Electroacupuncture Ameliorates Endometriosis-Associated Ovarian Dysfunction by Activating Nrf2 Pathway to Upregulate GPX4 Function and Inhibiting Ferroptosis

Electroacupuncture Ameliorates Endometriosis-Associated Ovarian Dysfunction by Activating Nrf2 Pathway to Upregulate GPX4 Function and Inhibiting Ferroptosis | 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 Electroacupuncture Ameliorates Endometriosis-Associated Ovarian Dysfunction by Activating Nrf2 Pathway to Upregulate GPX4 Function and Inhibiting Ferroptosis Yan Zan, Xiaoquan Huang, Yu Zhuang, Junwei Li, Qian Zhu, Tiantian Ma, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6928457/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 Endometriosis (EMs) often leads to ovarian dysfunction and infertility. Its mechanism is closely associated with oxidative stress and ferroptosis induced by pelvic iron overload. Electroacupuncture (EA) has potential in treating reproductive disorders, but its mechanism of action on ovarian ferroptosis in EMs remains unclear. This study aimed to investigate the protective effect of EA on ovarian function in an EMs mouse model and its underlying molecular mechanisms. Methods A mouse model simulating chronic hemorrhage in EMs was used. Mice were randomly divided into a Normal group, Sham group, EMs group, and EA group. Ovarian function (estrous cycle, ovarian weight/index, serum sex hormones, ovarian histopathology), iron metabolism levels (peritoneal fluid/ovarian Fe 2 ⁺, ferritin, Prussian blue staining), oxidative stress levels (ovarian ROS, GSH content, T-SOD activity, Nrf2 and its downstream HO-1/SOD2/NQO1 mRNA), and ferroptosis levels (ferroptosis markers GPX4, SLC7A11, FTH1, ACSL4, COX-2 protein and mRNA, and MDA levels) were assessed using qRT-PCR, Western blot, IHC, colorimetric methods, and histochemistry. Results EA intervention significantly improved ovarian function in EMs mice. This was reflected in the normalization of the estrous cycle, increased ovarian weight/index, restored serum sex hormone levels, increased number of primordial follicles, and reduced atretic follicles. EA effectively alleviated ovarian iron overload (reduced Fe 2 ⁺, ferritin, iron deposition) and oxidative stress (inhibited ROS, increased GSH, enhanced T-SOD activity). Mechanistically, EA activated the Nrf2 pathway (upregulated Nrf2, HO-1, SOD2, NQO1), upregulated key anti-ferroptosis molecules (GPX4, SLC7A11) and ferritin (FTH1). Furthermore, EA downregulated pro-ferroptosis factors (ACSL4, COX-2) and reduced lipid peroxidation (MDA). The results demonstrated that EA effectively blocked the ferroptosis process in ovarian granulosa cells by activating the Nrf2 pathway to upregulate GPX4 activity and suppressing ACSL4-mediated lipid peroxidation. Conclusions This study confirms that iron overload–oxidative stress–granulosa cell ferroptosis is a key pathological mechanism of EMs-induced ovarian damage. EA effectively inhibits ovarian granulosa cell ferroptosis by activating the Nrf2 pathway to upregulate GPX4 activity and suppress ACSL4-mediated lipid peroxidation, thereby ameliorating EMs-associated ovarian dysfunction. This provides important experimental evidence supporting EA as a complementary therapy for EMs-related infertility. Endometriosis Ferroptosis Oxidative stress Electroacupuncture Ovarian reserve Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Endometriosis (EMs) is an estrogen-dependent chronic inflammatory disease characterized by the implantation and invasion of ectopic endometrial tissue within the pelvic cavity. It affects approximately 10–15% of reproductive-aged women globally, and among them, 30–50% suffer from infertility, posing a serious threat to female reproductive health [ 1 – 3 ]. Common clinical manifestations include chronic pelvic pain and infertility [ 4 , 5 ]. Clinical management primarily involves pharmacological and surgical interventions, often combined with in vitro fertilization-embryo transfer (IVF-ET) for patients with infertility [ 6 ]. However, studies indicate that pharmacological interventions often fail to improve the decline in ovarian reserve function, and surgical intervention may even exacerbate ovarian damage [ 7 , 8 ]. In EMs patients, cyclic bleeding from ectopic lesions leads to the persistent accumulation of iron ions and disruption of iron metabolism homeostasis within the pelvic microenvironment [ 9 ]. Clinical examinations reveal significantly elevated iron levels in the peritoneal fluid, ectopic lesions, peritoneum, and macrophages of EMs patients. Excessive iron ions generate reactive oxygen species (ROS) via the Fenton reaction, triggering significant oxidative stress damage and lipid peroxidation, which is the core pathological mechanism of tissue injury induced by iron overload [ 10 – 12 ]. As a vital organ within the pelvic microenvironment, the ovary, particularly its granulosa cells and follicular microenvironment, are chronically exposed to this high-iron-induced oxidative stress environment. This results in impaired follicular development, reduced oocyte quality, and decreased ovarian reserve function [ 13 ]. Clinical studies observe reduced serum anti-Müllerian hormone (AMH) levels, elevated estradiol (E 2 ) and follicle-stimulating hormone (FSH) in EMs patients [ 14 , 15 ], with ovarian granulosa cells (GCs) exhibiting pathological alterations such as enhanced apoptosis, inflammation, and oxidative stress [ 16 – 18 ]. Although the classic “Retrograde Menstruation-Ectopic Implantation-Inflammatory Response” theory partially explains the pathological basis of EMs [ 19 ], the key molecular pathways underlying its induction of ovarian damage, particularly how iron metabolism dysregulation triggers cell death programs, require further exploration. In recent years, ferroptosis, an iron-dependent, lipid peroxidation-driven form of programmed cell death, has garnered significant attention in research on the pathological mechanisms of endometriosis (EMs) [ 20 ] [ 21 , 22 ]. Its key regulatory mechanisms involve the collapse of the antioxidant defense system and ferroptotic lipid peroxidation induced by molecules such as long-chain acyl-CoA synthetase 4 (ACSL4) under oxidative stress [ 20 ]. Notably, glutathione peroxidase 4 (GPX4), the core enzyme inhibiting lipid peroxidation, relies on sufficient reduced glutathione (GSH) supply for its function. The nuclear factor erythroid 2-related factor 2 (Nrf2) pathway maintains GSH biosynthesis by directly regulating the cystine transporter SLC7A11 (a component of System Xc⁻), thereby providing the essential substrate for GPX4 and indirectly sustaining its antioxidant function [ 23 ]. Studies indicate that follicular fluid and peritoneal fluid from EMs patients can induce ferroptosis in murine ovarian granulosa cells and follicles, manifested by significantly elevated levels of malondialdehyde (MDA), a terminal product of lipid peroxidation, and downregulated GPX4 protein expression [ 24 , 25 ]. However, the regulatory role of the Nrf2 pathway in this process and the precise role of ACSL4 require further validation. Based on this, we propose the hypothesis that iron ions abnormally accumulate locally in the ovary within the pelvic microenvironment of EMs, triggering intense oxidative stress. This inhibits the functional support of GPX4 by the Nrf2 pathway and activates ACSL4-mediated lipid peroxidation, inducing ferroptosis in granulosa cells, ultimately leading to ovarian dysfunction. Electroacupuncture (EA), as a non-pharmacological intervention, possesses unique advantages in improving reproductive endocrine function. Studies show that EA can ameliorate ovarian function impairment through multi-target regulation [ 26 ]. However, whether EA alleviates oxidative stress and inhibits ferroptosis by activating the Nrf2 pathway, thereby improving EMs-related ovarian dysfunction, requires systematic investigation. Utilizing an established murine model that simulates the chronic bleeding characteristics of ectopic lesions in EMs patients, this study aims to validate the aforementioned key hypothesis regarding iron overload-oxidative stress-ovarian granulosa cell ferroptosis. Furthermore, it will delve into the specific mechanisms by which EA intervention improves ovarian function through regulating iron metabolism, ameliorating oxidative stress, and antagonizing granulosa cell ferroptosis. The findings are expected to provide new perspectives for elucidating the pathological mechanisms of EMs-related ovarian dysfunction and offer important experimental evidence for developing non-pharmacological intervention strategies. Methods Animals This study utilized 6-week-old specific pathogen-free (SPF) female C57BL/6 mice (initial body weight: 18 2 g) provided by Jiangsu Huachuang Sino Medical Technology Co., Ltd. (License No.: SCXK (Su) 2020-0009). All mice were housed in the barrier facility of the Experimental Animal Center at Nanjing University of Chinese Medicine under an individually ventilated cage (IVC) system (5 mice per cage), with controlled environmental conditions: temperature 22 ± 2℃, humidity 50 ± 5%, and a 12 h/12 h light-dark cycle. Food and water were provided ad libitum. Body weight was recorded weekly throughout the experiment. A total of 88 mice were randomly divided into five groups using a random number table: normal group ( n = 8), sham surgery group ( n = 8), donor group ( n = 8), recipient group ( n = 16), and blood supply group ( n = 48). At the end of the experimental period, mice were euthanized by cervical dislocation under anesthesia, and organ tissues, blood, and peritoneal fluid samples were collected. This study was approved by the Animal Ethics Committee of Nanjing University of Chinese Medicine (Approval No.: 202403A036). Modeling methods Prior to modeling, all mice except the normal and blood supply groups were subcutaneously injected with estradiol benzoate (150 µ g/kg, once every 4 days for two doses) to synchronize estrous cycles. For modeling, donor mice were euthanized under isoflurane anesthesia, and uterine horns were excised aseptically, rinsed with PBS, longitudinally incised, and minced into fragments smaller than 1 mm 3 . The fragments were suspended in 0.5 mL PBS for subsequent use. Recipient mice then received an intraperitoneal injection of the uterine fragment suspension (0.5 cm above the urethral orifice), with each donor uterus allocated to two recipient mice. After injection, recipient mice were randomly assigned to the EMs group ( n = 8) or electroacupuncture (EA) group ( n = 8), while the sham surgery group received an equivalent volume of PBS. All procedures were completed within 5 minutes under sterile conditions.Post-modeling, estradiol benzoate (150 µ g/kg, once every 4 days for two doses) was continuously administered, and allogeneic whole blood (0.2 mL/mouse, once every 3 days for 24 days) was intraperitoneally injected starting from day 1 after modeling; the sham group received equivalent saline. Laparotomy was performed to validate successful modeling, defined by the presence of cystic lesions containing yellowish/whitish/reddish fluid accompanied by vascularization and tissue adhesion [ 27 ]. Electroacupuncture intervention The EA group commenced treatment on day 14 post-modeling. Daily, mice were anesthetized with isoflurane and fixed in position. The following acupoints were punctured: CV4 (Guanyuan) with a perpendicular insertion of 3 mm, bilateral SP6 (Sanyinjiao)at 4 mm depth, and bilateral ST36 (Zusanli) at 5 mm depth. SP6 and ST36 on the same side were connected to an electroacupuncture apparatus (SDZ-II, Suzhou Medical Appliance Factory, China) using a dense-disperse wave, pulse width 0.2–0.6 ms, and current intensity of 1 mA, with stimulation intensity adjusted to induce slight toe twitching. Each session lasted 15 minutes and was administered once daily for 12 consecutive days. The blank control group received standard housing without intervention, while the EMs and sham surgery groups were subjected to identical anesthesia and fixation procedures (without electroacupuncture stimulation) for 12 days. Estrous cycle monitoring Vaginal smears were collected daily at 9:00 AM and examined under a light microscope (IX73; Olympus, Japan) to observe cytomorphological characteristics. Based on vaginal epithelial cell composition, the estrous cycle was classified into four stages [ 28 ]: proestrus (P, nucleated epithelial cells > 75%), estrus (E, anucleated keratinized cells > 80%), metestrus (M, keratinized cells and leukocytes at a ratio of approximately 1:1), and diestrus (D, leukocytes > 75%). Estrous cycle irregularities were defined as meeting any of the following criteria: Abnormal cycle duration ( 7 days; the normal cycle for C57BL/6 mice is 4–5 days); Cycle stagnation (persistence of the same stage for ≥ 3 days); Loss of sequential stage alternation. Serum hormone measurement Serum levels of estradiol (E 2 ), follicle-stimulating hormone (FSH), anti-Müllerian hormone (AMH), and luteinizing hormone (LH) were measured using commercial ELISA kits (AiFang Biology, AF02566-A, AF02555-A, AF09402-A, AF02582-A). All assays were performed according to the manufacturer’s instructions, with samples assayed in duplicate. The experimental procedure included the generation of standard curves and inter-assay variability controls (coefficient of variation < 10%). Ovarian histological analysis and follicle counting Ovarian tissues were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned into 5- µ m-thick slices. After hematoxylin and eosin (H&E) staining, ovarian morphological structures were observed under a light microscope at 200× magnification. Following established criteria [ 29 ], follicles at various developmental stages (primordial, primary, secondary, antral, and atretic follicles) were classified and counted in every fifth serial section. Follicles were quantified only if they contained a visible intact oocyte nucleus. Iron metabolism analysis Serum ferritin levels were measured using an ELISA kit (AiFang Biology, AF02312-A). Iron ion content in peritoneal fluid and ovarian tissues was determined via a ferrous ion colorimetric assay (Elabscience, E-BC-K773-M) by measuring absorbance at 593 nm. Ovarian iron deposition was histologically localized using a Prussian blue staining kit (Servicebio, G1029), in which Fe 3+ reacted with potassium ferrocyanide (K 4 [Fe(CN) 6 ]) to form characteristic blue precipitates. All procedures were strictly performed according to the manufacturer’s protocols, with standard curves and duplicate measurements included (intra-assay coefficient of variation < 10%). Detection of oxidative stress-related indicators Ovarian tissues were homogenized in pre-cooled PBS, and supernatants were collected after centrifugation for subsequent