N-acetylcysteine Supplementation Improves Endocrine-Metabolism Profiles and Ovulation Induction Efficacy in Polycystic Ovary Syndrome

N-acetylcysteine Supplementation Improves Endocrine-Metabolism Profiles and Ovulation Induction Efficacy in Polycystic Ovary Syndrome | 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 N-acetylcysteine Supplementation Improves Endocrine-Metabolism Profiles and Ovulation Induction Efficacy in Polycystic Ovary Syndrome Yu-Qing Fang, Hui Ding, Tao Li, Xiao-Jie Zhao, Dan Luo, Yi Liu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4485542/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Oct, 2024 Read the published version in Journal of Ovarian Research → Version 1 posted 10 You are reading this latest preprint version Abstract Background Polycystic ovary syndrome (PCOS) affects 6–20% of women worldwide, with insulin resistance and hyperinsulinemia occurring in 50%-70% of patients. Hyperinsulinemia exacerbates oxidative stress, contributing to PCOS pathogenesis. N-acetylcysteine (NAC) is an antioxidant and insulin sensitizer that shows promise as a therapeutic for PCOS. Our current study aimed to investigate the effects of NAC supplementation on endocrine-metabolic parameters in PCOS mice and its effect on ovulation induction (OI) efficacy in women with PCOS. Methods: Female C57BL/6 mice were orally administered letrozole (LE) to induce PCOS and then randomly divided into groups receiving daily oral administration of 160 mg/kg NAC (PCOS + NAC group), 200 mg/kg metformin (PCOS + Met group), or 0.5% carboxymethyl cellulose (drug solvent) (pure PCOS group) for 12 days. Healthy female mice served as pure controls. Estrous cycles were monitored during the intervention. Metabolic and hormone levels, ovarian phenotypes, antioxidant activity in ovarian tissues, and oxidative stress levels in oocytes were assessed postintervention. This pragmatic, randomized, controlled clinical study included 230 women with PCOS who were randomly assigned to the NAC group (1.8 g/day oral NAC, n = 115) or the control group (n = 115). Patients in both groups underwent ≤ 3 cycles of OI with sequential LE and urinary follicle-stimulating hormone (uFSH). Cycle characteristics and pregnancy outcomes were compared between groups. Results Similar to metformin, NAC supplementation significantly improved the estrous cycles and ovarian phenotypes of PCOS mice; reduced the LH concentration, LH/FSH ratio, and T level; and increased glucose clearance and insulin sensitivity. Notably, NAC significantly reduced oocyte ROS levels and increased the mitochondrial membrane potential in PCOS mice. Additionally, NAC significantly enhanced enzymatic and nonenzymatic antioxidant activities in PCOS mouse ovaries, whereas metformin had no such effect. In the clinical trial, compared to women in the control group, women receiving NAC had significantly lower average uFSH dosage and duration (p < 0.005) and significantly greater clinical pregnancy rates per OI cycle and cumulative clinical pregnancy rates per patient (p < 0.005). Conclusion NAC supplementation improved endocrine-metabolic parameters in PCOS mice and significantly enhanced OI efficacy with sequential LE and uFSH in women with PCOS. Therefore, NAC could be a valuable adjuvant in OI for women with PCOS. Polycystic ovary syndrome N-acetylcysteine Insulin resistance Metformin Oxidative stress Ovulation induction Pregnancy rate Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Polycystic ovary syndrome (PCOS) is a heterogeneous familial disorder [ 1 ] . Depending on different diagnostic criteria, the prevalence among reproductive-age women fluctuates between 6% and 20% [ 2 ] . From an evolutionary medicine perspective, the overlap of risk alleles for PCOS between European and Chinese women suggests that PCOS is an ancient disorder [ 3 ] . A considerable proportion of PCOS patients have defects in insulin secretion and insulin resistance, resulting in hyperinsulinemia in more than 50% of cases. Hyperinsulinemia stimulates testosterone secretion from the ovaries and adrenal glands and inhibits hepatic synthesis of sex hormone-binding globulin, thereby increasing circulating free testosterone levels. Hyperinsulinemia also promotes oxidative stress by generating reactive oxygen species (ROS), which is another important pathogenic factor in PCOS. Currently, metformin is the most commonly recommended drug for treating insulin resistance associated with PCOS [ 4 ] . Although metformin has been demonstrated to be an effective treatment for PCOS, it is associated with significant adverse effects. Over 30% of patients experience noticeable gastrointestinal side effects during the initial treatment period, including bloating, diarrhea, constipation, nausea, vomiting, and an increase in serum homocysteine levels, which may lead to intolerance and discontinuation of the drug [ 5 ] . Therefore, clinicians have been exploring new insulin sensitizers with improved tolerability for treating PCOS. Among them, N-acetylcysteine (NAC) is considered a promising drug with potential applications [ 6 ] . NAC is the acetylated precursor of L-cysteine and reduced glutathione. It has been established as a potent cell-permeable antioxidant that effectively prevents cell apoptosis and promotes cell survival through antioxidant stress [ 7 , 8 ] . The antioxidant activity of NAC is attributed to its thiol group, which enhances the activity of glutathione S-transferase, thereby protecting target cells and cell membranes [ 9 ] . In animal experiments, Fan et al. showed that NAC protects against oxidative stress toxicity and mitochondrial functional damage induced by repeated ovulation stimulation [ 10 ] . Furthermore, in vitro studies have demonstrated that NAC activates insulin secretion in pancreatic cells [ 11 ] and modulates insulin receptors in human erythrocytes [ 12 ] . However, there is limited research on the effects of NAC on endocrine-metabolic profiles and ovarian antioxidant enzymatic systems in PCOS animal models. In addition, several clinical studies have explored the effects of NAC supplementation in various induction ovulation (OI) protocols for women with PCOS. In a randomized controlled trial (RCT) involving 150 PCOS women with clomiphene citrate (CC)-resistant, Rizk AY et al. [ 13 ] reported that the use of NAC (1.2 g/day) as an adjuvant to CC significantly increased ovulation and pregnancy rates compared to the use of a placebo (1.3% vs. 49.3%, 0 vs. 21.3%) [ 13 ] . However, in a 2017 RCT, Behrouzi Lak et al. reported that NAC supplementation did not improve clinical pregnancy rates in CC combined with LE induction of ovulation followed by intrauterine insemination. However, it is important to note that the study had a small sample size (only 97 participants), and the NAC supplementation group already showed a trend toward higher clinical pregnancy rates (32.7% vs. 18.8%) [ 14 ] . Therefore, further clinical studies with sufficient sample sizes are needed to explore the application of NAC supplementation in OI for women with PCOS. In this study, we first investigated the effects of NAC supplementation on endocrine-metabolic parameters and ovarian/oocyte oxidative stress responses in LE-induced PCOS mice through in vivo experiments. After confirming that NAC supplementation significantly improved endocrine metabolism profiles and ovarian/oocyte antioxidant capacity in PCOS mice, a pragmatic clinical trial (PCT) was conducted. PCOS patients with anovulation or oligo-ovulation were recruited to observe the effects of NAC supplementation on the characteristics of OI cycles and clinical pregnancy outcomes following sequential LE and urinary follicle-stimulating hormone (uFSH) treatment. The aim of this study was to elucidate the potential benefits of NAC supplementation in OI for PCOS patients with anovulation or oligo-ovulation and to provide more foundational and clinical data for implementing NAC supplementation therapy in PCOS patients. Materials and methods PCOS mouse model and grouping intervention Three-week-old SPF-grade C57BL/6 female mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and housed in the Laboratory Animal Center of Huazhong University of Science and Technology. The mice were maintained at a constant temperature of 26°C with a 12-hour light/dark cycle (light period: 07:00 to 19:00) and had ad libitum access to food and water. After one week of acclimatization, 15 mice were randomly selected to constitute the control group. The mice were administered 0.5% carboxymethyl cellulose (CMC; Shanghai Biochemical Technology Co., Ltd., Shanghai, China) solution (1 mg/kg) via gavage for 21 consecutive days, followed by no further treatment for the next 12 days. The remaining mice were used to construct the PCOS model using LE, following methods reported in previous literature [ 15 , 16 ] . Briefly, LE (Furui, Jiangsu Hengrui Medicine Co., Ltd., Lianyungang, China) was dissolved in a 0.5% CMC solution and administered by gavage at a dosage of 1 mg/kg/day for 21 consecutive days to induce the PCOS model. During the last 10 days of LE administration, the estrous cycles of the mice were monitored daily. After 21 days of LE treatment, three mice from the control group and three from the PCOS model group were randomly selected at the diestrus stage and anesthetized with isoflurane, and blood samples were collected from the retro-orbital plexus. The mice were then euthanized by cervical dislocation, and ovarian tissues were collected to evaluate the success of the PCOS model. The successfully generated PCOS model mice were then randomly divided into three groups: a pure PCOS group (model control), a PCOS + metformin (Met) group, and a PCOS + NAC group. Starting on day 22, the three groups of PCOS mice were administered 0.5% CMC, 200 mg/kg/day metformin (Shanghai Squibb Pharmaceutical Co., Ltd.), or 160 mg/kg/day NAC [ 17 ] via gavage for 12 days. During the intervention period, estrous cycle monitoring continued. After the intervention, six mice from each group were euthanized in the same manner as described above, and blood samples and ovarian tissues were collected. The remaining six mice in each group were treated with pregnant mare serum gonadotropin (PMSG) to obtain oocytes. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Huazhong University of Science and Technology (approval number: 2585) and were conducted in accordance with international ethical guidelines and the relevant requirements of the ethics committee of Huazhong University of Science and Technology. Mouse estrous cycle assessment Vaginal secretions were collected daily between 12:00 and 13:00 from each group of mice and smeared onto slides. These slides were then examined under an optical microscope to determine the stage of the estrous cycle for each mouse. Diestrus was characterized by predominantly leukocytic vaginal smears, while proestrus exhibited a high concentration of nucleated cells. The estrus was typified by vaginal smears primarily composed of cornified epithelial cells, whereas metestrus displayed the presence of both cornified epithelial cells and leukocytes. Glucose tolerance test (GTT) and insulin tolerance test (ITT) Prior to the glucose tolerance test, the mice were fasted for 16 hours (from 17:00 to 09:00 the next day) with ad libitum access to water. Blood samples were drawn from the tail tips of the mice before and 30–60, 90, and 120 minutes after intraperitoneal administration of D-glucose (2.0 g/kg body weight). The blood glucose levels were then tested with an Accu-Chek glucose monitoring system (Roche Diagnostics). In the insulin tolerance test (ITT), mice were fasted for four hours (with ad libitum access to water) before receiving an intraperitoneal injection of insulin (1 U/kg body weight). Blood glucose levels were monitored at 0, 15, 30, and 45 minutes after insulin infusion. Infrared thermography and core body temperature measurement Mice were individually housed in cages and subjected to a 4°C cold chamber for a maximum of 4 hours, with continuous access to food and water. Images were captured using an infrared digital thermal camera (E60: compact infrared thermal imaging camera; FLIR), and the data were analyzed using FLIR Quick Report software (FLIR ResearchIR Max 3.4; FLIR). The core body temperature of each group of mice was assessed using rectal probes connected to digital thermometers. Assessment of hormone levels in mouse blood Isoflurane was administered to anesthetize the mice before blood samples were collected from the retro-orbital plexus. Following a two-hour incubation period at room temperature, the samples were centrifuged at 3000 rpm for 15 minutes. The resulting supernatant (plasma) was collected and stored at -80°C for subsequent analysis. Plasma concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone (T) were determined using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions (Quanzhou RuiXin Biotechnology Co., Ltd., Fujian, China). The sensitivities of the ELISA kits for LH, FSH, and T were 0.1 mIU/L, 0.1 mIU/mL, and 0.1 ng/mL, respectively. The intra- and interassay coefficients of variation for all three kits were less than 10%. Mouse ovarian tissue sectioning and staining analysis Ovarian tissues from each group of mice were preserved overnight in 4% formaldehyde at room temperature. Following embedding in paraffin, the tissues were sectioned serially and then stained with hematoxylin and eosin (H&E). H&E staining was carried out as previously described [ 18 ] . Briefly, the mouse ovaries were halved and fixed in 4% paraformaldehyde. Following embedding in paraffin wax, the tissue was sliced into 5 µm thick sections and mounted onto glass slides. These sections then underwent a series of steps, including deparaffinization, hydration, H&E staining, dehydration, and mounting. Subsequently, images were captured under a microscope, and the number of follicles and corpora lutea at each stage was determined. Ovarian follicles and corpora lutea were classified using Pederson's classification system [ 19 ] . Briefly, primordial follicles are described as having a compact oocyte surrounded by a single layer of flattened granulosa cells (GCs). Primary follicles are identified by an enlarged oocyte encircled by a single layer of cuboidal GCs. Secondary follicles are defined by an enlarged oocyte surrounded by at least a partial or complete second layer of cuboidal GCs. Antral follicles are characterized by the presence of areas of follicular fluid or a single large antral space. To avoid double counting, primordial, primary, and secondary follicles were counted once every 10 consecutive sections, while antral follicles were counted once every 40 consecutive sections [ 20 ] . Mouse oocyte collection After intraperitoneal injection of 10 IU PMSG, mice from each group received an intraperitoneal injection of 5 IU human chorionic gonadotropin (HCG) 48 hours later. Approximately 14–16 hours post-HCG administration, the mice were euthanized by cervical dislocation. Cumulus-oocyte complexes (COCs) were then retrieved from the ampullary region of the fallopian tubes. The collected COCs were transferred to growth medium containing hyaluronidase (Solarbia, catalog number H8030) to remove the surrounding GCs. Mature mouse oocytes at the metaphase II (MII) stage were obtained, washed, and placed in M16 medium (Sigma‒Aldrich). Subsequently, the oocytes were cultured at 37°C in a 5% CO2 incubator. Quantification of mitochondrial membrane potential (MMP) The oocyte MMP was measured as described in previous studies [ 21 ] . Oocytes were exposed to a JC-1 working solution (Beyotime Biotech, Shanghai, China) and maintained at 37°C for 20 minutes. After two washes with JC-1 staining buffer (Beyotime Biotech, Shanghai, China), the oocytes were examined using a fluorescence microscope. In mitochondria with low membrane potential, the JC-1 probe exists in its monomeric form, emitting green fluorescence. Conversely, in mitochondria with high membrane potential, the JC-1 probe underwent J-aggregation, emitting red fluorescence. The degree of mitochondrial depolarization was assessed by the ratio of red to green fluorescence. Quantification of ROS levels Oocytes were incubated in a dark environment with a diluted solution of the fluorescence probe 2',7'-dichlorofluorescin diacetate (DCFH-DA) (Beyotime Biotech, Shanghai, China) at 37°C for 20 minutes. After being washed with M2 media, images were captured using a fluorescence microscope. Upon exposure to reactive oxygen species (ROS), DCFH emits green fluorescence, and the intensity of the fluorescence directly correlates with the ROS level. Biochemical analysis Mouse ovarian tissue was rapidly homogenized in an ice-cold 0.9% NaCl solution (10% w/v). The supernatant from centrifugation (3500 rpm, 15 minutes, 25°C) was utilized for biochemical analysis. The tissue protein concentration was determined using bovine serum albumin as a standard. The activities of reduced glutathione (GSH), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT) in mouse ovarian tissue homogenates were measured using assay kits (Beyotime Biotech, Shanghai, China) following the protocols provided by the manufacturer. Pragmatic clinical trial and participants PCT was conducted at Union Hospital, Tongji Medical College, Huazhong University of Science and Technology to evaluate the effects of NAC supplementation in OI for PCOS patients with anovulation or oligo-ovulation. Participants were responsible for covering the costs of examinations and medications, with no additional compensation provided. The study received approval from the medical ethics committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (approval number 2023 − 0353; approval date June 6, 2023) and was registered on the Chinese Clinical Trial Registry ( www.chictr.org.cn ; trial registration number: ChiCTR2300077709, registration date: July 1, 2023.). All participants provided informed consent before enrolling in the study. Detailed information about the trial protocol is available in the Chinese Clinical Trial Registry ( https://www.chictr.org.cn/bin/project/edit?pid=205777 ). The study recruited PCOS patients suffering from anovulation or oligo-ovulation. The diagnosis of PCOS was based on the modified Rotterdam criteria [ 22 ] , requiring the fulfillment of any two of the following three criteria: oligo-ovulation or anovulation, clinical or biochemical signs of hyperandrogenism, and polycystic ovaries, while excluding diseases such as congenital adrenal hyperplasia or tumors, folliculogenesis abnormalities, Cushing's syndrome, and androgen-secreting ovarian tumors. Additionally, the subjects met the following inclusion criteria: ① aged ≥ 21 years and ≤ 38 years. ② Observation of at least one patent fallopian tube during hysterosalpingography or laparoscopy. ③ A BMI < 35 kg/m 2 . ④ A male partner must have a minimum sperm concentration of 15 million per milliliter, based on the World Health Organization's criteria. ⑤ Neither the patients nor their male partners experienced any sexual dysfunction, and both agreed to engage in regular intercourse to achieve pregnancy. The exclusion criteria were as follows: ① Infertility due to causes other than PCOS-related ovulatory disorders. ② Abnormal thyroid-stimulating hormone levels. ③ Use of oral contraceptives, metformin, inositol, N-acetylcysteine, or other medications in the past three months. ④ Presence of large ovarian cysts (≥ 5 cm), uterine malformations, intrauterine adhesions, submucosal fibroids, etc. ⑤ Presence of concurrent pregnancy or surgical or medical conditions, including but not limited to uncontrolled diabetes, hypertension, liver disease, kidney disease, thyroid disease, adrenal disorders, autoimmune diseases, etc. PCOS women who met the inclusion criteria and did not have the exclusion criteria were provided with detailed information about the study's objectives, procedures, potential benefits, and risks. After providing fully informed consent, they were enrolled in the study by signing an informed consent form. Patient interventions Using a stratified randomization method, eligible participants were assigned in a 1:1 ratio to either the control group or the NAC group. Both groups underwent a sequential LE and urinary follicle-stimulating hormone (uFSH) protocol for OI as described below. In the control group, all patients underwent baseline ultrasound examination starting from the 2nd to the 4th day of spontaneous or induced menstruation. Those meeting the criteria for OI initiated LE (Furui, Jiangsu Hengrui Medicine Co., Ltd., Lianyungang, China) at a dose of 2.5 mg daily for 5 consecutive days. The day after completing LE, they received intramuscular injections of uFSH (Urofollitropin, Lizhu Pharmaceutical Factory, Zhuhai, China) at a dosage of 75–150 IU daily. Regular vaginal ultrasound examinations were conducted to monitor follicular growth. Upon the attainment of at least one follicle with a diameter of 18–20 mm, recombinant human chorionic gonadotropin (rhCG) (Ovidrel, Merck Serono, Aubonne, Switzerland) was administered at a dose of 250 µg to induce ovulation, with instructions for timed intercourse 12–36 hours after rhCG injection. Subsequent vaginal ultrasounds were performed every other day postrhCG injection to assess ovulation. Following ovulation, patients were prescribed oral progesterone capsules (Laiting, Zhejiang Medicine Co., Ltd., Xinchang, China) for 14 days. Urine pregnancy testing was subsequently conducted to confirm pregnancy. In the NAC group, women with PCOS were administered oral NAC (Jinkangsuli, Zhejiang Jinhua Kang'enbei Biopharmaceutical Co., Ltd., Jinhua, China) at a dose of 1.8 g/day (dosage: 0.6 g, orally, three times a day) from the 2nd to the 4th day of the menstrual cycle for five consecutive days. The remaining OI procedures mirrored those of the control group. For both groups, pregnancy outcomes were documented after each OI cycle, and the intervention study was considered complete upon clinical pregnancy. The follow-up period for both groups was limited to ≤ 3 OI cycles. Randomization and masking in the clinical trial The study utilized block randomization with a block size of 4. Randomized sequences were generated using SPSS software (Version 26, IBM, Armonk, NY) and were securely stored in sealed opaque envelopes. Eligible participants who met the inclusion and exclusion criteria and provided informed consent were assigned by a third party (nurse) in a 1:1 ratio to either the NAC group or the control group based on their enrollment order. Ultrasound examiners, statisticians, and outcome assessors remained blinded to the allocation, while treating physicians and participants were not blinded. Calculation of the sample size for the clinical trial The current study is a pragmatic, randomized, parallel, noncontrolled superiority trial. Sample size calculations for both groups were performed using PASS software version 11.0 (NCSS, LLC. Kaysville, Utah, USA). Our aim was to investigate whether NAC supplementation enhances the clinical pregnancy rate in women with PCOS who have undergone OI compared to women in the control group. Based on prior clinical research conducted at our center, the clinical pregnancy rate per OI cycle for PCOS-related ovulatory disorders using sequential LE + uFSH therapy was determined to be 23% [ 23 ] . Our objective was to assess the difference in pregnancy rates between the two groups at a significance level of 10%, with α = 0.05 (one-sided) and a test power of 0.8, resulting in a minimum required induction cycle count of 219 per group. This calculation accounts for a dropout rate of 10%. Consequently, 239 cycles of patients were planned for each group in this study. Outcome measures of the clinical trial The primary outcome measure was the clinical pregnancy rate per OI cycle. Clinical pregnancy was defined as the presence of one or more gestational sacs observed via ultrasound, including intrauterine pregnancy, ectopic pregnancy, and combined intra- and extrauterine pregnancy, with or without visible fetal heartbeats. Multiple gestational sacs were counted as one clinical pregnancy. Secondary outcome measures included the ongoing pregnancy rate, early miscarriage rate, biochemical pregnancy rate, ovulation rate, incidence of ovarian hyperstimulation syndrome (OHSS) and treatment discontinuation rate. Ongoing pregnancy is defined as an intrauterine pregnancy with at least one visible fetal heartbeat observed after 12 weeks of gestation. Biochemical pregnancy was defined as a serum level of human chorionic gonadotropin greater than 10 IU/L. OHSS was defined by the Golan criteria [ 24 ] . Mild OHSS involves abdominal distension and discomfort, with possible nausea, vomiting, or diarrhea. Moderate OHSS includes these symptoms plus ultrasonographic ascites. Severe OHSS features clinical ascites and/or hydrothorax, breathing difficulties, and may involve hemoconcentration, coagulation issues, and reduced renal function. Statistical analysis In animal experiments, comparisons between groups were conducted using independent samples t tests or one-way analysis of variance (ANOVA) with Tukey's post hoc test (for continuous variables) or the chi-square test (for categorical variables). In clinical PCT studies, comparisons of outcome measures between the two study groups were conducted using intention-to-treat (ITT) analyses, with per-protocol (PP) analysis also performed as a sensitivity analysis ( Supplementary Tables S1 and S2 ). Descriptive analysis was employed to compare baseline data between the two groups, while balance analysis between the two groups was assessed by analyzing different baseline data. For continuous variables, the normality of the distribution was initially estimated using frequency histograms and the Kolmogorov‒Smirnov test. Normally distributed continuous variables are presented as the means ± SDs, and statistical comparisons were made using Student's t test. Alternatively, if continuous variables did not follow a normal distribution, they are presented as the median and interquartile range (IQR), and differences between groups were analyzed using the Mann‒Whitney U test. Categorical variables were described in terms of n (%), with the chi-square test used for difference testing and the Fisher exact probability test employed when expected frequencies were less than 5. Statistical significance was determined using one-sided tests, and the primary outcome measure was reported using superiority tests. All the statistical analyses were conducted using the statistical software package SPSS V.25.0 (SPSS). Statistical significance was defined as p < 0.05. Results NAC supplementation reverses estrous cycle irregularities and the ovarian phenotype in PCOS mice An irregular menstrual cycle is one of the primary diagnostic criteria for PCOS [ 25 ] . In the present study, vaginal cytology of female mice after 21 days of LE intervention revealed a lack of estrous cycle regularity. Compared with those in the control group, the serum LH levels and LH/FSH ratios were considerably greater in the LE intervention mice. H&E staining of ovarian tissue sections revealed that the LE intervention mice (PCOS model mice) had considerably more follicles and fewer corpora lutea ( Supplementary Figure S1 ), confirming successful modeling of PCOS in mice induced by LE intervention. Following a 12-day random group intervention in the PCOS mice (Fig. 1 A), vaginal cytology revealed that 83.33% (9/12) of the mice in the PCOS + NAC group and 75.00% (9/12) of the mice in the PCOS + Met group had normalized estrous cycles. In contrast, the pure PCOS group showed no cyclicity throughout the 12-day intervention period, but the 12 control mice had a normal estrous cycle (Fig. 1 B-E). Notably, H&E staining of ovarian tissue sections revealed that, compared to the pure PCOS group, the PCOS + NAC and PCOS + Met groups had considerably more corpora lutea and fewer follicles (Fig. 1 F-I). This finding suggested that NAC intervention can dramatically improve estrous cycle abnormalities and the ovarian phenotype in PCOS mice, with effects similar to those of Met. NAC supplementation alleviates endocrine and metabolic disorders in LE-induced PCOS mice. PCOS patients often have endocrine and metabolic disorders. To determine the effect of NAC intervention on the endocrine status of PCOS mice, we measured sex hormone levels in each group after the intervention. Although the serum FSH levels did not differ significantly among the groups, the serum LH levels and LH/FSH ratios were significantly lower in the PCOS + NAC and PCOS + Met groups than in the pure PCOS group, returning to levels similar to those in the pure control group (Fig. 2 A-C). Furthermore, the serum T levels in the PCOS + NAC and PCOS + Met groups were considerably lower than those in the PCOS group following the intervention (Fig. 2 D). These results suggest that NAC supplementation can alleviate endocrine disorders in LE-induced PCOS mice. To investigate whether NAC intervention affects glucose metabolism in LE-induced PCOS mice, we conducted GTTs and ITTs. After 12 days of intervention, the PCOS + NAC and PCOS + Met groups had significantly lower fasting blood glucose (4.98 ± 0.39, 4.92 ± 0.71 vs 6.59 ± 0.64; both p < 0.01) and fasting insulin (7.20 ± 0.42, 7.84 ± 0.31 vs 8.94 ± 0.52; both p < 0.05) levels than the pure PCOS group and were similar to the pure control group. The area under the GTT/ITT curve indicated that the pure PCOS group exhibited significant insulin resistance compared to the pure control group, while NAC and Met intervention improved insulin sensitivity and enhanced plasma glucose clearance in LE-induced PCOS mice (Fig. 2 E-H). Studies have reported that adaptive thermogenesis in response to stimuli such as postprandial or cold exposure is reduced in PCOS patients [ 26 ] . In mice, most energy expenditure is used to maintain core body temperature, and measuring core body temperature can directly reflect energy expenditure [ 27 ] . In this study, compared with the control group, the pure PCOS group exhibited a significant decrease in body temperature under cold stimulation. However, NAC and Met significantly stimulated thermogenesis in PCOS mice, as indicated by a significant increase in body temperature in these two groups (Fig. 2 I-J). Due to the glucose metabolism and thermogenesis-inducing effects observed with NAC intervention, the average body weight of the PCOS + NAC group was significantly lower than that of the pure PCOS group after 12 days of intervention and was comparable to the average body weight of the PCOS + Met and pure control groups (Fig. 2 K). NAC supplementation improves the oxidative stress response in oocytes of LE-induced PCOS mice To evaluate the effect of NAC supplementation on the oxidative stress response in the oocytes of LE-induced PCOS mice, we used JCI and DCFH-DA fluorescent probes to analyze the MMP and ROS levels in the oocytes from each group after the intervention. Compared to those in the pure control group, oocytes from the pure PCOS group had significantly lower MMP and significantly higher intracellular ROS levels at the end of the intervention. However, NAC intervention, similar to Met intervention, significantly increased the MMP and decreased intracellular ROS levels in oocytes from LE-induced PCOS mice. These findings suggest that NAC has the potential to counteract the excessive oxidative stress response in the oocytes of PCOS mice, possibly improving oocyte quality and pregnancy outcomes. (Fig. 3 A-D). NAC supplementation increased the activity levels of antioxidant enzymes in mouse ovarian tissue Compared to those in the pure control group, the ovarian tissues of mice in the LE-induced PCOS group exhibited significantly lower activity of the enzymatic antioxidants SOD, GSH-Px, and CAT, as well as the nonenzymatic antioxidant GSH. In contrast, the activities of GSH-Px, SOD, and GSH in the ovarian tissue of the PCOS + NAC group were significantly greater than those in the ovarian tissue of the PCOS group. However, the activity levels of these enzymatic and nonenzymatic antioxidants in the ovarian tissue of the PCOS + Met group did not differ significantly from those in the ovarian tissue of the PCOS group (Fig. 3 E-H). This shows that NAC treatment can boost the activity of the antioxidant system in ovarian tissue. Comparisons of baseline characteristics between the NAC group and control group Based on promising results from in vivo animal studies, we designed a pragmatic randomized, parallel-group controlled clinical study to determine the therapeutic efficacy of NAC supplementation in improving pregnancy outcomes for PCOS-related infertility patients. In the current PCT, 252 women with PCOS were recruited and screened, 230 of whom eventually enrolled in the research. Among them, 115 PCOS women were randomly assigned to the NAC group, with the remaining 115 PCOS women allocated to the control group. The patients' baseline characteristics were similar across the two study groups ( see Table 1 ). During the intervention period, the NAC group had two incidences of natural conception, while the control group had three (these five participants were omitted from the final statistical analysis). Overall, the NAC group had 113 subjects and 284 complete OI cycles, whereas 112 patients in the control group had 279 complete OI cycles. (Fig. 4 ) Table 1 Baseline characteristics of PCOS women in the NAC group and control groups. Characteristic NAC group n = 113 Control group n = 112 p value t or U value / χ2 value Risk ratio (95% CI) Age (years) 28.50 (2.81) 28.02(2.99) 0.2092 1.259 - BMI (kg/m 2 ) 23.36(20.70, 26.24) 24.17(22.04, 27.04) 0.1748 5665 - Infertility duration (months) 12.00(6.00, 23.50) 12.00(6.00, 23.50) 0.6573 6113 - Serum basal FSH (mIU/ml) 6.05(1.50) 5.79(1.63) 0.2284 1.208 - Serum basal LH(mIU/ml) 8.12(4.75 to 12.08) 9.61(6.13 to 13.79) 0.0724 5451 - LH/FSH ratio 1.66(1.25, 2.45) 1.64(1.10, 2.41) 0.3366 5805 - Serum basal E2(pg/ml) 33.71(26.00 to 45.33) 35.35(26.00 to 46.08) 0.4381 5841 - Testosterone (ng/mL) 0.54(0.40 to 0.78) 0.58(0.42 to 0.84) 0.3098 5516 - Prolactin (ng/ml) 13.89(10.96 to 19.04) 13.19(10.45 to 19.35) 0.4698 5186 - The proportion of hyperprolactinemia (> 30 ng/ml) %(n) 7.96% (9/113) 6.25% (7/112) 0.617 0.25 1.30(0.47–3.62) AMH (ng/ml) 8.76(6.17 to 11.76) 8.40(6.04 to 11.13) 0.7751 6188 - HOMA-IR 3.27(2.42 to 4.73) 3.28(2.21(to 4.83) 0.8925 6262 - The proportion of women with insulin resistance (HOMA-IR > 2.69) 67.26% (76/113) 65.18% (73/112) 0.742 0.109 1.10(0.63–1.91) Fasting insulin (mIU/L) 14.14(10.87 to 20.58) 14.02(10.20 to 21.00) 0.8633 6188 - Fasting glucose (mmol/L) 5.20 (4.90 to 5.50) 5.20 (4.90 to 5.50) 0.7856 6195 - Menses, %(n) Oligomenorrhoea 40.71% (46/113) 42.86% (48/112) 0.353 -0.062 - Amenorrhoea 7.08% (8/113) 10.71% (12/112) Irregular 41.59% (47/113) 41.07% (46/112) Regular 10.61% (12/113) 5.36% (6/112) Primary infertility, %(n) 80.53% (91/113) 75.89% (85/112) 0.399 0.71 1.31(0.70–2.48) Primiparity, %(n) 92.04% (104/113) 91.96% (103/112) 0.984 0 1.01(0.39–2.65) NAC supplementation shortened the days of uFSH stimulation and the total days of sequential OI with LE/uFSH. There were no significant differences between the two groups in terms of ovulation rate per