Advanced glycation end products promote polycystic ovary syndrome by regulating AMH via PI3K/Akt/SF1 signaling pathway

Advanced glycation end products promote polycystic ovary syndrome by regulating AMH via PI3K/Akt/SF1 signaling pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Advanced glycation end products promote polycystic ovary syndrome by regulating AMH via PI3K/Akt/SF1 signaling pathway Jingling Zhu, Minqi Liu, Di Cheng, Zhaoming Zeng, Yuanjie Xie, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3916965/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background： Advanced glycation end products (AGEs) are involved in the pathogenesis of polycystic ovary syndrome (PCOS)and high concentrations of anti-Müllerian hormone (AMH) are considered one of the primary causes of anovulation in women with PCOS. However, the specific mechanism of action remains unclear. Method and Results: Ovarian granulosa cells (KGN cells) were treated with AGEs at different concentrations and times. The results showed that pretreatment with AGEs concentration-dependent and time-dependent affected the expression of AMH and SF1. PCNA expression was suppressed by AGEs treatment, and the ratio of the apoptosis-related protein Bax/Bcl2 was elevated. Tetrazolium colorimetric assay shows the same results. Granulosa cells caused by AGE to induce apoptosis could be significantly reversed in KGN cells transfected with AMH siRNA. AMH and the Bax/Bcl2 ratio expression were reduced after SF1 expression was inhibited. Inhibition of the PI3K upregulated PCNA, SF1 and AMH expression. Additionally, in ovarian tissues of AGEs group and PCOS group, the expression of AMH and SF1 increased, accompanied with marked up-regulation of the phosphorylation of PI3K and Akt expression. And the ratio of Bax/Bcl2 increased, while the expression of PCNA was opposite. Conclusions: AGEs increase the level of AMH (Anti-Mullerian Hormone) in ovarian granulosa cells, promoting apoptosis and restricting cell proliferation, thus leading to PCOS in rats. Studies suggest that SF1 may be a crucial target for AGE-mediated AMH production, involving the phosphorylation of the PI3K/Akt signaling pathway. polycystic ovarian syndrome (PCOS) advanced glycation end products (AGEs) anti-müllerian hormone (AMH) apoptosis proliferation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Backgroung Polycystic ovary syndrome (PCOS) is one of the most common endocrine diseases leading to infertility in women of reproductive age, which affects 6–10% of women globally [ 1 ]. The clinical signs and symptoms of PCOS vary greatly. The key factor behind PCOS-afflicted women's high abortion rate and poor conception rate is ovulation disruption. Ovarian granulosa cell plays an important role in the development and maturation of oocytes. Granulosa cell apoptosis is thought to have close relevance to follicular atresia and anovulation [ 2 ]. Anti-Mullerian hormone (AMH), a member of the transforming growth factor superfamily, is secreted only by ovarian granulosa cells [ 3 ]. The level of circulating AMH reflects the reserve function of the ovary, and low levels of AMH indicate a decrease in ovarian reserve function, thus affecting the conception rate [ 4 ]. Clinically, AMH is often used as one of the important indicators to evaluate ovarian function. Compared to fertile women with ovulatory cycles, women with follicular arrest have higher concentrations of AMH in follicular fluid with luminal follicles [ 5 ]. Abnormal follicular growth is one of the main features of PCOS. Serum AMH levels are 2–3 times higher in patients with polycystic ovary syndrome compared to normal women [ 6 ]. Recent studies have demonstrated that high AMH concentrations in PCOS may lead to apoptosis of ovarian granulosa cells, resulting in loss of follicular processes, which may be one of the main causes of anovulation in women with PCOS [ 7 ]. According to recent studies, elevated levels of serum and follicular fluid advanced glycosylation end products (AGEs) have been found in women with polycystic ovary syndrome [ 8 , 9 ]. AGEs are a complex group of compounds that are mainly produced by non-enzymatic glycosylation of reducing sugars on proteins, lipids and nucleic acids and are associated with various diseases such as diabetes, Alzheimer's disease and atherosclerosis [ 10 , 11 ]. A clinical study showed that the expression of AGEs and AMH was increased in both ovulatory and anovulatory PCOS women, and the more prominent elevated was found in non-ovulating PCOS [ 12 ]. In addition, there is a significant positive correlation between age and AMH. However, the mechanism of the interaction remains unclear. Excessive ingestion of AGEs has been shown to affect the metabolic, hormonal and oxidative stress status of women with polycystic ovary syndrome [ 13 ]. In consideration of the above data, the interaction between AGEs and AMH may be related to ovulatory dysfunction in PCOS. Steroidogenic factor-1 (SF1), also known as NR5A1, is a member of the nuclear receptor superfamily, which is an orphan nuclear receptor encoded by FTZ-F1 gene [ 14 ]. SF1 is highly expressed in the adrenal cortex and gonads and is essential for the maintenance of ovarian function [ 14 ]. SF1 binds to the AMH promoter at two sites and influences follicular development by promoting AMH expression in ovarian granulosa cells [ 15 ]. Specific knockdown of the SF1 gene revealed reduced expression of genes related to ovarian granulosa cell proliferation, as well as infertility, incomplete ovarian development, reduced oocyte numbers, and complete absence of the corpus luteum in female mice [ 16 ], indicating a role for SF1 in ovarian follicle development. However, to date, few studies have been reported on the regulatory effects of SF1 on PCOS. Based on these data, we hypothesized that AGEs may be involved in the development of PCOS by regulating the expression of AMH in granulosa cells. Consequently, the present study aimed to investigate whether AGEs affect apoptosis and proliferation of granulosa cells by regulating the expression of AMH, and further understand the possible mechanisms of effects. Materials and Methods Animals and treatment Female Sprague-Dawley rats (5 weeks old) were purchased from Changsha Tianqin Biotechnology Co. Ltd. All of the animals were maintained under standard laboratory conditions, and the experiments were conducted in accordance with the National Institutes of Health guidelines. After 1 week of adjustment, rats were randomly divided into control, AGEs, PCOS, and PCOS + AGEs groups. The PCOS model was induced by 3 weeks of letrozole administration (1 mg/kg dissolved in 1% CMC solutions once a day). AGEs and PCOS + AGEs groups were fed with high AGEs diets (AIN-93G baked for 3h at 125°C) and other two groups were fed with normal diets. Estrous cycle was determined by daily vaginal smears. Cell culture Human granulosa tumor cell line KGN were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin at 37°C in a humidified environment with 5% CO2. Cell viability assay KGN cell viability was assessed using MTT (Solarbio Life Sciences, Beijing). Cells were seeded in a 96-well plate with 1×10 4 cells/wells and incubated in a 37°C incubator for 6h. Cells were incubated in AGEs (ABCAM, USA) at different concentrations (0-200 mg/ml) for 48 h. Each well was incubated for 4 h at 37°C after the addition of 10 µl of tetramethylazole salt reagent, and measured at 490 nm with gentle shaking. TUNEL staining TUNEL assays kit (Beijing 4A Biotech Co, China) was used to treat the Paraffin sections ovrian tissue according to manufacturer's instructions. Images were captured at 200× by Carl Zeiss fluorescence microscope. Apoptotic cells are labeled with bright red fluorescence. Histology staining Ovaries were fixed with the 4% paraformaldehyde and embedded with paraffin, which were sliced to 4 µm for histology staining. We performed haematoxylin and eosin (HE) to observe the organization morphology and masson's trichrome to measure the level of fibrosis. Immunofluorescence KGN Cells were seeded into in 6-well plates and incubated with 100 µg/ml BSA or AGE separately for 24h.Fixed with and 0.25% Triton X-100 transparency. 