cytokine profiles that further strengthens their ability to model PCOS. Presentation: Thursday, June 15, 2023

OR35-02 Novel Androgen Receptor PROTAC Degrader ARV-110 Ameliorates Androgen Mediated Renal Injury in a Mouse Model of Polycystic Ovary Syndrome

Abstract Disclosure: J. Basnet: None. S. Rezq: None. A.M. Huffman: None. T.E. Asala: None. L.L. Yanes Cardozo: None. D.G. Romero: None. Background: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in reproductive-age women. PCOS women often have increased central adiposity, insulin resistance, and renal injury. Hyperandrogenism is prevalent in PCOS women and is associated with renal injury and dysfunction. Androgens bind to the androgen receptor (AR) to impart downstream transcriptional activity. Targeting the AR may alleviate PCOS complications; however, the use of AR blockers is limited by their severe adverse effects in females; thus, safer alternative approaches are highly needed. The novel AR Proteolysis Targeting Chimera (PROTAC) degrader ARV-110 has shown high efficacy to prevent AR signaling in prostate cancer; however, its efficacy in PCOS is unknown. Using a well-established hyperandrogenemic mouse model of PCOS, we aim to test the hypothesis that decreasing AR protein levels with ARV-110 ameliorates androgen-mediated derangements in PCOS. Methods: Three-week old female mice (C57BL/6N) were implanted with Silastic tubes filled with the androgen dihydrotestosterone (DHT, 8mg, s.c., 90 days) or vehicle (n=6/grp). Four weeks post-DHT administration, mice were treated with ARV-110 (1 mg/kg/day, s.c.) for an additional 8 weeks. Body composition (EchoMRI) and oral glucose tolerance test (OGTT) were assessed. Kidney weight was measured by gravimetry. AR, extracellular matrix (ECM) remodeling marker MMP9, pro-fibrotic marker TGF-β, renal injury markers (urinary albumin to creatinine ration (UACR), KIM1, and NGAL) were assessed by Western-blot or ELISA. Results: DHT mice showed significant (p<0.05) increases in body weight (27.3±0.6 vs 23.9±0.26 g), fat mass (4.2±0.6 vs 2.1±0.1 g), lean mass (21.6±0.2 vs 19.8±0.2 g), as well as kidney weight (491.3±7.69 vs 303.1±5.5 mg) compared to controls. DHT increased fasting glucose (183.2±25.2 vs 141.5±9.9 mg/dL, p<0.05) and impaired OGTT (AUC:34557±6255 vs 23459±1896 mg.min/dL, p<0.05). ARV-110 treatment ameliorated all aforementioned DHT-mediated changes. At the molecular level, DHT-treated mice had higher renal AR (2.5-fold) and MMP9 (1.5-fold) protein expression. DHT upregulated renal TGF-β levels (2-fold, p<0.05), UACR (966.8±160.9 vs 381.5±41.2 ng albumin/mg creatinine), and urinary KIM1 (3.5-fold) and NGAL (6-fold). ARV-110 successfully decreased renal AR protein levels by 70% in DHT mice compared to vehicle-treated DHT. Excitingly, while ARV-110 did not modulate renal TGF-β levels, it completely abolished DHT-mediated increase in renal MMP9 while significantly lowering the levels of urinary renal injury markers UACR, KIM1 and NGAL. Conclusion and significance: Our findings suggest that DHT-mediated kidney hypertrophy, associated ECM remodeling and injury are ameliorated by ARV-110 in DHT-treated mice. AR PROTACs could be a novel therapeutic approach to treat renal dysfunction in PCOS. Supported by NIH P20GM121334. Presentation Date: Sunday, June 18, 2023

SAT387 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. 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. 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 Date: Saturday, June 17, 2023

Emodin improves glucose metabolism and ovarian function in PCOS mice via the HMGB1/TLR4/NF-ĸB molecular pathway.

Polycystic ovary syndrome (PCOS) is a reproductive disorder with an unclear etiology. It affects 5-10% of women worldwide and is largely associated with impaired glucose metabolism and obesity. High mobility group box 1 (HMGB1) is a nuclear protein associated with impaired glucose metabolism and PCOS. We sought to investigate the potential therapeutic effects of emodin on glucose metabolism and ovarian functions in PCOS mice via the HMGB1 molecular pathway. A high-fat diet (HFD) and dehydroepiandrosterone (DHEA) induced PCOS mouse model comprising four experimental groups was established: control, PCOS, PCOS plus emodin, and PCOS plus vehicle groups. Emodin administration attenuated obesity, elevated fasting glucose levels, impaired glucose tolerance, and insulin resistance, and improved the polycystic ovarian morphology of PCOS mice. Additionally, it lowered elevated serum HMGB1, LH, and testosterone levels in PCOS mice. Elevated ovarian protein and mRNA levels of HMGB1 and TLR4 in PCOS mice were also lowered following emodin treatment. Furthermore, emodin lowered high NF-ĸB/65 protein levels in the ovaries of PCOS mice. Immunohistochemical staining of the ovaries revealed strong HMGB1, TLR4, and AR expressions in PCOS mice, which were lowered by emodin treatment. Moreover, emodin significantly increased GLUT4, Irs2, and Insr levels that were PCOS. Overall, our study showed that emodin alleviated the impaired glucose metabolism and improved ovarian function in PCOS mice, possibly via the HMGB1/TLR4/NF-ĸB signaling pathway. Thus, emodin could be considered a potential therapeutic agent in the management of PCOS.