assays. Oxidative stress markers were measured using commercial kits: malondialdehyde (MDA) (Beyotime, S0131S), glutathione (GSH) (Nanjing Jiancheng, A006-2-1), and superoxide dismutase (SOD) (Nanjing Jiancheng, A001-3-2). All measurements were performed strictly following the manufacturer’s protocols using a microplate reader, and data were normalized to total protein concentration determined by a BCA assay (Beyotime, P0010). Each sample was assayed in duplicate with an intra-assay coefficient of variation < 10%. Gene expression analysis by qRT-PCR Total RNA was extracted from ovarian tissues using the TRIzol reagent, and reverse transcription was performed with the Hifair® II First Strand cDNA Synthesis Kit (Yeasen, 11141ES60). Quantitative real-time PCR was conducted using the Hieff® qPCR SYBR Green Master Mix (Low Rox Plus) (Yeasen, 11202ES08) on a real-time PCR system. GAPDH was used as the reference gene, and relative gene expression was calculated using the 2 −ΔΔCt method. Primer sequences are listed in Supplementary Table 1. Western blot analysis The ovarian tissues were lysed using RIPA lysis buffer containing protease inhibitors (Beyotime, P0013B). Protein concentration was determined by BCA assay. Equal amounts of protein (30 µ g per lane) were separated by SDS-PAGE electrophoresis and subsequently transferred onto PVDF membranes (Millipore, IPVH00010). The membranes were blocked with rapid blocking buffer (Yoche, YWB0501) at room temperature for 5 minutes, followed by overnight incubation with primary antibodies at 4℃. After TBST washing, the membranes were incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were visualized using ECL detection reagent (Yeasen, 36208ES60), and quantitative analysis was performed with ImageJ software using GAPDH as the internal control. The antibody sequences are provided in Supplementary Table 2. Immunohistochemical analysis of ovarian tissues Ovarian tissue sections (5 µ m) were dewaxed followed by antigen retrieval using citrate buffer (pH = 6.0, 95℃, 20 minutes). Endogenous peroxidase activity was blocked with 3% H 2 O 2 for 15 minutes, followed by blocking with 5% BSA at room temperature for 30 minutes. The sections were then incubated with primary antibodies overnight at 4℃. After PBS washing, the sections were incubated with HRP-conjugated secondary antibodies at 37℃ for 1 hour. Following DAB chromogenic reaction, the sections were counterstained with hematoxylin, dehydrated, cleared, and mounted. Five non-overlapping fields per section were randomly captured under an optical microscope (400 × magnification). Quantitative analysis of integrated optical density (IOD) was performed using ImageJ software, and the mean IOD value from five fields per section was used for intergroup comparisons. Detection of reactive oxygen Species (ROS) in ovarian tissues Ovarian tissues were embedded in OCT compound (Sakura, 4583) and snap-frozen in liquid nitrogen. Cryosections (8 µ m thickness) were prepared and stained using a reactive oxygen species (ROS) detection kit (Solarbio, G4817) according to the manufacturer’s instructions. The sections were mounted with antifade mounting medium containing DAPI. Imaging was performed under a confocal microscope with the following excitation/emission wavelength settings: DHE (535/610 nm) and DAPI (380/420 nm). Five non-overlapping fields per section were randomly selected at 400× magnification, and the mean fluorescence intensity of ROS-positive areas was quantified using ImageJ software. Statistical analysis All statistical analyses and graph generation were performed using GraphPad Prism software. Continuous data were expressed as mean ± standard error of the mean (SEM). For multiple group comparisons, one-way ANOVA was applied followed by Tukey’s multiple comparison test for normally distributed data, while Kruskal-Wallis test was used with Dunn’s post hoc analysis for non-normally distributed data. Correlation analyses were conducted using Pearson’s correlation coefficient (for normal distributions) or Spearman’s rank correlation coefficient (for non-normal distributions), based on data distribution characteristics. A two-tailed P -value < 0.05 was considered statistically significant. Results EA ameliorated ovarian functional impairment in EMs mice To simulate the estrogen-dependent nature and chronic hemorrhagic characteristics of EMs lesions, intraperitoneal injection of allogeneic uterine fragments combined with subcutaneous injection of estradiol benzoate in the neck region was employed (Fig. 1 A). Additionally, allogeneic whole blood was intraperitoneally injected every 3 days after modeling [ 30 ] (0.2 mL/injection, total of 8 injections) (Fig. 1 B). Twenty-four days post-modeling, mice in the EMs group developed typical multiple yellowish cystic lesions with hypervascularization and adhesions in the abdominal cavity (Fig. 1 C). Compared to the Normal group, the EMs group showed significantly decreased body weight, ovarian weight, and ovarian index (Fig. 1 D-F). The estrous cycle was disrupted, manifested as a disturbed rhythm and prolonged average cycle length and estrus phase duration (Fig. 1 G-I). EA intervention significantly reversed these pathological phenotypes ( P < 0.05), restoring body weight, ovarian weight, and ovarian index, and ameliorating estrous cycle disruption. Serum hormone assays detected significantly decreased AMH levels and significantly elevated E2 levels in the EMs group (Fig. 1 J&K), consistent with the “low AMH-high E 2 ” hormonal imbalance pattern observed in EMs patients [ 31 , 32 ]. Concurrently, FSH, LH levels, and the FSH/LH ratiowere all elevated, suggesting potential dysfunction in the hypothalamic-pituitary-ovarian (HPO) axis feedback regulation (Fig. 1 L&M). EA intervention effectively corrected these abnormalities in serum hormone levels ( P < 0.05). HE staining results revealed that the number of follicles at all stages was significantly reduced, while the number of atretic follicles was significantly increased in the ovaries of the EMs group, with granulosa cell structure destruction. In the EA group, the number of follicles at various stages was restored, the number of atretic follicles decreased, and the granulosa cell structure was repaired ( P < 0.05) (Fig. 1 N-P). EA reduced ovarian iron overload in EMs mice To investigate the association between ovarian damage and iron metabolism, we detected that Fe 2 ⁺ and Ferritin levels in the peritoneal fluid were significantly higher in the EMs group compared to the Normal group (Fig. 2 A&B). Ovarian Fe 2 ⁺ concentration was elevated and showed a positive correlation with peritoneal fluid Fe 2+ levels ( R 2 = 0.92, P < 0.001), indicating that ovarian iron overload was closely associated with iron metabolism dysregulation in the peritoneal fluid (Fig. 2 C&D). EA intervention significantly reduced these parameters ( P < 0.05). Further localization of iron deposition by Prussian blue staining revealed that the amount of iron deposition in the ovaries was significantly higher in the EMs group than in the Normal group. Iron deposition was primarily distributed around the theca folliculi, interstitial blood vessels, and within granulosa cells of atretic follicles, exhibiting a spatial distribution highly overlapping with atretic follicles. In the EA group, the amount of iron deposition was significantly reduced compared to the EMs group, and the deposition was confined mainly to the perivascular areas of interstitial blood vessels (Fig. 2 E&F). Correlation analysisconfirmed a positive correlation between ovarian Fe 2 ⁺ concentration and the number of atretic follicles ( R 2 = 0.94, P < 0.001) (Fig. 2 G). As free iron catalyzes ROS generation via the Fenton reaction, we hypothesized that iron overloadmight accelerate follicular atresia through oxidative stress pathways, leading to ovarian functional impairment, and that EA ameliorated ovarian damage by regulating iron metabolism. EA alleviated ovarian oxidative stress by activating the Nrf2 pathway To clarify the impact of EA-regulated iron metabolism on redox balance, we assessed ovarian oxidative stress levels and the antioxidant defense system. Compared with the Normal group, ovaries from the EMs group showed significant oxidative damage, manifested as a significant increase in ROS fluorescence intensity (Fig. 3 A&B), along with significant decreases in the levels of the key antioxidant molecule GSH and the activity of total superoxide dismutase (T-SOD). After EA intervention, ROS fluorescence intensity decreased, while GSH levels and T-SOD activity increased ( P < 0.05) (Fig. 3 C&D), indicating partial restoration of endogenous antioxidant reserves. The mRNA expression level of the core antioxidant transcription factor Nrf2 was significantly downregulated compared to the Normal group. The transcriptional levels of its classic downstream target genes-mitochondrial superoxide dismutase (SOD2), heme oxygenase-1 (HO-1), and NAD(P)H quinone oxidoreductase 1 (NQO1)-were synchronously downregulated (Fig. 3 E-H). EA intervention significantly reversed the inhibition of these gene expressions ( P < 0.05). These findings indicated that the Nrf2 pathway inactivation in EMs mouse ovariesled to the collapse of antioxidant defense. EA activated the Nrf2 pathway, promoted the expression of its downstream antioxidant enzymes, enhanced GSH biosynthesis reserves and SOD activity, effectively neutralized excess ROS, and re-established redox homeostasis. EA inhibited ferroptosis by upregulating GPX4 function via the Nrf2 pathway Given that GPX4 is a core inhibitor of ferroptosis whose downregulation drives the ferroptosis process, and that the Nrf2 pathway plays a central regulatory role in maintaining cellular redox homeostasis and inhibiting ferroptosis [ 23 ], we hypothesized that Nrf2 pathway inactivation might lead to ferroptosis by affecting GPX4 function. To investigate whether EA regulates ferroptosis through the Nrf2 pathway, we detected key markers of ovarian ferroptosis. Compared with the Normal group, GPX4 protein and mRNA levels were significantly downregulated in the EMs group (Fig. 4 A, 4 E&J). Immunohistochemistry showed weakened expression in granulosa cells (Fig. 4 O). The mRNA level of the Nrf2 target gene SLC7A11 was significantly downregulated (Fig. 4 K), resulting in the loss of the key transporter for GSH synthesis and impaired maintenance of GPX4 activity. Decreased expression of ferritin heavy chain (FTH1) protein and mRNA corresponded with increased iron deposition, indicating impaired iron storage capacity and an expanded free iron pool (Fig. 4 B, F&L). EA intervention significantly reversed these changes ( P < 0.05). Additionally, the concentration of the lipid peroxidation product MDA was significantly higher in the EMs group than in the Normal group (Fig. 4 I). The protein and mRNA expression levels of pro-ferroptotic factors ACSL4 and cyclooxygenase-2 (COX-2) were significantly upregulated (Fig. 4 C&D, 4G&H, 4M&N), and both were highly enriched in granulosa cells of atretic follicles (Fig. 4 O). Their expression decreased after EA intervention ( P < 0.05). These results confirmed that ferroptosis occurs in EMs ovaries. The mechanism involved Nrf2 pathway inactivation leading to SLC7A11 downregulation, which impaired GSH synthesis and indirectly weakened GPX4 function, as well as ACSL4-mediated lipid metabolism disorder expanding the ferroptosis substrate pool. EA effectively inhibited the ferroptosis process. Discussion EMs-related ovarian dysfunction is an important cause of female infertility, and its pathological mechanism is complex. Oxidative stress caused by cyclic bleeding of ectopic lesions is widely considered one of the core links in the pathogenesis of EMs [ 33 ]. However, traditional hormonal or surgical therapies have shown limited efficacy in blocking the depletion of ovarian reserve caused by persistent oxidative damage. Moreover, some therapies themselves may have potential negative impacts on ovarian reserve or in vitro fertilization-embryo transfer (IVF-ET) outcomes [ 34 – 37 ]. Although numerous studies have focused on elevated oxidative stress levels in the peritoneal fluid microenvironment, changes in the intrinsic antioxidant capacity of ectopic endometrial cells, the effects of ROS on lesion proliferation, apoptosis, angiogenesis, and their association with clinical symptoms like pain and adhesion [ 38 – 41 ], systematic research remains insufficient regarding theoxidative stress damage experienced by the ovary—a gonad highly sensitive to oxidative damage [ 42 ]—under EMs conditions, particularly the precise molecular mechanisms driving follicle loss and reserve decline. This knowledge gap limits a comprehensive understanding of the key molecular mechanisms by which EMs leads to diminished ovarian reserve function. Furthermore, interventions targeting oxidative stress in ectopic lesions may not effectively alleviate local oxidative damage in the ovary. Therefore, in-depth investigation into the local oxidative stress status of the ovary in the context of EMs, its specific drivers, and its damaging effects on the follicular microenvironment is crucial for understanding EMs-related infertility and protecting fertility. Given these challenges, exploring new strategies capable of effectively protecting the ovary from EMs-related oxidative damage is particularly important. EA therapy, combining traditional acupuncture with modern electrical stimulation technology, demonstrates unique advantages in the field of assisted reproduction, particularly in treating infertility. It has been widely applied in the treatment and research of infertility conditions such as ovulation disorders [ 43 ], poor endometrial receptivity [ 44 ], and diminished ovarian reserve [ 45 , 46 ]. Notably, recent studies suggest that EA may exert protective effects in various pathological models by modulating oxidative stress and ferroptosis pathways. For example, EA can activate the Nrf2/SLC7A11/GPX4 signaling axis, directly enhancing glutathione synthesis and GPX4 antioxidant enzymeactivity, thereby inhibiting oxidative stress, lipid peroxidation, and ferroptosis [ 47 , 48 ]. Regarding ovarian function, EA has also been shown to improve granulosa cell apoptosis, oxidative stress, and mitochondrial dysfunction in polycystic ovary syndrome (PCOS) models [ 49 ]. Based on the potential role of EA in regulating oxidative stress/ferroptosis and improving ovarian function, and the current lack of clarity on the mechanisms of local ovarian oxidative damage in EMs, we utilized established EMs animal models, successfully replicated the ovarian dysfunction phenotype and ovarian iron overload phenomenon, and focused on investigating the protective effects and mechanisms of EA intervention. Our study found