5% goat serum was used for nonspecific blocking.Incubated overnight at 4°C with primary anti-AMH antibody (1:250, Abcam) and anti-SF1 antibody (1:100, Abcam).C ells were incubated with fluorescent secondary antibody at 37°C for 1 h in the dark, and the nucleus was stained with DAPI (Solarbio, Beijing). Images were captured by a Carl Zeiss fluorescence microscope (ZEISS, Germany). Western blotting Total protein was extracted from cultured KGN cells and rat ovarian tissue using RIPA buffer (RIPA: PMSF = 100:1) containing phosphatase and protease inhibitor 4°C for 30 min, centrifuging at 12,000 rmp for 20 min 4°C, and collecting supernatant. the protein concen-tration was measured using a BCA kit (Pierce, Rockford, USA). Proteins were separated by 10% or 12% SDS-PAGE and transferred to PVDF membrane. After blocking in 5% skim milk powder for 2 h at room temperature, and the membranes were washed 3 times with TBST and incubated overnight at 4°C with one of the following primary antibodies: AMH ( 1:1000, Abcam, USA), SF1 (1:1000, Affinity, China), PI3K (1:1000, Fine Biotech Co., China), p-PI3K (1:1000, Abcam, USA), Akt (1:1000, Fine Biotech Co., China), p-Akt (1:1000, Abcam, USA), PCNA (1:1000, Fine Biotech Co., China), Bax (1:1000, Boster Biological Technology, China), Bcl-2 (1:1000, Boster Biological Technology, China), \({\beta }\) -actin (1:1000, Proteintech, China). After washing 3 times, the membranes were incubated 2 h at room temperature with a secondary antibody (either HRP-conjugated affinipure goat anti-rabbit IgG or anti-mouse secondary antibodies, 1:5000, Proteintech, China). The membranes were treated with ECL luminescent working solution and the relative expression levels of the target proteins were measured by Image Lab software. Quantitative real-time RT-PCR (qPCR) Total RNA was extracted from KGN cells using Trizol reagent (Solarbio Life Sciences, Beijing, China) and cDNA was synthesized using the reverse transcription kit (ABM, Canada) according to the manufacturer’s instructions. Quantitative real-time PCR was performed with Eva Green 2 \(\times\) qPCR MasterMix (ABM, Canada). The target genes were calculated with the 2 −ΔΔCT method. The primer sequences were purchased from Sangon Biotech (Shanghai, China) and listed in Supplementary Table 1. 18S was used as an internal control. Statistical analysis The measurement data are expressed as the means ± SEM. Prism 8 software was used to analyze the data. Statistical significance was evaluated using Student’s t-test or one-way analysis of variance (ANOVA) for comparison between groups. Differences were considered statistically significant when P < 0.05. Results AGEs induce apoptosis and inhibits proliferation in KGN cells. KGN cells cultured in vitro were treated with different concentrations of AGEs (0, 25, 50, 100, 200µg/ml) for 24 hours, or with 100µg/ml AGEs for different times (0, 24, 48, 72, 96h). The results showed that compared with the control group, the viability of KGN cells was significantly reduced after AGEs treatment in a concentration-dependent and time-dependent manner (Fig. 1 A). Subsequently, we examined the effect of AGEs on the apoptosis and proliferation levels in KGN cells. As demonstrated, with the extension of time and concentration, the results revealed that AGEs significantly increased the ratio of Bax/Bcl2 and inhibited the expression of PCNA (Fig. 1 B and C). AGEs inhibit KGN cell proliferation and induces apoptosis by up-regulating the expression of AMH. Serum AMH has been suggested as a monitoring indicator of female ovarian reserve function and treatment in women with PCOS. Therefore, we examined the effect of AGEs on AMH expression in KGN cells. The results showed that the expression of AMH was up-regulate in a dose- and time-dependent manner of AGEs (Fig. 2 A and B). Consistent with our findings, the immunofluorescence and immunocytochemistry results showed that, AGEs could increase the expression of AMH in KGN cells compared with the control group (Fig. 2 C and D). We next assessed the effects whether AGEs affect the apoptosis and proliferation in KGN cells through AMH by interfering with the expression of AMH with siRNA. The results showed that AMH protein levels were lower in the siRNA group than in the control group (Fig. 2 E). Furthermore, the ratio of apoptosis-related protein Bax/Bcl2 was significantly decreased after inhibition of AMH expression, while the expression of proliferation-related protein PCNA was upregulated (Fig. 2 F and G), indicating that AGEs may promote the apoptosis and inhibit the proliferation of KGN cells by increasing the expression of AMH. AGEs facilitate the expression of AMH through PI3K/AKT/SF1 pathway in KGN cells. We investigated the potential signaling pathway of AGEs promoting AMH expression. We have examined the change of SF1, PI3K and Akt expression after AGE treatment. It was found that the mRNA and protein expression levels of SF1 were increased in a concentration- and time-dependent manner compared with the control in KGN cells (Fig. 3 A and B). Immunofluorescence results further confirmed that AGEs (100 µg/ml, 24 hours) up-regulated the expression of SF1 in KGN cells (Fig. 3 C). Simultaneously, the results showed that AGEs facilitated the phosphorylation of PI3K and Akt in KGN cells (Fig. 3 E and F). To further study whether AGEs promoted the expression of AMH by via PI3K/Ak/SF1 pathway, we used PI3K inhibitors (Wortmannin 100nM) and cells were transfected with a SF1 siRNA. The results showed that AMH expression was significantly down-regulated after knockdown the SF1 (Fig. 3 D). The results showed that the expression levels of AMH and SF1 were down-regulated in Wortmannin and AGEs co-treatment group comparison with the AGEs alone treatment group (Fig. 3 J). Concurrently, the Bax/Bcl2 ratio was decreased and PCNA levels were increased in the AGEs alone group (Fig. 3 G-I). Taken together, these findings provide evidence of the involvement of PI3K/Akt/SF1 pathway in AGE mediated stimulation of AMH expression in KGN cells. High AGEs diet s promote the development of PCOS in rats. Female rats had 4–5 days of estrous cycle, comprising preoestrus, estrum, metaoestrus and diestrus phases. Vaginal smears were monitored for 7 consecutive days and analyzed microscopically to assess the effects of AGEs treatment on the estrous cycle. The results showed that both AGEs group and letrozole-induced PCOS model group showed estrous cycle disorder. The PCOS model group stayed in the diestrus phases, and the AGEs group did not have a normal estrous cycle (Fig. 4 A). Weight gain was significantly increased in both the AGEs and PCOS groups as compared to the control group. However, the AGEs group had not change in ovary/body weight index and the ovaries size (Fig. 4 B and C). The histological features in the control ovaries were normal, whereas the ovaries were morphologically disorganized, with an increased number of follicular cysts, a thinner granulosa cell layer and a lack of corpus luteum in the PCOS and AGEs groups (Fig. 4 D). Masson staining showed a significantly higher degree of ovarian cortical fibrosis in the AGEs and PCOS groups (Fig. 4 E). These results indicate that phenotypes induced by AGEs were similar morphological changes to PCOS in ovarian tissue, such as polycystic changes in the ovaries, inhibited normal ovulation in rats, and increased ovarian cortical fibrosis. AGEs-induced elevated AMH promote the development of