Alteration of the N6-methyladenosine methylation landscape in a mouse model of polycystic ovary syndrome

ObjectiveTo explore the N6-methyladenosine (m6A) methylation abnormality of mRNAs and its potential roles in the mouse model of polycystic ovary syndrome (PCOS).MethodsThe mouse model of PCOS were induced by injecting dehydroepiandrosterone (DHEA), and confirmed by observing the morphological structures of ovarian follicles. Subsequently, m6A-tagged mRNAs were identified via m6A epitranscriptomic microarray and its potential functional pathways were predicted in KEGG database. The expression and modification levels of key mRNAs in the most enriched pathway were evaluated and compared using western blot and methylated RNA immunoprecipitation-quantitative PCR (MeRIP-qPCR).ResultsCompared with the control group, 415 hypermethylated and downregulated mRNAs, 8 hypomethylated and upregulated mRNAs, and 14 hypermethylated and upregulated mRNAs were identified in the PCOS group (Fold change ≥ 1.5). Those mRNAs were mainly involved in insulin signaling pathway, type II diabetes mellitus, Fc epsilon RI signaling pathway, inositol phosphate metabolism, and GnRH secretion. In insulin signaling pathway, the expression levels of phosphorylated protein kinase B (p-AKT) were decreased, whereas that of upstream phosphorylated phosphatidylinositol 3-kinase (p-PI3K) were increased in PCOS group. Moreover, skeletal muscle and kidney-enriched inositol polyphosphate 5-phosphatease (SKIP), one of PIP3 phosphatases, was verified to be overexpressed, and Skip mRNAs were hypermethylated in PCOS group.ConclusionThe altered m6A modification of mRNAs might play a critical role in PCOS process. The PI3K/AKT pathway is inhibited in the mouse model of PCOS. Whether it is caused by the m6A modification of Skip mRNAs is worthy of further exploration.

Serum granulosa cell-derived TNF-α promotes inflammation and apoptosis of renal tubular cells and PCOS-related kidney injury through NF-κB signaling.

Polycystic ovary syndrome (PCOS) is a disorder with endocrinal and metabolic problems in reproductive aged women. Evidence shows that PCOS is in a high prone trend to develop kidney diseases. In this study, we investigated the mediators responsible for PCOS-related kidney injury. We found that tumor necrosis factor (TNF-α) levels were significantly increased in serum and primary cultured granulosa cells (GCs) from PCOS patients. Serum TNF-α levels were positively correlated with serum testosterone and luteinizing hormone (LH)/follicle-stimulating hormone (FSH) ratio, suggesting its positive role in the severity of PCOS. Serum TNF-α levels were also positively correlated with the levels of urinary KapU, LamU,α1-MU and β2-MU, the markers for renal tubular cell-derived proteinuria. We established a PCOS mouse model by resection of the right kidney, followed by daily administration of dihydrotestosterone (DHT, 27.5 μg, i.p.) from D7 for 90 days. We found that TNF-α levels were significantly increased in the ovary and serum of the mice, accompanied by increased renal tubular cell apoptosis, inflammation and fibrosis in kidneys. Furthermore, the receptor of TNF-α, tumor necrosis factor receptor 1 (TNFR1), was significantly upregulated in renal tubular cells. We treated human ovarian granulosa-like tumor cells (KGN) with DHT (1 μg/ml) in vitro, the conditioned medium derived from the granulosa cellculture greatly accelerated apoptotic injury in human proximal tubular epithelial cells (HKC-8), which was blocked after knockdown of TNF-α in KGN cells. Furthermore, knockdown of TNFR1 in renal tubular epithelial cells greatly ameliorated cell injury induced by granulosa cell-derived conditioned medium. These results suggest that serum TNF-α plays a key role in mediating inflammation and apoptosis in renal tubular cells associated with PCOS-related kidney injury.

Abnormal Expression of Prolyl Oligopeptidase (POP) and Its Catalytic Products Ac-SDKP Contributes to the Ovarian Fibrosis Change in Polycystic Ovary Syndrome (PCOS) Mice.

