Synergistic modulation of insulin resistance and ovarian oxidative stress by alpha-lipoic acid and vitamin D in an experimental rat model of polycystic ovary syndrome

Abstract

Polycystic ovary syndrome (PCOS) is characterized by insulin resistance, hyperandrogenism, oxidative stress, and disrupted folliculogenesis. The present study aimed to investigate whether combined alpha-lipoic acid (ALA) and vitamin D therapy exerts synergistic effects on metabolic, oxidative, endocrine, and histomorphological parameters in a letrozole-induced rat model of PCOS. Sixty female Wistar Albino rats were randomly divided into six groups: Control, PCOS, PCOS+Metformin (500 mg/kg/day), PCOS+Vitamin D (1000 IU/kg/day), PCOS + ALA (100 mg/kg/day), and PCOS + ALA+Vitamin D. PCOS was induced with letrozole (1 mg/kg/day) for 21 days, followed by 30 days of treatment. Fasting blood glucose (FBG), insulin, and HOMA-IR were assessed to evaluate metabolic status. Ovarian oxidative stress markers (MDA, SOD, CAT, GSH), serum hormonal parameters (testosterone, LH, FSH, LH/FSH ratio), and detailed histomorphometric analyses were performed. Statistical analyses included one-way and two-way ANOVA. Letrozole administration induced persistent diestrus, hyperandrogenemia, increased ovarian weight, elevated HOMA-IR (6.61 ± 1.18 vs. 2.24 ± 0.42, p < 0.001), and marked oxidative stress (MDA: 5.84 ± 0.72 vs. 2.31 ± 0.34 nmol/mg, p < 0.001). ALA and vitamin D monotherapies significantly improved metabolic, oxidative, and endocrine parameters compared with untreated PCOS rats (p < 0.05). The combination therapy group demonstrated the most pronounced improvements, with HOMA-IR (2.43 ± 0.47), MDA (2.52 ± 0.39 nmol/mg), testosterone (1.29 ± 0.27 ng/mL), and LH/FSH ratio (1.03 ± 0.19) values approaching control levels (all p < 0.01 vs. PCOS). Histologically, combined treatment markedly reduced cystic follicles and restored granulosa and theca thickness. Two-way ANOVA revealed significant interaction effects for HOMA-IR, MDA, testosterone, and LH/FSH ratio (p < 0.05). Combined ALA and vitamin D therapy produced enhanced improvements in insulin resistance, oxidative stress, endocrine imbalance, and ovarian morphology in experimental PCOS. Simultaneous targeting of mitochondrial redox dysfunction and endocrine–metabolic signaling pathways may represent a promising multidimensional therapeutic approach in PCOS.

Introduction

Polycystic ovary syndrome (PCOS) is the most common endocrine–metabolic disorder affecting women of reproductive age, with a reported prevalence ranging between 6 and 20% depending on diagnostic criteria^1,2. It is characterized by chronic anovulation, hyperandrogenism, and polycystic ovarian morphology. Beyond reproductive dysfunction, PCOS is now considered a complex systemic disorder characterized by profound metabolic and endocrine disturbances, particularly insulin resistance and chronic low-grade inflammation^3,4.

Insulin resistance is considered a central pathogenic mechanism in PCOS. Approximately 50–70% of affected women exhibit impaired insulin sensitivity independent of obesity^5–7. Hyperinsulinemia exacerbates ovarian theca cell androgen production by stimulating cytochrome P450c17α activity and synergizing with luteinizing hormone (LH), thereby contributing to hyperandrogenism and follicular arrest^8,9. Furthermore, insulin resistance promotes hepatic sex hormone–binding globulin (SHBG) suppression, increasing free circulating androgens^10–12. Thus, metabolic and reproductive abnormalities in PCOS are tightly interconnected.

Emerging evidence suggests that oxidative stress plays a critical role in the pathophysiology of PCOS. Oxidative stress arises from an imbalance between reactive oxygen species (ROS) generation and antioxidant defense systems^13,14. Women with PCOS demonstrate elevated markers of lipid peroxidation, such as malondialdehyde (MDA), and reduced antioxidant enzyme activity including superoxide dismutase (SOD) and glutathione peroxidase (GPx)^15–17. Excess ROS can impair insulin receptor signaling by promoting serine phosphorylation of insulin receptor substrate-1 (IRS-1), thereby worsening insulin resistance^18,19. In the ovary, oxidative stress disrupts folliculogenesis, induces granulosa cell apoptosis, and contributes to oocyte dysfunction^20,21. Therefore, oxidative injury may represent a mechanistic bridge between metabolic dysfunction and ovarian pathology in PCOS.

Beyond metabolic dysfunction, increasing evidence suggests that PCOS is also characterized by profound alterations in ovarian cellular homeostasis, follicular dynamics, and endocrine signaling pathways. Oxidative stress within the ovarian microenvironment contributes to granulosa cell apoptosis, mitochondrial dysfunction, impaired follicular maturation, and disruption of normal folliculogenesis²². Recent studies have highlighted the importance of autophagy regulation, apoptotic signaling, and redox-sensitive endocrine pathways in maintaining the primordial follicular pool and ovarian function^23,24. Dysregulation of these mechanisms may accelerate follicular atresia and impair ovulatory competence. Furthermore, endocrine and inflammatory signaling pathways closely interact with oxidative stress responses, thereby contributing to persistent hyperandrogenism and reproductive dysfunction in PCOS. Therefore, therapeutic strategies targeting both oxidative and endocrine–metabolic pathways may provide broader protective effects on ovarian physiology and reproductive function.

Alpha-lipoic acid (ALA) is a naturally occurring mitochondrial cofactor involved in oxidative decarboxylation reactions and is recognized for its potent antioxidant properties^25–27. ALA and its reduced form, dihydrolipoic acid, can directly scavenge reactive oxygen species and regenerate other antioxidants such as vitamins C and E^28,29. Beyond its redox-modulating effects, ALA enhances insulin sensitivity by activating AMP-activated protein kinase (AMPK) and promoting glucose transporter type 4 (GLUT4) translocation in skeletal muscle and adipose tissue³⁰. Clinical studies in women with PCOS have demonstrated that ALA supplementation improves insulin sensitivity indices, reduces serum androgen levels, and ameliorates metabolic parameters^25,26,31. Experimental models further support its ability to reduce oxidative damage and restore ovarian morphology^32,33.

Vitamin D, traditionally known for its role in calcium–phosphate homeostasis, has emerged as an important modulator of metabolic and reproductive function. Vitamin D receptors (VDR) are expressed in ovarian tissue, including granulosa and theca cells. Vitamin D influences steroidogenesis, follicular development, and anti-Müllerian hormone (AMH) expression^34,35. Hypovitaminosis D is highly prevalent in women with PCOS and has been associated with increased insulin resistance, hyperandrogenism, and inflammatory markers^36–38. Mechanistically, vitamin D may improve insulin sensitivity by enhancing insulin receptor expression and modulating inflammatory cytokine production. Supplementation studies suggest that vitamin D may reduce HOMA-IR levels and improve menstrual regularity in PCOS patients^39,40.

Notably, both ALA and vitamin D target overlapping yet distinct mechanisms implicated in PCOS pathogenesis—namely oxidative stress, inflammation, and insulin resistance. ALA primarily acts as a mitochondrial antioxidant and insulin-sensitizing agent, while vitamin D exerts endocrine–immunomodulatory effects that influence steroidogenesis and metabolic regulation. Given the multifactorial nature of PCOS, a combined therapeutic approach targeting both redox imbalance and metabolic dysregulation may provide synergistic benefits.