that EA intervention significantly ameliorated EMs-induced ovarian injury. Its core mechanism lies in activating the Nrf2 signaling pathway to upregulate GPX4 activity and inhibiting ACSL4 expression, synergistically blocking the ferroptosis process in ovarian granulosa cells. Specifically, EA intervention effectively improved the reproductive endocrine homeostasis in model animals, alleviated local ovarian iron overload, and reduced oxidative stress levels. At the molecular mechanism level (Fig. 5 ), EA significantly reversed the suppression of the Nrf2 pathway in the EMs model and upregulated its downstream target gene SLC7A11 to promote GSH synthesis, thereby maintaining GPX4 enzymatic activity. Concurrently, it upregulated FTH1 to ameliorate iron metabolism disorders. These changes collectively enhanced cellular antioxidant capacity and helped mitigate iron overload. Furthermore, EA significantly downregulated key pro-ferroptotic factors ACSL4 and COX-2. A significant reduction in the lipid peroxidation product MDA levels further confirmed the inhibition of ferroptosis. In summary, this study revealed that EA synergistically inhibits ovarian granulosa cell ferroptosis through dual pathways: (1) The Nrf2 pathway-dependent route: activating Nrf2, upregulating SLC7A11, upregulating GPX4 functional activity, and enhancing anti-lipid peroxidation capacity; (2) A potentially non-Nrf2-dependent pathway: inhibiting ACSL4 to reduce the accumulation of pro-ferroptotic lipid substrates. This multi-target action effectively blocked the ferroptosis process, constituting the key mechanism by which EA improves follicular atresia and ovarian structural/functional damage. In conclusion, this study demonstrated for the first time that EA can effectively improve EMs-related ovarian dysfunction and revealed that its core protective mechanism lies in reactivating the Nrf2 pathway to restore GPX4 function while inhibiting ACSL4 expressionikding new and important insights into the molecular mechanisms underlying EMs-induced ovarian reserve decline. Concurrently, the results provide crucial experimental evidence for developing complementary therapies targeting EMs-related infertility, especially those aimed at protecting ovarian reserve. EA’s non-invasive nature and multi-target regulatory characteristics may confer unique potential advantages over traditional therapies, particularly for EMs patients with fertility desires or at risk of diminished ovarian reserve. However, this study has certain limitations. Primarily based on a mouse model, although it can simulate key pathological features of human EMs (e.g., ovarian injury, iron overload), inherent differences exist in reproductive physiology, anatomy, and disease progression between species. Future research needs deeper validation and mechanistic exploration in models closer to humans, such as primates. Conclusions This study revealed that EA effectively ameliorates EMs-associated ovarian injury, with its core mechanism lying in the inhibition of granulosa cell ferroptosis. EA activates the Nrf2 pathway to upregulate GPX4 activity and inhibits the pro-ferroptotic protein ACSL4 (potentially Nrf2-independent), synergistically reducing lipid peroxidation levels, thereby protecting follicular and ovarian function. This study was the first to reveal EA targeting ferroptosis to protect ovarian function in EMs, providing important experimental evidence for its use as a complementary therapy for EMs-related infertility. Abbreviations ACSL4 Acyl-CoA synthetase long-chain family member 4 AMH Anti-Müllerian Hormone COX-2 Cyclooxygenase-2 CV4 Guanyuan EMs Endometriosis EA Electroacupuncture E 2 Estradiol FSH Follicle-Stimulating Hormone FTH1 Ferritin Heavy Chain 1 GPX4 Glutathione Peroxidase 4 GCs Granulosa Cells GSH Glutathione HO-1 Heme oxygenase-1 IVF-ET In Vitro Fertilization and Embryo Transfer MDA Malondialdehyde Nrf2 Nuclear factor erythroid 2-related factor 2 NQO1 NAD(P)H quinone oxidoreductase 1 ROS Reactive Oxygen Species SLC7A11 Solute Carrier Family 7 Member 11 (xCT) SOD2 Superoxide dismutase 2 ST36 Zusanli SP6 Sanyinjiao Declarations Acknowledgements The study was supported by the Key Laboratory of Acupuncture and Medicine Research of Ministry of Education. And we would like to sincerely thank all those who participated in this study. Author contributions Youbing Xia and Liangjun Xia conceived and designed the study. Yan Zan, Xiaoquan Huang, Yu Zhuang, Junwei Li, Qian Zhu, and Tiantian Ma performed animal experiments. Yan Zan analyzed the data and wrote the manuscript. Liangjun Xia revised the manuscript. Youbing Xia and Liangjun Xia supervised the project. All authors read and approved the final manuscript. Funding This work was supported by the National Natural Science Foundation of China (grant numbers: 82205251 and 82274638). Data availability All data collected or analyzed in the study are included in this published article and can be acquired from the corresponding author on reasonable request. Ethics approval and Consent to Participate The Nanjing University of Traditional Chinese Medicine Ethics Committee reviewed and approved the experimental procedure, which complied with Guiding Opinions on the Treatment of Experimental Animals (Approval Number: 202403A036) Consent for publication Not applicable. Competing interests The authors declare no competing interests. References Taylor HS, Kotlyar AM, Flores VA. Endometriosis is a chronic systemic disease: clinical challenges and novel innovations. Lancet. 2021;397(10276):839–52. 10.1016/S0140-6736(21)00389-5 . Zondervan KT, Becker CM, Missmer SA, Endometriosis. N Engl J Med. 2020;382(13):1244–56. 10.1056/NEJMra1810764 . Horne AW, Missmer SA. Pathophysiology, diagnosis, and management of endometriosis. BMJ. 2022;379:e070750. 10.1136/bmj-2022-070750 . Saunders PTK, Horne AW. Endometriosis: etiology, pathobiology, and therapeutic prospects. Cell. 2021;184(11):2807–24. 10.1016/j.cell.2021.04.041 . Bulun SE, Yilmaz BD, Sison C, et al. Endometriosis. Endocr Rev. 2019;40(4):1048–79. 10.1210/er.2018-00242 . Allaire C, Bedaiwy MA, Yong PJ. Diagnosis and management of endometriosis. CMAJ. 2023;195(10):E363–71. 10.1503/cmaj.220637 . Zakhari A, Delpero E, McKeown S, Tomlinson G, Bougie O, Murji A. Endometriosis recurrence following postoperative hormonal suppression: a systematic review and meta-analysis. Hum Reprod Update. 2021;27(1):96–107. 10.1093/humupd/dmaa033 . Ata B, Somigliana E. Endometriosis, staging, infertility and assisted reproductive technology: time for a rethink. Reproductive Biomed Online. 2024;49(1):103943. 10.1016/j.rbmo.2024.103943 . Sampson JA. Peritoneal endometriosis due to the menstrual dissemination of endometrial tissue into the peritoneal cavity. Am J Obstet Gynecol. 1927;14(4):422–69. 10.1016/S0002-9378(15)30003-X . Ganz T, Macrophages, Metabolism I. Microbiol Spectr. 2016;4(5). 10.1128/microbiolspec.MCHD-0037-2016 . Nanda A, K T, Banerjee P, et al. Cytokines, Angiogenesis, and Extracellular Matrix Degradation are Augmented by Oxidative Stress in Endometriosis. Ann Lab Med. 2020;40(5):390–7. 10.3343/alm.2020.40.5.390 . Luo Y, Li L, Hu Q, et al. Iron overload increases the sensitivity of endometriosis stromal cells to ferroptosis via a PRC2-independent function of EZH2. Int J Biochem Cell Biol. 2024;169:106553. 10.1016/j.biocel.2024.106553 . Clower L, Fleshman T, Geldenhuys WJ, Santanam N. Targeting oxidative stress involved in endometriosis and its pain. Biomolecules. 2022;12(8):1055. 10.3390/biom12081055 . Pedachenko N et al. Serum anti-Müllerian hormone, prolactin and estradiol concentrations in infertile women with endometriosis. Gynecol Endocrinol . 2021; 37(2): 162–165. doi: 10.1080/09513590. 2020.1855634. Ata B, Turkgeldi E, Seyhan A, Urman B. Effect of hemostatic method on ovarian reserve following laparoscopic endometrioma excision: comparison of suture, hemostatic sealant, and bipolar desiccation. A systematic review and meta-analysis. J Minim Invasive Gynecol. 2015;22(3):363–72. 10.1016/j.jmig.2014.12.168 . Li A, Ni Z, Zhang J, Cai Z, Kuang Y, Yu C. Transferrin insufficiency and iron overload in follicular fluid contribute to oocyte dysmaturity in infertile women with advanced endometriosis. Front Endocrinol (Lausanne). 2020;11:391. 10.3389/fendo.2020.00391 . Fan W, Yuan Z, Li M, Zhang Y, Nan F. Decreased oocyte quality in patients with endometriosis is closely related to abnormal granulosa cells. Front Endocrinol (Lausanne). 2023;14:1226687. 10.3389/fendo.2023.1226687 . Mao J, Zhang J, Cai L, Cui Y, Liu J, Mao Y. Elevated prohibitin 1 expression mitigates glucose metabolism defects in granulosa cells of infertile patients with endometriosis. Mol Hum Reprod. 2022;28(6):gaac018. 10.1093/molehr/gaac018 . Sampson JA. Peritoneal endometriosis due to the menstrual dissemination of endometrial tissue into the peritoneal cavity. Am J Obstet Gynecol. 1927;14(4):422–69. 10.1016/S0002-9378(15)30003-X . Jiang X, Stockwell BR, Conrad M. Ferroptosis: mechanisms, biology and role in disease. Nat Rev Mol Cell Biol. 2021;22(4):266–82. 10.1038/s41580-020-00324-8 . Li G, Lin Y, Zhang Y, et al. Endometrial stromal cell ferroptosis promotes angiogenesis in endometriosis. Cell Death Discov. 2022;8(1):29. 10.1038/s41420-022-00821-z . Wan Y, Gu C, Kong J, et al. Long noncoding RNA ADAMTS9-AS1 represses ferroptosis of endometrial stromal cells by regulating the miR-6516-5p/GPX4 axis in endometriosis. Sci Rep. 2022;12(1):2618. 10.1038/s41598-022-04963-z . Liang D, Minikes AM, Jiang X. Ferroptosis at the intersection of lipid met-abolism and cellular signaling. Mol Cell. 2022;82(12):2215–27. 10.1016/j.molcel.2022.03.022 . Ni Z, Li Y, Song D, et al. Iron-overloaded follicular fluid increases the risk of endometriosis-related infertility by triggering granulosa cell ferroptosis and oocyte dysmaturity. Cell Death Dis. 2022;13(7):579. 10.1038/s41419-022-05037-8 . Li S, Zhou Y, Huang Q, et al. Iron overload in endometriosis peritoneal fluid induces early embryo ferroptosis mediated by HMOX1. Cell Death Discov. 2021;7(1):355. 10.1038/s41420-021-00751-2 . Leonhardt H, Hellström M, Gull B, et al. Serum anti-Müllerian hormone and ovarian morphology assessed by magnetic resonance imaging in response to acupuncture and exercise in women with polycystic ovary syndrome: secondary analyses of a randomized controlled trial. Acta Obstet Gynecol Scand. 2015;94(3):279–87. 10.1111/aogs.12571 . Tapmeier TT, Rahmioglu N, Lin J, et al. Neuropeptide S receptor 1 is a nonhormonal treatment target in endometriosis. Sci Transl Med. 2021;13:eabd6469. 10.1126/scitranslmed.abd6469 . Cora MC, Kooistra L, Travlos G. Vaginal Cytology of the Laboratory Rat and Mouse: Review and Criteria for the Staging of the Estrous Cycle Using Stained Vaginal Smears. Toxicol Pathol. 2015;43(6):776–93. 10.1177/0192623315570339 . Myers M, Britt KL, Wreford NG, Ebling FJ, Kerr JB. Methods for quantifying follicular numbers within the mouse ovary. Reproduction. 2004;127(5):569–80. 10.1530/rep.1.00095 . Ding J, Zhao Q, Zhou Z, et al. Huayu Jiedu Fang Protects Ovarian Function in Mouse with Endometriosis Iron Overload by Inhibiting Ferroptosis. Evid Based Complement Alternat Med. 2022;2022:1406820. 10.1155/2022/1406820 . Poirier D, Nyachieo A, Romano A, et al. An irreversible inhibitor of 17β-hydroxysteroid dehydrogenase type 1 inhibits estradiol synthesis in human endometriosis lesions and induces regression of the non-human primate endometriosis. J Steroid Biochem Mol Biol. 2022;222:106136. 10.1016/j.jsbmb.2022.106136 . Han SJ, Jung SY, Wu SP, et al. Estrogen Receptor β Modulates Apoptosis Complexes and the Inflammasome to Drive the Pathogenesis of Endometriosis. Cell. 2015;163(4):960–74. 10.1016/j.cell.2015.10.034 . Huang L, Shi L, Li M, Yin X, Ji X. Oxidative stress in endometriosis: Sources, mechanisms and therapeutic potential of antioxidants (Review). Int J Mol Med. 2025;55(5):72. 10.3892/ijmm.2025.5513 . Younis JS, Shapso N, Ben-Sira Y, Nelson SM, Izhaki I. Endometrioma surgery: a systematic review and meta-analysis of the effect on antral follicle count and anti-Müllerian hormone. Am J Obstet Gynecol . 2022;226(1):33–51.e7. 10.1016/j.ajog .2021.06.102 Erratum in: Am J Obstet Gynecol . 2023;229(1):88. doi:10.1016/j.ajog.2022.03.055. Muzii L, Galati G, Mattei G, et al. Expectant, Medical, and Surgical Management of Ovarian Endometriomas. J Clin Med. 2023;12(5):1858. 10.3390/jcm12051858 . Published 2023 Feb 26. Mircea O, Bartha E, Gheorghe M, Irimia T, Vlădăreanu R, Puşcaşiu L. Ovarian Damage after Laparoscopic Cystectomy for Endometrioma. Chirurgia (Bucur). 2016;111(1):54–7. Zhou L, Wang L, Geng Q, et al. Endometriosis is associated with a lower-ed cumulative live birth rate: A retrospective matched cohort study including 3071 in vitro fertilization cycles. J Reprod Immunol. 2022;151:103631. 10.1016/j.jri.2022.103631 . Clower L, Fleshman T, Geldenhuys WJ, Santanam N. Targeting Oxidative Stress Involved in Endometriosis and Its Pain. Biomolecules. 2022;12(8):1055. 10.3390/biom12081055 . Donnez J, Binda MM, Donnez O, Dolmans MM. Oxidative stress in the pelvic cavity and its role in the pathogenesis of endometriosis. Fertil Steril. 2016;106(5):1011–7. 10.1016/j.fertnstert.2016.07.1075 . Nanda A, K T, Banerjee P, et al. Cytokines, Angiogenesis, and Extracellular Matrix Degradation are Augmented by Oxidative Stress in Endometriosis. Ann Lab Med. 2020;40(5):390–7. 10.3343/alm.2020.40.5.390 . Ding J, Mei S, Wang K, Cheng W, Sun S, Ni Z, Wang X, Yu C. Curcumin modulates oxidative stress to inhibit pyroptosis and improve the inflammatory microenvironment to treat endometriosis. Genes Dis. 2024;11(3):101053. 10.1016/j.gendis.2023.06.022 . Ammar OF, Massarotti C, Mincheva M, et al. Oxidative stress and ovarian aging: from cellular mechanisms to diagnostics and treatment. Hum Reprod. 2024;39(7):1582–6. 10.1093/humrep/deae082 . Li Y, Zhi W, Haoxu D, et al. Effects of electroacupuncture on the expression of hypothalamic neuropeptide Y and ghrelin in pubertal rats with polycystic ovary syndrome. PLoS ONE. 2022;17(6):e0259609. 10.1371/journal.pone.0259609 . Xia L, Meng Q, Xi J et al. The synergistic effect of electroacupuncture and bone mesenchymal stem cell transplantation on repairing thin endometrial injury in rats. Stem Cell Res Ther. 2019;10(1):244. 10.1186/s13287 -019-1326-6 Erratum in: Stem Cell Res Ther. 2023;14(1):196. doi:10.1186/s13287-023-03421-5. Shi Y, Li L, Zhou J, et al. Efficacy of electroacupuncture in regulating the imbalance of AMH and FSH to improve follicle development and hyperandrogenism in PCOS rats. Biomed Pharmacother. 2019;113:108687. 10.1016/j.biopha.2019.108687 . Zhu H, Nan S, Suo C, et al. Electro-Acupuncture Affects the Activity of the Hypothalamic-Pituitary-Ovary Axis in Female Rats. Front Physiol. 2019;10:466. 