PCOS in rats. We further explore the effects of AGEs on expression of AMH and the progression of PCOS in ovarian granulosa cells of rats. The results showed that AMH expression was upregulated in the ovaries of both AGEs and PCOS groups compared with the control group ( P < 0.05). Meanwhile, the upregulation of AMH expression was significantly higher in the letrozole and AGEs co-treatment group ( P < 0.05) (Fig. 5 A and B). The results indicated that high AGEs up-regulated the expression of AMH in the ovary of PCOS and non-PCOS rats. TUNEL assay and Western blot assays revealed that the combined group of AGEs and PCOS had more increased apoptosis in ovarian granulosa cells, markedly increased Bax/Bcl2 ratio and decreased PCNA protein expression ( P < 0.05) (Fig. 5 C and D). These findings implicate that high AGEs diet further exacerbates the level of apoptosis and inhibits cell proliferation ( P < 0.05) (Fig. 5 E and F). Consistent with the cells, we observed a significant increase of SF1 protein levels and phosphorylation levels of PI3K and Akt proteins in the tissues of rats in the AGEs group ( P < 0.05). (Fig. 5 G and H). These results thus suggest that AGEs may be contribute to PCOS via activating SF1/PI3K/Akt signaling pathway. Discussion PCOS is the most common form of anovulatory infertility with high prevalence among women of reproductive age. Ovulation disorder is the most important factor leading to low pregnancy rate and high miscarriage rate in patients with PCOS [ 17 ]. AMH, an important regulator of follicular function, has been reported as a diagnostic indicator of ovulation disorders [ 18 ]. AMH inhibits early follicular recruitment and prevents the growth of primordial follicles/ovarian cells. It has been reported that the apoptosis of ovarian granulosa cells and atretic follicles increased in patients with PCOS, and AMH is the key factor in regulating follicular atresia [ 18 , 19 ]. AMH levels reflect the number of small follicles undetectable by ultrasound, and therefore AMH is considered to be a better indicator for the diagnosis of PCOS than an atrial follicle count (AFC) [ 20 – 22 ]. Ovarian granulosa cells play an important role in the development and maturation of oocytes. Granulosa cell apoptosis is considered to be closely related to follicular atresia and anovulation [ 2 ] It was found that the apoptosis rate of follicular granulosa cells was significantly higher in patients with PCOS than in normal women [ 19 ] and the high concentration of AMH in follicular fluid may be an important factor in promoting the development of PCOS. The role of AGEs in the pathophysiology of PCOS and its adverse effects on ovarian function are still unclear. High AGEs diet is thought to lead to reproductive and metabolic effects similar to PCOS [ 23 ]. In anovulatory and hyper-androgenemic PCOS patients, AGEs levels were significantly elevated and positively correlated with AMH expression [ 4 , 12 ], suggesting that AGEs may participate in the occurrence and development of PCOS by regulating the expression of AMH. Therefore, understanding the effects of AGEs on granulosa cells will help to further understand the pathological mechanism of PCOS and propose new clinical treatments. In our study, we found that AGEs can up-regulate the expression of AMH in KGN cells in a time-and concentration-dependent manner. AGEs promoted apoptosis and inhibited the proliferation in KGN cells, whereas AGEs-induced apoptosis was inhibited after silencing AMH, suggesting that AGEs may promote the apoptosis of KGN cells through AMH. In animal models, we also observed the same results, the expression of AMH in AGEs and letrozole groups was up-regulated, and the apoptosis of granulosa cells was increased. Furthermore, the AMH expression and apoptosis rate of granulosa cells in the combined AGEs and letrozole-treated group were considerably higher than that in the letrozole group. Notably, rats in the AGEs group had certain similar changes to the PCOS model group, such as disturbed estrous cycle, ovarian polycystic changes, and Masson staining showed a significant increase in ovarian cortical fibrosis, indicating that high AGEs diets could induce PCOS changes in rats. It was suggested that AGEs may promote the development of PCOS by promoting the expression of AMH in granulosa cells and promoting apoptosis of granulosa cells. SF1 is expressed in ovarian granulosa cells and closely regulates the expression of AMH during follicular development [ 24 ]. To further understand the mechanism of regulation of AMH by AGEs, we examined the expression level of SF1. The results showed that AGEs upregulated the expression of SF1 in a concentration-and time-dependent manner. It was consistent with the expression trend of AMH. After silencing the expression of SF1, the expression of AMH in KGN cells was down-regulated, suggesting that SF1 may be the key factor for AGEs to regulate the expression of AMH. PI3K/Akt signaling pathway plays an important role in controlling cell growth and metabolic regulation, it is hyperactivated in ovarian granulosa cells of patients with PCOS [ 25 ], which is involved in oocyte survival and the pathogenesis of PCOS [ 26 , 27 ]. In addition, PI3K/Akt signaling pathway is also thought to be involved in the pathogenesis of insulin resistance, hyperandrogenism and obesity in PCOS [ 27 ]. This study confirmed that AGEs can increase the phosphorylated protein level of PI3K/Akt signal molecules in KGN cells and rat ovaries. And the expression of SF1 and AMH was suppressed after inhibition of PI3K in KGN cells, indicating that PI3K/Akt signal pathway is involved in the regulation of SF1 and AMH by AGEs, along with the regulation of granulosa cell proliferation and apoptosis levels. In conclusion, our experiment confirmed that AGEs promoted the expression of AMH and the apoptosis of granulocytes through SF1, thereby promoting the occurrence of PCOS, which is related to the PI3K/Akt pathway. These findings contribute to our further understanding of the mechanisms underlying anovulation in PCOS. AGEs increase the apoptosis of follicular granulosa cells, hinder the process of ovulation, and lead to ovulation abnormalities. A diet high in AGEs resulted in PCOS-like changes in the ovaries of rats. Therefore, we should be aware that a long-term diet high in AGEs may increase the risk of PCOS and infertility. Furthermore, now that assisted reproductive techniques are the most common method to address infertility, should we also be aware of whether high levels of serum AGEs in patients with polycystic ovary syndrome may adversely affect the outcome of assisted reproduction? Abbreviations AGEs Advanced glycation end products PCOS Polycystic ovarian syndrome AMH Anti-müllerian hormone SF1 Steroidogenic factor-1 AFC Antral follicle count FBSF Fetal bovine serum HE Haematoxylin and eosin. Declarations Acknowledgements Not applicable. Authors' contributions JL Z, MQ L contributed to participated in the experimental work, data analysis, and drafting the article; DC, ZM Z, YJ X, LY C and YJ H collected data and participated in data analysis, reviewed the draft thoroughly; Y T and ZC M provided insightful comments, editing and approved the final version to be submitted. All authors read and approved the final manuscript. Funding This work was supported by the Horizontal Cooperation Project with Guidong People’s Hospital (2023GDHX03), the Guangxi Natural Science Foundation (No. 2020GXNSFAA238008) and the Horizontal Cooperation Project with Hunan Mingshun Pharmaceutical Co., LTD. (2021GLHX01). Availability of data and materials Data are available upon reasonable request from corresponding authors. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate The animal experiments of the project met the requirements of animal welfare ethics and passed the animal welfare ethics investigation by IACUC of Guilin Medical University (Investigation Number: GLMC-IACUC-20241015). References Bozdag G, Mumusoglu S, Zengin D, Karabulut E, Yildiz BO. The prevalence and phenotypic features of polycystic ovary syndrome: a systematic review and meta-analysis. Hum Reprod. 2016;31:2841–55. Yu YS, et al. 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Functional roles of the phosphatidylinositol 3-kinases (PI3Ks) signaling in the mammalian ovary. Mol Cell Endocrinol. 2012;356:24–30. Li T, et al. Role of the PI3K-Akt Signaling Pathway in the Pathogenesis of Polycystic Ovary Syndrome. Reprod Sci. 2017;24:646–55. Additional Declarations No competing interests reported. Supplementary Files S1TableQuantitativereal.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3916965","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":271423212,"identity":"5cbb9e31-00c4-42fa-8e64-044697b02888","order_by":0,"name":"Jingling Zhu","email":"","orcid":"","institution":"Guidong People’s Hospital of Guangxi Zhang Autonomous Region, Affiliated Guidong People’s Hospital of Guilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jingling","middleName":"","lastName":"Zhu","suffix":""},{"id":271423213,"identity":"c8ac2743-e982-46a3-a7a9-770dd5ddc53e","order_by":1,"name":"Minqi Liu","email":"","orcid":"","institution":"Guidong People’s Hospital of Guangxi Zhang Autonomous Region, Affiliated Guidong People’s Hospital of Guilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Minqi","middleName":"","lastName":"Liu","suffix":""},{"id":271423214,"identity":"1cf2e5cd-6053-4b13-af22-a7e6bc645e91","order_by":2,"name":"Di Cheng","email":"","orcid":"","institution":"Guidong People’s Hospital of Guangxi Zhang Autonomous Region, Affiliated Guidong People’s Hospital of Guilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Di","middleName":"","lastName":"Cheng","suffix":""},{"id":271423215,"identity":"a5555d94-1b2a-4a8f-8f0f-49f82ca8da5f","order_by":3,"name":"Zhaoming Zeng","email":"","orcid":"","institution":"Joint Laboratory of Chronic Disease Prevention and Research in Guilin Medical University \u0026 Hunan Mingshun Pharmaceutical Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Zhaoming","middleName":"","lastName":"Zeng","suffix":""},{"id":271423216,"identity":"0518911f-6326-4a99-ba9b-3da50e4cf4dd","order_by":4,"name":"Yuanjie Xie","email":"","orcid":"","institution":"Guidong People’s Hospital of Guangxi Zhang Autonomous Region, Affiliated Guidong People’s Hospital of Guilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuanjie","middleName":"","lastName":"Xie","suffix":""},{"id":271423217,"identity":"92bf14fc-3b13-4e38-b876-5fb22b7d82d2","order_by":5,"name":"Liying Cen","email":"","orcid":"","institution":"Guidong People’s Hospital of Guangxi Zhang Autonomous Region, Affiliated Guidong People’s Hospital of Guilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Liying","middleName":"","lastName":"Cen","suffix":""},{"id":271423218,"identity":"6760387c-9616-4c48-b08f-f6c6e93b8a39","order_by":6,"name":"Yuanjiao Huang","email":"","orcid":"","institution":"Guidong People’s Hospital of Guangxi Zhang Autonomous Region, Affiliated Guidong People’s Hospital of Guilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuanjiao","middleName":"","lastName":"Huang","suffix":""},{"id":271423219,"identity":"25de91e7-7452-4df2-af88-379dae49c922","order_by":7,"name":"Yi Tan","email":"","orcid":"","institution":"Guidong People’s Hospital of Guangxi Zhang Autonomous Region, Affiliated Guidong People’s Hospital of Guilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Tan","suffix":""},{"id":271423220,"identity":"e6ba27f9-876e-42ec-946b-f8f17373ecd1","order_by":8,"name":"Zhongcheng Mo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvUlEQVRIiWNgGAWjYDACCQY2IGmTAOYkFBCvJS0BTCUYEK/lMEQLAzFa5Gc3P3t0o+J8Hr98d+KHBwYM8vxiB/BrYZxzzNw458ztYsk23s0SQIcZzpydgF8Ls0QOm3Ru2+3EDcd4N4C0JBjcJqCFDaLlHEjL5h9EaeGBaDkA0rKNOFskJNLMpHPOJCfObMvdZpFgIEHYL/Izkp9J51TYJfYzn91880eFjTy/NAEtGLaSpnwUjIJRMApGAXYAAAFrPk+u9oo/AAAAAElFTkSuQmCC","orcid":"","institution":"Guidong People’s Hospital of Guangxi Zhang Autonomous Region, Affiliated Guidong People’s Hospital of Guilin Medical University","correspondingAuthor":true,"prefix":"","firstName":"Zhongcheng","middleName":"","lastName":"Mo","suffix":""}],"badges":[],"createdAt":"2024-02-01 10:14:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3916965/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3916965/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50813331,"identity":"353f5727-6d6f-460c-8add-1419917811bb","added_by":"auto","created_at":"2024-02-07 19:21:05","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":182386,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAGEs promote apoptosis and inhibit proliferation of KGN cells. \u003c/strong\u003e(A）MTT detection of the cell viability in KGN cells. (B-C) Western blot analysis and relative quantitative analysis of PCNA, Bax and Bcl-2 in KGN cells. (In all experiments, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3916965/v1/c3f679fcac51e51ee7715010.jpeg"},{"id":50813336,"identity":"6e4a637a-8e09-44b1-971a-915d2134365f","added_by":"auto","created_at":"2024-02-07 19:21:06","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":179373,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAGEs inhibit cell proliferation and induce apoptosis by up-regulating the expression of AMH in KGN cells.\u003c/strong\u003e (A) Western blot analysis and relative quantitative analysis of AMH in KGN cells;(B) Quantitative real-time PCR analysis of the mRNA levels of AMH in KGN cells;(C-D) Immunofluorescence analysis and Immunocytochemical analysis to detect AMH in KGN cells; (E-G) Western blot analysis and relative quantitative analysis of AMH, Bax, Bcl-2 and PCNA in KGN cells. (In all experiments, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3916965/v1/ef77c09f3a5deba150fc1601.jpeg"},{"id":50813332,"identity":"a6418016-d681-4871-87e0-6f23560aa161","added_by":"auto","created_at":"2024-02-07 19:21:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":111104,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAGEs promote the expression of AMH through PI3K/AKT/SF1 pathway.\u003c/strong\u003e (A) Western blot analysis and relative quantitative analysis of SF1 in KGN cells;(B) Quantitative real-time PCR analysis of the mRNA levels of SF1 in KGN cells; (C) Immunocytochemical analysis to detect SF1 in KGN cells. The KGN cells were transfected with SF1 siRNA. (D) Western blot analysis and relative quantitative analysis of AMH and SF1 in KGN cells; (E-F) Western blot analysis and relative quantitative analysis of p-PI3K, PI3K, p-Akt and Akt in KGN cells. Then, the KGN cells pretreated with Wortmannin (100nM). (G-J) Western blot analysis and relative quantitative analysis of p-Akt, Akt, PCNA, Bax, Bcl-2, SF1 and AMH in KGN cells. (In all experiments, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3916965/v1/c7c68d0015aea915b9c37ead.png"},{"id":50813335,"identity":"026955ae-f276-4aa6-bac1-05f08ea5bea6","added_by":"auto","created_at":"2024-02-07 19:21:06","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":246479,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eH igh AGEs diet induces PCOS in rats. \u003c/strong\u003e(A)The changes of estrous cycle of rats in each group were monitored by vaginal smear for 7 days. P: proestrus, E: estrus, M: diestrus, D: diestrus; (B-C) Effects of AGEs on body weight and ovarian body mass index in rats; (D) Morphological changes of ovarian volume in rats; (E) HE staining shows morphological changes of rat ovary;(F) Masson staining shows morphological changes of rat ovary. (In all experiments, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3916965/v1/03052c48a82c405f06a3ca7f.jpeg"},{"id":50813334,"identity":"8dcf8be9-9571-4733-90fb-15215b10df99","added_by":"auto","created_at":"2024-02-07 