Preclinical rat models, particularly those induced by letrozole or dehydroepiandrosterone (DHEA), replicate key reproductive and metabolic features of human PCOS, including hyperandrogenism, insulin resistance, and cystic ovarian morphology^41,42. These models provide a controlled platform for investigating mechanistic interventions and evaluating biochemical, hormonal, and histopathological outcomes.

Although previous studies have investigated the individual effects of alpha-lipoic acid or vitamin D in PCOS models, evidence regarding their combined administration remains extremely limited. Moreover, most earlier investigations focused primarily on isolated metabolic or hormonal parameters without simultaneously evaluating oxidative stress, endocrine dysfunction, and detailed ovarian histomorphology within a single experimental framework. The present study differs from previous reports by specifically investigating the potential synergistic interaction between ALA and vitamin D through two-way ANOVA interaction analysis, thereby providing a multidimensional assessment of combined metabolic and ovarian protective effects in experimental PCOS.

Despite accumulating evidence regarding the individual benefits of ALA and vitamin D, limited data exist regarding their combined effects in experimental PCOS. The potential synergistic modulation of insulin resistance and ovarian oxidative stress has not been thoroughly explored in a controlled preclinical setting. Therefore, the present study aimed to investigate the combined effects of alpha-lipoic acid and vitamin D on insulin resistance, oxidative stress parameters, and ovarian histopathology in an experimental rat model of PCOS. We hypothesized that co-administration would exert synergistic protective effects by attenuating metabolic dysfunction and restoring ovarian redox homeostasis.

Materials and methods

Study design and experimental groups

Sixty female Wistar Albino rats (8–10 weeks old, weighing 200–230 g) were obtained from the Experimental Research Center of Mustafa Kemal University, Hatay, Turkey. Animals were housed under standardized laboratory conditions (temperature 21–22 °C, relative humidity 55 ± 5%, 12 h light/dark cycle) with unrestricted access to standard pellet diet and water.

A 12-day acclimatization period was provided prior to initiation of the experimental protocol. During this phase, vaginal smears were collected once daily between 08:00 and 10:00 a.m. to monitor estrous cyclicity. Only animals demonstrating regular 4–5-day estrous cycles were included. Rats exhibiting irregular cycles during acclimatization were excluded.

All experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of Mustafa Kemal University (Approval No: 2024-11/9, 11 May 2024).

The animals were randomly allocated into six equal groups (n = 10 per group):

• Group 1 (Control): Healthy rats receiving vehicle treatment without PCOS induction.

• Group 2 (PCOS): Rats with letrozole-induced PCOS receiving no therapeutic intervention.

• Group 3 (PCOS + MET): PCOS-induced rats treated with metformin (500 mg/kg/day) as a reference insulin-sensitizing therapy.

• Group 4 (PCOS + Vitamin D): PCOS-induced rats treated with vitamin D3 (1000 IU/kg/day) to evaluate its endocrine and metabolic effects.

• Group 5 (PCOS + ALA): PCOS-induced rats treated with alpha-lipoic acid (100 mg/kg/day) to assess its antioxidant and insulin-sensitizing properties.

• Group 6 (PCOS + ALA + Vitamin D): PCOS-induced rats treated with combined alpha-lipoic acid and vitamin D3 to investigate potential synergistic metabolic and ovarian protective effects.

Randomization was performed using a computer-generated sequence. Investigators responsible for histopathological and biochemical assessments were blinded to group allocation. Body weight was recorded weekly throughout both the induction and treatment phases to monitor general metabolic status and treatment-related changes. However, adiposity indices and detailed food intake measurements were not systematically evaluated.

Induction of PCOS and treatment protocol

PCOS was induced using letrozole (1 mg/kg/day) administered orally via gavage for 21 consecutive days. Letrozole, a nonsteroidal aromatase inhibitor, suppresses estrogen synthesis and induces hyperandrogenism, anovulation, and cystic ovarian morphology consistent with human PCOS^1,4. The control group received the vehicle solution only. At the end of the 21-day induction period, estrous cycle disruption was confirmed by persistent diestrus phase in vaginal cytology, and PCOS phenotype was verified prior to initiation of treatment interventions.

Following confirmation of PCOS induction, treatment was administered for 30 consecutive days as follows: Metformin: 500 mg/kg/day, orally (positive control comparator), Vitamin D3 (cholecalciferol): 1000 IU/kg/day (~ 25 µg/kg/day), orally^43,44, and ALA: 100 mg/kg/day, orally^45,46. In the combination group (Group 6), ALA and vitamin D were administered concomitantly at the same doses. Dose selection was based on previously published experimental studies demonstrating metabolic, antioxidant, and ovarian protective efficacy within these ranges in rodent models of endocrine and oxidative stress–related disorders^43–46. The selected doses were also chosen to achieve biologically relevant effects without evidence of systemic toxicity in prior preclinical investigations.

Estrous cycle monitoring and sample collection

Vaginal smears were collected daily throughout both induction and treatment phases. Samples were stained using methylene blue and examined under light microscopy (×10 and ×40 magnification). Estrous phases (proestrus, estrus, metestrus, diestrus) were identified according to established cytological criteria⁴⁷. Restoration of cyclicity during the treatment phase was recorded as a functional reproductive outcome.

At the end of the treatment period, rats were fasted overnight (12 h). Anesthesia was induced with ketamine (50 mg/kg) and xylazine (10 mg/kg) intraperitoneally. Blood samples were collected via cardiac puncture and centrifuged at 3000 rpm for 10 min to obtain serum, which was stored at − 80 °C until analysis. Both ovaries were excised, weighed, and divided into two portions. One portion was fixed in 10% neutral buffered formalin for histopathological analysis and the remaining tissue was snap-frozen in liquid nitrogen and stored at − 80 °C for biochemical assays (Fig. 1).

Fig. 1.

Experimental timeline and confirmation of PCOS induction. (A) Study design schematic. (B) Representative vaginal smear cytology images (Control vs. PCOS). (C) Estrous cycle distribution across groups.

Biochemical and hormonal analysis

Fasting blood glucose (FBG) levels were measured using an automated biochemical analyzer. Serum insulin concentrations were determined by ELISA kits specific for rat insulin. Insulin resistance was calculated using the Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) formula as “HOMA-IR = (Fasting Glucose × Fasting Insulin)/405”.

Ovarian tissues were homogenized in ice-cold phosphate-buffered saline (PBS, pH 7.4) using a glass–Teflon homogenizer (IKA-Werke GmbH, Staufen, Germany) (1:10 w/v). Homogenates were centrifuged at 10,000 × g for 15 min at 4 °C (Eppendorf 5804R, Eppendorf AG, Hamburg, Germany), and the supernatants were collected for biochemical analyses. Lipid peroxidation was assessed by measuring MDA levels using the thiobarbituric acid reactive substances (TBARS) method, with results expressed as nmol/mg protein. Superoxide dismutase (SOD) activity was determined based on its ability to inhibit nitroblue tetrazolium reduction and expressed as U/mg protein. Catalase (CAT) activity was measured spectrophotometrically by monitoring the decomposition rate of hydrogen peroxide at 240 nm and expressed as U/mg protein. Reduced glutathione (GSH) levels were quantified using Ellman’s reagent (5,5′-dithiobis-2-nitrobenzoic acid; DTNB) and expressed as µmol/g protein^12,35,44,45. Total protein concentrations were determined using the Bradford assay. Absorbance measurements were obtained using a microplate spectrophotometer (BioTek Epoch 2 Microplate Reader, Agilent Technologies Inc., Santa Clara, CA, USA). Commercial assay kits were obtained from Elabscience Biotechnology Co., Ltd. (Wuhan, China) and applied according to the manufacturer’s instructions.