10.3389/fphys.2019.00466 . Published 2019 Apr 24. Yang XC, Jin YJ, Ning R, et al. Electroacupuncture attenuates ferroptosis by promoting Nrf2 nuclear translocation and activating Nrf2/SLC7A11/GPX4 pathway in ischemic stroke. Chin Med. 2025;20(1):4. 10.1186/s13020-024-01047-0 . Jiang H, Shang Z, You L, et al. Electroacupuncture Pretreatment at Zusanli (ST36) Ameliorates Hepatic Ischemia/Reperfusion Injury in Mice by Reducing Oxidative Stress via Activating Vagus Nerve-Dependent Nrf2 Pathway. J Inflamm Res. 2023;16:1595–610. 10.2147/JIR.S404087 . Cong J, Li M, Wang Y, et al. Protective effects of electroacupuncture on polycystic ovary syndrome in rats: Down-regulating Alas2 to inhibit apoptosis, oxidative stress, and mitochondrial dysfunction in ovarian granulosa cells. Tissue Cell. 2023;82:102090. 10.1016/j.tice.2023.102090 . Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial.docx WesternBlotSupplementaryMaterial.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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6928457","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":475499005,"identity":"aa829eaa-fb73-466b-8ec4-724b658b678c","order_by":0,"name":"Yan Zan","email":"","orcid":"","institution":"Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Zan","suffix":""},{"id":475499006,"identity":"12ede3ed-ef13-4b79-8b65-9d64b7c9ded5","order_by":1,"name":"Xiaoquan Huang","email":"","orcid":"","institution":"Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xiaoquan","middleName":"","lastName":"Huang","suffix":""},{"id":475499007,"identity":"e99db2a6-91d4-4c91-acea-67f2cad54ea7","order_by":2,"name":"Yu Zhuang","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Zhuang","suffix":""},{"id":475499008,"identity":"ce6d7ba9-368b-4b4f-b15b-98f46c1fd8e1","order_by":3,"name":"Junwei Li","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Junwei","middleName":"","lastName":"Li","suffix":""},{"id":475499009,"identity":"3c290afa-5119-4cbf-8260-33a56af52af7","order_by":4,"name":"Qian Zhu","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Zhu","suffix":""},{"id":475499010,"identity":"0f7c9694-a075-4700-846a-a071220c74a9","order_by":5,"name":"Tiantian Ma","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Tiantian","middleName":"","lastName":"Ma","suffix":""},{"id":475499011,"identity":"578a5086-4bd6-4026-939b-110b08be137d","order_by":6,"name":"Liangjun Xia","email":"","orcid":"","institution":"Nanjing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Liangjun","middleName":"","lastName":"Xia","suffix":""},{"id":475499012,"identity":"488c07c6-ca33-4f8f-8b52-52a05334dd32","order_by":7,"name":"Youbing Xia","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYFACHgaJhAobOX4GhgSIwAFitDw4k2Ys2UCKFsmHbYcSN8BVEtJicCP34I3EtgPGxrcbnkn8zGGQ47uRwPi5AI8WyRl5yRYJ5+7Imd05kCbZu43BWPJGArP0DDxa+CVyzCQSyp4Zm91ISJNm3MaQuOFGAhszDx4tbGAtbIcTN8+AaKknqAViS9vhxA0SEC0JBoS0SPa8A/oFGMgSNxKSLXu3SRjOPPOwWRqfFoPjuQdv/gBF5YycxBs/t9nI8x1PPvgZnxYGgQQYiwfEkgBixgZ8GoCeOQBjsR/ArWoUjIJRMApGNAAAkOZREbgHIrEAAAAASUVORK5CYII=","orcid":"","institution":"Nanjing University of Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Youbing","middleName":"","lastName":"Xia","suffix":""}],"badges":[],"createdAt":"2025-06-19 07:23:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6928457/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6928457/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85428299,"identity":"0c92d210-db89-4f5c-adce-bd050483912f","added_by":"auto","created_at":"2025-06-25 17:29:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":619435,"visible":true,"origin":"","legend":"\u003cp\u003eConstruction of the EMs mouse model and EMs-induced ovarian functional impairment. \u003cstrong\u003e(A) \u003c/strong\u003eSchematic diagram illustrating the establishment of the EMs mouse model. \u003cstrong\u003e(B)\u003c/strong\u003e Schematic diagram of the experimental groups and workflow: Normal group (no surgery, \u003cem\u003en = \u003c/em\u003e8), Sham group (sham surgery, \u003cem\u003en = \u003c/em\u003e8), Donor group (uterus donation, \u003cem\u003en = \u003c/em\u003e8), Recipient group (divided into EMs group and EA group, \u003cem\u003en = \u003c/em\u003e8 per group). \u003cstrong\u003e(C)\u003c/strong\u003e Indicators of successful EMs model establishment: Formation of ectopic cysts containing yellow/white/red fluid within the cavity (black arrows indicate neovascularization; white arrows indicate tissue adhesions). \u003cstrong\u003e(D-F) \u003c/strong\u003eMouse body weight, ovarian weight, and ovarian index (Ovarian index (%) = Ovarian weight (mg) / Body weight (g) × 100%, \u003cem\u003en = \u003c/em\u003e8). \u003cstrong\u003e(G)\u003c/strong\u003e Incidence of estrous cycle disruption (Percentage of disrupted days (%) = Disrupted days / Total observation days × 100%, \u003cem\u003en = \u003c/em\u003e8). \u003cstrong\u003e(H)\u003c/strong\u003e Duration of the estrous cycle (days, \u003cem\u003en = \u003c/em\u003e8). \u003cstrong\u003e(I) \u003c/strong\u003eDistribution of estrous cycle phases (\u003cem\u003en = \u003c/em\u003e8). \u003cstrong\u003e(J-M) \u003c/strong\u003eSerum levels of AMH, E\u003csub\u003e2\u003c/sub\u003e, FSH, and LH (\u003cem\u003en = \u003c/em\u003e8). \u003cstrong\u003e(N) \u003c/strong\u003eCounts of follicles at various stages: Primordial follicle (PMF, \u003cem\u003en = \u003c/em\u003e5), Primary follicle (PF, \u003cem\u003en = \u003c/em\u003e5), Secondary follicle (SF, \u003cem\u003en = \u003c/em\u003e5), Antral follicle (ANF, \u003cem\u003en = \u003c/em\u003e5), Atretic follicle (ATF, \u003cem\u003en = \u003c/em\u003e5). \u003cstrong\u003e(O)\u003c/strong\u003e Total follicle count per ovary (\u003cem\u003en = \u003c/em\u003e5). \u003cstrong\u003e(P)\u003c/strong\u003e Ovarian tissue structure (HE staining). a: Primary follicle, b: Primary follicle, c: Secondary follicle, d: Antral follicle. The ovarian cortex in the Normal group shows numerous morphologically normal follicles at various stages, with clear follicular antrum structure, intact oocyte zona pellucida, and tightly arranged granulosa cell layers. Bar: 200, 100 \u003cem\u003eµ\u003c/em\u003em. *\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6928457/v1/9d842e7150e876a61e1a17e7.png"},{"id":85428301,"identity":"b4fcdd06-985f-40c6-a0ed-51f2faa974c0","added_by":"auto","created_at":"2025-06-25 17:29:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":427729,"visible":true,"origin":"","legend":"\u003cp\u003eOvarian iron overload in EMs mice. \u003cstrong\u003e(A)\u003c/strong\u003e Peritoneal fluid Fe\u003csup\u003e2\u003c/sup\u003e⁺ concentration (\u003cem\u003en = \u003c/em\u003e4).\u003cstrong\u003e (B)\u003c/strong\u003e Peritoneal fluid Ferritin level (\u003cem\u003en = \u003c/em\u003e4).\u003cstrong\u003e (C)\u003c/strong\u003e Ovarian tissue iron content (\u003cem\u003en = \u003c/em\u003e4).\u003cstrong\u003e (D)\u003c/strong\u003e Correlation analysis between peritoneal fluid Fe\u003csup\u003e2\u003c/sup\u003e⁺ concentration and ovarian iron content. Pearson correlation test.\u003cstrong\u003e (E)\u003c/strong\u003e Correlation analysis between ovarian Fe\u003csup\u003e2\u003c/sup\u003e⁺ content and the number of atretic follicles. Pearson correlation test.\u003cstrong\u003e (F and G)\u003c/strong\u003e Prussian blue staining of ovarian tissue (black arrows indicate sites of iron deposition; circles mark atretic follicles. Bar: 50 \u003cem\u003eµ\u003c/em\u003em, \u003cem\u003en = \u003c/em\u003e3). *\u003cem\u003eP \u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003eP \u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP \u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6928457/v1/767e154ec2c71f3c527cb2ee.png"},{"id":85428302,"identity":"7f464093-7fc7-4047-9ca1-fceea7dec075","added_by":"auto","created_at":"2025-06-25 17:29:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":534499,"visible":true,"origin":"","legend":"\u003cp\u003eOxidative stress levels in ovarian tissue. \u003cstrong\u003e(A)\u003c/strong\u003eRepresentative images of immunofluorescence co-localization analysis of reactive oxygen species (ROS) in ovarian tissue (red: ROS signal; blue: DAPI nuclear stain. Bar: 50 \u003cem\u003eµ\u003c/em\u003em).\u003cstrong\u003e (B)\u003c/strong\u003e Quantitative analysis of ROS fluorescence intensity (\u003cem\u003en = \u003c/em\u003e3).\u003cstrong\u003e (C)\u003c/strong\u003eOvarian tissue GSH level (\u003cem\u003en = \u003c/em\u003e3).\u003cstrong\u003e (D)\u003c/strong\u003e Ovarian tissue T-SOD activity (\u003cem\u003en = \u003c/em\u003e3). \u003cstrong\u003e(E-H)\u003c/strong\u003e mRNA expression of oxidative stress regulatory genes in ovarian tissue: Quantitative real-time PCR (qRT-PCR) analysis of \u003cem\u003eNrf2\u003c/em\u003e, \u003cem\u003eHmox1\u003c/em\u003e, \u003cem\u003eNqo1\u003c/em\u003e and \u003cem\u003eSod2 \u003c/em\u003e(\u003cem\u003en = \u003c/em\u003e3). *\u003cem\u003eP \u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP \u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6928457/v1/ead7236a56280581ba013a50.png"},{"id":85428491,"identity":"028b1f5d-02e2-4f0b-9b7e-987244826d21","added_by":"auto","created_at":"2025-06-25 17:37:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":810079,"visible":true,"origin":"","legend":"\u003cp\u003eExpression levels and spatial localization of ferroptosis-related markers in ovarian tissue. \u003cstrong\u003e(A-H)\u003c/strong\u003e Expression levels of ferroptosis-related proteins: Western blot analysis of GPX4, FTH1, ACSL4, and COX-2 protein levels (\u003cem\u003en = \u003c/em\u003e3). \u003cstrong\u003e(I)\u003c/strong\u003e Content of malondialdehyde (MDA), the terminal product of lipid peroxidation, in ovarian tissue (\u003cem\u003en = \u003c/em\u003e3). \u003cstrong\u003e(J-N)\u003c/strong\u003e mRNA expression levels of ferroptosis-related genes: Real-time quantitative PCR (qPCR) detection of \u003cem\u003eGpx4\u003c/em\u003e, \u003cem\u003eSlc7a11\u003c/em\u003e, \u003cem\u003eFth1\u003c/em\u003e, \u003cem\u003eAcsl4\u003c/em\u003e, and\u003cem\u003e Ptgs2\u003c/em\u003e (\u003cem\u003en = \u003c/em\u003e3). \u003cstrong\u003e(O)\u003c/strong\u003e Immunohistochemical localization of ferroptosis-related proteins: Representative staining images of GPX4, FTH1, ACSL4, and COX-2 (Bar: 50 \u003cem\u003eµ\u003c/em\u003em). \u003cstrong\u003e(P-S)\u003c/strong\u003e Quantitative analysis of target protein integrated optical density (IOD): Immunohistochemical signal intensity of FTH1, GPX4, ACSL4 and COX-2 (\u003cem\u003en = \u003c/em\u003e5). *\u003cem\u003eP \u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP \u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6928457/v1/7d87677c0e47e6af38fd3923.png"},{"id":85428306,"identity":"5e866d0a-65c6-4623-923d-bf3688dd0384","added_by":"auto","created_at":"2025-06-25 17:29:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":308519,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram illustrating the mechanism of iron overload-induced ovarian ferroptosis in EMs and its restoration by electroacupuncture. Pelvic iron metabolic imbalance in EMs leads to ovarian iron overload. Excess iron triggers lipid peroxidation via the Fenton reaction, driving ferroptosis. Electroacupuncture activates the ovarian Nrf2 pathway, upregulating GPX4 to suppress ferroptosis, thereby protecting EMs-affected ovaries.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6928457/v1/f5fbfcdc3a2a1236a9777074.png"},{"id":91415701,"identity":"d92dc91a-9cc4-4de3-92f2-4c53306a81c8","added_by":"auto","created_at":"2025-09-16 09:23:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3447728,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6928457/v1/b16a98c8-bda7-4cf9-a37e-8e9d9eca16c3.pdf"},{"id":85428298,"identity":"a68913f8-2c20-4610-8a28-cb61cf9adf91","added_by":"auto","created_at":"2025-06-25 17:29:18","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":18329,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6928457/v1/9038e14f483751797f65ca59.docx"},{"id":85428315,"identity":"a95b6f90-d956-40bc-85f2-4cf100f3cf6e","added_by":"auto","created_at":"2025-06-25 17:29:18","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5277319,"visible":true,"origin":"","legend":"","description":"","filename":"WesternBlotSupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6928457/v1/f534f0c2d9328b6a49c670ec.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Electroacupuncture Ameliorates Endometriosis-Associated Ovarian Dysfunction by Activating Nrf2 Pathway to Upregulate GPX4 Function and Inhibiting Ferroptosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEndometriosis (EMs) is an estrogen-dependent chronic inflammatory disease characterized by the implantation and invasion of ectopic endometrial tissue within the pelvic cavity. It affects approximately 10\u0026ndash;15% of reproductive-aged women globally, and among them, 30\u0026ndash;50% suffer from infertility, posing a serious threat to female reproductive health [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Common clinical manifestations include chronic pelvic pain and infertility [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Clinical management primarily involves pharmacological and surgical interventions, often combined with in vitro fertilization-embryo transfer (IVF-ET) for patients with infertility [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, studies indicate that pharmacological interventions often fail to improve the decline in ovarian reserve function, and surgical intervention may even exacerbate ovarian damage [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn EMs patients, cyclic bleeding from ectopic lesions leads to the persistent accumulation of iron ions and disruption of iron metabolism homeostasis within the pelvic microenvironment [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Clinical examinations reveal significantly elevated iron levels in the peritoneal fluid, ectopic lesions, peritoneum, and macrophages of EMs patients. Excessive iron ions generate reactive oxygen species (ROS) via the Fenton reaction, triggering significant oxidative stress damage and lipid peroxidation, which is the core pathological mechanism of tissue injury induced by iron overload [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. As a vital organ within the pelvic microenvironment, the ovary, particularly its granulosa cells and follicular microenvironment, are chronically exposed to this high-iron-induced oxidative stress environment. This results in impaired follicular development, reduced oocyte quality, and decreased ovarian reserve function [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Clinical studies observe reduced serum anti-M\u0026uuml;llerian hormone (AMH) levels, elevated estradiol (E\u003csub\u003e2\u003c/sub\u003e) and follicle-stimulating hormone (FSH) in EMs patients [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], with ovarian granulosa cells (GCs) exhibiting pathological alterations such as enhanced apoptosis, inflammation, and oxidative stress [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Although the classic \u0026ldquo;Retrograde Menstruation-Ectopic Implantation-Inflammatory Response\u0026rdquo; theory partially explains the pathological basis of EMs [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], the key molecular pathways underlying its induction of ovarian damage, particularly how iron metabolism dysregulation triggers cell death programs, require further exploration.