OI cycle (96.13% vs 96.77%, p = 0.678), mono-ovulation rate (82.04% vs 81.36%, p = 0.835), or multiple-ovulation rate (14.08% vs 13.62%, p = 0.109). However, patients in the NAC group required fewer days of uFSH injection per OI cycle [5.00 (4.00, 6.75) vs 6.00 (5.00, 7.00), p = 0.017] and had a significantly shorter total duration of OI [10.00 (9.00, 12.00) vs 11.00 (10.00, 13.00), p = 0.032] than did patients in the control group. Furthermore, there were no significant differences between the two groups in terms of OHSS or endometrial thickness on the trigger day. (See Table 2) Table 2. Cycle characteristics for NAC group versus control group—intention-to-treat analysis Outcome NAC group N = 113 Control group N = 112 Risk ratio (95% CI) p u value/ χ 2 value Underwent single-cycle OI 10.62% (12/113) 8.92% (10/112) - 0.067 -0.017 Underwent two-cycle OI 27.43% (31/113) 33.04% (37/112) Underwent three-cycle OI 61.95% (70/113) 58.04% (65/112) Total no. of OI cycles 284 279 - - - OI cycle characteristics Ovulation per cycle, %(n) 94.37% (268/284) 93.55% (261/279) 1.16(0.58, 2.31) 0.684 0.166 No. of anovulatory and follicular atresia cycles, %(n) 3.87% (11/284) 5.02% (14/279) 0.76 (0.34, 1.71) 0.510 0.435 No of Mono-ovulation cycles, %(n) 82.04% (233/284) 81.36% (227/279) 1.05 (0.68, 1.61) 0.835 0.044 No of Multiple-ovulation cycles, %(n) 14.08% (40/284) 13.62% (38/279) 1.47 (0.92, 2.36) 0.109 2.576 Days of uFSH stimulation, (d) 5.00 (4.00, 6.75) 6.00 (5.00, 7.00) - 0.017 2748 Total days of ovulation induction, (d) 10.00 (9.00, 12.00) 11.00 (10.00, 13.00) - 0.032 2409 Endometrial thickness on hCG trigger day (mm) 9.00 (8.00, 9.00) 9.00 (8.00, 9.00) - 0.856 1286 OHSS rate per cycle # , %(n) 3.53% (10/284) 3.94% (11/279) 0.89 (0.37, 2,13) 0.792 0.070 NAC Supplementation Improves Clinical Pregnancy Rates in LE/uFSH Sequential Therapy In the comparison of clinical pregnancy rates, both per OI cycle and cumulative per patient, the rates in the NAC group were significantly greater than those in the control group (30.99% vs. 23.30%, p = 0.040; and 77.88% vs. 58.04%, p = 0.001, respectively). The ongoing pregnancy rate per patient was also significantly greater in the NAC group than in the control group (69.91% vs. 53.57%, p = 0.012). However, there were no significant differences between the two groups in terms of multiple pregnancy rate, early miscarriage rate, or ectopic pregnancy rate (all p > 0.05). (Table 3 ). Table 3 Pregnancy outcomes for NAC group versus control group—intention-to-treat analysis Outcome NAC group N = 113 Control group N = 112 Risk ratio (95% CI) p χ 2 value Pregnancy outcomes (per cycle) Biochemical pregnancy rate per cycles, %(n) 33.45% (95/284) 25.09% (70/279) 1.50(1.04, 2.16) 0.029 4.749 Clinical pregnancy rate per cycle, %(n) 30.99% (88/284) 23.30% (65/279) 1.48(1.02, 2.15) 0.040 4.204 Clinical pregnancy rate per cycle—strata 1 (BMI < 24), %(n) 31.05% (50/161) 25.00% (38/152) 1.35(0.82, 2.22) 0.234 1.419 Clinical pregnancy rate per cycle—strata 1 (BMI ≥ 24), %(n) 30.89% (38/123) 21.26% (27/127) 1.66(0.94, 2.93) 0.083 3.015 Cumulative clinical pregnancy (per patient) Cumulative clinical pregnancy rate per patient 77.88% (88/113) 58.04% (65/112) 2.55(1.42, 4.55) 0.001 10.175 Cumulative clinical pregnancy rate—strata 1 (BMI < 24), %(n) 81.97% (50/61) 73.08% (38/52) 1.68(0.68, 4.10) 0.256 1.288 Cumulative clinical pregnancies—strata 2 (BMI ≥ 24) 73.08% (38/52) 45.00% (27/60) 3.32(1.50, 7.36) 0.003 9.017 Singleton pregnancy rate, %(n) 0.820 0.052 Multiple pregnancy rate, %(n) 0.718 0.131 Early miscarriage rate, %(n) 7.95% (7/88) 6.15% (4/65) 1.32(0.37, 4.71) 0.670 0.182 Ectopic pregnancy rate, %(n) 2.27% (2/88) 1.54% (1/65) 1.49(0.13, 16.77) 1.000 $ / Cumulative ongoing pregnancy rate per patient 69.91% (79/113) 53.57% (60/112) 0.17(0.07, 2.55) 0.012 6.36 Our current PCT included 225 participants, 49.78% (112/225) of whom were overweight or obese. Overweight or obesity negatively impacts both natural and assisted conception cycles in women with PCOS [ 28 ] . Notably, our stratified analysis revealed that among participants with a BMI ≥ 24, the cumulative clinical pregnancy rate was significantly greater in the NAC supplementation group than in the control group (73.08% vs. 45.00%, p = 0.003). These pregnancy outcomes suggest that NAC supplementation can improve clinical pregnancy rates after subsequent LE/uFSH therapy, with particularly pronounced benefits for overweight or obese women. Discussion PCOS, a highly prevalent and incurable disease affecting female reproductive, endocrine, metabolic, and mental health, demands the urgent exploration of more effective, safer, and cost-effective drugs or therapies to improve symptoms and enhance fertility. Insulin resistance, hyperandrogenemia, and oxidative stress imbalance are the three critical pathological mechanisms involved in the development of PCOS, forming a vicious cycle [ 29 , 30 ] that perpetuates the disease's progression. NAC, an effective antioxidant, has recently shown potent insulin-sensitizing effects in vitro [ 11 , 12 ] and in animal models of obesity [ 31 ] and diabetes mellitus [ 32 ] . Several studies [ 17 , 18 , 38 , 39 ] , including our current study, have explored the use of NAC as a daily medication for PCOS management or as an adjuvant for fertility treatments. In the present study, we are the first to verify that NAC intervention reverses endocrine-metabolic parameters and ovarian pathological phenotypes in LE-induced PCOS mouse models. We demonstrated that NAC has the ability to counteract oxidative stress damage to oocytes and enhance the activity of enzymatic and nonenzymatic antioxidants in ovarian tissues. Furthermore, through a clinical PCT study, we revealed that NAC supplementation significantly improved the efficacy of OI with sequential LE/uFSH. This research not only includes multilayered in vitro and in vivo validation but also incorporates clinical studies, further expanding the potential and application prospects of NAC in PCOS management. Insulin resistance is the core pathological mechanism involved in the development of PCOS [ 33 ] . Treating patients with insulin sensitizers is essential for alleviating symptoms, signs, and concomitant complications [ 4 ] . Currently, metformin is the most commonly used insulin sensitizer for treating PCOS. However, its gastrointestinal side effects limit its use in some patients [ 5 ] . Therefore, there is a need for alternate drugs with greater acceptance and fewer side effects. NAC has recently gained attention as a novel insulin sensitizer for PCOS [ 34 ] . In the present study, we first confirmed that NAC reversed the abnormal estrous cycles and ovarian phenotypes in LE-induced PCOS mice. NAC also significantly improved glucose homeostasis, insulin sensitivity and energy expenditure in PCOS mice. A recent stereopathological study on the uterus and ovaries of LE-induced PCOS mice also demonstrated that NAC, similar to metformin, could significantly reverse the pathological phenotypes to those of normal mice [ 17 ] . However, there are still limited studies on the effects of NAC on insulin sensitivity and endocrine-metabolic parameters in PCOS model mice. As early as 2002, Fulghesu et al. [ 35 ] reported that oral administration of NAC at 1.8 g/day (or 3.0 g/day for a BMI > 30) improved insulin sensitivity, testosterone levels, and lipid profiles in women with PCOS. Subsequent studies have shown that daily oral intake of 1.8 g of NAC had similar [ 36 ] or superior [ 37 ] effects compared to daily oral intake of 1.5 g of metformin in improving BMI, fasting insulin, homeostasis model assessment of insulin resistance (HOMA-IR), and menstrual irregularities. A meta-analysis in 2020 also demonstrated that NAC had comparable effects to metformin in improving serum LH levels, the LH/FSH ratio, and fasting insulin in women with PCOS [ 38 ] . In the current study, we also found that NAC supplementation had effects similar to those of metformin in enhancing the glucose clearance rate and insulin sensitivity. Although many researchers have studied the insulin-sensitizing effects of NAC, the mechanisms by which NAC reverses insulin resistance in individuals with PCOS remain unclear. These mechanisms may be closely related to its antioxidant and anti-inflammatory properties [ 39 ] and its ability to improve mitochondrial function [ 40 ] . In this study, we found that the activities of enzymatic antioxidants (SOD, GSH-Px, and CAT) and nonenzymatic antioxidants (GSH) were significantly decreased in the ovarian tissues of LE-induced PCOS mice. Additionally, these mice exhibited reduced MMP and increased ROS levels in their oocytes, indicating mitochondrial dysfunction and oxidative stress imbalance. This observation is consistent with that observed in clinical PCOS patients [ 41 , 42 ] . Oxidative stress can impact various physiological and pathological processes, leading to reproductive disorders such as PCOS, endometriosis, and recurrent spontaneous abortion [ 43 ] . Oxidative stress disrupts follicular development and maturation by damaging oocytes and granulosa cells. It also causes mitochondrial dysfunction, impairing insulin signaling and disrupting glucose metabolism, leading to insulin resistance [ 42 ] . The oxidative stress imbalance in PCOS primarily stems from impaired antioxidant capacity and the resulting overproduction of ROS. Our study demonstrated a decrease in the enzyme and nonenzyme antioxidants SOD, GSH-Px, CAT, and GSH in the ovarian tissues of pure PCOS mice, thus confirming this hypothesis. In our research, supplementation with NAC significantly increased the activities of GSH-Px, SOD, and GSH in the ovarian tissues of PCOS mice. Concurrently, the MMP of oocytes was enhanced, and ROS levels were reduced, indicating that NAC treatment enhanced antioxidant system activity in ovarian tissues, improved mitochondrial function, and ameliorated oxidative stress (OS) in PCOS mice. Cai et al. demonstrated in vitro that adding 0.5 mg/mL N-acetylcysteine (NAC) to a rabbit granulosa cell damage model induced by D-galactose (D-gal) significantly inhibited granulosa cell apoptosis and promoted proliferation. Moreover, NAC intervention in vitro was observed to suppress the release of cytochrome C, a marker of oxidative stress, while significantly increasing the activities of antioxidants such as CAT, GSH, and SOD [ 44 ] . The mechanism by which NAC enhances antioxidant activity in cells and tissues is considered to be the result of multiple pathways: first, as a precursor of glutathione (GSH), NAC transforms into GSH, which exhibits significant antioxidant effects; second, under conditions of significant depletion of endogenous cysteine (Cys) and GSH, NAC can also act as a direct antioxidant against certain oxidants (nitrogen dioxide and hypochlorous acid); third, NAC has the ability to decompose disulfide proteins, thereby releasing free thiols and reducing proteins to exert antioxidant effects [ 45 ] . The above findings collectively suggest that NAC supplementation has the potential to improve oocyte quality and fertility in women with PCOS. To validate this hypothesis, we further conducted a PCT study. The results showed that NAC supplementation reduced the total dose and duration of gonadotropins required for OI in women with PCOS. Importantly, NAC supplementation significantly increased the clinical pregnancy rate per OI cycle (30.99% vs. 23.30%) and the cumulative clinical pregnancy rate per enrolled patient (77.88% vs. 58.04%). In a 2018 study involving 130 women with PCOS who underwent OI with LE (5 mg/day), oral administration of NAC (1.2 g/day) for 5 days significantly improved ovulation and pregnancy rates compared to placebo [ 46 ] . Notably, the ovulation rates in both groups (16.1% vs. 33.3%) were significantly lower than the typically reported ovulation rates of approximately 90% [ 47 ] . A meta-analysis including 15 RCTs with a total of 2,330 women showed that oral NAC at doses of 1200–1600 mg/day tended to increase ovulation rates, clinical pregnancy rates, and live birth rates compared to placebo or no-treatment controls, although the differences did not reach statistical significance [ 48 ] . However, it is important to note that among the 15 RCTs included, 10 studies used CCs for OI, one used LE, one used laparoscopic ovarian drilling, one used oral contraceptives, and one did not report the intervention method. Given that the pregnancy rates in women with PCOS who use CCs for OI are significantly lower than those who use CCs for LE [ 47 ] , this could be a reason for the inconsistency between our study results and this meta-analysis. NAC supplementation improves clinical pregnancy outcomes in PCOS women with OI, which we believe is closely associated with the insulin-sensitizing and antioxidant effects of NAC observed in our previous animal models. Researchers have shown that in older IVF women, oral administration of 1.8 g/day NAC starting from the beginning of the menstrual cycle preceding controlled ovarian hyperstimulation until the trigger day significantly reduces the dose of gonadotropins required and increases the number of high-quality blastocysts. Furthermore, NAC intervention significantly increases GSH levels in follicular fluid [ 49 ] . Other studies have shown that in PCOS patients treated with NAC for 6 weeks (1.8 g/day), the level of receptor tyrosine kinase c-kit protein in follicular fluid decreases, while the expression of growth differentiation factor-9 in mature oocytes increases, indicating that NAC intervention has the potential to improve oocyte maturation and embryo quality [ 50 ] . In addition, our current study revealed a more pronounced effect of NAC intervention on increasing the cumulative clinical pregnancy rate per enrolled patient after OI in overweight and obese PCOS women with a BMI ≥ 24 kg/m² (73.08% vs. 45.00%). This may be related to the fact that overweight and obese PCOS women often exhibit more significant insulin resistance and oxidative stress imbalance [ 51 , 52 ] . Although further research is needed to confirm the above hypothesis, these results suggest that overweight and obese PCOS women may be a priority population for NAC supplementation. This study has several limitations. First, we did not further explore the mechanism by which NAC enhances the activity of antioxidant enzymes, especially SOD and GSH-Px, in ovarian tissue. Current research shows that NAC can promote the nuclear translocation of Nrf2 [ 10 ] or activate NF-κB signaling pathways [ 53 ] , which in turn activate the expression of SOD and GSH-Px , thereby reducing excessive ROS that damage mitochondria in mouse oocytes. Whether these molecular pathways play a role in the improvement of oxidative stress by NAC in LE-induced PCOS mouse ovaries in our study or whether other molecular pathways are involved still needs further investigation. Second, in our current animal model study, we did not further evaluate the effect of NAC intervention on mouse fertility. Therefore, we cannot directly conclude that NAC intervention improves endocrine-metabolic profiles and oxidative stress in PCOS mouse ovaries, thereby enhancing fertility in PCOS mice. However, our clinical PCT study confirmed that NAC supplementation improves OI efficacy in PCOS patients at the clinical level, suggesting a beneficial effect of NAC on fertility in PCOS patients. Last, in our current PCT study, we did not perform a controlled assessment of changes in participants’ glucose, lipid metabolism, or hormone levels before and after NAC intervention. Therefore, we did not conclusively demonstrate whether short-term NAC intervention can improve these parameters in PCOS patients. This aspect requires further clarification in future studies. Conclusions In summary, this study demonstrated that NAC supplementation significantly reversed changes in endocrine-metabolic parameters and the ovarian tissue phenotype in LE-induced PCOS mice. The results also revealed the protective effect of NAC supplementation on oxidative stress-induced damage to oocytes and the enhancement of antioxidant enzyme and nonenzymatic activities in the ovarian tissue of PCOS mice. Importantly, through a pragmatic, randomized, parallel, controlled clinical study, our current study further demonstrated that NAC supplementation with sequential letrozole/uFSH significantly improved the clinical pregnancy outcomes of patients with OI. Therefore, NAC could be a valuable adjuvant for OI therapy for women with PCOS. Declarations Ethics approval and consent to participate All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Huazhong University of Science and Technology (IACUC approval number: 2585). The pragmatic clinical trial was approved by the medical ethics committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (approval number 2023-0353) and was registered in the Chinese Clinical Trial Registry (www.chictr.org.cn; identifier ChiCTR2300077709). Informed consent was obtained from all individual participants included in the study. Consent for publication Not applicable. Availability of data and materials The datasets used during the current study are available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by the Hubei Provincial Natural Science Foundation of China (No. 2024AFB639). Authors' contributions YF, HD, TL and YHL performed the experiments. XZ, DL and YHL analyzed and interpreted the data. YHL wrote and conceived and provided financial support. manuscript. YL reviewed and edited the manuscript. All the authors have read and agreed to the published version of the manuscript. Acknowledgments We would like to express our gratitude to Dr. Zhenyuan Chen for his statistical advice. We would also like to thank our colleagues at the Reproductive Medicine Center, Union Hospital, Tongji Medical College, and Huazhong University of Science and Technology for their assistance during the study. References Shrivastava S, Conigliaro RL. 