Serum concentrations of total testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were quantified using rat-specific ELISA kits (Elabscience Biotechnology Co., Ltd., Wuhan, China; Cat. Nos. E-OSEL-R0003 for testosterone, E-EL-R0026 for LH, and E-EL-R0391 for FSH). All assays were performed according to the manufacturer’s instructions. Briefly, serum samples were brought to room temperature and added to pre-coated microplates, followed by incubation with biotinylated detection antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies. After washing, substrate solution (TMB) was added, and the reaction was stopped with sulfuric acid. Optical density was measured at 450 nm using a microplate reader (BioTek Epoch 2 Microplate Reader, Agilent Technologies Inc., Santa Clara, CA, USA). The assay sensitivities were < 0.1 ng/mL for testosterone, < 0.5 mIU/mL for LH, and < 0.5 mIU/mL for FSH. Intra-assay and inter-assay coefficients of variation were < 8% and < 10%, respectively. Hormone concentrations were calculated from standard curves generated using four-parameter logistic regression. The LH/FSH ratio was calculated for each animal as an additional indicator of endocrine imbalance associated with PCOS.

Histopathological evaluation

Formalin-fixed ovarian tissues were processed through graded alcohols, cleared in xylene, and embedded in paraffin. Serial sections (4–5 μm thickness) were obtained using a rotary microtome (Leica RM2235, Leica Biosystems, Wetzlar, Germany), mounted on poly-l-lysine–coated slides, deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E). To minimize the risk of duplicate follicle counting, every tenth section throughout the ovary was evaluated. Histological examination was performed using a light microscope (Olympus BX53, Olympus Corporation, Tokyo, Japan), and digital images were captured for morphometric analysis using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Follicles were classified according to established morphological criteria^48,49 as primordial, primary, secondary, antral, or cystic follicles. Cystic follicles were defined as enlarged fluid-filled structures with attenuated granulosa cell layers, thickened theca interna, and absence of a visible oocyte. The number of cystic follicles, healthy antral follicles, and corpora lutea per ovary was recorded. The thickness of granulosa and theca interna layers was measured at three equidistant points per follicle and averaged. All histological assessments were conducted by a blinded pathologist unaware of group allocation, and results were expressed as mean values per ovary.

Statistical analysis

All statistical analyses were performed using SPSS software (IBM SPSS Statistics for Windows, Version XX.0, IBM Corp., Armonk, NY, USA). Data were expressed as mean ± standard deviation (SD). Normality of distribution was assessed using the Shapiro–Wilk test, and homogeneity of variances was evaluated with Levene’s test. For comparisons between two groups (Control vs. PCOS at Day 21), independent samples t-test was used for normally distributed variables, and the χ² test was applied for categorical data (persistent diestrus rates). For comparisons among multiple groups at Day 51, one-way analysis of variance (ANOVA) was performed, followed by Tukey’s post hoc test for pairwise multiple comparisons when overall significance was detected. In cases where normality assumptions were not met, the Kruskal–Wallis test followed by Dunn–Bonferroni correction was applied to control for multiple testing. These adjustment procedures were consistently used across biochemical, hormonal, oxidative stress, and histomorphometric outcome analyses to minimize type I error inflation. To evaluate potential synergistic effects of alpha-lipoic acid and vitamin D, interaction analysis was performed using two-way ANOVA, with ALA and vitamin D as fixed factors. Effect sizes were calculated using partial eta squared (η²) for ANOVA models. A p-value < 0.05 was considered statistically significant.

Results

Following 21 days of letrozole administration, 45 of 50 rats (90%) in the PCOS induction cohort exhibited persistent diestrus, whereas no rats in the control group showed persistent diestrus (0%) (p < 0.001). The proportion of days spent in diestrus was significantly higher in the PCOS group compared with controls (83.6 ± 6.2% vs. 24.8 ± 5.4%, p < 0.001). Ovarian weight was significantly increased in PCOS-induced rats relative to the control group (0.162 ± 0.018 g vs. 0.118 ± 0.014 g, p < 0.001). Serum total testosterone levels were also markedly elevated in the PCOS group compared with controls (2.84 ± 0.41 ng/mL vs. 1.12 ± 0.26 ng/mL, p < 0.001) (Table 1).

Table 1.

Confirmation of PCOS induction (Day 21, before treatment).

Parameter	Control (n = 10)	PCOS (n = 50)*	p-value
Rats with persistent diestrus, n (%)	0 (0%)	45 (90%)	< 0.001
Diestrus days (% of 21 days)	24.8 ± 5.4	83.6 ± 6.2	< 0.001
Ovarian weight (g)	0.118 ± 0.014	0.162 ± 0.018	< 0.001
Serum total testosterone (ng/mL)	1.12 ± 0.26	2.84 ± 0.41	< 0.001

Values are mean ± SD unless otherwise stated. *PCOS induction cohort includes all letrozole-exposed rats prior to randomization into treatment arms (Groups 2–6). independent samples t-test and χ² test were used.

At the end of the treatment period (Day 51), ovarian weight remained significantly higher in the PCOS group compared with controls (0.165 ± 0.019 g vs. 0.118 ± 0.014 g, p < 0.001). Treatment with metformin, vitamin D, and ALA significantly reduced ovarian weight compared with the untreated PCOS group (0.132 ± 0.016 g, 0.144 ± 0.017 g, and 0.136 ± 0.015 g, respectively; all p < 0.05 vs. PCOS). The combination therapy group demonstrated the lowest ovarian weight among treated groups (0.123 ± 0.013 g, p < 0.01 vs. PCOS), approaching control values. Serum total testosterone levels were significantly elevated in the PCOS group compared with controls (2.91 ± 0.44 ng/mL vs. 1.12 ± 0.26 ng/mL, p < 0.001). Metformin, vitamin D, and ALA treatments each significantly decreased testosterone levels relative to the PCOS group (1.73 ± 0.33 ng/mL, 2.03 ± 0.37 ng/mL, and 1.87 ± 0.31 ng/mL, respectively; all p < 0.05 vs. PCOS). The combined ALA and vitamin D treatment resulted in a further reduction in testosterone levels (1.29 ± 0.27 ng/mL, p < 0.01 vs. PCOS), nearing control concentrations (Table 2).

Table 2.

Ovarian weight and serum total testosterone after treatment (Day 51).

Parameter	Control (n = 10)	PCOS (n = 10)	PCOS + MET (n = 10)	PCOS + Vit D (n = 10)	PCOS + ALA (n = 10)	PCOS + ALA+ Vit D (n = 10)	p-value
Ovarian weight (g)	0.118 ± 0.014	0.165 ± 0.019†	0.132 ± 0.016*	0.144 ± 0.017*	0.136 ± 0.015*	0.123 ± 0.013**	< 0.001
Serum total testosterone (ng/mL)	1.12 ± 0.26	2.91 ± 0.44†	1.73 ± 0.33*	2.03 ± 0.37*	1.87 ± 0.31*	1.29 ± 0.27**	< 0.001

Values are mean ± SD unless otherwise stated. ^†p < 0.001 vs. Control; *p < 0.05 vs. PCOS; **p < 0.01 vs. PCOS.

