Toll-like Receptor 4 Contributes to PCOS-like Metabolic and Reproductive Pathogenesis

Abstract

Polycystic ovary syndrome (PCOS) is a reproductive disorder with heterogeneous symptoms and severity. Despite extensive research documenting chronic immune dysfunction as a hallmark of PCOS, the specific molecular mechanisms driving immune activation and its connection to the syndrome's diverse symptoms remain poorly understood. Emerging evidence suggests that gut-derived bacterial endotoxins, particularly lipopolysaccharide (LPS), may breach the intestinal barriers in PCOS patients and trigger systemic inflammation through Toll-like receptor 4 (TLR4), a pattern recognition receptor of the innate immune system. This study investigated whether TLR4 serves as a critical mechanistic driver of PCOS pathogenesis by examining the effect of genetic TLR4 knockout (TLR4^-/-) in a letrozole (LET)-induced mouse model of PCOS. Our results demonstrate that TLR4 deficiency reduces many PCOS-like symptoms, including elevated luteinizing hormone, anovulation, and metabolic dysfunction. TLR4 knockout also preserved estrous cycling and fertility, improved glucose tolerance, maintained gut barrier integrity, and reduced inflammatory markers in LET-treated females. These findings establish TLR4 as a key mediator orchestrating PCOS's multi-system pathology, positioning TLR4 as a critical convergence point rather than affecting individual symptoms in isolation. This novel work reveals that TLR4-mediated inflammation drives multiple PCOS pathologies, opening avenues for targeted anti-inflammatory treatments in women with this disorder.

Fig. 1.. TLR4 Knockout Ameliorates LET-Induced Metabolic and Pancreatic Hormone Dysregulation

(A) Overview of experimental groups, colors, and symbols (WT Placebo=10, WT LET=10, TLR4^−/− Placebo=10, TLR4^−/− LET=10). (B) Timeline of LET or placebo pellet implantation at week 4, with in vivo assessments (glucose tolerance, body composition, estrous cycling) at weeks 8–9 and tissue collection at week 9. (C) Total body weight during the 5 weeks of treatment. (D) Area of the curve for weight gain over a 5-week period. (E) Fat-to-lean mass ratio (% body weight). (F) Glucose tolerance test (GTT) curve and (G) Area of the curve derived from GTT measurements. (H–J) Serum pancreatic hormones: (H) insulin, (I) C-peptide 2, (J) glucagon. Data are presented as mean ± SEM and analyzed with two-way ANOVA followed by Tukey’s post-hoc test. Statistical significance was accepted at p < 0.05 and differences among groups are denoted by a connecting letter system, where groups sharing the same letter are not significantly different from each other, while groups with different letters are significantly different (p < 0.05).

Fig. 2.. Genetic Knockout of TLR4 Prevents LET-Induced Reproductive Hormone Imbalances and Estrous Cycling Disruption

(A) Representative mouse estrous cycling patterns from each experimental group for 9 days. (B) Percentage of time spent in each estrous cycle stage (proestrus, estrus, metestrus, diestrus). (C) Serum testosterone levels measured by Luminex and displayed as mean fluorescence intensity (MFI). (D) Testosterone fold change of WT female mice relative to TLR4^−/− female mice. (E) Serum luteinizing hormone (LH) concentration (ng/mL). (F) LH fold change fold change of WT female mice relative to TLR4^−/− female mice. (G) Serum follicle-stimulating hormone (FSH) concentration (ng/mL). (H) LH:FSH ratio calculated from serum hormone measurements. Data are presented as mean ± SEM and analyzed with two-way ANOVA followed by Tukey’s post-hoc test. Statistical significance was accepted at p < 0.05 and differences among groups are denoted by a connecting letter system, where groups sharing the same letter are not significantly different from each other, while groups with different letters are significantly different (p < 0.05).

Fig. 3.. LET-Induced PCOS-Like Ovarian Morphology and Function is Preserved in TLR4 Knockout Mice

(A) Representative hematoxylin and eosin (H&E) stained ovarian sections (10 μm) from each experimental group with morphological features labeled: CL = corpus luteum, CF = cystic follicle. (B) Quantification of CL and CF per ovarian section. (C) Timeline of fertility assessment protocol following treatment period. (D) Percentage of fertile females by group (E) Average number of offspring per litter from fertile females. Data are presented as mean ± SEM and analyzed with two-way ANOVA followed by Tukey’s post-hoc test. Statistical significance was accepted at p < 0.05 and differences among groups are denoted by a connecting letter system, where groups sharing the same letter are not significantly different from each other, while groups with different letters are significantly different (p < 0.05).

Fig. 4.. TLR4 Promotes Intestinal Barrier Compromise and Immune Dysfunction

(A-B) Fecal lipocalin-2 (A) and calprotectin (B) concentration (μg/mg fecal sample). (C) Serum FITC-Dextran fluorescence pre and post 4-hour oral-gavage. (D) Immunoglobulin M (IgM) concentration (ng/mL) in serum (top) and fecal supernatant (μg/mg fecal sample) (bottom). (E) Immunoglobulin A (IgA) concentration (ng/mL) in serum (top) and fecal supernatant (μg/mg fecal sample) (bottom). (F) Immunoglobulin G3 (IgG3) concentration (ng/mL) in serum (top) and fecal supernatant (μg/mg fecal sample) (bottom). Data are presented as mean ± SEM and analyzed with two-way ANOVA followed by Tukey’s post-hoc test. Statistical significance was accepted at p < 0.05 and differences among groups are denoted by a connecting letter system, where groups sharing the same letter are not significantly different from each other, while groups with different letters are significantly different (p < 0.05).

Fig. 5.. TLR4 Drives Elevated Cytokine Levels in LET-Induced PCOS Mouse Model

(A-B) Heatmap of serum cytokine expression profiles from 25-cytokine multiplex panel across experimental groups. (C-D) Partial least squares discriminant analysis (PLS-DA) of serum cytokines comparing WT (C) and TLR4^−/− (D) responses to LET treatment. (E-F) VIP analysis of cytokine contribution to PLS-DA models in (C and D). (G-H) TNF-α (G) and TNF-β (H) logarithmic ratio of fecal versus serum concentrations (values closer to −1 indicate fecal predominance; values closer to 1 indicate serum predominance). Groups which significantly deviate from 0 are annotated with #. (I) IL-22 concentration values in serum (ng/mL) and fecal samples (μg/mg fecal sample). Data are presented as mean ± SEM and analyzed with two-way ANOVA followed by Tukey’s post-hoc test. Statistical significance was accepted at p < 0.05 and differences among groups are denoted by a connecting letter system, where groups sharing the same letter are not significantly different from each other, while groups with different letters are significantly different (p < 0.05).