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Supplementary Files additionalfile1supplementaryfigureS1.docx Additionalfile2supplementarytables.docx Cite Share Download PDF Status: Published Journal Publication published 16 Oct, 2024 Read the published version in Journal of Ovarian Research → Version 1 posted Editorial decision: Revision requested 06 Sep, 2024 Reviews received at journal 19 Aug, 2024 Reviewers agreed at journal 12 Aug, 2024 Reviewers agreed at journal 09 Aug, 2024 Reviews received at journal 23 Jul, 2024 Reviewers agreed at journal 01 Jul, 2024 Reviewers invited by journal 06 Jun, 2024 Editor assigned by journal 29 May, 2024 Submission checks completed at journal 29 May, 2024 First submitted to journal 27 May, 2024 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4485542","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":312644031,"identity":"319bd929-f8de-4103-b018-9236f5edfcf2","order_by":0,"name":"Yu-Qing Fang","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yu-Qing","middleName":"","lastName":"Fang","suffix":""},{"id":312644032,"identity":"4e48ab3e-7ec8-4024-994f-0d52fb35f5b3","order_by":1,"name":"Hui Ding","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Ding","suffix":""},{"id":312644034,"identity":"67649f5f-7cdc-41ac-af02-6059a94456e7","order_by":2,"name":"Tao Li","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Li","suffix":""},{"id":312644035,"identity":"cbd1f17d-ba37-4638-b36e-5896cbf9efc7","order_by":3,"name":"Xiao-Jie Zhao","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Xiao-Jie","middleName":"","lastName":"Zhao","suffix":""},{"id":312644036,"identity":"cb9cf75e-07b8-4dbb-bbc8-636648780ffc","order_by":4,"name":"Dan Luo","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Luo","suffix":""},{"id":312644044,"identity":"4f557d0f-981e-4219-aa87-8a08b6a18e68","order_by":5,"name":"Yi Liu","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Liu","suffix":""},{"id":312644047,"identity":"42fac594-a43c-494b-b176-e598b2c0da84","order_by":6,"name":"Yan-Hui Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYBACPmYgwdjAwMMvf7DxQUJFDWEtbDAtkjOYmw0enDlGhBYGiBYGgxvsbZIPW5iJ0MLOY/i5cMdhGYbbjW0ViQ1sDPzt3QkEHMZjLD3zzGEexjkH224k7pBhkDhzdgMhLQbSvG2HeZgZEoFazrAxGEjkEtRi/BukhQ2opSCxjZkoLWZgW3gkEtsYiNTCVmbN25bOI8FzsFki4cwxHoJ+4ec/vPk2b5u1vf3x9ocff1TUyPG39+LXwsDAYYDC5SGgHATYHxChaBSMglEwCkY0AACQn0JWpmh0awAAAABJRU5ErkJggg==","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Yan-Hui","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-05-27 13:56:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4485542/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4485542/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13048-024-01528-8","type":"published","date":"2024-10-16T15:57:56+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58230980,"identity":"6f9fa612-dc1e-458c-af2e-fdc1d405a7da","added_by":"auto","created_at":"2024-06-12 19:22:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3338652,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of the animal study in which C57BL/6 female mice were administered letrozole (LE) by gavage to establish a PCOS model and then randomly divided into groups for intervention\u003cstrong\u003e (A)\u003c/strong\u003e. Representative estrous cycle curves for pure control group\u003cstrong\u003e (B)\u003c/strong\u003e, PCOS + Met \u003cstrong\u003e(C)\u003c/strong\u003e, PCOS + NAC\u003cstrong\u003e (D)\u003c/strong\u003e, and pure PCOS group\u003cstrong\u003e (E)\u003c/strong\u003e during the intervention period (n=12); Representative ovarian histopathological images of pure control group \u003cstrong\u003e(F)\u003c/strong\u003e, PCOS + Met \u003cstrong\u003e(G)\u003c/strong\u003e, PCOS + NAC\u003cstrong\u003e (H)\u003c/strong\u003e, and pure PCOS group \u003cstrong\u003e(I)\u003c/strong\u003e after 12 days of intervention (n=6). Scale bar = 200 µm. LE, letrozole; Met, metformin; NAC, N-acetylcysteine; CMC, carboxymethyl cellulose.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4485542/v1/d13ca41568714e28b27cb929.png"},{"id":58230982,"identity":"7d986c2c-6549-4b88-aeda-06677a5e5644","added_by":"auto","created_at":"2024-06-12 19:22:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":658188,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of serum levels of \u003cstrong\u003e(A)\u003c/strong\u003e follicle-stimulating hormone (FSH), \u003cstrong\u003e(B)\u003c/strong\u003eluteinizing hormone (LH), \u003cstrong\u003e(C)\u003c/strong\u003e testosterone (T), and \u003cstrong\u003e(D)\u003c/strong\u003e LH/FSH ratio in each group of mice after 12 days of intervention. Comparison of glucose tolerance test \u003cstrong\u003e(E)\u003c/strong\u003e and insulin tolerance test \u003cstrong\u003e(G)\u003c/strong\u003e results at the end of the intervention, with \u003cstrong\u003e(F)\u003c/strong\u003e and \u003cstrong\u003e(H)\u003c/strong\u003e representing the area under the curve of GTT and ITT, respectively. Infrared thermographic images\u003cstrong\u003e (I) \u003c/strong\u003eand core body temperature\u003cstrong\u003e (J)\u003c/strong\u003e of each group of mice at the end of the study, and body weight change curves \u003cstrong\u003e(K)\u003c/strong\u003e of each group of mice during the study. Data are presented as M±SD and analyzed by one-way ANOVA with Tukey’s post hoc test. n=4~6. *, p\u0026lt;0.05; **, p\u0026lt;0.01; ***, p\u0026lt;0.001. Met, metformin; NAC, N-acetylcysteine.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4485542/v1/53fd2e17042f40563034485b.png"},{"id":58230984,"identity":"c9f50791-cb08-44ed-bbb3-87b27720cae8","added_by":"auto","created_at":"2024-06-12 19:22:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4555185,"visible":true,"origin":"","legend":"\u003cp\u003eAfter group intervention, MII oocytes from each group of mice were collected. Representative images of the MMP measured with a JC-1 fluorescent probe \u003cstrong\u003e(A)\u003c/strong\u003e and ROS levels measured with a DCFH-DA fluorescent probe \u003cstrong\u003e(C)\u003c/strong\u003e and statistical analysis of the MMP (red/green) \u003cstrong\u003e(B)\u003c/strong\u003eand ROS signal \u003cstrong\u003e(D)\u003c/strong\u003e. Comparison of the levels of the nonenzymatic antioxidant glutathione (GSH) \u003cstrong\u003e(E)\u003c/strong\u003e and the enzymatic antioxidants superoxide dismutase (SOD)\u003cstrong\u003e (F)\u003c/strong\u003e, glutathione peroxidase (GSH-Px)\u003cstrong\u003e (G)\u003c/strong\u003e, and catalase (CAT) \u003cstrong\u003e(H)\u003c/strong\u003e in the ovarian tissues of each group of mice after 12 days of intervention using the corresponding assay kits. The data are presented as M±SD and were analyzed by one-way ANOVA with Tukey’s post hoc test. n=4~6. *, p\u0026lt;0.05; ***, p\u0026lt;0.001. Scale bar = 200 µm. BF, bright field; ROS, reactive oxygen species; Met, metformin; NAC, N-acetylcysteine.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4485542/v1/1200413ef26c6d65787b7aaa.png"},{"id":58230981,"identity":"7c3e3e34-f5be-4b4e-8bc5-0658e1a44d11","added_by":"auto","created_at":"2024-06-12 19:22:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":467968,"visible":true,"origin":"","legend":"\u003cp\u003eCONSORT flow diagram revealing the participation process for each stage of the study. CONSORT (Consolidated Standards of Reporting Trials). \u003csup\u003e# \u003c/sup\u003eDuring the intervention stage, patients who conceived naturally were excluded from the final statistical analyses. *Incomplete OI cycles were not included in the final statistics. NAC, N-acetyl-L-cysteine; OI, ovulation induction; ITT, intention-to-treat; PP, per-protocol.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4485542/v1/c43cfb6aee98af32d71f4d65.png"},{"id":67149575,"identity":"b6000e5c-285c-4204-bd03-370ed4062a93","added_by":"auto","created_at":"2024-10-21 16:13:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11802420,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4485542/v1/c194d54c-45f4-471e-aedd-d52569e688ee.pdf"},{"id":58231862,"identity":"108e43f0-5d31-4442-90a2-fa503e4f3fdc","added_by":"auto","created_at":"2024-06-12 19:30:58","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":375217,"visible":true,"origin":"","legend":"","description":"","filename":"additionalfile1supplementaryfigureS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4485542/v1/eef1a449a8408a05cd5d8542.docx"},{"id":58230979,"identity":"23d3692d-c98f-496a-a404-28602769c72e","added_by":"auto","created_at":"2024-06-12 19:22:58","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":23616,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile2supplementarytables.docx","url":"https://assets-eu.researchsquare.com/files/rs-4485542/v1/01705883642cae55fd186705.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"N-acetylcysteine Supplementation Improves Endocrine-Metabolism Profiles and Ovulation Induction Efficacy in Polycystic Ovary Syndrome","fulltext":[{"header":"Background","content":"\u003cp\u003ePolycystic ovary syndrome (PCOS) is a heterogeneous familial disorder\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Depending on different diagnostic criteria, the prevalence among reproductive-age women fluctuates between 6% and 20%\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. From an evolutionary medicine perspective, the overlap of risk alleles for PCOS between European and Chinese women suggests that PCOS is an ancient disorder\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. A considerable proportion of PCOS patients have defects in insulin secretion and insulin resistance, resulting in hyperinsulinemia in more than 50% of cases. Hyperinsulinemia stimulates testosterone secretion from the ovaries and adrenal glands and inhibits hepatic synthesis of sex hormone-binding globulin, thereby increasing circulating free testosterone levels. Hyperinsulinemia also promotes oxidative stress by generating reactive oxygen species (ROS), which is another important pathogenic factor in PCOS.\u003c/p\u003e \u003cp\u003eCurrently, metformin is the most commonly recommended drug for treating insulin resistance associated with PCOS\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Although metformin has been demonstrated to be an effective treatment for PCOS, it is associated with significant adverse effects. Over 30% of patients experience noticeable gastrointestinal side effects during the initial treatment period, including bloating, diarrhea, constipation, nausea, vomiting, and an increase in serum homocysteine levels, which may lead to intolerance and discontinuation of the drug\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Therefore, clinicians have been exploring new insulin sensitizers with improved tolerability for treating PCOS. Among them, N-acetylcysteine (NAC) is considered a promising drug with potential applications\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNAC is the acetylated precursor of L-cysteine and reduced glutathione. It has been established as a potent cell-permeable antioxidant that effectively prevents cell apoptosis and promotes cell survival through antioxidant stress\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. The antioxidant activity of NAC is attributed to its thiol group, which enhances the activity of glutathione S-transferase, thereby protecting target cells and cell membranes\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. In animal experiments, Fan et al. showed that NAC protects against oxidative stress toxicity and mitochondrial functional damage induced by repeated ovulation stimulation\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Furthermore, in vitro studies have demonstrated that NAC activates insulin secretion in pancreatic cells\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e and modulates insulin receptors in human erythrocytes\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. However, there is limited research on the effects of NAC on endocrine-metabolic profiles and ovarian antioxidant enzymatic systems in PCOS animal models.\u003c/p\u003e \u003cp\u003eIn addition, several clinical studies have explored the effects of NAC supplementation in various induction ovulation (OI) protocols for women with PCOS. In a randomized controlled trial (RCT) involving 150 PCOS women with clomiphene citrate (CC)-resistant, Rizk AY et al.\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e reported that the use of NAC (1.2 g/day) as an adjuvant to CC significantly increased ovulation and pregnancy rates compared to the use of a placebo (1.3% vs. 49.3%, 0 vs. 21.3%)\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. However, in a 2017 RCT, Behrouzi Lak et al. reported that NAC supplementation did not improve clinical pregnancy rates in CC combined with LE induction of ovulation followed by intrauterine insemination. However, it is important to note that the study had a small sample size (only 97 participants), and the NAC supplementation group already showed a trend toward higher clinical pregnancy rates (32.7% vs. 18.8%)\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Therefore, further clinical studies with sufficient sample sizes are needed to explore the application of NAC supplementation in OI for women with PCOS.\u003c/p\u003e \u003cp\u003eIn this study, we first investigated the effects of NAC supplementation on endocrine-metabolic parameters and ovarian/oocyte oxidative stress responses in LE-induced PCOS mice through in vivo experiments. After confirming that NAC supplementation significantly improved endocrine metabolism profiles and ovarian/oocyte antioxidant capacity in PCOS mice, a pragmatic clinical trial (PCT) was conducted. PCOS patients with anovulation or oligo-ovulation were recruited to observe the effects of NAC supplementation on the characteristics of OI cycles and clinical pregnancy outcomes following sequential LE and urinary follicle-stimulating hormone (uFSH) treatment. The aim of this study was to elucidate the potential benefits of NAC supplementation in OI for PCOS patients with anovulation or oligo-ovulation and to provide more foundational and clinical data for implementing NAC supplementation therapy in PCOS patients.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePCOS mouse model and grouping intervention\u003c/h2\u003e \u003cp\u003eThree-week-old SPF-grade C57BL/6 female mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and housed in the Laboratory Animal Center of Huazhong University of Science and Technology. The mice were maintained at a constant temperature of 26\u0026deg;C with a 12-hour light/dark cycle (light period: 07:00 to 19:00) and had ad libitum access to food and water. After one week of acclimatization, 15 mice were randomly selected to constitute the control group. The mice were administered 0.5% carboxymethyl cellulose (CMC; Shanghai Biochemical Technology Co., Ltd., Shanghai, China) solution (1 mg/kg) via gavage for 21 consecutive days, followed by no further treatment for the next 12 days. The remaining mice were used to construct the PCOS model using LE, following methods reported in previous literature\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Briefly, LE (Furui, Jiangsu Hengrui Medicine Co., Ltd., Lianyungang, China) was dissolved in a 0.5% CMC solution and administered by gavage at a dosage of 1 mg/kg/day for 21 consecutive days to induce the PCOS model. During the last 10 days of LE administration, the estrous cycles of the mice were monitored daily. After 21 days of LE treatment, three mice from the control group and three from the PCOS model group were randomly selected at the diestrus stage and anesthetized with isoflurane, and blood samples were collected from the retro-orbital plexus. The mice were then euthanized by cervical dislocation, and ovarian tissues were collected to evaluate the success of the PCOS model.\u003c/p\u003e \u003cp\u003eThe successfully generated PCOS model mice were then randomly divided into three groups: a pure PCOS group (model control), a PCOS\u0026thinsp;+\u0026thinsp;metformin (Met) group, and a PCOS\u0026thinsp;+\u0026thinsp;NAC group. Starting on day 22, the three groups of PCOS mice were administered 0.5% CMC, 200 mg/kg/day metformin (Shanghai Squibb Pharmaceutical Co., Ltd.), or 160 mg/kg/day NAC\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e via gavage for 12 days. During the intervention period, estrous cycle monitoring continued. After the intervention, six mice from each group were euthanized in the same manner as described above, and blood samples and ovarian tissues were collected. The remaining six mice in each group were treated with pregnant mare serum gonadotropin (PMSG) to obtain oocytes. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Huazhong University of Science and Technology (approval number: 2585) and were conducted in accordance with international ethical guidelines and the relevant requirements of the ethics committee of Huazhong University of Science and Technology.