At Day 51, fasting blood glucose (FBG) levels were significantly elevated in the PCOS group compared with controls (136.7 ± 11.2 mg/dL vs. 92.4 ± 6.8 mg/dL, p < 0.001). Treatment with metformin, vitamin D, and ALA significantly reduced FBG levels relative to the untreated PCOS group (101.3 ± 8.4 mg/dL, 112.6 ± 9.7 mg/dL, and 105.8 ± 8.9 mg/dL, respectively; all p < 0.05 vs. PCOS). The combination therapy group demonstrated the lowest FBG levels among the treated groups (94.7 ± 7.2 mg/dL, p < 0.01 vs. PCOS). Serum insulin levels were also significantly higher in the PCOS group compared with controls (19.6 ± 2.8 µIU/mL vs. 9.8 ± 1.5 µIU/mL, p < 0.001). Metformin, vitamin D, and ALA treatments significantly decreased insulin levels compared with PCOS (11.7 ± 1.9 µIU/mL, 14.8 ± 2.3 µIU/mL, and 12.9 ± 2.1 µIU/mL, respectively; all p < 0.05 vs. PCOS). The combined ALA and vitamin D group showed further reduction (10.4 ± 1.6 µIU/mL, p < 0.01 vs. PCOS). HOMA-IR values were markedly increased in the PCOS group relative to controls (6.61 ± 1.18 vs. 2.24 ± 0.42, p < 0.001). All treatment groups exhibited significantly lower HOMA-IR values compared with PCOS (metformin: 2.92 ± 0.53; vitamin D: 4.12 ± 0.78; ALA: 3.36 ± 0.64; combination: 2.43 ± 0.47; all p < 0.05 vs. PCOS), with the combination therapy group demonstrating values closest to the control group (p < 0.01 vs. PCOS) (Table 3).

Table 3.

Metabolic parameters across experimental groups (Day 51).

Parameter	Control (n = 10)	PCOS (n = 10)	PCOS + MET (n = 10)	PCOS + Vit D (n = 10)	PCOS + ALA (n = 10)	PCOS + ALA+ Vit D (n = 10)	p-value (ANOVA)
FBG (mg/dL)	92.4 ± 6.8	136.7 ± 11.2†	101.3 ± 8.4*	112.6 ± 9.7*	105.8 ± 8.9*	94.7 ± 7.2**	< 0.001
Insulin (µIU/mL)	9.8 ± 1.5	19.6 ± 2.8†	11.7 ± 1.9*	14.8 ± 2.3*	12.9 ± 2.1*	10.4 ± 1.6**	< 0.001
HOMA-IR	2.24 ± 0.42	6.61 ± 1.18†	2.92 ± 0.53*	4.12 ± 0.78*	3.36 ± 0.64*	2.43 ± 0.47**	< 0.001

Values are mean ± SD unless otherwise stated. ^†p < 0.001 vs. Control; *p < 0.05 vs. PCOS; **p < 0.01 vs. PCOS.

Since insulin resistance and oxidative stress are closely interconnected in PCOS pathophysiology, oxidative stress parameters were subsequently evaluated to determine whether the observed metabolic improvements were accompanied by restoration of ovarian redox balance. At Day 51, ovarian malondialdehyde (MDA) levels were significantly elevated in the PCOS group compared with controls (5.84 ± 0.72 vs. 2.31 ± 0.34 nmol/mg, p < 0.001). Treatment with metformin, vitamin D, and ALA significantly reduced MDA levels relative to the untreated PCOS group (3.48 ± 0.51, 4.12 ± 0.63, and 3.01 ± 0.46 nmol/mg, respectively; all p < 0.05 vs. PCOS). The combination therapy group demonstrated the lowest MDA levels among treated groups (2.52 ± 0.39 nmol/mg, p < 0.01 vs. PCOS), approaching control values.

Superoxide dismutase (SOD) activity was significantly decreased in the PCOS group compared with controls (9.6 ± 1.8 vs. 18.7 ± 2.4 U/mg, p < 0.001). All treatment groups showed significantly higher SOD activity compared with PCOS (metformin: 15.2 ± 2.1; vitamin D: 13.4 ± 1.9; ALA: 16.8 ± 2.3; combination: 17.9 ± 2.2 U/mg; all p < 0.05 vs. PCOS), with the combination group exhibiting values closest to control. Catalase (CAT) activity was also significantly reduced in PCOS rats compared with controls (21.6 ± 3.9 vs. 42.3 ± 4.8 U/mg, p < 0.001). Metformin, vitamin D, and ALA treatments significantly increased CAT activity relative to PCOS (34.7 ± 4.2, 30.5 ± 3.7, and 37.9 ± 4.4 U/mg, respectively; all p < 0.05 vs. PCOS). The combination group demonstrated the highest CAT activity among treated groups (40.8 ± 4.6 U/mg, p < 0.01 vs. PCOS). Reduced glutathione (GSH) levels were significantly lower in the PCOS group compared with controls (3.71 ± 0.82 vs. 8.42 ± 1.01 µmol/g, p < 0.001). All treatments significantly increased GSH levels compared with PCOS (metformin: 6.54 ± 0.93; vitamin D: 5.82 ± 0.88; ALA: 7.36 ± 0.95; combination: 8.05 ± 0.97 µmol/g; all p < 0.05 vs. PCOS), with the combination therapy group showing values closest to control (Table 4).

Table 4.

Ovarian oxidative stress parameters across experimental groups (Day 51).

Parameter	Control (n = 10)	PCOS (n = 10)	PCOS + MET (n = 10)	PCOS + Vit D (n = 10)	PCOS + ALA (n = 10)	PCOS + ALA+ Vit D (n = 10)	p-value (ANOVA)
MDA (nmol/mg)	2.31 ± 0.34	5.84 ± 0.72†	3.48 ± 0.51*	4.12 ± 0.63*	3.01 ± 0.46**	2.52 ± 0.39**	< 0.001
SOD (U/mg)	18.7 ± 2.4	9.6 ± 1.8†	15.2 ± 2.1*	13.4 ± 1.9*	16.8 ± 2.3**	17.9 ± 2.2**	< 0.001
CAT (U/mg)	42.3 ± 4.8	21.6 ± 3.9†	34.7 ± 4.2*	30.5 ± 3.7*	37.9 ± 4.4**	40.8 ± 4.6**	< 0.001
GSH (µmol/g)	8.42 ± 1.01	3.71 ± 0.82†	6.54 ± 0.93*	5.82 ± 0.88*	7.36 ± 0.95**	8.05 ± 0.97**	< 0.001

Values are mean ± SD unless otherwise stated. ^†p < 0.001 vs. Control; *p < 0.05 vs. PCOS; **p < 0.01 vs. PCOS.

Given the close relationship between endocrine imbalance and follicular dysfunction in PCOS, histomorphometric analyses were performed to determine whether the hormonal improvements translated into structural ovarian recovery. At Day 51, serum total testosterone levels were significantly elevated in the PCOS group compared with controls (2.91 ± 0.44 ng/mL vs. 1.12 ± 0.26 ng/mL, p < 0.001). Treatment with metformin, vitamin D, and ALA significantly reduced testosterone levels relative to the untreated PCOS group (1.73 ± 0.33, 2.03 ± 0.37, and 1.87 ± 0.31 ng/mL, respectively; all p < 0.05 vs. PCOS). The combination therapy group demonstrated the lowest testosterone levels among treated groups (1.29 ± 0.27 ng/mL, p < 0.01 vs. PCOS). Luteinizing hormone (LH) levels were significantly higher in the PCOS group compared with controls (7.96 ± 1.14 vs. 3.84 ± 0.62 mIU/mL, p < 0.001). All treatment groups showed significantly reduced LH levels compared with PCOS (metformin: 5.02 ± 0.88; vitamin D: 6.11 ± 0.93; ALA: 5.48 ± 0.81; combination: 4.21 ± 0.69 mIU/mL; all p < 0.05 vs. PCOS). Follicle-stimulating hormone (FSH) levels were significantly lower in the PCOS group compared with controls (3.18 ± 0.64 vs. 4.21 ± 0.73 mIU/mL, p < 0.001). Metformin, ALA, and combination therapy significantly increased FSH levels relative to PCOS (3.87 ± 0.71, 3.94 ± 0.76, and 4.08 ± 0.68 mIU/mL, respectively; all p < 0.05 vs. PCOS), whereas vitamin D treatment did not demonstrate a statistically significant increase. The LH/FSH ratio was markedly increased in the PCOS group compared with controls (2.51 ± 0.42 vs. 0.91 ± 0.18, p < 0.001). All treatment groups exhibited significantly lower LH/FSH ratios compared with PCOS (metformin: 1.30 ± 0.24; vitamin D: 1.69 ± 0.31; ALA: 1.39 ± 0.27; combination: 1.03 ± 0.19; all p < 0.05 vs. PCOS), with the combination therapy group showing values closest to control (Table 5).

