Lower Serum ATG7 Levels Linked to Insulin Resistance in Women with Polycystic Ovary Syndrome

Introduction

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders among women of reproductive age, with a prevalence of 8–13% [1,2]. It is characterized by ovulatory dysfunction, polycystic ovarian morphology, insulin resistance, and elevated androgen levels [3,4]. Common symptoms include irregular menstrual cycles, infertility, obesity, hirsutism, and acne. Additionally, women with PCOS are at increased risk of various complications such as metabolic, obstetric, psychological, and tumor-related diseases, which significantly impact their health throughout their lives [5,6]. The heterogeneity of PCOS complicates the understanding of its etiology, and despite extensive research, the details of its pathophysiological mechanisms remain elusive.

Previous studies have linked PCOS with impaired autophagy [7–9]. ATG7 is a crucial protein in the autophagy pathway and plays a pivotal role in initiating the process that removes degraded macromolecules and damaged organelles [10]. Dysfunctions in ATG7 have been associated with various human diseases, including neurodegenerative diseases, cancers, pregnancy complications, and infectious diseases [11]. ATG7 is involved in metabolic regulation, a key aspect of PCOS’s pathogenesis. Studies have shown that impaired ATG7 function leads to dysregulated insulin signaling and glucose homeostasis [9]. In mouse models of ATG7 deficiency, there is a clear manifestation of metabolic disturbances such as insulin resistance, hyperglycemia, and obesity, which are hallmark features of PCOS [9]. Moreover, reduced ATG7 activity has been linked to chronic inflammatory responses, which is a known contributor to PCOS. Therefore, these findings show a potential role that ATG7 plays in the development of PCOS.

This study hypothesized that serum ATG7 levels reflect impaired autophagic and metabolic functions in women with PCOS. To investigate this, we evaluated serum ATG7 levels in women diagnosed with PCOS and compared them to levels in women without the condition. We also explored potential associations between serum ATG7 levels and the risk of developing PCOS, and assessed the utility of serum ATG7 levels in predicting this disorder. The findings from our study could enhance the understanding of PCOS’s pathogenesis and potentially influence its diagnostic criteria. Additionally, these insights may enable clinicians to tailor interventions more precisely based on the underlying metabolic state indicated by ATG7 levels, thereby enhancing personalized treatment approaches for women suffering from PCOS.

Material and Methods

STUDY POPULATION:

The study was conducted in adherence to the Declaration of Helsinki and received approval from the Ethical Committee of The First Affiliated Hospital of Chengdu Medical College, a large tertiary teaching hospital in China (No.2021CYFYIRB-BA-Dec02).

Informed written consents were obtained from all the participants before the study and any identifiable personal information was not included in the manuscript.

The study involved 188 patients who were diagnosed with PCOS at the hospital’s reproductive endocrinology outpatient clinic between January 2022 and December 2023. The diagnosis of PCOS was based on the Rotterdam consensus criteria, requiring at least 2 out of the following 3 conditions: (1) evidence of hyperandrogenism, either clinical or biochemical; (2) Oligo-anovulation; (3) polycystic ovarian morphology identified by ultrasound [12,13].

Participants were selected based on the following criteria: they had both ovaries, were non-smokers, maintained normal blood pressure, and abstained from regular alcohol consumption. Additionally, they were free from cancer, pelvic infections, diabetes, and endometriosis. They had not taken medications such as oral contraceptives, glucocorticoids, or drugs affecting lipid levels, weight, diabetes, androgen levels, or ovulation for at least 3 months before the study.

The sample size was calculated based on the formula described in previous studies: n=Z2×P×(1-P)/e2, n=required sample size; Z=1.96 at 95% confidence interval (CI); P=prevalence of PCOS (8–13%); e=margin of error (5%) [14]. The calculation determined a required sample size of 174 participants. Nevertheless, a total of 188 women who met the recruitment criteria were ultimately enrolled in the study. The study also included 188 healthy women as a control group.

LABORATORY MEASUREMENTS:

Fasting blood samples were collected from all participants and centrifuged to isolate serum for assessing various parameters, including ATG7, sex hormones, and biochemical parameters. These included fasting glucose, fasting insulin, triglycerides (TGs), low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) in the participants. Serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) were measured using electrochemiluminescence immunoassay on COBASE immunoassay analyzers (Roche Diagnostics GmbH). Dehydroepiandrosterone (DHEA) levels were determined using an automated analyzer.