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMouse estrous cycle assessment\u003c/h2\u003e \u003cp\u003eVaginal secretions were collected daily between 12:00 and 13:00 from each group of mice and smeared onto slides. These slides were then examined under an optical microscope to determine the stage of the estrous cycle for each mouse. Diestrus was characterized by predominantly leukocytic vaginal smears, while proestrus exhibited a high concentration of nucleated cells. The estrus was typified by vaginal smears primarily composed of cornified epithelial cells, whereas metestrus displayed the presence of both cornified epithelial cells and leukocytes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eGlucose tolerance test (GTT) and insulin tolerance test (ITT)\u003c/h2\u003e \u003cp\u003ePrior to the glucose tolerance test, the mice were fasted for 16 hours (from 17:00 to 09:00 the next day) with ad libitum access to water. Blood samples were drawn from the tail tips of the mice before and 30\u0026ndash;60, 90, and 120 minutes after intraperitoneal administration of D-glucose (2.0 g/kg body weight). The blood glucose levels were then tested with an Accu-Chek glucose monitoring system (Roche Diagnostics). In the insulin tolerance test (ITT), mice were fasted for four hours (with ad libitum access to water) before receiving an intraperitoneal injection of insulin (1 U/kg body weight). Blood glucose levels were monitored at 0, 15, 30, and 45 minutes after insulin infusion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eInfrared thermography and core body temperature measurement\u003c/h2\u003e \u003cp\u003eMice were individually housed in cages and subjected to a 4\u0026deg;C cold chamber for a maximum of 4 hours, with continuous access to food and water. Images were captured using an infrared digital thermal camera (E60: compact infrared thermal imaging camera; FLIR), and the data were analyzed using FLIR Quick Report software (FLIR ResearchIR Max 3.4; FLIR). The core body temperature of each group of mice was assessed using rectal probes connected to digital thermometers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of hormone levels in mouse blood\u003c/h2\u003e \u003cp\u003eIsoflurane was administered to anesthetize the mice before blood samples were collected from the retro-orbital plexus. Following a two-hour incubation period at room temperature, the samples were centrifuged at 3000 rpm for 15 minutes. The resulting supernatant (plasma) was collected and stored at -80\u0026deg;C for subsequent analysis. Plasma concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone (T) were determined using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions (Quanzhou RuiXin Biotechnology Co., Ltd., Fujian, China). The sensitivities of the ELISA kits for LH, FSH, and T were 0.1 mIU/L, 0.1 mIU/mL, and 0.1 ng/mL, respectively. The intra- and interassay coefficients of variation for all three kits were less than 10%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMouse ovarian tissue sectioning and staining analysis\u003c/h2\u003e \u003cp\u003eOvarian tissues from each group of mice were preserved overnight in 4% formaldehyde at room temperature. Following embedding in paraffin, the tissues were sectioned serially and then stained with hematoxylin and eosin (H\u0026amp;E). H\u0026amp;E staining was carried out as previously described\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Briefly, the mouse ovaries were halved and fixed in 4% paraformaldehyde. Following embedding in paraffin wax, the tissue was sliced into 5 \u0026micro;m thick sections and mounted onto glass slides. These sections then underwent a series of steps, including deparaffinization, hydration, H\u0026amp;E staining, dehydration, and mounting. Subsequently, images were captured under a microscope, and the number of follicles and corpora lutea at each stage was determined. Ovarian follicles and corpora lutea were classified using Pederson's classification system\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Briefly, primordial follicles are described as having a compact oocyte surrounded by a single layer of flattened granulosa cells (GCs). Primary follicles are identified by an enlarged oocyte encircled by a single layer of cuboidal GCs. Secondary follicles are defined by an enlarged oocyte surrounded by at least a partial or complete second layer of cuboidal GCs. Antral follicles are characterized by the presence of areas of follicular fluid or a single large antral space. To avoid double counting, primordial, primary, and secondary follicles were counted once every 10 consecutive sections, while antral follicles were counted once every 40 consecutive sections\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMouse oocyte collection\u003c/h2\u003e \u003cp\u003eAfter intraperitoneal injection of 10 IU PMSG, mice from each group received an intraperitoneal injection of 5 IU human chorionic gonadotropin (HCG) 48 hours later. Approximately 14\u0026ndash;16 hours post-HCG administration, the mice were euthanized by cervical dislocation. Cumulus-oocyte complexes (COCs) were then retrieved from the ampullary region of the fallopian tubes. The collected COCs were transferred to growth medium containing hyaluronidase (Solarbia, catalog number H8030) to remove the surrounding GCs. Mature mouse oocytes at the metaphase II (MII) stage were obtained, washed, and placed in M16 medium (Sigma‒Aldrich). Subsequently, the oocytes were cultured at 37\u0026deg;C in a 5% CO2 incubator.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of mitochondrial membrane potential (MMP)\u003c/h2\u003e \u003cp\u003eThe oocyte MMP was measured as described in previous studies\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Oocytes were exposed to a JC-1 working solution (Beyotime Biotech, Shanghai, China) and maintained at 37\u0026deg;C for 20 minutes. After two washes with JC-1 staining buffer (Beyotime Biotech, Shanghai, China), the oocytes were examined using a fluorescence microscope. In mitochondria with low membrane potential, the JC-1 probe exists in its monomeric form, emitting green fluorescence. Conversely, in mitochondria with high membrane potential, the JC-1 probe underwent J-aggregation, emitting red fluorescence. The degree of mitochondrial depolarization was assessed by the ratio of red to green fluorescence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of ROS levels\u003c/h2\u003e \u003cp\u003eOocytes were incubated in a dark environment with a diluted solution of the fluorescence probe 2',7'-dichlorofluorescin diacetate (DCFH-DA) (Beyotime Biotech, Shanghai, China) at 37\u0026deg;C for 20 minutes. After being washed with M2 media, images were captured using a fluorescence microscope. Upon exposure to reactive oxygen species (ROS), DCFH emits green fluorescence, and the intensity of the fluorescence directly correlates with the ROS level.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical analysis\u003c/h2\u003e \u003cp\u003eMouse ovarian tissue was rapidly homogenized in an ice-cold 0.9% NaCl solution (10% w/v). The supernatant from centrifugation (3500 rpm, 15 minutes, 25\u0026deg;C) was utilized for biochemical analysis. The tissue protein concentration was determined using bovine serum albumin as a standard. The activities of reduced glutathione (GSH), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT) in mouse ovarian tissue homogenates were measured using assay kits (Beyotime Biotech, Shanghai, China) following the protocols provided by the manufacturer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePragmatic clinical trial and participants\u003c/h2\u003e \u003cp\u003ePCT was conducted at Union Hospital, Tongji Medical College, Huazhong University of Science and Technology to evaluate the effects of NAC supplementation in OI for PCOS patients with anovulation or oligo-ovulation. Participants were responsible for covering the costs of examinations and medications, with no additional compensation provided. The study received approval from the medical ethics committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (approval number 2023\u0026thinsp;\u0026minus;\u0026thinsp;0353; approval date June 6, 2023) and was registered on the Chinese Clinical Trial Registry (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.chictr.org.cn\" target=\"_blank\"\u003ewww.chictr.org.cn\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.chictr.org.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; trial registration number: ChiCTR2300077709, registration date: July 1, 2023.). All participants provided informed consent before enrolling in the study. Detailed information about the trial protocol is available in the Chinese Clinical Trial Registry (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.chictr.org.cn/bin/project/edit?pid=205777\u003c/span\u003e\u003cspan address=\"https://www.chictr.org.cn/bin/project/edit?pid=205777\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe study recruited PCOS patients suffering from anovulation or oligo-ovulation. The diagnosis of PCOS was based on the modified Rotterdam criteria \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e, requiring the fulfillment of any two of the following three criteria: oligo-ovulation or anovulation, clinical or biochemical signs of hyperandrogenism, and polycystic ovaries, while excluding diseases such as congenital adrenal hyperplasia or tumors, folliculogenesis abnormalities, Cushing's syndrome, and androgen-secreting ovarian tumors. Additionally, the subjects met the following inclusion criteria: ① aged\u0026thinsp;\u0026ge;\u0026thinsp;21 years and \u0026le;\u0026thinsp;38 years. ② Observation of at least one patent fallopian tube during hysterosalpingography or laparoscopy. ③ A BMI\u0026thinsp;\u0026lt;\u0026thinsp;35 kg/m\u003csup\u003e2\u003c/sup\u003e. ④ A male partner must have a minimum sperm concentration of 15\u0026nbsp;million per milliliter, based on the World Health Organization's criteria. ⑤ Neither the patients nor their male partners experienced any sexual dysfunction, and both agreed to engage in regular intercourse to achieve pregnancy. The exclusion criteria were as follows: ① Infertility due to causes other than PCOS-related ovulatory disorders. ② Abnormal thyroid-stimulating hormone levels. ③ Use of oral contraceptives, metformin, inositol, N-acetylcysteine, or other medications in the past three months. ④ Presence of large ovarian cysts (\u0026ge;\u0026thinsp;5 cm), uterine malformations, intrauterine adhesions, submucosal fibroids, etc. ⑤ Presence of concurrent pregnancy or surgical or medical conditions, including but not limited to uncontrolled diabetes, hypertension, liver disease, kidney disease, thyroid disease, adrenal disorders, autoimmune diseases, etc. PCOS women who met the inclusion criteria and did not have the exclusion criteria were provided with detailed information about the study's objectives, procedures, potential benefits, and risks. After providing fully informed consent, they were enrolled in the study by signing an informed consent form.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePatient interventions\u003c/h2\u003e \u003cp\u003eUsing a stratified randomization method, eligible participants were assigned in a 1:1 ratio to either the control group or the NAC group. Both groups underwent a sequential LE and urinary follicle-stimulating hormone (uFSH) protocol for OI as described below. In the control group, all patients underwent baseline ultrasound examination starting from the 2nd to the 4th day of spontaneous or induced menstruation. Those meeting the criteria for OI initiated LE (Furui, Jiangsu Hengrui Medicine Co., Ltd., Lianyungang, China) at a dose of 2.5 mg daily for 5 consecutive days. The day after completing LE, they received intramuscular injections of uFSH (Urofollitropin, Lizhu Pharmaceutical Factory, Zhuhai, China) at a dosage of 75\u0026ndash;150 IU daily. Regular vaginal ultrasound examinations were conducted to monitor follicular growth. Upon the attainment of at least one follicle with a diameter of 18\u0026ndash;20 mm, recombinant human chorionic gonadotropin (rhCG) (Ovidrel, Merck Serono, Aubonne, Switzerland) was administered at a dose of 250 \u0026micro;g to induce ovulation, with instructions for timed intercourse 12\u0026ndash;36 hours after rhCG injection. Subsequent vaginal ultrasounds were performed every other day postrhCG injection to assess ovulation. Following ovulation, patients were prescribed oral progesterone capsules (Laiting, Zhejiang Medicine Co., Ltd., Xinchang, China) for 14 days. Urine pregnancy testing was subsequently conducted to confirm pregnancy. In the NAC group, women with PCOS were administered oral NAC (Jinkangsuli, Zhejiang Jinhua Kang'enbei Biopharmaceutical Co., Ltd., Jinhua, China) at a dose of 1.8 g/day (dosage: 0.6 g, orally, three times a day) from the 2nd to the 4th day of the menstrual cycle for five consecutive days. The remaining OI procedures mirrored those of the control group. For both groups, pregnancy outcomes were documented after each OI cycle, and the intervention study was considered complete upon clinical pregnancy. The follow-up period for both groups was limited to \u0026le;\u0026thinsp;3 OI cycles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRandomization and masking in the clinical trial\u003c/h2\u003e \u003cp\u003eThe study utilized block randomization with a block size of 4. Randomized sequences were generated using SPSS software (Version 26, IBM, Armonk, NY) and were securely stored in sealed opaque envelopes. Eligible participants who met the inclusion and exclusion criteria and provided informed consent were assigned by a third party (nurse) in a 1:1 ratio to either the NAC group or the control group based on their enrollment order. Ultrasound examiners, statisticians, and outcome assessors remained blinded to the allocation, while treating physicians and participants were not blinded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCalculation of the sample size for the clinical trial\u003c/h2\u003e \u003cp\u003eThe current study is a pragmatic, randomized, parallel, noncontrolled superiority trial. Sample size calculations for both groups were performed using PASS software version 11.0 (NCSS, LLC. Kaysville, Utah, USA). Our aim was to investigate whether NAC supplementation enhances the clinical pregnancy rate in women with PCOS who have undergone OI compared to women in the control group. Based on prior clinical research conducted at our center, the clinical pregnancy rate per OI cycle for PCOS-related ovulatory disorders using sequential LE\u0026thinsp;+\u0026thinsp;uFSH therapy was determined to be 23%\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Our objective was to assess the difference in pregnancy rates between the two groups at a significance level of 10%, with α\u0026thinsp;=\u0026thinsp;0.05 (one-sided) and a test power of 0.8, resulting in a minimum required induction cycle count of 219 per group. This calculation accounts for a dropout rate of 10%. Consequently, 239 cycles of patients were planned for each group in this study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eOutcome measures of the clinical trial\u003c/h2\u003e \u003cp\u003eThe primary outcome measure was the clinical pregnancy rate per OI cycle. Clinical pregnancy was defined as the presence of one or more gestational sacs observed via ultrasound, including intrauterine pregnancy, ectopic pregnancy, and combined intra- and extrauterine pregnancy, with or without visible fetal heartbeats. Multiple gestational sacs were counted as one clinical pregnancy. Secondary outcome measures included the ongoing pregnancy rate, early miscarriage rate, biochemical pregnancy rate, ovulation rate, incidence of ovarian hyperstimulation syndrome (OHSS) and treatment discontinuation rate. Ongoing pregnancy is defined as an intrauterine pregnancy with at least one visible fetal heartbeat observed after 12 weeks of gestation. Biochemical pregnancy was defined as a serum level of human chorionic gonadotropin greater than 10 IU/L. OHSS was defined by the Golan criteria\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Mild OHSS involves abdominal distension and discomfort, with possible nausea, vomiting, or diarrhea. Moderate OHSS includes these symptoms plus ultrasonographic ascites. Severe OHSS features clinical ascites and/or hydrothorax, breathing difficulties, and may involve hemoconcentration, coagulation issues, and reduced renal function.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eIn animal experiments, comparisons between groups were conducted using independent samples t tests or one-way analysis of variance (ANOVA) with Tukey's post hoc test (for continuous variables) or the chi-square test (for categorical variables).\u003c/p\u003e \u003cp\u003eIn clinical PCT studies, comparisons of outcome measures between the two study groups were conducted using intention-to-treat (ITT) analyses, with per-protocol (PP) analysis also performed as a sensitivity analysis (\u003cb\u003eSupplementary Tables S1 and S2\u003c/b\u003e). Descriptive analysis was employed to compare baseline data between the two groups, while balance analysis between the two groups was assessed by analyzing different baseline data. For continuous variables, the normality of the distribution was initially estimated using frequency histograms and the Kolmogorov‒Smirnov test. Normally distributed continuous variables are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SDs, and statistical comparisons were made using Student's t test. Alternatively, if continuous variables did not follow a normal distribution, they are presented as the median and interquartile range (IQR), and differences between groups were analyzed using the Mann‒Whitney U test. Categorical variables were described in terms of n (%), with the chi-square test used for difference testing and the Fisher exact probability test employed when expected frequencies were less than 5. Statistical significance was determined using one-sided tests, and the primary outcome measure was reported using superiority tests.