Table 5.

Hormonal parameters across experimental groups (Day 51).

Parameter	Control (n = 10)	PCOS (n = 10)	PCOS + MET (n = 10)	PCOS + Vit D (n = 10)	PCOS + ALA (n = 10)	PCOS + ALA+ Vit D (n = 10)	p-value (ANOVA)
Testosterone (ng/mL)	1.12 ± 0.26	2.91 ± 0.44†	1.73 ± 0.33*	2.03 ± 0.37*	1.87 ± 0.31*	1.29 ± 0.27**	< 0.001
LH (mIU/mL)	3.84 ± 0.62	7.96 ± 1.14†	5.02 ± 0.88*	6.11 ± 0.93*	5.48 ± 0.81*	4.21 ± 0.69**	< 0.001
FSH (mIU/mL)	4.21 ± 0.73	3.18 ± 0.64†	3.87 ± 0.71*	3.62 ± 0.69	3.94 ± 0.76*	4.08 ± 0.68**	< 0.001
LH/FSH Ratio	0.91 ± 0.18	2.51 ± 0.42†	1.30 ± 0.24*	1.69 ± 0.31*	1.39 ± 0.27*	1.03 ± 0.19**	< 0.001

Values are mean ± SD unless otherwise stated. ^†p < 0.001 vs. Control; *p < 0.05 vs. PCOS; **p < 0.01 vs. PCOS.

At Day 51, the number of cystic follicles was significantly increased in the PCOS group compared with controls (8.6 ± 1.7 vs. 0.8 ± 0.9, p < 0.001). Treatment with metformin, vitamin D, and ALA significantly reduced cystic follicle counts relative to PCOS (4.2 ± 1.3, 5.1 ± 1.5, and 3.6 ± 1.2, respectively; all p < 0.05 vs. PCOS). The combination therapy group demonstrated the lowest number of cystic follicles among treated groups (1.4 ± 1.0, p < 0.01 vs. PCOS).

Antral follicle numbers were significantly decreased in the PCOS group compared with controls (3.2 ± 1.1 vs. 6.9 ± 1.6, p < 0.001). All treatment groups showed significantly higher antral follicle counts compared with PCOS (metformin: 5.4 ± 1.4; vitamin D: 4.7 ± 1.3; ALA: 5.8 ± 1.5; combination: 6.5 ± 1.4; all p < 0.05 vs. PCOS). The number of corpora lutea was markedly reduced in the PCOS group compared with controls (1.1 ± 0.7 vs. 4.8 ± 1.2, p < 0.001). Metformin, vitamin D, and ALA treatments significantly increased corpora lutea counts relative to PCOS (3.6 ± 1.0, 2.9 ± 0.9, and 3.9 ± 1.1, respectively; all p < 0.05 vs. PCOS). The combination therapy group exhibited 4.5 ± 1.0 corpora lutea (p < 0.01 vs. PCOS). Theca thickness was significantly increased in the PCOS group compared with controls (86.3 ± 8.7 μm vs. 42.6 ± 4.9 μm, p < 0.001). All treatments significantly reduced theca thickness relative to PCOS (metformin: 59.4 ± 6.8 μm; vitamin D: 66.8 ± 7.4 μm; ALA: 54.7 ± 6.1 μm; combination: 46.9 ± 5.3 μm; all p < 0.05 vs. PCOS). Granulosa layer thickness was significantly decreased in the PCOS group compared with controls (38.7 ± 5.8 μm vs. 71.2 ± 6.5 μm, p < 0.001). Treatment with metformin, vitamin D, and ALA significantly increased granulosa thickness compared with PCOS (60.3 ± 6.2 μm, 54.1 ± 5.9 μm, and 63.8 ± 6.7 μm, respectively; all p < 0.05 vs. PCOS). The combination therapy group demonstrated granulosa thickness values closest to control (69.4 ± 6.1 μm, p < 0.01 vs. PCOS) (Table 6; Figs. 2 and 3).

Table 6.

Histomorphometric analysis of ovarian tissue (Day 51).

Parameter	Control (n = 10)	PCOS (n = 10)	PCOS + MET (n = 10)	PCOS + Vit D (n = 10)	PCOS + ALA (n = 10)	PCOS + ALA+ Vit D (n = 10)	p-value (ANOVA)
Cystic follicles	0.8 ± 0.9	8.6 ± 1.7†	4.2 ± 1.3*	5.1 ± 1.5*	3.6 ± 1.2**	1.4 ± 1.0**	< 0.001
Antral follicles	6.9 ± 1.6	3.2 ± 1.1†	5.4 ± 1.4*	4.7 ± 1.3*	5.8 ± 1.5**	6.5 ± 1.4**	< 0.001
Corpora lutea	4.8 ± 1.2	1.1 ± 0.7†	3.6 ± 1.0*	2.9 ± 0.9*	3.9 ± 1.1**	4.5 ± 1.0**	< 0.001
Theca thickness (µm)	42.6 ± 4.9	86.3 ± 8.7†	59.4 ± 6.8*	66.8 ± 7.4*	54.7 ± 6.1**	46.9 ± 5.3**	< 0.001
Granulosa thickness (µm)	71.2 ± 6.5	38.7 ± 5.8†	60.3 ± 6.2*	54.1 ± 5.9*	63.8 ± 6.7**	69.4 ± 6.1**	< 0.001

Values are mean ± SD unless otherwise stated. ^†p < 0.001 vs. Control; *p < 0.05 vs. PCOS; **p < 0.01 vs. PCOS.

Fig. 2.

H&E-stained ovarian sections (×100 magnification). Control vs. PCOS vs. MET vs. Vitamin D vs. ALA vs. ALA + Vitamin D.

Fig. 3.

Quantitative histological comparison. (A) Number of cystic follicles; (B) Number of corpora lutea; (C) Theca thickness; (D) Granulosa thickness.

Two-way ANOVA demonstrated significant main effects of ALA and vitamin D as well as significant interaction effects for multiple outcome variables. For HOMA-IR, significant main effects were observed for ALA (F = 18.42, p < 0.001, partial η² = 0.41) and vitamin D (F = 9.76, p = 0.003, partial η² = 0.23), along with a significant ALA × vitamin D interaction (F = 6.58, p = 0.014, partial η² = 0.17). For MDA levels, significant main effects were detected for ALA (F = 22.63, p < 0.001, partial η² = 0.46) and vitamin D (F = 11.12, p = 0.002, partial η² = 0.26), with a significant interaction effect (F = 7.21, p = 0.011, partial η² = 0.19). Testosterone levels also showed significant main effects of ALA (F = 14.88, p < 0.001, partial η² = 0.36) and vitamin D (F = 6.95, p = 0.012, partial η² = 0.18), as well as a significant interaction (F = 5.02, p = 0.031, partial η² = 0.14). Similarly, LH/FSH ratio demonstrated significant main effects for ALA (F = 12.41, p = 0.001, partial η² = 0.32) and vitamin D (F = 5.87, p = 0.019, partial η² = 0.16), with a significant interaction effect (F = 4.66, p = 0.037, partial η² = 0.13) (Table 7).