19:21:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":221656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUpregulation of AMH expression promote the development of PCOS in rats. \u003c/strong\u003e(A-B) Western blot analysis and relative quantitative analysis of AMH and SF1 in rat ovary; (C) Immunohistochemical detection of the effect of AGEs on the expression of AMH protein in rat ovary;(D) Immunofluorescence detection of the effect of AGEs on ovarian apoptosis in rat ovary; (E-H) Western blot analysis and relative quantitative analysis of Bax, Bcl-2, PCNA, p-PI3K, PI3K, p-Akt and Akt in rat ovary. (In all experiments, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3916965/v1/90198fbdf0d9596917740568.png"},{"id":50957794,"identity":"aba38375-bf52-47d4-8b74-da1eb2e28e45","added_by":"auto","created_at":"2024-02-10 20:54:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1655259,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3916965/v1/e0f26df4-2015-410e-aed1-b770fe99f7fa.pdf"},{"id":50813333,"identity":"3d8aada4-e201-4aa4-b366-69c1fda61903","added_by":"auto","created_at":"2024-02-07 19:21:06","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13500,"visible":true,"origin":"","legend":"","description":"","filename":"S1TableQuantitativereal.docx","url":"https://assets-eu.researchsquare.com/files/rs-3916965/v1/de5b4344ff94d4ac18047b7f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Advanced glycation end products promote polycystic ovary syndrome by regulating AMH via PI3K/Akt/SF1 signaling pathway","fulltext":[{"header":"Backgroung","content":"\u003cp\u003ePolycystic ovary syndrome (PCOS) is one of the most common endocrine diseases leading to infertility in women of reproductive age, which affects 6\u0026ndash;10% of women globally [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The clinical signs and symptoms of PCOS vary greatly. The key factor behind PCOS-afflicted women's high abortion rate and poor conception rate is ovulation disruption. Ovarian granulosa cell plays an important role in the development and maturation of oocytes. Granulosa cell apoptosis is thought to have close relevance to follicular atresia and anovulation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnti-Mullerian hormone (AMH), a member of the transforming growth factor superfamily, is secreted only by ovarian granulosa cells [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The level of circulating AMH reflects the reserve function of the ovary, and low levels of AMH indicate a decrease in ovarian reserve function, thus affecting the conception rate [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Clinically, AMH is often used as one of the important indicators to evaluate ovarian function. Compared to fertile women with ovulatory cycles, women with follicular arrest have higher concentrations of AMH in follicular fluid with luminal follicles [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Abnormal follicular growth is one of the main features of PCOS. Serum AMH levels are 2\u0026ndash;3 times higher in patients with polycystic ovary syndrome compared to normal women [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Recent studies have demonstrated that high AMH concentrations in PCOS may lead to apoptosis of ovarian granulosa cells, resulting in loss of follicular processes, which may be one of the main causes of anovulation in women with PCOS [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccording to recent studies, elevated levels of serum and follicular fluid advanced glycosylation end products (AGEs) have been found in women with polycystic ovary syndrome [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. AGEs are a complex group of compounds that are mainly produced by non-enzymatic glycosylation of reducing sugars on proteins, lipids and nucleic acids and are associated with various diseases such as diabetes, Alzheimer's disease and atherosclerosis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. A clinical study showed that the expression of AGEs and AMH was increased in both ovulatory and anovulatory PCOS women, and the more prominent elevated was found in non-ovulating PCOS [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In addition, there is a significant positive correlation between age and AMH. However, the mechanism of the interaction remains unclear. Excessive ingestion of AGEs has been shown to affect the metabolic, hormonal and oxidative stress status of women with polycystic ovary syndrome [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In consideration of the above data, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ethe interaction\u003c/span\u003e between AGEs and AMH may be related to ovulatory dysfunction in PCOS.\u003c/p\u003e \u003cp\u003eSteroidogenic factor-1 (SF1), also known as NR5A1, is a member of the nuclear receptor superfamily, which is an orphan nuclear receptor encoded by FTZ-F1 gene [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. SF1 is highly expressed in the adrenal cortex and gonads and is essential for the maintenance of ovarian function [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. SF1 binds to the AMH promoter at two sites and influences follicular development by promoting AMH expression in ovarian granulosa cells [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Specific knockdown of the SF1 gene revealed reduced expression of genes related to ovarian granulosa cell proliferation, as well as infertility, incomplete ovarian development, reduced oocyte numbers, and complete absence of the corpus luteum in female mice [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], indicating a role for SF1 in ovarian follicle development. However, to date, few studies have been reported on the regulatory effects of SF1 on PCOS.\u003c/p\u003e \u003cp\u003eBased on these data, we hypothesized that AGEs may be involved in the development of PCOS by regulating the expression of AMH in granulosa cells. Consequently, the present study aimed to investigate whether AGEs affect apoptosis and proliferation of granulosa cells by regulating the expression of AMH, and further understand the possible mechanisms of effects.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and treatment\u003c/h2\u003e \u003cp\u003e Female Sprague-Dawley rats (5 weeks old) were purchased from Changsha Tianqin Biotechnology Co. Ltd. All of the animals were maintained under standard laboratory conditions, and the experiments were conducted in accordance with the National Institutes of Health guidelines. After 1 week of adjustment, rats were randomly divided into control, AGEs, PCOS, and PCOS\u0026thinsp;+\u0026thinsp;AGEs groups. The PCOS model was induced by 3 weeks of letrozole administration (1 mg/kg dissolved in 1% CMC solutions once a day). AGEs and PCOS\u0026thinsp;+\u0026thinsp;AGEs groups were fed with high AGEs diets (AIN-93G baked for 3h at 125\u0026deg;C) and other two groups were fed with normal diets. Estrous cycle was determined by daily vaginal smears.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eHuman granulosa tumor cell line KGN were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin at 37\u0026deg;C in a humidified environment with 5% CO2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell viability assay\u003c/h2\u003e \u003cp\u003eKGN cell viability was assessed using MTT (Solarbio Life Sciences, Beijing). Cells were seeded in a 96-well plate with 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/wells and incubated in a 37\u0026deg;C incubator for 6h. Cells were incubated in AGEs (ABCAM, USA) at different concentrations (0-200 mg/ml) for 48 h. Each well was incubated for 4 h at 37\u0026deg;C after the addition of 10 \u0026micro;l of tetramethylazole salt reagent, and measured at 490 nm with gentle shaking.