\u003c/p\u003e \u003cp\u003eAll the statistical analyses were conducted using the statistical software package SPSS V.25.0 (SPSS). Statistical significance was defined as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n\u003ch2\u003eNAC supplementation reverses estrous cycle irregularities and the ovarian phenotype in PCOS mice\u003c/h2\u003e\n\u003cp\u003eAn irregular menstrual cycle is one of the primary diagnostic criteria for PCOS\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. In the present study, vaginal cytology of female mice after 21 days of LE intervention revealed a lack of estrous cycle regularity. Compared with those in the control group, the serum LH levels and LH/FSH ratios were considerably greater in the LE intervention mice. H\u0026amp;E staining of ovarian tissue sections revealed that the LE intervention mice (PCOS model mice) had considerably more follicles and fewer corpora lutea (\u003cstrong\u003eSupplementary Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/strong\u003e), confirming successful modeling of PCOS in mice induced by LE intervention.\u003c/p\u003e\n\u003cp\u003eFollowing a 12-day random group intervention in the PCOS mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA), vaginal cytology revealed that 83.33% (9/12) of the mice in the PCOS\u0026thinsp;+\u0026thinsp;NAC group and 75.00% (9/12) of the mice in the PCOS\u0026thinsp;+\u0026thinsp;Met group had normalized estrous cycles. In contrast, the pure PCOS group showed no cyclicity throughout the 12-day intervention period, but the 12 control mice had a normal estrous cycle (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB-E). Notably, H\u0026amp;E staining of ovarian tissue sections revealed that, compared to the pure PCOS group, the PCOS\u0026thinsp;+\u0026thinsp;NAC and PCOS\u0026thinsp;+\u0026thinsp;Met groups had considerably more corpora lutea and fewer follicles (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF-I). This finding suggested that NAC intervention can dramatically improve estrous cycle abnormalities and the ovarian phenotype in PCOS mice, with effects similar to those of Met.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNAC supplementation alleviates endocrine and metabolic disorders in LE-induced PCOS mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePCOS patients often have endocrine and metabolic disorders. To determine the effect of NAC intervention on the endocrine status of PCOS mice, we measured sex hormone levels in each group after the intervention. Although the serum FSH levels did not differ significantly among the groups, the serum LH levels and LH/FSH ratios were significantly lower in the PCOS\u0026thinsp;+\u0026thinsp;NAC and PCOS\u0026thinsp;+\u0026thinsp;Met groups than in the pure PCOS group, returning to levels similar to those in the pure control group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). Furthermore, the serum T levels in the PCOS\u0026thinsp;+\u0026thinsp;NAC and PCOS\u0026thinsp;+\u0026thinsp;Met groups were considerably lower than those in the PCOS group following the intervention (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). These results suggest that NAC supplementation can alleviate endocrine disorders in LE-induced PCOS mice.\u003c/p\u003e\n\u003cp\u003eTo investigate whether NAC intervention affects glucose metabolism in LE-induced PCOS mice, we conducted GTTs and ITTs. After 12 days of intervention, the PCOS\u0026thinsp;+\u0026thinsp;NAC and PCOS\u0026thinsp;+\u0026thinsp;Met groups had significantly lower fasting blood glucose (4.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39, 4.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71 vs 6.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64; both p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and fasting insulin (7.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42, 7.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31 vs 8.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52; both p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) levels than the pure PCOS group and were similar to the pure control group. The area under the GTT/ITT curve indicated that the pure PCOS group exhibited significant insulin resistance compared to the pure control group, while NAC and Met intervention improved insulin sensitivity and enhanced plasma glucose clearance in LE-induced PCOS mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE-H).\u003c/p\u003e\n\u003cp\u003eStudies have reported that adaptive thermogenesis in response to stimuli such as postprandial or cold exposure is reduced in PCOS patients\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. In mice, most energy expenditure is used to maintain core body temperature, and measuring core body temperature can directly reflect energy expenditure\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. In this study, compared with the control group, the pure PCOS group exhibited a significant decrease in body temperature under cold stimulation. However, NAC and Met significantly stimulated thermogenesis in PCOS mice, as indicated by a significant increase in body temperature in these two groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eI-J).\u003c/p\u003e\n\u003cp\u003eDue to the glucose metabolism and thermogenesis-inducing effects observed with NAC intervention, the average body weight of the PCOS\u0026thinsp;+\u0026thinsp;NAC group was significantly lower than that of the pure PCOS group after 12 days of intervention and was comparable to the average body weight of the PCOS\u0026thinsp;+\u0026thinsp;Met and pure control groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eK).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n\u003ch2\u003eNAC supplementation improves the oxidative stress response in oocytes of LE-induced PCOS mice\u003c/h2\u003e\n\u003cp\u003eTo evaluate the effect of NAC supplementation on the oxidative stress response in the oocytes of LE-induced PCOS mice, we used JCI and DCFH-DA fluorescent probes to analyze the MMP and ROS levels in the oocytes from each group after the intervention. Compared to those in the pure control group, oocytes from the pure PCOS group had significantly lower MMP and significantly higher intracellular ROS levels at the end of the intervention. However, NAC intervention, similar to Met intervention, significantly increased the MMP and decreased intracellular ROS levels in oocytes from LE-induced PCOS mice. These findings suggest that NAC has the potential to counteract the excessive oxidative stress response in the oocytes of PCOS mice, possibly improving oocyte quality and pregnancy outcomes. (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA-D).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n\u003ch2\u003eNAC supplementation increased the activity levels of antioxidant enzymes in mouse ovarian tissue\u003c/h2\u003e\n\u003cp\u003eCompared to those in the pure control group, the ovarian tissues of mice in the LE-induced PCOS group exhibited significantly lower activity of the enzymatic antioxidants SOD, GSH-Px, and CAT, as well as the nonenzymatic antioxidant GSH. In contrast, the activities of GSH-Px, SOD, and GSH in the ovarian tissue of the PCOS\u0026thinsp;+\u0026thinsp;NAC group were significantly greater than those in the ovarian tissue of the PCOS group. However, the activity levels of these enzymatic and nonenzymatic antioxidants in the ovarian tissue of the PCOS\u0026thinsp;+\u0026thinsp;Met group did not differ significantly from those in the ovarian tissue of the PCOS group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE-H). This shows that NAC treatment can boost the activity of the antioxidant system in ovarian tissue.\u003c/p\u003e\n\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n\u003ch2\u003eComparisons of baseline characteristics between the NAC group and control group\u003c/h2\u003e\n\u003cp\u003eBased on promising results from in vivo animal studies, we designed a pragmatic randomized, parallel-group controlled clinical study to determine the therapeutic efficacy of NAC supplementation in improving pregnancy outcomes for PCOS-related infertility patients. In the current PCT, 252 women with PCOS were recruited and screened, 230 of whom eventually enrolled in the research. Among them, 115 PCOS women were randomly assigned to the NAC group, with the remaining 115 PCOS women allocated to the control group. The patients' baseline characteristics were similar across the two study groups (\u003cstrong\u003esee\u003c/strong\u003e Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). During the intervention period, the NAC group had two incidences of natural conception, while the control group had three (these five participants were omitted from the final statistical analysis). Overall, the NAC group had 113 subjects and 284 complete OI cycles, whereas 112 patients in the control group had 279 complete OI cycles. (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"char\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eBaseline characteristics of PCOS women in the NAC group and control groups.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCharacteristic\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eNAC group\u003c/p\u003e\n\u003cp\u003en\u0026thinsp;=\u0026thinsp;113\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eControl group\u003c/p\u003e\n\u003cp\u003en\u0026thinsp;=\u0026thinsp;112\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ep value\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003et\u003c/em\u003e or \u003cem\u003eU\u003c/em\u003e value\u003c/p\u003e\n\u003cp\u003e/\u003cem\u003e\u0026chi;2\u003c/em\u003e value\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eRisk ratio\u003c/p\u003e\n\u003cp\u003e(95% CI)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAge (years)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e28.50 (2.81)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e28.02(2.99)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.2092\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.259\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBMI (kg/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e23.36(20.70, 26.24)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e24.17(22.04, 27.04)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.1748\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5665\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eInfertility duration (months)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12.00(6.00, 23.50)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12.00(6.00, 23.50)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.6573\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6113\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSerum basal FSH (mIU/ml)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.05(1.50)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.79(1.63)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.2284\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.208\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSerum basal LH(mIU/ml)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.12(4.75 to 12.08)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.61(6.13 to 13.79)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.0724\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5451\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLH/FSH ratio\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.66(1.25, 2.45)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.64(1.10, 2.41)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.3366\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5805\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSerum basal E2(pg/ml)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e33.71(26.00 to 45.33)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e35.35(26.00 to 46.08)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.4381\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5841\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTestosterone (ng/mL)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.54(0.40 to 0.78)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.58(0.42 to 0.84)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.3098\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5516\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eProlactin (ng/ml)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e13.89(10.96 to 19.04)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e13.19(10.45 to 19.35)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.4698\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5186\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eThe proportion of hyperprolactinemia (\u0026gt;\u0026thinsp;30 ng/ml) %(n)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.96% (9/113)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.25% (7/112)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.617\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.30(0.47\u0026ndash;3.62)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAMH (ng/ml)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.76(6.17 to 11.76)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.40(6.04 to 11.13)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.7751\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6188\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHOMA-IR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.27(2.42 to 4.73)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.28(2.21(to 4.83)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.8925\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6262\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eThe proportion of women with insulin resistance (HOMA-IR\u0026thinsp;\u0026gt;\u0026thinsp;2.69)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e67.26% (76/113)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e65.18% (73/112)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.742\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.109\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.10(0.63\u0026ndash;1.91)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFasting insulin (mIU/L)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14.14(10.87 to 20.58)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14.02(10.20 to 21.00)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.8633\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6188\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFasting glucose (mmol/L)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.20 (4.90 to 5.50)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.20 (4.90 to 5.50)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.7856\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6195\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"3\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eMenses, %(n)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eOligomenorrhoea\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e40.71% (46/113)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e42.86% (48/112)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"4\" align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.353\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"4\" align=\"left\"\u003e\n\u003cp\u003e-0.062\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"4\" align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAmenorrhoea\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.08% (8/113)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10.71% (12/112)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eIrregular\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e41.59% (47/113)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e41.07% (46/112)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRegular\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10.61% (12/113)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.36% (6/112)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePrimary infertility, %(n)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e80.53% (91/113)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e75.89% (85/112)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.399\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.71\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.31(0.70\u0026ndash;2.48)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePrimiparity, %(n)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e92.04% (104/113)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e91.96% (103/112)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.984\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.01(0.39\u0026ndash;2.65)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNAC supplementation shortened the days of uFSH stimulation and the total days of sequential OI with LE/uFSH.