\u003c/p\u003e \u003cp\u003eIn recent years, ferroptosis, an iron-dependent, lipid peroxidation-driven form of programmed cell death, has garnered significant attention in research on the pathological mechanisms of endometriosis (EMs) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Its key regulatory mechanisms involve the collapse of the antioxidant defense system and ferroptotic lipid peroxidation induced by molecules such as long-chain acyl-CoA synthetase 4 (ACSL4) under oxidative stress [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Notably, glutathione peroxidase 4 (GPX4), the core enzyme inhibiting lipid peroxidation, relies on sufficient reduced glutathione (GSH) supply for its function. The nuclear factor erythroid 2-related factor 2 (Nrf2) pathway maintains GSH biosynthesis by directly regulating the cystine transporter SLC7A11 (a component of System Xc⁻), thereby providing the essential substrate for GPX4 and indirectly sustaining its antioxidant function [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Studies indicate that follicular fluid and peritoneal fluid from EMs patients can induce ferroptosis in murine ovarian granulosa cells and follicles, manifested by significantly elevated levels of malondialdehyde (MDA), a terminal product of lipid peroxidation, and downregulated GPX4 protein expression [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, the regulatory role of the Nrf2 pathway in this process and the precise role of ACSL4 require further validation.\u003c/p\u003e \u003cp\u003eBased on this, we propose the hypothesis that iron ions abnormally accumulate locally in the ovary within the pelvic microenvironment of EMs, triggering intense oxidative stress. This inhibits the functional support of GPX4 by the Nrf2 pathway and activates ACSL4-mediated lipid peroxidation, inducing ferroptosis in granulosa cells, ultimately leading to ovarian dysfunction.\u003c/p\u003e \u003cp\u003eElectroacupuncture (EA), as a non-pharmacological intervention, possesses unique advantages in improving reproductive endocrine function. Studies show that EA can ameliorate ovarian function impairment through multi-target regulation [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, whether EA alleviates oxidative stress and inhibits ferroptosis by activating the Nrf2 pathway, thereby improving EMs-related ovarian dysfunction, requires systematic investigation.\u003c/p\u003e \u003cp\u003eUtilizing an established murine model that simulates the chronic bleeding characteristics of ectopic lesions in EMs patients, this study aims to validate the aforementioned key hypothesis regarding iron overload-oxidative stress-ovarian granulosa cell ferroptosis. Furthermore, it will delve into the specific mechanisms by which EA intervention improves ovarian function through regulating iron metabolism, ameliorating oxidative stress, and antagonizing granulosa cell ferroptosis. The findings are expected to provide new perspectives for elucidating the pathological mechanisms of EMs-related ovarian dysfunction and offer important experimental evidence for developing non-pharmacological intervention strategies.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eThis study utilized 6-week-old specific pathogen-free (SPF) female C57BL/6 mice (initial body weight: 18 2 g) provided by Jiangsu Huachuang Sino Medical Technology Co., Ltd. (License No.: SCXK (Su) 2020-0009). All mice were housed in the barrier facility of the Experimental Animal Center at Nanjing University of Chinese Medicine under an individually ventilated cage (IVC) system (5 mice per cage), with controlled environmental conditions: temperature 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2℃, humidity 50\u0026thinsp;\u0026plusmn;\u0026thinsp;5%, and a 12 h/12 h light-dark cycle. Food and water were provided ad libitum. Body weight was recorded weekly throughout the experiment. A total of 88 mice were randomly divided into five groups using a random number table: normal group (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8), sham surgery group (\u003cem\u003en\u0026thinsp;=\u003c/em\u003e\u0026thinsp;8), donor group (\u003cem\u003en\u0026thinsp;=\u003c/em\u003e\u0026thinsp;8), recipient group (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16), and blood supply group (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;48). At the end of the experimental period, mice were euthanized by cervical dislocation under anesthesia, and organ tissues, blood, and peritoneal fluid samples were collected. This study was approved by the Animal Ethics Committee of Nanjing University of Chinese Medicine (Approval No.: 202403A036).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eModeling methods\u003c/h3\u003e\n\u003cp\u003ePrior to modeling, all mice except the normal and blood supply groups were subcutaneously injected with estradiol benzoate (150 \u003cem\u003e\u0026micro;\u003c/em\u003eg/kg, once every 4 days for two doses) to synchronize estrous cycles. For modeling, donor mice were euthanized under isoflurane anesthesia, and uterine horns were excised aseptically, rinsed with PBS, longitudinally incised, and minced into fragments smaller than 1 mm\u003csup\u003e3\u003c/sup\u003e. The fragments were suspended in 0.5 mL PBS for subsequent use. Recipient mice then received an intraperitoneal injection of the uterine fragment suspension (0.5 cm above the urethral orifice), with each donor uterus allocated to two recipient mice. After injection, recipient mice were randomly assigned to the EMs group (\u003cem\u003en\u0026thinsp;=\u003c/em\u003e\u0026thinsp;8) or electroacupuncture (EA) group (\u003cem\u003en\u0026thinsp;=\u003c/em\u003e\u0026thinsp;8), while the sham surgery group received an equivalent volume of PBS. All procedures were completed within 5 minutes under sterile conditions.Post-modeling, estradiol benzoate (150 \u003cem\u003e\u0026micro;\u003c/em\u003eg/kg, once every 4 days for two doses) was continuously administered, and allogeneic whole blood (0.2 mL/mouse, once every 3 days for 24 days) was intraperitoneally injected starting from day 1 after modeling; the sham group received equivalent saline. Laparotomy was performed to validate successful modeling, defined by the presence of cystic lesions containing yellowish/whitish/reddish fluid accompanied by vascularization and tissue adhesion [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eElectroacupuncture intervention\u003c/h3\u003e\n\u003cp\u003eThe EA group commenced treatment on day 14 post-modeling. Daily, mice were anesthetized with isoflurane and fixed in position. The following acupoints were punctured: CV4 (Guanyuan) with a perpendicular insertion of 3 mm, bilateral SP6 (Sanyinjiao)at 4 mm depth, and bilateral ST36 (Zusanli) at 5 mm depth. SP6 and ST36 on the same side were connected to an electroacupuncture apparatus (SDZ-II, Suzhou Medical Appliance Factory, China) using a dense-disperse wave, pulse width 0.2\u0026ndash;0.6 ms, and current intensity of 1 mA, with stimulation intensity adjusted to induce slight toe twitching. Each session lasted 15 minutes and was administered once daily for 12 consecutive days. The blank control group received standard housing without intervention, while the EMs and sham surgery groups were subjected to identical anesthesia and fixation procedures (without electroacupuncture stimulation) for 12 days.\u003c/p\u003e\n\u003ch3\u003eEstrous cycle monitoring\u003c/h3\u003e\n\u003cp\u003eVaginal smears were collected daily at 9:00 AM and examined under a light microscope (IX73; Olympus, Japan) to observe cytomorphological characteristics. Based on vaginal epithelial cell composition, the estrous cycle was classified into four stages [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]: proestrus (P, nucleated epithelial cells\u0026thinsp;\u0026gt;\u0026thinsp;75%), estrus (E, anucleated keratinized cells\u0026thinsp;\u0026gt;\u0026thinsp;80%), metestrus (M, keratinized cells and leukocytes at a ratio of approximately 1:1), and diestrus (D, leukocytes\u0026thinsp;\u0026gt;\u0026thinsp;75%). Estrous cycle irregularities were defined as meeting any of the following criteria: Abnormal cycle duration (\u0026lt;\u0026thinsp;3 days or \u0026gt;\u0026thinsp;7 days; the normal cycle for C57BL/6 mice is 4\u0026ndash;5 days); Cycle stagnation (persistence of the same stage for \u0026ge;\u0026thinsp;3 days); Loss of sequential stage alternation.\u003c/p\u003e\n\u003ch3\u003eSerum hormone measurement\u003c/h3\u003e\n\u003cp\u003eSerum levels of estradiol (E\u003csub\u003e2\u003c/sub\u003e), follicle-stimulating hormone (FSH), anti-M\u0026uuml;llerian hormone (AMH), and luteinizing hormone (LH) were measured using commercial ELISA kits (AiFang Biology, AF02566-A, AF02555-A, AF09402-A, AF02582-A). All assays were performed according to the manufacturer\u0026rsquo;s instructions, with samples assayed in duplicate. The experimental procedure included the generation of standard curves and inter-assay variability controls (coefficient of variation\u0026thinsp;\u0026lt;\u0026thinsp;10%).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eOvarian histological analysis and follicle counting\u003c/h2\u003e \u003cp\u003eOvarian tissues were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned into 5-\u003cem\u003e\u0026micro;\u003c/em\u003em-thick slices. After hematoxylin and eosin (H\u0026amp;E) staining, ovarian morphological structures were observed under a light microscope at 200\u0026times; magnification. Following established criteria [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], follicles at various developmental stages (primordial, primary, secondary, antral, and atretic follicles) were classified and counted in every fifth serial section. Follicles were quantified only if they contained a visible intact oocyte nucleus.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIron metabolism analysis\u003c/h3\u003e\n\u003cp\u003eSerum ferritin levels were measured using an ELISA kit (AiFang Biology, AF02312-A). Iron ion content in peritoneal fluid and ovarian tissues was determined via a ferrous ion colorimetric assay (Elabscience, E-BC-K773-M) by measuring absorbance at 593 nm. Ovarian iron deposition was histologically localized using a Prussian blue staining kit (Servicebio, G1029), in which Fe\u003csup\u003e3+\u003c/sup\u003e reacted with potassium ferrocyanide (K\u003csub\u003e4\u003c/sub\u003e[Fe(CN)\u003csub\u003e6\u003c/sub\u003e]) to form characteristic blue precipitates. All procedures were strictly performed according to the manufacturer\u0026rsquo;s protocols, with standard curves and duplicate measurements included (intra-assay coefficient of variation\u0026thinsp;\u0026lt;\u0026thinsp;10%).\u003c/p\u003e\n\u003ch3\u003eDetection of oxidative stress-related indicators\u003c/h3\u003e\n\u003cp\u003eOvarian tissues were homogenized in pre-cooled PBS, and supernatants were collected after centrifugation for subsequent assays. Oxidative stress markers were measured using commercial kits: malondialdehyde (MDA) (Beyotime, S0131S), glutathione (GSH) (Nanjing Jiancheng, A006-2-1), and superoxide dismutase (SOD) (Nanjing Jiancheng, A001-3-2). All measurements were performed strictly following the manufacturer\u0026rsquo;s protocols using a microplate reader, and data were normalized to total protein concentration determined by a BCA assay (Beyotime, P0010). Each sample was assayed in duplicate with an intra-assay coefficient of variation\u0026thinsp;\u0026lt;\u0026thinsp;10%.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGene expression analysis by qRT-PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from ovarian tissues using the TRIzol reagent, and reverse transcription was performed with the Hifair\u0026reg; II First Strand cDNA Synthesis Kit (Yeasen, 11141ES60). Quantitative real-time PCR was conducted using the Hieff\u0026reg; qPCR SYBR Green Master Mix (Low Rox Plus) (Yeasen, 11202ES08) on a real-time PCR system. GAPDH was used as the reference gene, and relative gene expression was calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method. Primer sequences are listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eThe ovarian tissues were lysed using RIPA lysis buffer containing protease inhibitors (Beyotime, P0013B). Protein concentration was determined by BCA assay. Equal amounts of protein (30 \u003cem\u003e\u0026micro;\u003c/em\u003eg per lane) were separated by SDS-PAGE electrophoresis and subsequently transferred onto PVDF membranes (Millipore, IPVH00010). The membranes were blocked with rapid blocking buffer (Yoche, YWB0501) at room temperature for 5 minutes, followed by overnight incubation with primary antibodies at 4℃. After TBST washing, the membranes were incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were visualized using ECL detection reagent (Yeasen, 36208ES60), and quantitative analysis was performed with ImageJ software using GAPDH as the internal control. The antibody sequences are provided in Supplementary Table\u0026nbsp;2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical analysis of ovarian tissues\u003c/h2\u003e \u003cp\u003eOvarian tissue sections (5 \u003cem\u003e\u0026micro;\u003c/em\u003em) were dewaxed followed by antigen retrieval using citrate buffer (pH\u0026thinsp;=\u0026thinsp;6.0, 95℃, 20 minutes). Endogenous peroxidase activity was blocked with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 15 minutes, followed by blocking with 5% BSA at room temperature for 30 minutes. The sections were then incubated with primary antibodies overnight at 4℃. After PBS washing, the sections were incubated with HRP-conjugated secondary antibodies at 37℃ for 1 hour. Following DAB chromogenic reaction, the sections were counterstained with hematoxylin, dehydrated, cleared, and mounted. Five non-overlapping fields per section were randomly captured under an optical microscope (400 \u0026times; magnification). Quantitative analysis of integrated optical density (IOD) was performed using ImageJ software, and the mean IOD value from five fields per section was used for intergroup comparisons.