Table 7.

Effect size comparison and interaction analysis between treatment modalities (Two-Way ANOVA).

Outcome variable	Main effect: ALA (F, p)	Partial η2 (ALA)	Main effect: vitamin D (F, p)	Partial η2 (Vit D)	Interaction (ALA × Vit D) F, p	Partial η2 (interaction)
HOMA-IR	F = 18.42, p < 0.001	0.41	F = 9.76, p = 0.003	0.23	F = 6.58, p = 0.014	0.17
MDA (nmol/mg)	F = 22.63, p < 0.001	0.46	F = 11.12, p = 0.002	0.26	F = 7.21, p = 0.011	0.19
Testosterone (ng/mL)	F = 14.88, p < 0.001	0.36	F = 6.95, p = 0.012	0.18	F = 5.02, p = 0.031	0.14
LH/FSH Ratio	F = 12.41, p = 0.001	0.32	F = 5.87, p = 0.019	0.16	F = 4.66, p = 0.037	0.13

Discussion

In the present study, combined administration of alpha-lipoic acid and vitamin D produced the most pronounced improvement in metabolic, oxidative, endocrine, and histomorphological parameters in a letrozole-induced rat model of PCOS. Letrozole induction successfully generated a hyperandrogenic, insulin-resistant, and cystic ovarian phenotype characterized by increased ovarian weight, elevated testosterone levels, impaired estrous cyclicity, marked elevation of HOMA-IR, increased malondialdehyde levels, reduced antioxidant enzyme activities, and disrupted follicular architecture. While metformin, vitamin D, and ALA monotherapies each ameliorated several of these abnormalities, the combination treatment consistently restored fasting glucose, insulin resistance indices, oxidative stress markers, LH/FSH ratio, and ovarian morphology to levels closest to those observed in the control group. Interaction analyses further indicated that the combined intervention exerted effects that exceeded those of either agent alone for key outcomes, including HOMA-IR, MDA, testosterone, and LH/FSH ratio.

PCOS is increasingly recognized as a systemic metabolic disorder in which insulin resistance and hyperandrogenism mutually reinforce each other^1,6. In our model, letrozole successfully induced persistent diestrus, ovarian hypertrophy, hyperandrogenemia, and severe insulin resistance, consistent with previously validated preclinical models^37,38. Ercan et al. reported that letrozole-induced PCOS rats exhibit elevated HOMA-IR, increased testosterone levels, and cystic ovarian morphology comparable to the findings observed in our PCOS group⁵. The magnitude of metabolic impairment in our untreated PCOS rats (approximately threefold increase in HOMA-IR) supports the robustness of the model.

Insulin resistance is a central driver of ovarian androgen excess^17,36. In our study, ALA significantly reduced fasting glucose, insulin levels, and HOMA-IR, consistent with its known role in enhancing insulin signaling via AMPK activation and GLUT4 translocation^25,30. Capece et al. emphasized that ALA improves glucose uptake and reduces oxidative stress–mediated insulin signaling impairment²⁵. Dajnowicz-Brzezik et al. demonstrated that ALA reduces oxidative stress and improves insulin sensitivity in insulin-resistant animal models³⁰. Our findings align with these mechanistic observations.

Vitamin D monotherapy also ameliorated insulin resistance, albeit to a lesser extent than ALA. Rashidi et al. showed that vitamin D supplementation reduces fasting glucose and improves HOMA-IR in women with PCOS³⁹. Trummer et al. similarly reported modest metabolic benefits in randomized clinical trials⁴⁰. The improvement observed in our vitamin D group parallels these findings. Notably, the combination therapy group demonstrated HOMA-IR values nearly indistinguishable from controls. Two-way ANOVA revealed significant interaction effects for HOMA-IR, suggesting that the metabolic improvement exceeded additive expectations. This supports a mechanistic synergy, likely attributable to simultaneous correction of mitochondrial oxidative dysfunction (ALA) and endocrine–inflammatory dysregulation (Vitamin D).

Metformin was included in the present study as a reference insulin-sensitizing therapy because it remains one of the most widely used pharmacological agents in PCOS management. Consistent with previous literature, metformin significantly ameliorated glucose metabolism, hormonal imbalance, and ovarian morphology in our experimental model. However, the combined ALA and vitamin D treatment demonstrated comparable or even greater improvements in several parameters, particularly oxidative stress markers, ovarian histomorphology, and LH/FSH ratio normalization. These findings suggest that multidimensional modulation targeting both mitochondrial oxidative stress and endocrine–metabolic dysfunction may provide broader biological benefits than insulin sensitization alone. Therefore, the observed efficacy of combination therapy supports the concept of polytherapeutic approaches in complex multifactorial disorders such as PCOS.

Collectively, these findings support the concept that concurrent modulation of oxidative and endocrine–metabolic pathways may represent a promising multidimensional therapeutic strategy in experimental PCOS^30,39. PCOS patients and experimental models exhibit elevated MDA levels and decreased antioxidant enzyme activity^31–33. Our PCOS group demonstrated marked elevation of MDA and suppression of SOD, CAT, and GSH, confirming a severe redox imbalance. ALA monotherapy significantly reduced lipid peroxidation and restored antioxidant enzyme activity. Elmosalamy et al. reported that ALA ameliorates oxidative stress and regulates steroidogenesis in letrozole-induced PCOS rats³². Abu-Zaid et al. demonstrated improvements in oxidative stress markers following ALA supplementation in PCOS patients³³. Our results are consistent with these data.

Vitamin D also ameliorated oxidative parameters, though less robustly than ALA. Morgante et al. described vitamin D as a modulator of inflammatory and oxidative pathways in PCOS³⁶. Narayanan et al. showed an association between vitamin D status and metabolic dysfunction³⁷. Similar antioxidant and insulin-regulatory effects of vitamin D have also been demonstrated in experimental diabetic models, where vitamin D supplementation reduced oxidative stress, apoptosis, and metabolic dysfunction in pancreatic tissues^50,51. The moderate improvement observed in our vitamin D group reflects this supportive but not dominant antioxidant role. The most striking finding was the near-complete normalization of MDA and antioxidant enzymes in the combination group. The significant interaction term for MDA indicates that combined therapy provides synergistic redox stabilization. This is biologically plausible: ALA directly scavenges ROS and regenerates endogenous antioxidants^28,29, while vitamin D modulates inflammatory cytokines and insulin receptor expression, thereby reducing upstream ROS production.

The beneficial effects observed in the present study may be explained through complementary yet interconnected molecular mechanisms involving mitochondrial redox regulation, insulin signaling, and ovarian steroidogenic modulation. Alpha-lipoic acid is known to improve insulin sensitivity through activation of AMP-activated protein kinase (AMPK), enhancement of GLUT4 translocation, and reduction of reactive oxygen species-mediated impairment of IRS-1 signaling^21,26. Previous experimental studies also demonstrated that modulation of oxidative stress and MAPK-related pathways may contribute to metabolic improvement and attenuation of apoptosis in insulin-resistant conditions^52–54. By restoring mitochondrial redox balance, ALA may reduce oxidative damage within granulosa cells and improve follicular microenvironment integrity. In contrast, vitamin D exerts endocrine and immunomodulatory effects primarily through activation of the VDR, which regulates steroidogenic enzyme activity, inflammatory cytokine production, and insulin receptor expression^30,32,36. Experimental evidence suggests that vitamin D signaling may attenuate ovarian androgen excess by modulating theca cell steroidogenesis and improving hypothalamic–pituitary–ovarian axis regulation. Therefore, the enhanced combined efficacy observed in our study may reflect simultaneous correction of oxidative stress–mediated insulin dysfunction and endocrine dysregulation. The observed effects may reflect convergence of complementary protective pathways rather than direct pharmacodynamic interaction. Although direct molecular analyses such as AMPK phosphorylation, IRS-1 activity, inflammatory cytokine profiling, or VDR expression were not performed, the integrated biochemical, hormonal, and histomorphological improvements observed in the combination group support the presence of coordinated metabolic and ovarian protective mechanisms.