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere were no significant differences between the two groups in terms of ovulation rate per OI cycle (96.13% vs 96.77%, p\u0026thinsp;=\u0026thinsp;0.678), mono-ovulation rate (82.04% vs 81.36%, p\u0026thinsp;=\u0026thinsp;0.835), or multiple-ovulation rate (14.08% vs 13.62%, p\u0026thinsp;=\u0026thinsp;0.109). However, patients in the NAC group required fewer days of uFSH injection per OI cycle [5.00 (4.00, 6.75) vs 6.00 (5.00, 7.00), p\u0026thinsp;=\u0026thinsp;0.017] and had a significantly shorter total duration of OI [10.00 (9.00, 12.00) vs 11.00 (10.00, 13.00), p\u0026thinsp;=\u0026thinsp;0.032] than did patients in the control group. Furthermore, there were no significant differences between the two groups in terms of OHSS or endometrial thickness on the trigger day. (See Table\u0026nbsp;2)\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003eTable\u0026nbsp;2. Cycle characteristics for NAC group versus control group\u0026mdash;intention-to-treat analysis\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Taba\" border=\"1\"\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eOutcome\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eNAC group\u003c/div\u003e\n\u003cdiv class=\"SimplePara\"\u003eN\u0026thinsp;=\u0026thinsp;113\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eControl group\u003c/div\u003e\n\u003cdiv class=\"SimplePara\"\u003eN\u0026thinsp;=\u0026thinsp;112\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eRisk ratio\u003c/div\u003e\n\u003cdiv class=\"SimplePara\"\u003e(95% CI)\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Italic\"\u003ep\u003c/span\u003e\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Italic\"\u003eu\u003c/span\u003e value/\u003c/div\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Italic\"\u003e\u0026chi;\u003c/span\u003e\u003csup\u003e\u003cspan class=\"Italic\"\u003e2\u003c/span\u003e\u003c/sup\u003e value\u003c/div\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eUnderwent single-cycle OI\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e10.62% (12/113)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e8.92% (10/112)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.067\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"3\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e-0.017\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eUnderwent two-cycle OI\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e27.43% (31/113)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e33.04% (37/112)\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eUnderwent three-cycle OI\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e61.95% (70/113)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e58.04% (65/112)\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eTotal no. of OI cycles\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e284\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e279\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Bold\"\u003e-\u003c/span\u003e\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"6\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Bold\"\u003eOI cycle characteristics\u003c/span\u003e\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eOvulation per cycle, %(n)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e94.37% (268/284)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e93.55% (261/279)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e1.16(0.58, 2.31)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.684\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.166\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eNo. of anovulatory and follicular atresia cycles, %(n)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e3.87% (11/284)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e5.02% (14/279)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.76 (0.34, 1.71)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.510\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.435\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eNo of Mono-ovulation cycles, %(n)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e82.04% (233/284)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e81.36% (227/279)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e1.05 (0.68, 1.61)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.835\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.044\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eNo of Multiple-ovulation cycles, %(n)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e14.08% (40/284)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e13.62% (38/279)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e1.47 (0.92, 2.36)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.109\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e2.576\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eDays of uFSH stimulation, (d)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e5.00 (4.00, 6.75)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e6.00 (5.00, 7.00)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.017\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e2748\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eTotal days of ovulation induction, (d)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e10.00 (9.00, 12.00)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e11.00 (10.00, 13.00)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.032\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e2409\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eEndometrial thickness on hCG trigger day (mm)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e9.00 (8.00, 9.00)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e9.00 (8.00, 9.00)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.856\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e1286\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eOHSS rate per cycle\u003csup\u003e#\u003c/sup\u003e, %(n)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e3.53% (10/284)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e3.94% (11/279)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.89 (0.37, 2,13)\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.792\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.070\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n\u003ch2\u003eNAC Supplementation Improves Clinical Pregnancy Rates in LE/uFSH Sequential Therapy\u003c/h2\u003e\n\u003cp\u003eIn the comparison of clinical pregnancy rates, both per OI cycle and cumulative per patient, the rates in the NAC group were significantly greater than those in the control group (30.99% vs. 23.30%, p\u0026thinsp;=\u0026thinsp;0.040; and 77.88% vs. 58.04%, p\u0026thinsp;=\u0026thinsp;0.001, respectively). The ongoing pregnancy rate per patient was also significantly greater in the NAC group than in the control group (69.91% vs. 53.57%, p\u0026thinsp;=\u0026thinsp;0.012). However, there were no significant differences between the two groups in terms of multiple pregnancy rate, early miscarriage rate, or ectopic pregnancy rate (all p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003ePregnancy outcomes for NAC group versus control group\u0026mdash;intention-to-treat analysis\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eOutcome\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eNAC group\u003c/p\u003e\n\u003cp\u003eN\u0026thinsp;=\u0026thinsp;113\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eControl group\u003c/p\u003e\n\u003cp\u003eN\u0026thinsp;=\u0026thinsp;112\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eRisk ratio (95% CI)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003e\u0026chi;\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e value\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth colspan=\"10\" align=\"left\"\u003e\n\u003cp\u003ePregnancy outcomes (per cycle)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBiochemical pregnancy rate per cycles, %(n)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e33.45% (95/284)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e25.09% (70/279)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e1.50(1.04, 2.16)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.029\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.749\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eClinical pregnancy rate per cycle, %(n)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e30.99% (88/284)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e23.30% (65/279)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e1.48(1.02, 2.15)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.040\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.204\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eClinical pregnancy rate per cycle\u0026mdash;strata 1 (BMI\u0026thinsp;\u0026lt;\u0026thinsp;24), %(n)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e31.05% (50/161)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e25.00% (38/152)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e1.35(0.82, 2.22)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.234\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.419\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eClinical pregnancy rate per cycle\u0026mdash;strata 1 (BMI\u0026thinsp;\u0026ge;\u0026thinsp;24), %(n)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e30.89% (38/123)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e21.26% (27/127)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e1.66(0.94, 2.93)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.083\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.015\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"10\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eCumulative clinical pregnancy (per patient)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCumulative clinical pregnancy rate per patient\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e77.88% (88/113)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e58.04% (65/112)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e2.55(1.42, 4.55)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.001\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10.175\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCumulative clinical pregnancy rate\u0026mdash;strata 1 (BMI\u0026thinsp;\u0026lt;\u0026thinsp;24), %(n)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e81.97% (50/61)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e73.08% (38/52)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e1.68(0.68, 4.10)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.256\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.288\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCumulative clinical pregnancies\u0026mdash;strata 2 (BMI\u0026thinsp;\u0026ge;\u0026thinsp;24)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e73.08% (38/52)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e45.00% (27/60)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e3.32(1.50, 7.36)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.003\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.017\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSingleton pregnancy rate, %(n)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.820\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.052\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMultiple pregnancy rate, %(n)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.718\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.131\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eEarly miscarriage rate, %(n)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e7.95% (7/88)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e6.15% (4/65)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e1.32(0.37, 4.71)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.670\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.182\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eEctopic pregnancy rate, %(n)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e2.27% (2/88)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e1.54% (1/65)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e1.49(0.13, 16.77)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e1.000\u003csup\u003e$\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e/\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCumulative ongoing pregnancy rate per patient\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e69.91% (79/113)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e53.57% (60/112)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e0.17(0.07, 2.55)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.012\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.36\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eOur current PCT included 225 participants, 49.78% (112/225) of whom were overweight or obese. Overweight or obesity negatively impacts both natural and assisted conception cycles in women with PCOS\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Notably, our stratified analysis revealed that among participants with a BMI\u0026thinsp;\u0026ge;\u0026thinsp;24, the cumulative clinical pregnancy rate was significantly greater in the NAC supplementation group than in the control group (73.08% vs. 45.00%, p\u0026thinsp;=\u0026thinsp;0.003). These pregnancy outcomes suggest that NAC supplementation can improve clinical pregnancy rates after subsequent LE/uFSH therapy, with particularly pronounced benefits for overweight or obese women.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePCOS, a highly prevalent and incurable disease affecting female reproductive, endocrine, metabolic, and mental health, demands the urgent exploration of more effective, safer, and cost-effective drugs or therapies to improve symptoms and enhance fertility. Insulin resistance, hyperandrogenemia, and oxidative stress imbalance are the three critical pathological mechanisms involved in the development of PCOS, forming a vicious cycle \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e that perpetuates the disease's progression. NAC, an effective antioxidant, has recently shown potent insulin-sensitizing effects in vitro \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e and in animal models of obesity \u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e and diabetes mellitus \u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Several studies \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e, including our current study, have explored the use of NAC as a daily medication for PCOS management or as an adjuvant for fertility treatments. In the present study, we are the first to verify that NAC intervention reverses endocrine-metabolic parameters and ovarian pathological phenotypes in LE-induced PCOS mouse models. We demonstrated that NAC has the ability to counteract oxidative stress damage to oocytes and enhance the activity of enzymatic and nonenzymatic antioxidants in ovarian tissues. Furthermore, through a clinical PCT study, we revealed that NAC supplementation significantly improved the efficacy of OI with sequential LE/uFSH. This research not only includes multilayered in vitro and in vivo validation but also incorporates clinical studies, further expanding the potential and application prospects of NAC in PCOS management.\u003c/p\u003e \u003cp\u003eInsulin resistance is the core pathological mechanism involved in the development of PCOS \u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Treating patients with insulin sensitizers is essential for alleviating symptoms, signs, and concomitant complications \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Currently, metformin is the most commonly used insulin sensitizer for treating PCOS. However, its gastrointestinal side effects limit its use in some patients \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Therefore, there is a need for alternate drugs with greater acceptance and fewer side effects. NAC has recently gained attention as a novel insulin sensitizer for PCOS \u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. In the present study, we first confirmed that NAC reversed the abnormal estrous cycles and ovarian phenotypes in LE-induced PCOS mice. NAC also significantly improved glucose homeostasis, insulin sensitivity and energy expenditure in PCOS mice. A recent stereopathological study on the uterus and ovaries of LE-induced PCOS mice also demonstrated that NAC, similar to metformin, could significantly reverse the pathological phenotypes to those of normal mice \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. However, there are still limited studies on the effects of NAC on insulin sensitivity and endocrine-metabolic parameters in PCOS model mice.