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDetection of reactive oxygen Species (ROS) in ovarian tissues\u003c/h2\u003e \u003cp\u003eOvarian tissues were embedded in OCT compound (Sakura, 4583) and snap-frozen in liquid nitrogen. Cryosections (8 \u003cem\u003e\u0026micro;\u003c/em\u003em thickness) were prepared and stained using a reactive oxygen species (ROS) detection kit (Solarbio, G4817) according to the manufacturer\u0026rsquo;s instructions. The sections were mounted with antifade mounting medium containing DAPI. Imaging was performed under a confocal microscope with the following excitation/emission wavelength settings: DHE (535/610 nm) and DAPI (380/420 nm). Five non-overlapping fields per section were randomly selected at 400\u0026times; magnification, and the mean fluorescence intensity of ROS-positive areas was quantified using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses and graph generation were performed using GraphPad Prism software. Continuous data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). For multiple group comparisons, one-way ANOVA was applied followed by Tukey\u0026rsquo;s multiple comparison test for normally distributed data, while Kruskal-Wallis test was used with Dunn\u0026rsquo;s post hoc analysis for non-normally distributed data. Correlation analyses were conducted using Pearson\u0026rsquo;s correlation coefficient (for normal distributions) or Spearman\u0026rsquo;s rank correlation coefficient (for non-normal distributions), based on data distribution characteristics. A two-tailed \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEA ameliorated ovarian functional impairment in EMs mice\u003c/h2\u003e \u003cp\u003eTo simulate the estrogen-dependent nature and chronic hemorrhagic characteristics of EMs lesions, intraperitoneal injection of allogeneic uterine fragments combined with subcutaneous injection of estradiol benzoate in the neck region was employed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Additionally, allogeneic whole blood was intraperitoneally injected every 3 days after modeling [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] (0.2 mL/injection, total of 8 injections) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Twenty-four days post-modeling, mice in the EMs group developed typical multiple yellowish cystic lesions with hypervascularization and adhesions in the abdominal cavity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Compared to the Normal group, the EMs group showed significantly decreased body weight, ovarian weight, and ovarian index (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-F). The estrous cycle was disrupted, manifested as a disturbed rhythm and prolonged average cycle length and estrus phase duration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-I). EA intervention significantly reversed these pathological phenotypes (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), restoring body weight, ovarian weight, and ovarian index, and ameliorating estrous cycle disruption. Serum hormone assays detected significantly decreased AMH levels and significantly elevated E2 levels in the EMs group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ\u0026amp;K), consistent with the \u0026ldquo;low AMH-high E\u003csub\u003e2\u003c/sub\u003e\u0026rdquo; hormonal imbalance pattern observed in EMs patients [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Concurrently, FSH, LH levels, and the FSH/LH ratiowere all elevated, suggesting potential dysfunction in the hypothalamic-pituitary-ovarian (HPO) axis feedback regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL\u0026amp;M). EA intervention effectively corrected these abnormalities in serum hormone levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). HE staining results revealed that the number of follicles at all stages was significantly reduced, while the number of atretic follicles was significantly increased in the ovaries of the EMs group, with granulosa cell structure destruction. In the EA group, the number of follicles at various stages was restored, the number of atretic follicles decreased, and the granulosa cell structure was repaired (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eN-P).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEA reduced ovarian iron overload in EMs mice\u003c/h2\u003e \u003cp\u003eTo investigate the association between ovarian damage and iron metabolism, we detected that Fe\u003csup\u003e2\u003c/sup\u003e⁺ and Ferritin levels in the peritoneal fluid were significantly higher in the EMs group compared to the Normal group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026amp;B). Ovarian Fe\u003csup\u003e2\u003c/sup\u003e⁺ concentration was elevated and showed a positive correlation with peritoneal fluid Fe\u003csup\u003e2+\u003c/sup\u003e levels (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.92, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), indicating that ovarian iron overload was closely associated with iron metabolism dysregulation in the peritoneal fluid (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u0026amp;D). EA intervention significantly reduced these parameters (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Further localization of iron deposition by Prussian blue staining revealed that the amount of iron deposition in the ovaries was significantly higher in the EMs group than in the Normal group. Iron deposition was primarily distributed around the theca folliculi, interstitial blood vessels, and within granulosa cells of atretic follicles, exhibiting a spatial distribution highly overlapping with atretic follicles. In the EA group, the amount of iron deposition was significantly reduced compared to the EMs group, and the deposition was confined mainly to the perivascular areas of interstitial blood vessels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE\u0026amp;F). Correlation analysisconfirmed a positive correlation between ovarian Fe\u003csup\u003e2\u003c/sup\u003e⁺ concentration and the number of atretic follicles (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.94, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). As free iron catalyzes ROS generation via the Fenton reaction, we hypothesized that iron overloadmight accelerate follicular atresia through oxidative stress pathways, leading to ovarian functional impairment, and that EA ameliorated ovarian damage by regulating iron metabolism.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eEA alleviated ovarian oxidative stress by activating the Nrf2 pathway\u003c/h2\u003e \u003cp\u003eTo clarify the impact of EA-regulated iron metabolism on redox balance, we assessed ovarian oxidative stress levels and the antioxidant defense system. Compared with the Normal group, ovaries from the EMs group showed significant oxidative damage, manifested as a significant increase in ROS fluorescence intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026amp;B), along with significant decreases in the levels of the key antioxidant molecule GSH and the activity of total superoxide dismutase (T-SOD). After EA intervention, ROS fluorescence intensity decreased, while GSH levels and T-SOD activity increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u0026amp;D), indicating partial restoration of endogenous antioxidant reserves. The mRNA expression level of the core antioxidant transcription factor Nrf2 was significantly downregulated compared to the Normal group. The transcriptional levels of its classic downstream target genes-mitochondrial superoxide dismutase (SOD2), heme oxygenase-1 (HO-1), and NAD(P)H quinone oxidoreductase 1 (NQO1)-were synchronously downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-H). EA intervention significantly reversed the inhibition of these gene expressions (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These findings indicated that the Nrf2 pathway inactivation in EMs mouse ovariesled to the collapse of antioxidant defense. EA activated the Nrf2 pathway, promoted the expression of its downstream antioxidant enzymes, enhanced GSH biosynthesis reserves and SOD activity, effectively neutralized excess ROS, and re-established redox homeostasis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eEA inhibited ferroptosis by upregulating GPX4 function via the Nrf2 pathway\u003c/h2\u003e \u003cp\u003eGiven that GPX4 is a core inhibitor of ferroptosis whose downregulation drives the ferroptosis process, and that the Nrf2 pathway plays a central regulatory role in maintaining cellular redox homeostasis and inhibiting ferroptosis [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], we hypothesized that Nrf2 pathway inactivation might lead to ferroptosis by affecting GPX4 function. To investigate whether EA regulates ferroptosis through the Nrf2 pathway, we detected key markers of ovarian ferroptosis. Compared with the Normal group, GPX4 protein and mRNA levels were significantly downregulated in the EMs group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE\u0026amp;J). Immunohistochemistry showed weakened expression in granulosa cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eO). The mRNA level of the Nrf2 target gene SLC7A11 was significantly downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK), resulting in the loss of the key transporter for GSH synthesis and impaired maintenance of GPX4 activity. Decreased expression of ferritin heavy chain (FTH1) protein and mRNA corresponded with increased iron deposition, indicating impaired iron storage capacity and an expanded free iron pool (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, F\u0026amp;L). EA intervention significantly reversed these changes (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, the concentration of the lipid peroxidation product MDA was significantly higher in the EMs group than in the Normal group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). The protein and mRNA expression levels of pro-ferroptotic factors ACSL4 and cyclooxygenase-2 (COX-2) were significantly upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u0026amp;D, 4G\u0026amp;H, 4M\u0026amp;N), and both were highly enriched in granulosa cells of atretic follicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eO). Their expression decreased after EA intervention (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These results confirmed that ferroptosis occurs in EMs ovaries. The mechanism involved Nrf2 pathway inactivation leading to SLC7A11 downregulation, which impaired GSH synthesis and indirectly weakened GPX4 function, as well as ACSL4-mediated lipid metabolism disorder expanding the ferroptosis substrate pool. EA effectively inhibited the ferroptosis process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eEMs-related ovarian dysfunction is an important cause of female infertility, and its pathological mechanism is complex. Oxidative stress caused by cyclic bleeding of ectopic lesions is widely considered one of the core links in the pathogenesis of EMs [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, traditional hormonal or surgical therapies have shown limited efficacy in blocking the depletion of ovarian reserve caused by persistent oxidative damage. Moreover, some therapies themselves may have potential negative impacts on ovarian reserve or in vitro fertilization-embryo transfer (IVF-ET) outcomes [\u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Although numerous studies have focused on elevated oxidative stress levels in the peritoneal fluid microenvironment, changes in the intrinsic antioxidant capacity of ectopic endometrial cells, the effects of ROS on lesion proliferation, apoptosis, angiogenesis, and their association with clinical symptoms like pain and adhesion [\u003cspan additionalcitationids=\"CR39 CR40\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], systematic research remains insufficient regarding theoxidative stress damage experienced by the ovary\u0026mdash;a gonad highly sensitive to oxidative damage [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u0026mdash;under EMs conditions, particularly the precise molecular mechanisms driving follicle loss and reserve decline. This knowledge gap limits a comprehensive understanding of the key molecular mechanisms by which EMs leads to diminished ovarian reserve function. Furthermore, interventions targeting oxidative stress in ectopic lesions may not effectively alleviate local oxidative damage in the ovary. Therefore, in-depth investigation into the local oxidative stress status of the ovary in the context of EMs, its specific drivers, and its damaging effects on the follicular microenvironment is crucial for understanding EMs-related infertility and protecting fertility.\u003c/p\u003e \u003cp\u003eGiven these challenges, exploring new strategies capable of effectively protecting the ovary from EMs-related oxidative damage is particularly important. EA therapy, combining traditional acupuncture with modern electrical stimulation technology, demonstrates unique advantages in the field of assisted reproduction, particularly in treating infertility. It has been widely applied in the treatment and research of infertility conditions such as ovulation disorders [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], poor endometrial receptivity [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], and diminished ovarian reserve [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Notably, recent studies suggest that EA may exert protective effects in various pathological models by modulating oxidative stress and ferroptosis pathways. For example, EA can activate the Nrf2/SLC7A11/GPX4 signaling axis, directly enhancing glutathione synthesis and GPX4 antioxidant enzymeactivity, thereby inhibiting oxidative stress, lipid peroxidation, and ferroptosis [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Regarding ovarian function, EA has also been shown to improve granulosa cell apoptosis, oxidative stress, and mitochondrial dysfunction in polycystic ovary syndrome (PCOS) models [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e Based on the potential role of EA in regulating oxidative stress/ferroptosis and improving ovarian function, and the current lack of clarity on the mechanisms of local ovarian oxidative damage in EMs, we utilized established EMs animal models, successfully replicated the ovarian dysfunction phenotype and ovarian iron overload phenomenon, and focused on investigating the protective effects and mechanisms of EA intervention. Our study found that EA intervention significantly ameliorated EMs-induced ovarian injury. Its core mechanism lies in activating the Nrf2 signaling pathway to upregulate GPX4 activity and inhibiting ACSL4 expression, synergistically blocking the ferroptosis process in ovarian granulosa cells.