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eTUNEL staining\u003c/h2\u003e \u003cp\u003eTUNEL assays kit (Beijing 4A Biotech Co, China) was used to treat the Paraffin sections ovrian tissue according to manufacturer's instructions. Images were captured at 200\u0026times; by Carl Zeiss fluorescence microscope. Apoptotic cells are labeled with bright red fluorescence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eHistology staining\u003c/h2\u003e \u003cp\u003eOvaries were fixed with the 4% paraformaldehyde and embedded with paraffin, which were sliced to 4 \u0026micro;m for histology staining. We performed haematoxylin and eosin (HE) to observe the organization morphology and masson's trichrome to measure the level of fibrosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eKGN Cells were seeded into in 6-well plates and incubated with 100 \u0026micro;g/ml BSA or AGE separately for 24h.Fixed with and 0.25% Triton X-100 transparency. 5% goat serum was used for nonspecific blocking.Incubated overnight at 4\u0026deg;C with primary anti-AMH antibody (1:250, Abcam) and anti-SF1 antibody (1:100, Abcam).C ells were incubated with fluorescent secondary antibody at 37\u0026deg;C for 1 h in the dark, and the nucleus was stained with DAPI (Solarbio, Beijing). Images were captured by a Carl Zeiss fluorescence microscope (ZEISS, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eTotal protein was extracted from cultured KGN cells and rat ovarian tissue using RIPA buffer (RIPA: PMSF\u0026thinsp;=\u0026thinsp;100:1) containing phosphatase and protease inhibitor 4\u0026deg;C for 30 min, centrifuging at 12,000 rmp for 20 min 4\u0026deg;C, and collecting supernatant. the protein concen-tration was measured using a BCA kit (Pierce, Rockford, USA). Proteins were separated by 10% or 12% SDS-PAGE and transferred to PVDF membrane. After blocking in 5% skim milk powder for 2 h at room temperature, and the membranes were washed 3 times with TBST and incubated overnight at 4\u0026deg;C with one of the following primary antibodies: AMH ( 1:1000, Abcam, USA), SF1 (1:1000, Affinity, China), PI3K (1:1000, Fine Biotech Co., China), p-PI3K (1:1000, Abcam, USA), Akt (1:1000, Fine Biotech Co., China), p-Akt (1:1000, Abcam, USA), PCNA (1:1000, Fine Biotech Co., China), Bax (1:1000, Boster Biological Technology, China), Bcl-2 (1:1000, Boster Biological Technology, China), \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\beta }\\)\u003c/span\u003e\u003c/span\u003e-actin (1:1000, Proteintech, China). After washing 3 times, the membranes were incubated 2 h at room temperature with a secondary antibody (either HRP-conjugated affinipure goat anti-rabbit IgG or anti-mouse secondary antibodies, 1:5000, Proteintech, China). The membranes were treated with ECL luminescent working solution and the relative expression levels of the target proteins were measured by Image Lab software.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eQuantitative real-time RT-PCR (qPCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from KGN cells using Trizol reagent (Solarbio Life Sciences, Beijing, China) and cDNA was synthesized using the reverse transcription kit (ABM, Canada) according to the manufacturer\u0026rsquo;s instructions. Quantitative real-time PCR was performed with Eva Green 2\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\times\\)\u003c/span\u003e\u003c/span\u003e qPCR MasterMix (ABM, Canada). The target genes were calculated with the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method. The primer sequences were purchased from Sangon Biotech (Shanghai, China) and listed in Supplementary Table\u0026nbsp;1. 18S was used as an internal control.\u003c/p\u003e \u003c/div\u003e\u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe measurement data are expressed as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Prism 8 software was used to analyze the data. Statistical significance was evaluated using Student\u0026rsquo;s t-test or one-way analysis of variance (ANOVA) for comparison between groups. Differences were considered statistically significant when \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eAGEs induce apoptosis and inhibits proliferation in KGN cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eKGN cells cultured in vitro were treated with different concentrations of AGEs (0, 25, 50, 100, 200\u0026micro;g/ml) for 24 hours, or with 100\u0026micro;g/ml AGEs for different times (0, 24, 48, 72, 96h). The results showed that compared with the control group, the viability of KGN cells was significantly reduced after AGEs treatment in a concentration-dependent and time-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Subsequently, we examined the effect of AGEs on the apoptosis and proliferation levels in KGN cells. As demonstrated, with the extension of time and concentration, the results revealed that AGEs significantly increased the ratio of Bax/Bcl2 and inhibited the expression of PCNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAGEs inhibit KGN cell proliferation and induces apoptosis by up-regulating the expression of AMH.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSerum AMH has been suggested as a monitoring indicator of female ovarian reserve function and treatment in women with PCOS. Therefore, we examined the effect of AGEs on AMH expression in KGN cells. The results showed that the expression of AMH was up-regulate in a dose- and time-dependent manner of AGEs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B). Consistent with our findings, the immunofluorescence and immunocytochemistry results showed that, AGEs could increase the expression of AMH in KGN cells compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next assessed the effects whether AGEs affect the apoptosis and proliferation in KGN cells through AMH by interfering with the expression of AMH with siRNA. The results showed that AMH protein levels were lower in the siRNA group than in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Furthermore, the ratio of apoptosis-related protein Bax/Bcl2 was significantly decreased after inhibition of AMH expression, while the expression of proliferation-related protein PCNA was upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF and G), indicating that AGEs may promote the apoptosis and inhibit the proliferation of KGN cells by increasing the expression of AMH.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAGEs facilitate the expression of AMH through PI3K/AKT/SF1 pathway in KGN cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe investigated the potential signaling pathway of AGEs promoting AMH expression. We have examined the change of SF1, PI3K and Akt expression after AGE treatment. It was found that the mRNA and protein expression levels of SF1 were increased in a concentration- and time-dependent manner compared with the control in KGN cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). Immunofluorescence results further confirmed that AGEs (100 \u0026micro;g/ml, 24 hours) up-regulated the expression of SF1 in KGN cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Simultaneously, the results showed that AGEs facilitated the phosphorylation of PI3K and Akt in KGN cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further study whether AGEs promoted the expression of AMH by via PI3K/Ak/SF1 pathway, we used PI3K inhibitors (Wortmannin 100nM) and cells were transfected with a SF1 siRNA. The results showed that AMH expression was significantly down-regulated after knockdown the SF1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). The results showed that the expression levels of AMH and SF1 were down-regulated in Wortmannin and AGEs co-treatment group comparison with the AGEs alone treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). Concurrently, the Bax/Bcl2 ratio was decreased and PCNA levels were increased in the AGEs alone group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-I). Taken together, these findings provide evidence of the involvement of PI3K/Akt/SF1 pathway in AGE mediated stimulation of AMH expression in KGN cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHigh AGEs diet\u003c/b\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003es\u003c/span\u003e \u003cb\u003epromote the development of PCOS in rats.