The observed synergistic effects may arise not from a single direct molecular interaction, but from complementary modulation of interconnected signaling networks involved in oxidative stress, insulin resistance, inflammation, and ovarian steroidogenesis. ALA primarily targets mitochondrial redox homeostasis and insulin signaling pathways, whereas vitamin D exerts broader endocrine–immunomodulatory effects through VDR-mediated transcriptional regulation. Reduction of oxidative stress by ALA may indirectly improve cellular responsiveness to vitamin D signaling by preserving intracellular redox-sensitive regulatory mechanisms, while vitamin D-mediated attenuation of inflammatory and endocrine dysregulation may enhance the metabolic stability required for effective mitochondrial function. Therefore, the “1 + 1 > 2” effect observed in the present study may reflect convergence of parallel protective pathways rather than direct pharmacodynamic interaction between the two agents. Additional pathway cross-talk involving PI3K/Akt signaling, redox-sensitive steroidogenic regulation, inflammatory mediator suppression, and mitochondrial-endocrine signaling interactions may also contribute to the enhanced combined efficacy observed in the present model. Nevertheless, these proposed molecular interactions remain hypothetical and require validation through future studies incorporating pathway-specific molecular analyses such as AMPK phosphorylation, VDR activity assays, inflammatory cytokine profiling, and transcriptomic evaluation.

Hyperandrogenism is a hallmark of PCOS and is closely linked to insulin resistance, wherein insulin-driven theca cell hyperactivity contributes to excessive ovarian androgen production, as highlighted by Rambaran et al.⁸. Consistent with this pathophysiological framework, our PCOS rats exhibited marked elevations in testosterone and LH/FSH ratio. ALA treatment significantly reduced testosterone levels, in agreement with previous findings by Jannatifar et al.³¹, who demonstrated improved hormonal profiles following ALA supplementation in PCOS patients undergoing assisted reproductive technologies, and by Elmosalamy et al.³², who reported modulation of steroidogenesis in experimental PCOS models. Likewise, Vitamin D monotherapy reduced LH and testosterone levels, consistent with its recognized role in ovarian VDR-mediated steroidogenic regulation. Moridi et al. further demonstrated that Vitamin D influences anti-Müllerian hormone (AMH) levels and ovarian endocrine function³⁴. Notably, the combination therapy group showed testosterone and LH/FSH ratio values closest to controls, suggesting that simultaneous improvement in insulin sensitivity by ALA and modulation of ovarian steroidogenic signaling by Vitamin D may collectively attenuate androgen overproduction more effectively than either intervention alone. The observed endocrine improvements may therefore reflect convergence of complementary metabolic and steroidogenic regulatory pathways rather than isolated hormonal modulation alone. Nevertheless, these findings should be interpreted cautiously, since single time-point serum hormone measurements and absence of direct molecular pathway analyses limit definitive mechanistic interpretation of hypothalamic–pituitary–ovarian axis regulation.

Morphologically, PCOS ovaries displayed increased cystic follicles, reduced corpora lutea, theca hyperplasia, and granulosa thinning, consistent with established models^48,49,55,56. Ueda et al. described similar cystic morphology and theca thickening in experimental PCOS rats⁴⁸. ALA significantly reduced cystic follicles and restored granulosa thickness. Dokuyucu et al. previously demonstrated antioxidant-mediated ovarian protection with lipoic acid in ischemia-reperfusion injury, suggesting a broader ovarian cytoprotective effect⁴⁵. Vitamin D moderately ameliorated follicular morphology. Karateke et al. reported protective ovarian effects of vitamin D in ischemia-reperfusion models³⁵. The combination therapy group exhibited near-normal follicular distribution and theca thickness, paralleling metabolic and endocrine normalization. The histological improvements likely reflect restoration of redox balance and insulin signaling, both of which influence granulosa cell survival and folliculogenesis.

The observed restoration of estrous cyclicity, normalization of follicular architecture, increase in corpora lutea counts, and improvement in endocrine parameters may collectively suggest partial recovery of reproductive ovarian function. Previous experimental and clinical studies have reported associations between improvement in folliculogenesis, endocrine balance, and reproductive outcomes in PCOS-related infertility settings, particularly following interventions targeting oxidative stress and metabolic dysfunction^31,39,40. Nevertheless, the present study did not directly evaluate functional fertility endpoints such as ovulation competence, mating success, implantation efficiency, pregnancy maintenance, litter size, or live birth outcomes. Therefore, although the histomorphological and endocrine improvements observed in the combination-treatment group may indicate enhanced reproductive potential, such interpretation should currently be considered inferential rather than definitive evidence of restored fertility.

Beyond systemic metabolic improvement, the ovarian protective effects observed in the present study may also involve preservation of granulosa cell integrity and attenuation of follicular apoptotic processes. Granulosa cells play a critical role in follicular maturation, steroidogenesis, and oocyte survival, and oxidative injury to these cells has been implicated in follicular atresia and reproductive dysfunction. Previous studies demonstrated that oxidative stress–mediated apoptosis and cytotoxic injury in granulosa cells contribute to impaired ovarian follicular dynamics and endocrine imbalance^{22–24,55,56}. Bhardwaj and colleagues reported that oxidative and toxic insults may induce ultrastructural degeneration, DNA fragmentation, and apoptotic alterations within granulosa cells, thereby disrupting normal folliculogenesis and ovarian function^23,56,57. In this context, the restoration of granulosa thickness and reduction in cystic follicle formation observed in the combination-treatment group may not merely reflect systemic metabolic recovery but could also indicate preservation of ovarian cellular microarchitecture through antioxidant and anti-apoptotic mechanisms. Additionally, although the present study focused primarily on oxidative stress and endocrine parameters, alternative mechanisms such as modulation of autophagy, inflammatory signaling pathways, mitochondrial stabilization, and local ovarian cytokine regulation may also contribute to the observed protective effects. These pathways warrant further molecular investigation in future studies.

It should also be acknowledged that the ovarian protective effects observed in the present in vivo model may not be exclusively mediated through direct ovarian mechanisms. Improvements in systemic insulin sensitivity, oxidative balance, inflammatory status, and metabolic regulation may indirectly contribute to restoration of ovarian morphology and endocrine function through metabolic cross-talk involving the liver, adipose tissue, and peripheral endocrine signaling pathways. Therefore, the observed histomorphological and hormonal improvements likely reflect integrated systemic and ovarian responses rather than isolated ovary-specific effects alone. Future studies employing ovarian cell-specific molecular analyses, isolated granulosa/theca cell models, or tissue-targeted pathway investigations are necessary to distinguish direct ovarian actions from secondary systemic metabolic effects.

PCOS represents an interplay between metabolic dysfunction, oxidative stress, and endocrine imbalance. ALA primarily targets mitochondrial oxidative stress and insulin signaling, whereas vitamin D modulates steroidogenesis, inflammation, and insulin receptor expression. The present findings suggest that simultaneous correction of mitochondrial redox imbalance and endocrine–metabolic dysregulation produces greater therapeutic efficacy than targeting either axis alone. The significant interaction effects observed for HOMA-IR, MDA, testosterone, and LH/FSH ratio support enhanced combined efficacy that may extend beyond simple additive effects, although definitive mechanistic synergy cannot be established without direct molecular validation. These results align with emerging therapeutic paradigms emphasizing multi-target modulation in complex endocrine–metabolic disorders^3,9.