Polycystic ovary syndrome (PCOS) is an endocrine disorder and metabolic syndrome. Ovarian fibrosis pathological change in PCOS has gradually attracted people's attention. In this study, we constructed a PCOS mouse model through the use of dehydroepiandrosterone. Sirius red staining showed that the ovarian tissues in PCOS mice had obvious fibrosis. Prolyl oligopeptidase (POP) is a serine protease and N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is its catalytic product. Studies show that abnormal expression and activity of POP and Ac-SDKP are closely related to tissue fibrosis. It was found that the expression of POP and Ac-SDKP was decreased in the ovaries of PCOS mice. Further studies showed that POP and Ac-SDKP promoted the expression of matrix metalloproteinases 2 (MMP-2) expression and decreased the expression of transforming growth factor beta 1 (TGF-β1) in granulosa cells. Hyperandrogenemia is a typical symptom of PCOS. We found that testosterone induced the low expression of POP and MMP2 and high expression of TGF-β1 in granulosa cells. POP overexpression and Ac-SDKP treatment inhibited the effect of testosterone on TGF-β1 and MMP2 in vitro and inhibited ovarian fibrosis in the PCOS mouse model. In conclusion, PCOS ovarian tissue showed obvious fibrosis. Low expression of POP and Ac-SDKP and changes in fibrotic factors contribute to the ovarian pathological fibrosis induced by androgen.

Therapeutic Potential of Mesenchymal Stem Cell-Derived Extracellular Vesicles to Treat PCOS.

Polycystic ovary syndrome (PCOS) is known as the most common endocrine disorder in women. Previously, we suggested that human mesenchymal stem cells (MSCs) can reverse the PCOS condition by secreting factors. Here, we evaluated the therapeutic capability of MSC-derived extracellular vesicles (EVs), also known as exosomes, in both in vitro and in vivo PCOS models. Exosomes were used to treat androgen-producing H293R cells and injected in a mouse model through intraovarian and intravenous injection into a letrozole (LTZ)-induced PCOS mouse model. We assessed the effects of the exosomes on androgen-producing cells or the PCOS mouse model by analyzing steroidogenic gene expression (quantitative real-time polymerase chain reaction (qRT-PCR)), body weight change, serum hormone levels, and fertility by pup delivery. Our data show the therapeutic effect of MSC-derived EVs for reversing PCOS conditions, including fertility issues. Interestingly, intravenous injection was more effective for serum glucose regulation, and an intraovarian injection was more effective for ovary restoration. Our study suggests that MSC-derived exosomes can be promising biopharmaceutics for treating PCOS conditions as a novel therapeutic option. Despite the fact that we need more validation in human patients, we may evaluate this novel treatment option for PCOS with the following clinical trials.

Protocatechuic acid alleviates polycystic ovary syndrome symptoms in mice by PI3K signaling in granulosa cells to relieve ROS pressure and apoptosis

Background Polycystic ovary syndrome (PCOS) is a complicated gynecological endocrine disease that occurs in women of childbearing age. Protocatechuic acid is a phenol-rich compound derived from herbs and owns vital functions in numerous diseases. Howbeit, protocatechuic acid’s impact on PCOS is unknown. Methods A combination of in vivo and in vitro models was examined in this study. C57BL/6 mice were injected subcutaneously daily with dehydroepiandrosterone to establish a PCOS mouse model, and protocatechuic acid was intraperitoneally injected into PCOS mice. Granulosa cells of PCOS ovaries were also isolated. The function of protocatechuic acid was appraised using enzyme-linked immunosorbent assay, hematoxylin–eosin staining, reactive oxygen species (ROS) and LC3 levels analysis, flow cytometry, quantitative real-time PCR, and western blot. Meanwhile, the mechanism of protocatechuic acid was assessed with a series of molecular experiments. Results Protocatechuic acid owned no apparent toxic effect on mice. Functionally, protocatechuic acid owned a function of mitigating PCOS in vivo. Meanwhile, protocatechuic acid repressed ROS, autophagy, and apoptosis of PCOS ovarian granulosa cells in vitro. Mechanistically, rescue assays elucidated that the protective function of protocatechuic acid against PCOS was interrelated to the activation of the PI3K/AKT/mTOR axis. Conclusion Protocatechuic acid alleviated PCOS symptoms in mice through PI3K signaling in granulosa cells to reduce ROS levels and apoptosis.