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFemale rats had 4\u0026ndash;5 days of estrous cycle, comprising preoestrus, estrum, metaoestrus and diestrus phases. Vaginal smears were monitored for 7 consecutive days and analyzed microscopically to assess the effects of AGEs treatment on the estrous cycle. The results showed that both AGEs group and letrozole-induced PCOS model group showed estrous cycle disorder. The PCOS model group stayed in the diestrus phases, and the AGEs group did not have a normal estrous cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWeight gain was significantly increased in both the AGEs and PCOS groups as compared to the control group. However, the AGEs group had not change in ovary/body weight index and the ovaries size (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and C). The histological features in the control ovaries were normal, whereas the ovaries were morphologically disorganized, with an increased number of follicular cysts, a thinner granulosa cell layer and a lack of corpus luteum in the PCOS and AGEs groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Masson staining showed a significantly higher degree of ovarian cortical fibrosis in the AGEs and PCOS groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). These results indicate that phenotypes induced by AGEs were similar morphological changes to PCOS in ovarian tissue, such as polycystic changes in the ovaries, inhibited normal ovulation in rats, and increased ovarian cortical fibrosis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAGEs-induced elevated AMH promote the development of PCOS in rats.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe further explore the effects of AGEs on expression of AMH and the progression of PCOS in ovarian granulosa cells of rats. The results showed that AMH expression was upregulated in the ovaries of both AGEs and PCOS groups compared with the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Meanwhile, the upregulation of AMH expression was significantly higher in the letrozole and AGEs co-treatment group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B). The results indicated that high AGEs up-regulated the expression of AMH in the ovary of PCOS and non-PCOS rats. TUNEL assay and Western blot assays revealed that the combined group of AGEs and PCOS had more increased apoptosis in ovarian granulosa cells, markedly increased Bax/Bcl2 ratio and decreased PCNA protein expression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and D). These findings implicate that high AGEs diet further exacerbates the level of apoptosis and inhibits cell proliferation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and F). Consistent with the cells, we observed a significant increase of SF1 protein levels and phosphorylation levels of PI3K and Akt proteins in the tissues of rats in the AGEs group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and H). These results thus suggest that AGEs may be contribute to PCOS via activating SF1/PI3K/Akt signaling pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePCOS is the most common form of anovulatory infertility with high prevalence among women of reproductive age. Ovulation disorder is the most important factor leading to low pregnancy rate and high miscarriage rate in patients with PCOS [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. AMH, an important regulator of follicular function, has been reported as a diagnostic indicator of ovulation disorders [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. AMH inhibits early follicular recruitment and prevents the growth of primordial follicles/ovarian cells. It has been reported that the apoptosis of ovarian granulosa cells and atretic follicles increased in patients with PCOS, and AMH is the key factor in regulating follicular atresia [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. AMH levels reflect the number of small follicles undetectable by ultrasound, and therefore AMH is considered to be a better indicator for the diagnosis of PCOS than an atrial follicle count (AFC) [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Ovarian granulosa cells play an important role in the development and maturation of oocytes. Granulosa cell apoptosis is considered to be closely related to follicular atresia and anovulation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] It was found that the apoptosis rate of follicular granulosa cells was significantly higher in patients with PCOS than in normal women [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and the high concentration of AMH in follicular fluid may be an important factor in promoting the development of PCOS.\u003c/p\u003e \u003cp\u003eThe role of AGEs in the pathophysiology of PCOS and its adverse effects on ovarian function are still unclear. High AGEs diet is thought to lead to reproductive and metabolic effects similar to PCOS [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In anovulatory and hyper-androgenemic PCOS patients, AGEs levels were significantly elevated and positively correlated with AMH expression [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], suggesting that AGEs may participate in the occurrence and development of PCOS by regulating the expression of AMH. Therefore, understanding the effects of AGEs on granulosa cells will help to further understand the pathological mechanism of PCOS and propose new clinical treatments.\u003c/p\u003e \u003cp\u003eIn our study, we found that AGEs can up-regulate the expression of AMH in KGN cells in a time-and concentration-dependent manner. AGEs promoted apoptosis and inhibited the proliferation in KGN cells, whereas AGEs-induced apoptosis was inhibited after silencing AMH, suggesting that AGEs may promote the apoptosis of KGN cells through AMH. In animal models, we also observed the same results, the expression of AMH in AGEs and letrozole groups was up-regulated, and the apoptosis of granulosa cells was increased. Furthermore, the AMH expression and apoptosis rate of granulosa cells in the combined AGEs and letrozole-treated group were considerably higher than that in the letrozole group. Notably, rats in the AGEs group had certain similar changes to the PCOS model group, such as disturbed estrous cycle, ovarian polycystic changes, and Masson staining showed a significant increase in ovarian cortical fibrosis, indicating that high AGEs diets could induce PCOS changes in rats. It was suggested that AGEs may promote the development of PCOS by promoting the expression of AMH in granulosa cells and promoting apoptosis of granulosa cells.\u003c/p\u003e \u003cp\u003eSF1 is expressed in ovarian granulosa cells and closely regulates the expression of AMH during follicular development [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. To further understand the mechanism of regulation of AMH by AGEs, we examined the expression level of SF1. The results showed that AGEs upregulated the expression of SF1 in a concentration-and time-dependent manner. It was consistent with the expression trend of AMH. After silencing the expression of SF1, the expression of AMH in KGN cells was down-regulated, suggesting that SF1 may be the key factor for AGEs to regulate the expression of AMH.