\u003c/p\u003e \u003cp\u003eSpecifically, EA intervention effectively improved the reproductive endocrine homeostasis in model animals, alleviated local ovarian iron overload, and reduced oxidative stress levels. At the molecular mechanism level (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), EA significantly reversed the suppression of the Nrf2 pathway in the EMs model and upregulated its downstream target gene SLC7A11 to promote GSH synthesis, thereby maintaining GPX4 enzymatic activity. Concurrently, it upregulated FTH1 to ameliorate iron metabolism disorders. These changes collectively enhanced cellular antioxidant capacity and helped mitigate iron overload. Furthermore, EA significantly downregulated key pro-ferroptotic factors ACSL4 and COX-2. A significant reduction in the lipid peroxidation product MDA levels further confirmed the inhibition of ferroptosis. In summary, this study revealed that EA synergistically inhibits ovarian granulosa cell ferroptosis through dual pathways: (1) The Nrf2 pathway-dependent route: activating Nrf2, upregulating SLC7A11, upregulating GPX4 functional activity, and enhancing anti-lipid peroxidation capacity; (2) A potentially non-Nrf2-dependent pathway: inhibiting ACSL4 to reduce the accumulation of pro-ferroptotic lipid substrates. This multi-target action effectively blocked the ferroptosis process, constituting the key mechanism by which EA improves follicular atresia and ovarian structural/functional damage.\u003c/p\u003e \u003cp\u003eIn conclusion, this study demonstrated for the first time that EA can effectively improve EMs-related ovarian dysfunction and revealed that its core protective mechanism lies in reactivating the Nrf2 pathway to restore GPX4 function while inhibiting ACSL4 expressionikding new and important insights into the molecular mechanisms underlying EMs-induced ovarian reserve decline. Concurrently, the results provide crucial experimental evidence for developing complementary therapies targeting EMs-related infertility, especially those aimed at protecting ovarian reserve. EA\u0026rsquo;s non-invasive nature and multi-target regulatory characteristics may confer unique potential advantages over traditional therapies, particularly for EMs patients with fertility desires or at risk of diminished ovarian reserve.\u003c/p\u003e \u003cp\u003eHowever, this study has certain limitations. Primarily based on a mouse model, although it can simulate key pathological features of human EMs (e.g., ovarian injury, iron overload), inherent differences exist in reproductive physiology, anatomy, and disease progression between species. Future research needs deeper validation and mechanistic exploration in models closer to humans, such as primates.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study revealed that EA effectively ameliorates EMs-associated ovarian injury, with its core mechanism lying in the inhibition of granulosa cell ferroptosis. EA activates the Nrf2 pathway to upregulate GPX4 activity and inhibits the pro-ferroptotic protein ACSL4 (potentially Nrf2-independent), synergistically reducing lipid peroxidation levels, thereby protecting follicular and ovarian function. This study was the first to reveal EA targeting ferroptosis to protect ovarian function in EMs, providing important experimental evidence for its use as a complementary therapy for EMs-related infertility.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eACSL4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAcyl-CoA synthetase long-chain family member 4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAMH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAnti-M\u0026uuml;llerian Hormone\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCOX-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCyclooxygenase-2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCV4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGuanyuan\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eEMs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eEndometriosis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eEA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eElectroacupuncture\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eE\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eEstradiol\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFSH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFollicle-Stimulating Hormone\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFTH1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFerritin Heavy Chain 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGPX4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGlutathione Peroxidase 4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGCs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGranulosa Cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGSH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGlutathione\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHO-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHeme oxygenase-1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eIVF-ET\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eIn Vitro Fertilization and Embryo Transfer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMDA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMalondialdehyde\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNrf2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNuclear factor erythroid 2-related factor 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNQO1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNAD(P)H quinone oxidoreductase 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eROS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eReactive Oxygen Species\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSLC7A11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSolute Carrier Family 7 Member 11 (xCT)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSOD2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSuperoxide dismutase 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eST36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eZusanli\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSP6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSanyinjiao\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was supported by the Key Laboratory of Acupuncture and Medicine Research of Ministry of Education. And we would like to sincerely thank all those who participated in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYoubing Xia and Liangjun Xia conceived and designed the study. Yan Zan, Xiaoquan Huang, Yu Zhuang, Junwei Li, Qian Zhu, and Tiantian Ma performed animal experiments. Yan Zan analyzed the data and wrote the manuscript. Liangjun Xia revised the manuscript. Youbing Xia and Liangjun Xia supervised the project. 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 National Natural Science Foundation of China (grant numbers: 82205251 and 82274638).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data collected or analyzed in the study are included in this published article and can be acquired from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Nanjing University of Traditional Chinese Medicine Ethics Committee reviewed and approved the experimental procedure, which complied with Guiding Opinions on the Treatment of Experimental Animals (Approval Number: 202403A036)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTaylor HS, Kotlyar AM, Flores VA. Endometriosis is a chronic systemic disease: clinical challenges and novel innovations. Lancet. 2021;397(10276):839\u0026ndash;52. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S0140-6736(21)00389-5\u003c/span\u003e\u003cspan address=\"10.1016/S0140-6736(21)00389-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZondervan KT, Becker CM, Missmer SA, Endometriosis. N Engl J Med. 2020;382(13):1244\u0026ndash;56. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1056/NEJMra1810764\u003c/span\u003e\u003cspan address=\"10.1056/NEJMra1810764\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHorne AW, Missmer SA. Pathophysiology, diagnosis, and management of endometriosis. BMJ. 2022;379:e070750. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1136/bmj-2022-070750\u003c/span\u003e\u003cspan address=\"10.1136/bmj-2022-070750\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaunders PTK, Horne AW. Endometriosis: etiology, pathobiology, and therapeutic prospects. Cell. 2021;184(11):2807\u0026ndash;24. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cell.2021.04.041\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2021.04.041\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBulun SE, Yilmaz BD, Sison C, et al. Endometriosis. Endocr Rev. 2019;40(4):1048\u0026ndash;79. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1210/er.2018-00242\u003c/span\u003e\u003cspan address=\"10.1210/er.2018-00242\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAllaire C, Bedaiwy MA, Yong PJ. Diagnosis and management of endometriosis. CMAJ. 2023;195(10):E363\u0026ndash;71. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1503/cmaj.220637\u003c/span\u003e\u003cspan address=\"10.1503/cmaj.220637\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZakhari A, Delpero E, McKeown S, Tomlinson G, Bougie O, Murji A. Endometriosis recurrence following postoperative hormonal suppression: a systematic review and meta-analysis. Hum Reprod Update. 2021;27(1):96\u0026ndash;107. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/humupd/dmaa033\u003c/span\u003e\u003cspan address=\"10.1093/humupd/dmaa033\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAta B, Somigliana E. Endometriosis, staging, infertility and assisted reproductive technology: time for a rethink. Reproductive Biomed Online. 2024;49(1):103943. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.rbmo.2024.103943\u003c/span\u003e\u003cspan address=\"10.1016/j.rbmo.2024.103943\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSampson JA. Peritoneal endometriosis due to the menstrual dissemination of endometrial tissue into the peritoneal cavity. Am J Obstet Gynecol. 1927;14(4):422\u0026ndash;69. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S0002-9378(15)30003-X\u003c/span\u003e\u003cspan address=\"10.1016/S0002-9378(15)30003-X\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGanz T, Macrophages, Metabolism I. Microbiol Spectr. 2016;4(5). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/microbiolspec.MCHD-0037-2016\u003c/span\u003e\u003cspan address=\"10.1128/microbiolspec.MCHD-0037-2016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNanda A, K T, Banerjee P, et al. Cytokines, Angiogenesis, and Extracellular Matrix Degradation are Augmented by Oxidative Stress in Endometriosis. Ann Lab Med. 2020;40(5):390\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3343/alm.2020.40.5.390\u003c/span\u003e\u003cspan address=\"10.3343/alm.2020.40.5.390\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo Y, Li L, Hu Q, et al. Iron overload increases the sensitivity of endometriosis stromal cells to ferroptosis via a PRC2-independent function of EZH2. Int J Biochem Cell Biol. 2024;169:106553. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.biocel.2024.106553\u003c/span\u003e\u003cspan address=\"10.1016/j.biocel.2024.106553\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClower L, Fleshman T, Geldenhuys WJ, Santanam N. Targeting oxidative stress involved in endometriosis and its pain. Biomolecules. 2022;12(8):1055. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/biom12081055\u003c/span\u003e\u003cspan address=\"10.3390/biom12081055\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePedachenko N et al. Serum anti-M\u0026uuml;llerian hormone, prolactin and estradiol concentrations in infertile women with endometriosis. \u003cem\u003eGynecol Endocrinol\u003c/em\u003e. 2021; 37(2): 162\u0026ndash;165. doi: 10.1080/09513590. 2020.1855634.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAta B, Turkgeldi E, Seyhan A, Urman B. Effect of hemostatic method on ovarian reserve following laparoscopic endometrioma excision: comparison of suture, hemostatic sealant, and bipolar desiccation. A systematic review and meta-analysis. J Minim Invasive Gynecol. 2015;22(3):363\u0026ndash;72. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jmig.2014.12.168\u003c/span\u003e\u003cspan address=\"10.1016/j.jmig.2014.12.168\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi A, Ni Z, Zhang J, Cai Z, Kuang Y, Yu C. Transferrin insufficiency and iron overload in follicular fluid contribute to oocyte dysmaturity in infertile women with advanced endometriosis. Front Endocrinol (Lausanne). 2020;11:391. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fendo.2020.00391\u003c/span\u003e\u003cspan address=\"10.3389/fendo.2020.00391\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan W, Yuan Z, Li M, Zhang Y, Nan F. Decreased oocyte quality in patients with endometriosis is closely related to abnormal granulosa cells. Front Endocrinol (Lausanne). 2023;14:1226687. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fendo.2023.1226687\u003c/span\u003e\u003cspan address=\"10.3389/fendo.2023.1226687\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMao J, Zhang J, Cai L, Cui Y, Liu J, Mao Y. Elevated prohibitin 1 expression mitigates glucose metabolism defects in granulosa cells of infertile patients with endometriosis. Mol Hum Reprod. 2022;28(6):gaac018. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/molehr/gaac018\u003c/span\u003e\u003cspan address=\"10.1093/molehr/gaac018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSampson JA. Peritoneal endometriosis due to the menstrual dissemination of endometrial tissue into the peritoneal cavity. Am J Obstet Gynecol. 1927;14(4):422\u0026ndash;69. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S0002-9378(15)30003-X\u003c/span\u003e\u003cspan address=\"10.1016/S0002-9378(15)30003-X\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang X, Stockwell BR, Conrad M. Ferroptosis: mechanisms, biology and role in disease. Nat Rev Mol Cell Biol. 2021;22(4):266\u0026ndash;82. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41580-020-00324-8\u003c/span\u003e\u003cspan address=\"10.1038/s41580-020-00324-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi G, Lin Y, Zhang Y, et al. Endometrial stromal cell ferroptosis promotes angiogenesis in endometriosis. Cell Death Discov. 2022;8(1):29. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41420-022-00821-z\u003c/span\u003e\u003cspan address=\"10.1038/s41420-022-00821-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWan Y, Gu C, Kong J, et al. Long noncoding RNA ADAMTS9-AS1 represses ferroptosis of endometrial stromal cells by regulating the miR-6516-5p/GPX4 axis in endometriosis. Sci Rep. 2022;12(1):2618. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41598-022-04963-z\u003c/span\u003e\u003cspan address=\"10.1038/s41598-022-04963-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang D, Minikes AM, Jiang X. Ferroptosis at the intersection of lipid met-abolism and cellular signaling. Mol Cell. 2022;82(12):2215\u0026ndash;27. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.molcel.2022.03.022\u003c/span\u003e\u003cspan address=\"10.1016/j.molcel.2022.03.022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNi Z, Li Y, Song D, et al. Iron-overloaded follicular fluid increases the risk of endometriosis-related infertility by triggering granulosa cell ferroptosis and oocyte dysmaturity. Cell Death Dis. 2022;13(7):579. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41419-022-05037-8\u003c/span\u003e\u003cspan address=\"10.1038/s41419-022-05037-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi S, Zhou Y, Huang Q, et al. Iron overload in endometriosis peritoneal fluid induces early embryo ferroptosis mediated by HMOX1. Cell Death Discov. 2021;7(1):355. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41420-021-00751-2\u003c/span\u003e\u003cspan address=\"10.1038/s41420-021-00751-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeonhardt H, Hellstr\u0026ouml;m M, Gull B, et al. Serum anti-M\u0026uuml;llerian hormone and ovarian morphology assessed by magnetic resonance imaging in response to acupuncture and exercise in women with polycystic ovary syndrome: secondary analyses of a randomized controlled trial. Acta Obstet Gynecol Scand. 2015;94(3):279\u0026ndash;87. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/aogs.12571\u003c/span\u003e\u003cspan address=\"10.1111/aogs.12571\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTapmeier TT, Rahmioglu N, Lin J, et al. Neuropeptide S receptor 1 is a nonhormonal treatment target in endometriosis. Sci Transl Med. 2021;13:eabd6469. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/scitranslmed.abd6469\u003c/span\u003e\u003cspan address=\"10.1126/scitranslmed.abd6469\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCora MC, Kooistra L, Travlos G. Vaginal Cytology of the Laboratory Rat and Mouse: Review and Criteria for the Staging of the Estrous Cycle Using Stained Vaginal Smears. Toxicol Pathol. 2015;43(6):776\u0026ndash;93. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1177/0192623315570339\u003c/span\u003e\u003cspan address=\"10.1177/0192623315570339\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMyers M, Britt KL, Wreford NG, Ebling FJ, Kerr JB. Methods for quantifying follicular numbers within the mouse ovary. Reproduction. 2004;127(5):569\u0026ndash;80. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1530/rep.1.00095\u003c/span\u003e\u003cspan address=\"10.1530/rep.1.00095\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing J, Zhao Q, Zhou Z, et al. Huayu Jiedu Fang Protects Ovarian Function in Mouse with Endometriosis Iron Overload by Inhibiting Ferroptosis. Evid Based Complement Alternat Med. 2022;2022:1406820. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1155/2022/1406820\u003c/span\u003e\u003cspan address=\"10.1155/2022/1406820\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoirier D, Nyachieo A, Romano A, et al. An irreversible inhibitor of 17β-hydroxysteroid dehydrogenase type 1 inhibits estradiol synthesis in human endometriosis lesions and induces regression of the non-human primate endometriosis. J Steroid Biochem Mol Biol. 2022;222:106136. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jsbmb.2022.106136\u003c/span\u003e\u003cspan address=\"10.1016/j.jsbmb.2022.106136\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan SJ, Jung SY, Wu SP, et al. Estrogen Receptor β Modulates Apoptosis Complexes and the Inflammasome to Drive the Pathogenesis of Endometriosis. Cell. 2015;163(4):960\u0026ndash;74. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cell.2015.10.034\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2015.10.034\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang L, Shi L, Li M, Yin X, Ji X. Oxidative stress in endometriosis: Sources, mechanisms and therapeutic potential of antioxidants (Review). Int J Mol Med. 2025;55(5):72. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3892/ijmm.2025.5513\u003c/span\u003e\u003cspan address=\"10.3892/ijmm.2025.5513\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYounis JS, Shapso N, Ben-Sira Y, Nelson SM, Izhaki I. Endometrioma surgery: a systematic review and meta-analysis of the effect on antral follicle count and anti-M\u0026uuml;llerian hormone. \u003cem\u003eAm J Obstet Gynecol\u003c/em\u003e. 2022;226(1):33\u0026ndash;51.e7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ajog\u003c/span\u003e\u003cspan address=\"10.1016/j.ajog\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.2021.06.102 Erratum in: \u003cem\u003eAm J Obstet Gynecol\u003c/em\u003e. 2023;229(1):88. doi:10.1016/j.ajog.2022.03.055.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMuzii L, Galati G, Mattei G, et al. Expectant, Medical, and Surgical Management of Ovarian Endometriomas. J Clin Med. 2023;12(5):1858. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/jcm12051858\u003c/span\u003e\u003cspan address=\"10.3390/jcm12051858\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Published 2023 Feb 26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMircea O, Bartha E, Gheorghe M, Irimia T, Vlădăreanu R, Puşcaşiu L. Ovarian Damage after Laparoscopic Cystectomy for Endometrioma. Chirurgia (Bucur). 2016;111(1):54\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou L, Wang L, Geng Q, et al. Endometriosis is associated with a lower-ed cumulative live birth rate: A retrospective matched cohort study including 3071 in vitro fertilization cycles. J Reprod Immunol. 2022;151:103631. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jri.2022.103631\u003c/span\u003e\u003cspan address=\"10.1016/j.jri.2022.103631\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClower L, Fleshman T, Geldenhuys WJ, Santanam N. Targeting Oxidative Stress Involved in Endometriosis and Its Pain. Biomolecules. 2022;12(8):1055. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/biom12081055\u003c/span\u003e\u003cspan address=\"10.3390/biom12081055\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDonnez J, Binda MM, Donnez O, Dolmans MM. Oxidative stress in the pelvic cavity and its role in the pathogenesis of endometriosis. Fertil Steril. 2016;106(5):1011\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.fertnstert.2016.07.1075\u003c/span\u003e\u003cspan address=\"10.1016/j.fertnstert.2016.07.1075\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNanda A, K T, Banerjee P, et al. Cytokines, Angiogenesis, and Extracellular Matrix Degradation are Augmented by Oxidative Stress in Endometriosis. Ann Lab Med. 2020;40(5):390\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3343/alm.2020.40.5.390\u003c/span\u003e\u003cspan address=\"10.3343/alm.2020.40.5.390\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing J, Mei S, Wang K, Cheng W, Sun S, Ni Z, Wang X, Yu C. Curcumin modulates oxidative stress to inhibit pyroptosis and improve the inflammatory microenvironment to treat endometriosis. Genes Dis. 2024;11(3):101053. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.gendis.2023.06.022\u003c/span\u003e\u003cspan address=\"10.1016/j.gendis.2023.06.022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmmar OF, Massarotti C, Mincheva M, et al. Oxidative stress and ovarian aging: from cellular mechanisms to diagnostics and treatment. Hum Reprod. 2024;39(7):1582\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/humrep/deae082\u003c/span\u003e\u003cspan address=\"10.1093/humrep/deae082\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Zhi W, Haoxu D, et al. Effects of electroacupuncture on the expression of hypothalamic neuropeptide Y and ghrelin in pubertal rats with polycystic ovary syndrome. PLoS ONE. 2022;17(6):e0259609. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0259609\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0259609\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXia L, Meng Q, Xi J et al. The synergistic effect of electroacupuncture and bone mesenchymal stem cell transplantation on repairing thin endometrial injury in rats. \u003cem\u003eStem Cell Res Ther.\u003c/em\u003e 2019;10(1):244. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13287\u003c/span\u003e\u003cspan address=\"10.1186/s13287\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e-019-1326-6 Erratum in: \u003cem\u003eStem Cell Res Ther.\u003c/em\u003e 2023;14(1):196. doi:10.1186/s13287-023-03421-5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi Y, Li L, Zhou J, et al. Efficacy of electroacupuncture in regulating the imbalance of AMH and FSH to improve follicle development and hyperandrogenism in PCOS rats. Biomed Pharmacother. 2019;113:108687. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.biopha.2019.108687\u003c/span\u003e\u003cspan address=\"10.1016/j.biopha.2019.108687\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu H, Nan S, Suo C, et al. Electro-Acupuncture Affects the Activity of the Hypothalamic-Pituitary-Ovary Axis in Female Rats. Front Physiol. 2019;10:466. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fphys.2019.00466\u003c/span\u003e\u003cspan address=\"10.3389/fphys.2019.00466\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Published 2019 Apr 24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang XC, Jin YJ, Ning R, et al. Electroacupuncture attenuates ferroptosis by promoting Nrf2 nuclear translocation and activating Nrf2/SLC7A11/GPX4 pathway in ischemic stroke. Chin Med. 2025;20(1):4. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13020-024-01047-0\u003c/span\u003e\u003cspan address=\"10.1186/s13020-024-01047-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang H, Shang Z, You L, et al. Electroacupuncture Pretreatment at Zusanli (ST36) Ameliorates Hepatic Ischemia/Reperfusion Injury in Mice by Reducing Oxidative Stress via Activating Vagus Nerve-Dependent Nrf2 Pathway. J Inflamm Res. 2023;16:1595\u0026ndash;610. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2147/JIR.S404087\u003c/span\u003e\u003cspan address=\"10.2147/JIR.S404087\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCong J, Li M, Wang Y, et al. Protective effects of electroacupuncture on polycystic ovary syndrome in rats: Down-regulating Alas2 to inhibit apoptosis, oxidative stress, and mitochondrial dysfunction in ovarian granulosa cells. Tissue Cell. 2023;82:102090. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.tice.2023.102090\u003c/span\u003e\u003cspan address=\"10.1016/j.tice.2023.102090\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\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":"Endometriosis, Ferroptosis, Oxidative stress, Electroacupuncture, Ovarian reserve","lastPublishedDoi":"10.21203/rs.3.rs-6928457/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6928457/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eEndometriosis (EMs) often leads to ovarian dysfunction and infertility. Its mechanism is closely associated with oxidative stress and ferroptosis induced by pelvic iron overload. Electroacupuncture (EA) has potential in treating reproductive disorders, but its mechanism of action on ovarian ferroptosis in EMs remains unclear. This study aimed to investigate the protective effect of EA on ovarian function in an EMs mouse model and its underlying molecular mechanisms.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA mouse model simulating chronic hemorrhage in EMs was used. Mice were randomly divided into a Normal group, Sham group, EMs group, and EA group. Ovarian function (estrous cycle, ovarian weight/index, serum sex hormones, ovarian histopathology), iron metabolism levels (peritoneal fluid/ovarian Fe\u003csup\u003e2\u003c/sup\u003e⁺, ferritin, Prussian blue staining), oxidative stress levels (ovarian ROS, GSH content, T-SOD activity, Nrf2 and its downstream HO-1/SOD2/NQO1 mRNA), and ferroptosis levels (ferroptosis markers GPX4, SLC7A11, FTH1, ACSL4, COX-2 protein and mRNA, and MDA levels) were assessed using qRT-PCR, Western blot, IHC, colorimetric methods, and histochemistry.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eEA intervention significantly improved ovarian function in EMs mice. This was reflected in the normalization of the estrous cycle, increased ovarian weight/index, restored serum sex hormone levels, increased number of primordial follicles, and reduced atretic follicles. EA effectively alleviated ovarian iron overload (reduced Fe\u003csup\u003e2\u003c/sup\u003e⁺, ferritin, iron deposition) and oxidative stress (inhibited ROS, increased GSH, enhanced T-SOD activity). Mechanistically, EA activated the Nrf2 pathway (upregulated Nrf2, HO-1, SOD2, NQO1), upregulated key anti-ferroptosis molecules (GPX4, SLC7A11) and ferritin (FTH1). Furthermore, EA downregulated pro-ferroptosis factors (ACSL4, COX-2) and reduced lipid peroxidation (MDA). The results demonstrated that EA effectively blocked the ferroptosis process in ovarian granulosa cells by activating the Nrf2 pathway to upregulate GPX4 activity and suppressing ACSL4-mediated lipid peroxidation.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis study confirms that iron overload\u0026ndash;oxidative stress\u0026ndash;granulosa cell ferroptosis is a key pathological mechanism of EMs-induced ovarian damage. EA effectively inhibits ovarian granulosa cell ferroptosis by activating the Nrf2 pathway to upregulate GPX4 activity and suppress ACSL4-mediated lipid peroxidation, thereby ameliorating EMs-associated ovarian dysfunction. This provides important experimental evidence supporting EA as a complementary therapy for EMs-related infertility.\u003c/p\u003e","manuscriptTitle":"Electroacupuncture Ameliorates Endometriosis-Associated Ovarian Dysfunction by Activating Nrf2 Pathway to Upregulate GPX4 Function and Inhibiting Ferroptosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-25 17:29:13","doi":"10.21203/rs.3.rs-6928457/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":"68550fe6-2ba8-43c2-8778-a4627483b9f1","owner":[],"postedDate":"June 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-16T09:23:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-25 17:29:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6928457","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6928457","identity":"rs-6928457","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}