Leptin Inhibition Ameliorates Renal Mitochondrial Dysfunction And Associated Renal Injury In Polycystic Ovary Syndrome

Background: Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism and polycystic ovaries. Renal injury, increased central adiposity and higher circulating levels of the adipokine leptin are common characteristics of PCOS. Leptin has direct renal fibrotic, hypertrophic, and albuminuric effects and can cause renal mitochondrial dysfunction and oxidative stress leading to chronic kidney disease development. Despite increased circulating leptin level in both lean and obese PCOS women, its role in PCOS-mediated renal damage remains unknown. In a well-characterized mouse model of PCOS, we aimed to test the hypothesis that hyperandrogenemia increases central adiposity leading to hyperleptinemia, which via leptin receptors induces intrarenal mitochondrial oxidative stress and dysfunction causing renal injury. Methods: Three-week-old peripubertal female mice were implanted with Silastic tubes filled with the non-aromatizable androgen dihydrotestosterone (DHT, 8 mg) or vehicle for 12 weeks. Eight weeks post-Silastic tubes implantation, the animals were treated with the leptin receptor antagonist (LepR-Ant) pegylated leptin (2 mg/kg, ip, 3x/week) for 4 weeks. Body weight, fat mass (EchoMRI), kidney weight (gravimetry), plasma leptin (ELISA), the glomerular filtration rate (GFR, transcutaneous fluorescence), as well as the renal injury markers urinary albumin to creatinine ratio (UACR, clinical chemistry analyzer), NGAL, and KIM1 (ELISA) were measured. Freshly isolated kidney mitochondria were used to measure mitochondrial reactive oxygen species (mtROS) by Amplex Red assay. Moreover, mitochondrial complex I and complex II-driven respiration and complex IV activity were assessed using Oroboros Fluorespirometer. Results: DHT significantly (p<0.05) increased body weight (31.6 ± 2.3 vs. 23.6 ± 0.5 g), fat mass (3.5 ± 0.6 vs. 1.9 ± 0.4 g), kidney weight (365.6 ± 13.1 vs. 257.1 ± 3.7 mg), leptin (3.7-fold), UACR (1013.3 ± 56.4 vs. 658.9 ± 103.6 μg albumin/g creatinine), NGAL (6-fold), and KIM1 (3.7-fold). Moreover, PCOS mice had significantly (p<0.05) lower GFR (1067.2 ± 59.9 vs. 1335.6 ± 60.3 uL/min/100g body weight), higher mtROS driven by complexes I (3.8 ± 0.8 vs. 2.0 ± 0.3 % electron leak) and III (8.0 ± 1.8 vs. 3.7 ± 0.9 % electron leak), as well as lower complexes I, II, and IV respiration (43-71%). The LepR-Ant had no effect on kidney weight or GFR; however, it abolished DHT-induced increases in body weight and fat mass. Excitingly, the LepR-Ant inhibited mtROS generation and completely restored complex I and II respiration, which was associated with a significant reduction in the urinary renal injury markers UACR, NGAL, and KIM-1 levels (40-60%, p<0.05). Conclusion and significance: Our findings suggest that leptin via its receptor activation plays a significant role in the renal outcomes in PCOS by inducing mitochondrial dysfunction. Leptin receptor blockade could be a novel therapeutic approach to ameliorate renal injury in PCOS. Supported by NIH grants NIGMS P20GM121334 to KSE, LLYC, and DGR, NIGMS P20GM104357, NHLBI P01HL51971, and American Heart Association Predoctoral Fellowship 903804 (A.M.H.). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effects of n-3 PUFA supplementation on oocyte in vitro maturation in mice with polycystic ovary syndrome

n-3 PUFAs are classic antioxidant that can be used to treat follicular dysplasia and hyperinsulinemia caused by excessive oxidative stress in PCOS women. To investigate the effect of n-3 PUFA supplementation on the oocyte quality of polycystic ovary syndrome (PCOS) mice during in vitro maturation, a PCOS mouse model was established by dehydroepiandrosterone (DHEA). The GV oocytes of the control and PCOS groups were collected and cultured in vitro with or without n-3 PUFAs. After 14 h, the oocytes were collected. Our data demonstrated that the oocyte maturation rate of PCOS mice significantly increased after the addition of 50 µM n-3 PUFAs. The results of immunofluorescence showed that the abnormal rates of spindles and chromosomes in the PCOS + n-3 PUFA group were lower than those in the PCOS group. The mRNA expression of an antioxidant-related gene (Sirt1) and DNA damage repair genes (Brca1/Msh2) was found to be significantly rescued after n-3 treatment. Additionally, the results of living cell staining showed that the addition of n-3 PUFAs could reduce the levels of reactive oxygen species and mitochondrial superoxide in PCOS oocytes. In conclusion, the addition of 50 µM n-3 PUFAs during the in vitro maturation of PCOS mouse oocytes can improve the maturation rate by reducing the level of oxidative stress and the rate of spindle/chromosome abnormalities, providing valuable support during the IVM process.