\u003c/p\u003e \u003cp\u003ePI3K/Akt signaling pathway plays an important role in controlling cell growth and metabolic regulation, it is hyperactivated in ovarian granulosa cells of patients with PCOS [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], which is involved in oocyte survival and the pathogenesis of PCOS [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In addition, PI3K/Akt signaling pathway is also thought to be involved in the pathogenesis of insulin resistance, hyperandrogenism and obesity in PCOS [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This study confirmed that AGEs can increase the phosphorylated protein level of PI3K/Akt signal molecules in KGN cells and rat ovaries. And the expression of SF1 and AMH was suppressed after inhibition of PI3K in KGN cells, indicating that PI3K/Akt signal pathway is involved in the regulation of SF1 and AMH by AGEs, along with the regulation of granulosa cell proliferation and apoptosis levels.\u003c/p\u003e \u003cp\u003eIn conclusion, our experiment confirmed that AGEs promoted the expression of AMH and the apoptosis of granulocytes through SF1, thereby promoting the occurrence of PCOS, which is related to the PI3K/Akt pathway. These findings contribute to our further understanding of the mechanisms underlying anovulation in PCOS. AGEs increase the apoptosis of follicular granulosa cells, hinder the process of ovulation, and lead to ovulation abnormalities. A diet high in AGEs resulted in PCOS-like changes in the ovaries of rats. Therefore, we should be aware that a long-term diet high in AGEs may increase the risk of PCOS and infertility. Furthermore, now that assisted reproductive techniques are the most common method to address infertility, should we also be aware of whether high levels of serum AGEs in patients with polycystic ovary syndrome may adversely affect the outcome of assisted reproduction?\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAGEs \u0026nbsp; \u0026nbsp;Advanced glycation end products\u003c/p\u003e\n\u003cp\u003ePCOS \u0026nbsp; Polycystic ovarian syndrome\u003c/p\u003e\n\u003cp\u003eAMH \u0026nbsp; Anti-m\u0026uuml;llerian hormone\u003c/p\u003e\n\u003cp\u003eSF1 \u0026nbsp; \u0026nbsp; Steroidogenic factor-1\u003c/p\u003e\n\u003cp\u003eAFC \u0026nbsp; \u0026nbsp;Antral follicle count\u003c/p\u003e\n\u003cp\u003eFBSF \u0026nbsp; Fetal bovine serum\u003c/p\u003e\n\u003cp\u003eHE \u0026nbsp; \u0026nbsp; Haematoxylin and eosin.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJL Z, MQ L contributed to participated in the experimental work, data analysis, and drafting the article; DC, ZM Z, YJ X, LY C and YJ H collected data and participated in data analysis, reviewed the draft thoroughly; Y T and ZC M provided insightful comments, editing and approved the final version to be submitted. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Horizontal Cooperation Project with Guidong People\u0026rsquo;s Hospital (2023GDHX03), the Guangxi Natural Science Foundation (No. 2020GXNSFAA238008) and the Horizontal Cooperation Project with Hunan Mingshun Pharmaceutical Co., LTD. (2021GLHX01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are available upon reasonable request from corresponding authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal experiments of the project met the requirements of animal welfare ethics and passed the animal welfare ethics investigation by IACUC of Guilin Medical University (Investigation Number: GLMC-IACUC-20241015).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBozdag G, Mumusoglu S, Zengin D, Karabulut E, Yildiz BO. 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Efficacy of three COS protocols and predictability of AMH and AFC in women with discordant ovarian reserve markers: a retrospective study on 19,239 patients. J Ovarian Res. 2021;14:111.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeede H, et al. Anti-M\u0026uuml;llerian Hormone in PCOS: A Review Informing International Guidelines. Trends Endocrinol Metab. 2019;30:467\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDiamanti-Kandarakis E, Christakou C, Marinakis E. Phenotypes and enviromental factors: their influence in PCOS. Curr Pharm Des. 2012;18:270\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePuttabyatappa M, et al. Developmental Programming: Prenatal Testosterone Excess on Ovarian SF1/DAX1/FOXO3. Reprod Sci. 2020;27:342\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNekoonam S, et al. Expression of AKT1 along with AKT2 in granulosa-lutein cells of hyperandrogenic PCOS patients. Arch Gynecol Obstet. 2017;295:1041\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng W, Nagaraju G, Liu Z, Liu K. Functional roles of the phosphatidylinositol 3-kinases (PI3Ks) signaling in the mammalian ovary. Mol Cell Endocrinol. 2012;356:24\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi T, et al. Role of the PI3K-Akt Signaling Pathway in the Pathogenesis of Polycystic Ovary Syndrome. Reprod Sci. 2017;24:646\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"polycystic ovarian syndrome (PCOS), advanced glycation end products (AGEs), anti-müllerian hormone (AMH), apoptosis, proliferation","lastPublishedDoi":"10.21203/rs.3.rs-3916965/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3916965/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground：\u003c/strong\u003eAdvanced glycation end products (AGEs) are involved in the pathogenesis of polycystic ovary syndrome (PCOS)and high concentrations of anti-Müllerian hormone (AMH) are considered one of the primary causes of anovulation in women with PCOS. However, the specific mechanism of action remains unclear.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod and Results: \u003c/strong\u003eOvarian granulosa cells (KGN cells) were treated with AGEs at different concentrations and times. The results showed that pretreatment with AGEs concentration-dependent and time-dependent affected the expression of AMH and SF1. PCNA expression was suppressed by AGEs treatment, and the ratio of the apoptosis-related protein Bax/Bcl2 was elevated. Tetrazolium colorimetric assay shows the same results. Granulosa cells caused by AGE to induce apoptosis could be significantly reversed in KGN cells transfected with AMH siRNA. AMH and the Bax/Bcl2 ratio expression were reduced after SF1 expression was inhibited. Inhibition of the PI3K upregulated PCNA, SF1 and AMH expression. Additionally, in ovarian tissues of AGEs group and PCOS group, the expression of AMH and SF1 increased, accompanied with marked up-regulation of the phosphorylation of PI3K and Akt expression. And the ratio of Bax/Bcl2 increased, while the expression of PCNA was opposite.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eAGEs increase the level of AMH (Anti-Mullerian Hormone) in ovarian granulosa cells, promoting apoptosis and restricting cell proliferation, thus leading to PCOS in rats. Studies suggest that SF1 may be a crucial target for AGE-mediated AMH production, involving the phosphorylation of the PI3K/Akt signaling pathway.\u003c/p\u003e","manuscriptTitle":"Advanced glycation end products promote polycystic ovary syndrome by regulating AMH via PI3K/Akt/SF1 signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-07 19:20:59","doi":"10.21203/rs.3.rs-3916965/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ea62cf49-29ca-4358-803b-66afdc241eaa","owner":[],"postedDate":"February 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-02-10T20:46:00+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-07 19:20:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3916965","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3916965","identity":"rs-3916965","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}