Insulin resistance index (HOMA-IR) was calculated by the formula of fasting insulin (FIns, μU/ml) × fasting blood glucose (FBG, mmol/L)/22.5 [15]. The serum ATG7 levels were determined through an enzyme-linked immunosorbent assay (ELISA), following the procedures provided by the manufacturer.

STATISTICAL ANALYSIS:

Data are reported as mean±standard deviation (SD) for continuous variables with a normal distribution, and as frequency for categorical variables. We used the independent samples t test to assess differences in normally distributed continuous variables between the 2 groups. When the continuous variables were not normally distributed, we used the Mann-Whitney U test to compare the differences. The statistical difference was evaluated by a chi-square test for categorical variables. Pearson correlation analysis was used to evaluate the association between serum ATG7 levels and both lipid profiles and glycemic indices.

To explore the association between serum ATG7 levels and PCOS, we calculated the adjusted odds ratio (OR) and 95% confidence interval (CI) using a multivariable logistic regression model after adjustment of various confounding factors. We used Hosmer-Lemeshow Test to assess the goodness-of-fit of the multivariable logistic regression model. All statistical analyses were performed using SPSS software, version 24.0 (Chicago, IL, USA). Statistical significance was established at a two-sided p-value of less than 0.05.

Results

DEMOGRAPHIC DATA AND BIOCHEMICAL PARAMETERS OF THE STUDY POPULATION:

The demographic data and biochemical parameters of both PCOS patients and the control group are displayed in Table 1. Owing to the matching criteria used, there were no notable differences in age between the 2 groups. The study found that women with PCOS had higher BMI, longer menstrual cycles, increased antral follicle count (AFC), and ratio of luteinizing hormone (LH)/follicle-stimulating hormone (FSH), testosterone levels, and AMH levels. Additionally, compared to the control group of healthy women, those with PCOS exhibited higher levels of fasting insulin, DHEA, TGs, LDL-C, TC, and HOMA-IR.

SERUM ATG7 LEVELS IN PCOS PATIENTS AND HEALTHY CONTROL WOMEN:

Our research revealed that women with PCOS had significantly lower serum ATG7 levels compared to their healthy counterparts (Figure 1A, Table 1). We categorized all the participants into 2 groups based on their body mass index (BMI): those with a BMI of less than 25 kg/m2 were classified as lean, while those with a BMI of 25 kg/m2 or more were considered overweight/obese. It was observed that ATG7 levels were lower in both lean and overweight/obese PCOS women than in the healthy control women (Figure 1B). Furthermore, we found no substantial difference in serum ATG7 levels between the overweight/obese group and lean group of healthy control women. In contrast, serum ATG7 levels were significantly lower in the overweight/obese PCOS patients than those in the lean PCOS patients (Figure 1B).

ASSOCIATIONS OF SERUM ATG7 LEVELS WITH HORMONE LEVELS, INSULIN RESISTANCE, AND LIPID METABOLIC INDICES OF PCOS PATIENTS:

Pearson’s correlation analyses were used to determine associations of serum ATG7 levels with hormone levels, glucose metabolic indices, and lipid metabolic indices of PCOS patients. The results showed that serum ATG7 levels negatively correlated with the levels of TG, TC, LDL-C, fasting insulin, LH, testosterone, AMH, and HOMA-IR (Table 2). The multiple linear logistic regression analyses were used to identify variables that had independent associations with serum ATG7. The results indicated that only testosterone and HOMA-IR were independently associated with serum ATG7 after adjustment for TG, TC, LDL-C, fasting insulin, LH, testosterone, AMH, and HOMA-IR (Table 2).

ASSOCIATIONS BETWEEN ATG7 LEVELS AND THE RISK OF PCOS:

We conducted a logistic regression analysis to evaluate the correlation between serum ATG7 levels and the risk of developing PCOS. Serum ATG7 levels were divided into 3 tertiles based on their concentrations, with the highest tertile serving as a reference for comparison. In the initial logistic regression analysis without adjustments for confounding factors, we observed that women with ATG7 levels in the lowest and middle tertiles had a higher risk of developing PCOS compared to those in the highest tertile. The ORs (95% CIs) were 5.27 (3.16–8.23) for the lowest, 4.38 (1.89–7.53) for the middle, and 1.00 (reference) for the highest tertile (Table 3, Model 1, P=0.002).