\u003c/p\u003e \u003cp\u003eAs early as 2002, Fulghesu et al. \u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e reported that oral administration of NAC at 1.8 g/day (or 3.0 g/day for a BMI\u0026thinsp;\u0026gt;\u0026thinsp;30) improved insulin sensitivity, testosterone levels, and lipid profiles in women with PCOS. Subsequent studies have shown that daily oral intake of 1.8 g of NAC had similar \u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e or superior \u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e effects compared to daily oral intake of 1.5 g of metformin in improving BMI, fasting insulin, homeostasis model assessment of insulin resistance (HOMA-IR), and menstrual irregularities. A meta-analysis in 2020 also demonstrated that NAC had comparable effects to metformin in improving serum LH levels, the LH/FSH ratio, and fasting insulin in women with PCOS \u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. In the current study, we also found that NAC supplementation had effects similar to those of metformin in enhancing the glucose clearance rate and insulin sensitivity. Although many researchers have studied the insulin-sensitizing effects of NAC, the mechanisms by which NAC reverses insulin resistance in individuals with PCOS remain unclear. These mechanisms may be closely related to its antioxidant and anti-inflammatory properties \u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e and its ability to improve mitochondrial function \u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we found that the activities of enzymatic antioxidants (SOD, GSH-Px, and CAT) and nonenzymatic antioxidants (GSH) were significantly decreased in the ovarian tissues of LE-induced PCOS mice. Additionally, these mice exhibited reduced MMP and increased ROS levels in their oocytes, indicating mitochondrial dysfunction and oxidative stress imbalance. This observation is consistent with that observed in clinical PCOS patients \u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. Oxidative stress can impact various physiological and pathological processes, leading to reproductive disorders such as PCOS, endometriosis, and recurrent spontaneous abortion \u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. Oxidative stress disrupts follicular development and maturation by damaging oocytes and granulosa cells. It also causes mitochondrial dysfunction, impairing insulin signaling and disrupting glucose metabolism, leading to insulin resistance \u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. The oxidative stress imbalance in PCOS primarily stems from impaired antioxidant capacity and the resulting overproduction of ROS. Our study demonstrated a decrease in the enzyme and nonenzyme antioxidants SOD, GSH-Px, CAT, and GSH in the ovarian tissues of pure PCOS mice, thus confirming this hypothesis. In our research, supplementation with NAC significantly increased the activities of GSH-Px, SOD, and GSH in the ovarian tissues of PCOS mice. Concurrently, the MMP of oocytes was enhanced, and ROS levels were reduced, indicating that NAC treatment enhanced antioxidant system activity in ovarian tissues, improved mitochondrial function, and ameliorated oxidative stress (OS) in PCOS mice. Cai et al. demonstrated in vitro that adding 0.5 mg/mL N-acetylcysteine (NAC) to a rabbit granulosa cell damage model induced by D-galactose (D-gal) significantly inhibited granulosa cell apoptosis and promoted proliferation. Moreover, NAC intervention in vitro was observed to suppress the release of cytochrome C, a marker of oxidative stress, while significantly increasing the activities of antioxidants such as CAT, GSH, and SOD \u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. The mechanism by which NAC enhances antioxidant activity in cells and tissues is considered to be the result of multiple pathways: first, as a precursor of glutathione (GSH), NAC transforms into GSH, which exhibits significant antioxidant effects; second, under conditions of significant depletion of endogenous cysteine (Cys) and GSH, NAC can also act as a direct antioxidant against certain oxidants (nitrogen dioxide and hypochlorous acid); third, NAC has the ability to decompose disulfide proteins, thereby releasing free thiols and reducing proteins to exert antioxidant effects \u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe above findings collectively suggest that NAC supplementation has the potential to improve oocyte quality and fertility in women with PCOS. To validate this hypothesis, we further conducted a PCT study. The results showed that NAC supplementation reduced the total dose and duration of gonadotropins required for OI in women with PCOS. Importantly, NAC supplementation significantly increased the clinical pregnancy rate per OI cycle (30.99% vs. 23.30%) and the cumulative clinical pregnancy rate per enrolled patient (77.88% vs. 58.04%). In a 2018 study involving 130 women with PCOS who underwent OI with LE (5 mg/day), oral administration of NAC (1.2 g/day) for 5 days significantly improved ovulation and pregnancy rates compared to placebo \u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. Notably, the ovulation rates in both groups (16.1% vs. 33.3%) were significantly lower than the typically reported ovulation rates of approximately 90% \u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. A meta-analysis including 15 RCTs with a total of 2,330 women showed that oral NAC at doses of 1200\u0026ndash;1600 mg/day tended to increase ovulation rates, clinical pregnancy rates, and live birth rates compared to placebo or no-treatment controls, although the differences did not reach statistical significance \u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e. However, it is important to note that among the 15 RCTs included, 10 studies used CCs for OI, one used LE, one used laparoscopic ovarian drilling, one used oral contraceptives, and one did not report the intervention method. Given that the pregnancy rates in women with PCOS who use CCs for OI are significantly lower than those who use CCs for LE \u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e, this could be a reason for the inconsistency between our study results and this meta-analysis. NAC supplementation improves clinical pregnancy outcomes in PCOS women with OI, which we believe is closely associated with the insulin-sensitizing and antioxidant effects of NAC observed in our previous animal models. Researchers have shown that in older IVF women, oral administration of 1.8 g/day NAC starting from the beginning of the menstrual cycle preceding controlled ovarian hyperstimulation until the trigger day significantly reduces the dose of gonadotropins required and increases the number of high-quality blastocysts. Furthermore, NAC intervention significantly increases GSH levels in follicular fluid \u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. Other studies have shown that in PCOS patients treated with NAC for 6 weeks (1.8 g/day), the level of receptor tyrosine kinase c-kit protein in follicular fluid decreases, while the expression of growth differentiation factor-9 in mature oocytes increases, indicating that NAC intervention has the potential to improve oocyte maturation and embryo quality \u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn addition, our current study revealed a more pronounced effect of NAC intervention on increasing the cumulative clinical pregnancy rate per enrolled patient after OI in overweight and obese PCOS women with a BMI\u0026thinsp;\u0026ge;\u0026thinsp;24 kg/m\u0026sup2; (73.08% vs. 45.00%). This may be related to the fact that overweight and obese PCOS women often exhibit more significant insulin resistance and oxidative stress imbalance \u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e. Although further research is needed to confirm the above hypothesis, these results suggest that overweight and obese PCOS women may be a priority population for NAC supplementation.\u003c/p\u003e \u003cp\u003eThis study has several limitations. First, we did not further explore the mechanism by which NAC enhances the activity of antioxidant enzymes, especially SOD and GSH-Px, in ovarian tissue. Current research shows that NAC can promote the nuclear translocation of Nrf2 \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e or activate NF-κB signaling pathways \u003csup\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e, which in turn activate the expression of \u003cem\u003eSOD\u003c/em\u003e and \u003cem\u003eGSH-Px\u003c/em\u003e, thereby reducing excessive ROS that damage mitochondria in mouse oocytes. Whether these molecular pathways play a role in the improvement of oxidative stress by NAC in LE-induced PCOS mouse ovaries in our study or whether other molecular pathways are involved still needs further investigation. Second, in our current animal model study, we did not further evaluate the effect of NAC intervention on mouse fertility. Therefore, we cannot directly conclude that NAC intervention improves endocrine-metabolic profiles and oxidative stress in PCOS mouse ovaries, thereby enhancing fertility in PCOS mice. However, our clinical PCT study confirmed that NAC supplementation improves OI efficacy in PCOS patients at the clinical level, suggesting a beneficial effect of NAC on fertility in PCOS patients. Last, in our current PCT study, we did not perform a controlled assessment of changes in participants\u0026rsquo; glucose, lipid metabolism, or hormone levels before and after NAC intervention. Therefore, we did not conclusively demonstrate whether short-term NAC intervention can improve these parameters in PCOS patients. This aspect requires further clarification in future studies.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, this study demonstrated that NAC supplementation significantly reversed changes in endocrine-metabolic parameters and the ovarian tissue phenotype in LE-induced PCOS mice. The results also revealed the protective effect of NAC supplementation on oxidative stress-induced damage to oocytes and the enhancement of antioxidant enzyme and nonenzymatic activities in the ovarian tissue of PCOS mice. Importantly, through a pragmatic, randomized, parallel, controlled clinical study, our current study further demonstrated that NAC supplementation with sequential letrozole/uFSH significantly improved the clinical pregnancy outcomes of patients with OI. Therefore, NAC could be a valuable adjuvant for OI therapy for women with PCOS.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Huazhong University of Science and Technology (IACUC approval number: 2585). The pragmatic clinical trial was approved by the medical ethics committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (approval number 2023-0353) and was registered in the Chinese Clinical Trial Registry (www.chictr.org.cn; identifier ChiCTR2300077709). Informed consent was obtained from all individual participants included in the study.\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\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Hubei Provincial Natural Science Foundation of China (No. 2024AFB639).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYF, HD, TL and YHL\u003c/strong\u003e performed the experiments. XZ, DL and YHL analyzed and\u003c/p\u003e\n\u003cp\u003einterpreted the data. YHL wrote and conceived and provided financial support.\u003c/p\u003e\n\u003cp\u003emanuscript. YL reviewed and edited the manuscript. All the authors have read\u003c/p\u003e\n\u003cp\u003eand agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our gratitude to Dr. Zhenyuan Chen for his statistical advice. We would also like to thank our colleagues at the Reproductive Medicine Center, Union Hospital, Tongji Medical College, and Huazhong University of Science and Technology for their assistance during the study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eShrivastava S, Conigliaro RL. Polycystic Ovarian Syndrome. 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Front Med (Lausanne). 2022;9:917146.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheraghi E, et al. N-Acetylcysteine Compared to Metformin, Improves The Expression Profile of Growth Differentiation Factor-9 and Receptor Tyrosine Kinase c-Kit in The Oocytes of Patients with Polycystic Ovarian Syndrome. Int J Fertil Steril. 2018;11(4):270\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSatyaraddi A, et al. Body composition, metabolic characteristics, and insulin resistance in obese and nonobese women with polycystic ovary syndrome. J Hum reproductive Sci. 2019;12(2):78\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBannigida DM, Nayak BS, Vijayaraghavan R. Insulin resistance and oxidative marker in women with PCOS. Arch Physiol Biochem. 2020;126(2):183\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartinez PF, et al. Modulation of MAPK and NF-κB signaling pathways by antioxidant therapy in skeletal muscle of heart failure rats. Cell Physiol Biochem. 2016;39(1):371\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-ovarian-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jovr","sideBox":"Learn more about [Journal of Ovarian Research](http://ovarianresearch.biomedcentral.com)","snPcode":"13048","submissionUrl":"https://submission.nature.com/new-submission/13048/3","title":"Journal of Ovarian Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Polycystic ovary syndrome, N-acetylcysteine, Insulin resistance, Metformin, Oxidative stress, Ovulation induction, Pregnancy rate","lastPublishedDoi":"10.21203/rs.3.rs-4485542/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4485542/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePolycystic ovary syndrome (PCOS) affects 6\u0026ndash;20% of women worldwide, with insulin resistance and hyperinsulinemia occurring in 50%-70% of patients. Hyperinsulinemia exacerbates oxidative stress, contributing to PCOS pathogenesis. N-acetylcysteine (NAC) is an antioxidant and insulin sensitizer that shows promise as a therapeutic for PCOS. Our current study aimed to investigate the effects of NAC supplementation on endocrine-metabolic parameters in PCOS mice and its effect on ovulation induction (OI) efficacy in women with PCOS.\u003c/p\u003e\u003ch2\u003eMethods:\u003c/h2\u003e \u003cp\u003eFemale C57BL/6 mice were orally administered letrozole (LE) to induce PCOS and then randomly divided into groups receiving daily oral administration of 160 mg/kg NAC (PCOS\u0026thinsp;+\u0026thinsp;NAC group), 200 mg/kg metformin (PCOS\u0026thinsp;+\u0026thinsp;Met group), or 0.5% carboxymethyl cellulose (drug solvent) (pure PCOS group) for 12 days. Healthy female mice served as pure controls. Estrous cycles were monitored during the intervention. Metabolic and hormone levels, ovarian phenotypes, antioxidant activity in ovarian tissues, and oxidative stress levels in oocytes were assessed postintervention. This pragmatic, randomized, controlled clinical study included 230 women with PCOS who were randomly assigned to the NAC group (1.8 g/day oral NAC, n\u0026thinsp;=\u0026thinsp;115) or the control group (n\u0026thinsp;=\u0026thinsp;115). Patients in both groups underwent\u0026thinsp;\u0026le;\u0026thinsp;3 cycles of OI with sequential LE and urinary follicle-stimulating hormone (uFSH). Cycle characteristics and pregnancy outcomes were compared between groups.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSimilar to metformin, NAC supplementation significantly improved the estrous cycles and ovarian phenotypes of PCOS mice; reduced the LH concentration, LH/FSH ratio, and T level; and increased glucose clearance and insulin sensitivity. Notably, NAC significantly reduced oocyte ROS levels and increased the mitochondrial membrane potential in PCOS mice. Additionally, NAC significantly enhanced enzymatic and nonenzymatic antioxidant activities in PCOS mouse ovaries, whereas metformin had no such effect. In the clinical trial, compared to women in the control group, women receiving NAC had significantly lower average uFSH dosage and duration (p\u0026thinsp;\u0026lt;\u0026thinsp;0.005) and significantly greater clinical pregnancy rates per OI cycle and cumulative clinical pregnancy rates per patient (p\u0026thinsp;\u0026lt;\u0026thinsp;0.005).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eNAC supplementation improved endocrine-metabolic parameters in PCOS mice and significantly enhanced OI efficacy with sequential LE and uFSH in women with PCOS. Therefore, NAC could be a valuable adjuvant in OI for women with PCOS.\u003c/p\u003e","manuscriptTitle":"N-acetylcysteine Supplementation Improves Endocrine-Metabolism Profiles and Ovulation Induction Efficacy in Polycystic Ovary Syndrome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-12 19:22:54","doi":"10.21203/rs.3.rs-4485542/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-06T08:47:45+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-19T19:21:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"54757426350507155009837488915535431850","date":"2024-08-12T13:54:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"99504223626501523806751634146320315469","date":"2024-08-09T10:11:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-23T04:08:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"120834467706916987598173857702571358621","date":"2024-07-01T04:03:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-06T14:37:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-29T14:08:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-29T07:02:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Ovarian Research","date":"2024-05-27T13:55:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-ovarian-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jovr","sideBox":"Learn more about [Journal of Ovarian Research](http://ovarianresearch.biomedcentral.com)","snPcode":"13048","submissionUrl":"https://submission.nature.com/new-submission/13048/3","title":"Journal of Ovarian Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cef9f342-b434-4b23-b735-a81ebe1d5ccd","owner":[],"postedDate":"June 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-21T16:08:16+00:00","versionOfRecord":{"articleIdentity":"rs-4485542","link":"https://doi.org/10.1186/s13048-024-01528-8","journal":{"identity":"journal-of-ovarian-research","isVorOnly":false,"title":"Journal of Ovarian Research"},"publishedOn":"2024-10-16 15:57:56","publishedOnDateReadable":"October 16th, 2024"},"versionCreatedAt":"2024-06-12 19:22:54","video":"","vorDoi":"10.1186/s13048-024-01528-8","vorDoiUrl":"https://doi.org/10.1186/s13048-024-01528-8","workflowStages":[]},"version":"v1","identity":"rs-4485542","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4485542","identity":"rs-4485542","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}