Unlike previously published studies that evaluated either alpha-lipoic acid or vitamin D as monotherapy in experimental or clinical PCOS settings, the present study simultaneously targeted mitochondrial oxidative stress and endocrine–metabolic signaling within the same controlled model and formally tested interaction effects. Prior reports such as Elmosalamy et al. and Abu-Zaid et al. primarily focused on the antioxidant and metabolic benefits of ALA alone, while Trummer et al. and Rashidi et al. examined vitamin D supplementation mainly in relation to insulin resistance and hormonal modulation^32,33,39,40. In contrast, our experimental design incorporated a dedicated combination-treatment arm together with two-way ANOVA interaction analysis, enabling evaluation of whether the combined intervention exerted effects beyond those expected from individual therapies alone. Furthermore, the study simultaneously integrated metabolic, oxidative stress, endocrine, and detailed histomorphometric endpoints within a single experimental framework, thereby providing a multidimensional evaluation that extends beyond the scope of most prior investigations, which generally focused on isolated metabolic or hormonal domains. Collectively, these features strengthen the translational and mechanistic relevance of the present findings and support the concept that concurrent modulation of oxidative stress and endocrine–metabolic dysregulation may provide enhanced therapeutic benefit in experimental PCOS.

This study is limited by its preclinical design and absence of direct molecular pathway analyses such as AMPK activation, IRS-1 phosphorylation, inflammatory cytokine profiling, or VDR expression. Therefore, the proposed mechanistic interpretations regarding oxidative stress modulation, insulin signaling, and endocrine regulation should be considered biologically plausible but inferential rather than directly validated. Additionally, estradiol concentrations were not measured. Although estradiol is an important reproductive hormone in PCOS pathophysiology, the present study primarily focused on insulin resistance, oxidative stress, hyperandrogenism, and ovarian histomorphological alterations. Future studies incorporating comprehensive steroid hormone profiling, including estradiol and progesterone measurements, may provide further mechanistic insight into endocrine modulation associated with combined ALA and vitamin D therapy. Although body weight was monitored throughout the study, detailed adiposity analysis and quantitative food intake assessment were not performed, which may have influenced metabolic and oxidative outcomes. Furthermore, the ovarian protective effects observed in the present model may reflect both direct ovarian actions and indirect improvements secondary to systemic metabolic regulation involving liver–adipose–ovarian cross-talk. The present study also did not evaluate direct reproductive outcomes such as ovulation competence, mating success, implantation efficiency, pregnancy maintenance, litter size, or live birth outcomes. Therefore, although restoration of estrous cyclicity, follicular architecture, and corpora lutea formation may suggest partial recovery of reproductive ovarian function, these findings should not be interpreted as definitive evidence of restored fertility. In addition, the letrozole-induced PCOS model may not fully recapitulate the heterogeneity and chronicity of human PCOS phenotypes, particularly obesity-associated and chronic inflammatory subtypes. Thus, the present findings should be interpreted as preclinical proof-of-concept evidence rather than direct evidence supporting clinical efficacy or therapeutic recommendation in human PCOS populations. Finally, although the observed enhanced combined efficacy suggests interactive modulation of complementary metabolic and endocrine pathways, definitive mechanistic synergy cannot be established in the absence of pathway-specific molecular validation. Future studies employing sub-therapeutic monotherapy doses combined into biologically effective combination regimens, together with molecular signaling analyses and long-term reproductive assessment, may provide a more rigorous framework for distinguishing true pharmacological synergy from enhanced additive efficacy.

From a translational perspective, the present findings support the concept that targeting multiple interconnected pathogenic pathways may be more effective than isolated monotherapeutic approaches in PCOS. Given the multifactorial nature of the syndrome, combined antioxidant and endocrine–metabolic modulation may represent a promising adjunctive therapeutic strategy. However, extrapolation of these findings to human disease should be approached cautiously, and well-designed clinical studies are required before therapeutic recommendations can be established.

Nevertheless, potential systemic and off-target effects of combined antioxidant and endocrine modulation should also be considered. Although attenuation of oxidative stress may improve metabolic and ovarian dysfunction in PCOS, excessive suppression of physiological reactive oxygen species signaling could theoretically interfere with normal redox-dependent cellular pathways involved in follicular maturation, steroidogenesis, and ovulatory signaling. Similarly, supraphysiological vitamin D exposure may influence calcium metabolism, immune regulation, and endocrine homeostasis beyond the intended therapeutic targets. Therefore, the beneficial effects observed in the present study should be interpreted within the context of controlled experimental dosing conditions, and future studies are warranted to determine optimal therapeutic exposure ranges, long-term safety, and potential systemic consequences of combined ALA and vitamin D administration.

Conclusion

In conclusion, the present study demonstrated that combined alpha-lipoic acid and vitamin D therapy provided substantial protection against metabolic, oxidative, endocrine, and ovarian histopathological alterations in a letrozole-induced rat model of PCOS. The combination treatment showed greater overall efficacy than monotherapies in improving insulin resistance, restoring antioxidant defense systems, reducing hyperandrogenism, and normalizing follicular architecture. Importantly, the observed improvements extended beyond systemic metabolic correction and were accompanied by restoration of ovarian microenvironment integrity and follicular dynamics, suggesting coordinated ovarian protective effects. These findings support the concept that simultaneous targeting of oxidative stress and endocrine–metabolic dysregulation may represent a more comprehensive therapeutic strategy in complex multifactorial disorders such as PCOS. Nevertheless, the present findings should be interpreted as preclinical proof-of-concept evidence, since direct molecular pathway analyses and functional fertility outcomes were not evaluated. Future studies incorporating molecular signaling analyses, inflammatory and apoptotic pathway assessment, and long-term reproductive outcomes are warranted to clarify the mechanistic basis and translational applicability of combined ALA and vitamin D therapy in PCOS.

Abbreviations

ALA

Alpha-lipoic acid

AMPK

AMP-activated protein kinase

ANOVA

Analysis of variance

CAT

Catalase

CL

Corpus luteum

DHEA

Dehydroepiandrosterone

ELISA

Enzyme-linked immunosorbent assay

FBG

Fasting blood glucose

FSH

Follicle-stimulating hormone

GSH

Reduced glutathione

H&E

Hematoxylin and eosin

HOMA-IR

Homeostatic model assessment of insulin resistance

HRP

Horseradish peroxidase

IR

Insulin resistance

LH

Luteinizing hormone

MDA

Malondialdehyde

MET

Metformin

PBS

Phosphate-buffered saline

PCOS

Polycystic ovary syndrome

ROS

Reactive oxygen species

SOD

Superoxide dismutase

TBARS

Thiobarbituric acid reactive substances

TMB

Tetramethylbenzidine

VDR

Vitamin D receptor

AMH

Anti-müllerian hormone

GLUT4

Glucose transporter type 4

IRS-1

Insulin receptor substrate-1

Author contributions

S.S. and R.D.: concept and design, acquisition of data, analysis and interpretation of data, writing the article and critical review of the article. H.C. and S.C.: conception and design and critical review of the article. H.C., S.C. and R.D.: analysis and interpretation of histomorphology data and critical review of the article. All authors read and approved the final manuscript.

Funding

No external funding was either sought or obtained for this study.

Data availability

Data are available upon request.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval

All experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of Mustafa Kemal University (Approval No: 2024-11/9, 11 May 2024). All animal procedures were conducted in accordance with institutional and international guidelines for the care and use of laboratory animals.