N-acetylcysteine supplementation improves endocrine-metabolism profiles and ovulation induction efficacy in polycystic ovary syndrome

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 post-intervention. Furthermore, a pragmatic, randomized, controlled clinical study was conducted with 230 PCOS women, 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.

Keywords: Insulin resistance; Metformin; N-acetylcysteine; Ovulation induction; Oxidative stress; Polycystic ovary syndrome; Pregnancy rate.

Fig. 1

NAC supplementation reverses estrous cycle irregularities and ovarian phenotypes in PCOS mice. A Schematic diagram of the animal study, where C57BL/6 female mice were administered letrozole (LE) by gavage to establish a PCOS model, and subsequently randomly divided into intervention groups. B-E Representative estrous cycle curves for the pure control group, PCOS + Met, PCOS + NAC, and the pure PCOS group during the intervention period (n = 12). F-I Representative ovarian histopathological images for the pure control group, PCOS + Met, PCOS + NAC, and the pure PCOS group after 12 days of intervention (n=6). Scale bar = 200 μm. LE, letrozole; Met, metformin; NAC, N-acetylcysteine; CMC, carboxymethyl cellulose

Fig. 2

Effects of NAC supplementation on endocrine and metabolic profiles in LE-induced PCOS mice. Serum levels of (A) follicle-stimulating hormone (FSH), (B) luteinizing hormone (LH), (C) testosterone (T), and (D) LH/FSH ratio were compared among different groups of mice after 12 days of intervention. Glucose tolerance test (E) and insulin tolerance test (G) results were also compared at the end of the intervention, with (F) and (H) representing the area under the curve for GTT and ITT, respectively. Infrared thermographic images (I) and core body temperature (J) for each group of mice at the conclusion of the study, along with body weight change curves (K) for each group throughout the study. Data are presented as mean ± SD and analyzed using one-way ANOVA with Tukey’s post hoc test (n = 4-6). *, p < 0.05; **, p < 0.01; ***, p < 0.001. Met, metformin; NAC, N-acetylcysteine

Fig. 3

NAC supplementation enhances the oxidative stress response in oocytes and increases antioxidant enzyme activity levels in the ovaries. After group intervention, MII oocytes were collected from each group of mice. Representative images of mitochondrial membrane potential (MMP) measured with a JC-1 fluorescent probe (A) and reactive oxygen species (ROS) levels measured with a DCFH-DA fluorescent probe (C), along with statistical analysis of MMP (red/green) (B) and ROS signal (D). Comparison of nonenzymatic antioxidant glutathione (GSH) levels (E) and enzymatic antioxidants superoxide dismutase (SOD) (F), glutathione peroxidase (GSH-Px) (G), and catalase (CAT) (H) in the ovarian tissues of each group after 12 days of intervention using the corresponding assay kits. Data are presented as mean ± SD and analyzed by one-way ANOVA with Tukey’s post hoc test (n = 4-6). *, p < 0.05; ***, p < 0.001. Scale bar = 200 μm. BF, bright field; ROS, reactive oxygen species; Met, metformin; NAC, N-acetylcysteine

Fig. 4

Enrollment, allocation, and follow-up of participants and the CONSORT diagram of the pragmatic clinical trial. CONSORT (Consolidated Standards of Reporting Trials). # During the intervention stage, patients who conceived naturally were excluded from the final statistical analyses. *Incomplete ovulation induction cycles were not included in the final statistics. NAC, N-acetyl-L-cysteine; OI, ovulation induction; ITT, intention-to-treat; PP, per-protocol