After adjusting confounding factors such as age, BMI, TC, TG, AMH, HDL-C, LDL-C, LH, FSH, DHEA, fasting insulin, and testosterone, women with ATG7 levels in the lowest and middle tertiles still had a higher risk of developing PCOS compared to those in the highest tertile. The adjusted ORs (95% CIs) were 4.57 (2.15–7.56) for the lowest, 3.81 (1.68–6.36) for the middle, and 1.00 (reference) for the highest tertile (Table 3, Model 2, P=0.001). However, after further adjusting the insulin resistance index of HOMA-IR, the association between ATG7 levels and the risk of developing PCOS no longer existed. This was evidenced by the lack of statistical significance in the ORs (95% CIs), which were 1.07 (0.81–1.27) for the lowest, 0.94 (0.85–1.28) for the middle, and 1.00 (reference) for the highest tertile (Table 3, Model 3, P=0.115).

PERFORMANCE OF SERUM ATG7 LEVELS IN PREDICTION OF PCOS:

We used ROC curves to evaluate the performance of serum ATG7 in predicting PCOS. The AUC for ATG7 was 73.7%, with a cutoff value of 11.58 ng/mL, which demonstrated a sensitivity of 77.5% and a specificity of 72.6% (Figure 2). The AUC for the insulin resistance index of HOMA-IR was 85.8%, with a sensitivity of 75.1% and a specificity of 73.5% (Figure 2). Notably, the combination of ATG7 with the insulin resistance index of HOMA-IR yielded the most effective results, achieving the highest AUC of 92.3%, along with a sensitivity of 88.3% and a specificity of 85.3% in the prediction of PCOS (Figure 2).

Discussion

In this study, we found that serum ATG7 levels were significantly lower in women with PCOS compared with healthy women. Furthermore, we found that decreased serum ATG7 levels were associated with an increased risk of PCOS, even after adjusting for various confounding factors. By analyzing the ROC curve, we found that the combination of ATG7 with the insulin resistance index of HOMA-IR had excellent performance in predicting PCOS. These findings suggest that serum ATG7 could be an effective biomarker for the prediction of PCOS, which could help gynecologists in the diagnosis and management of PCOS in clinical practice.

PCOS has distinct features, including polycystic ovaries, ovulation dysfunctions, elevated androgen levels, and metabolic complications [16,17]. Previous research indicated that factors such as insulin resistance, obesity, chronic inflammation, genetic predispositions, oxidative stress, and high androgen levels contribute to the onset of PCOS [18,19]. However, the exact mechanisms leading to PCOS are still not fully understood. Our research found that women with PCOS have significantly lower ATG7 levels compared to those without the condition, suggesting that ATG7 deficiency plays a role in the development of PCOS.

ATG7 is a critical protein in the process of autophagy, which contributes to the regulation of lysosomal degradation and recycling in eukaryotic cells [10,20,21]. Autophagy is essential for maintaining cellular functionality and homeostasis by balancing cell survival and death. During the stage of follicular development, the great majority of follicles eventually become atretic follicles, and only a small number of follicles continue to develop and reach the size necessary for ovulation [22,23]. Appropriate functional autophagy is essential from the origination of oocytes to follicular development. Dysfunction of autophagy can significantly impact follicular development by reducing the number of oocytes, hindering the degeneration of atretic follicles, and impairing the formation of mature follicles, which are characteristics of PCOS [7,24,25]. These findings suggest that the decreased ATG7 levels in PCOS patients contribute to the pathogenesis of the disease through disruption of autophagy.

Previous studies have established that insulin resistance is a critical factor in the development of PCOS [26,27]. It has been shown to worsen hyperandrogenemia, disrupt regulation of the hypothalamic-pituitary-ovarian axis, and contribute to the progression of PCOS [28–30]. Elevated androgen levels in the bloodstream result in fat accumulation in various organs, leading to insulin resistance and hyperinsulinemia. This, in turn, stimulates the production of androgens in the ovaries and adrenal glands. The reciprocal relationship between insulin resistance and hyperandrogenemia can further impair ovarian function, leading to issues like anovulation, irregular menstrual cycles, infertility, obesity, and hirsutism [31]. Therefore, insulin resistance may act as a connector linking various physiological disorders in individuals with PCOS.

Our study found that serum ATG7 levels were negatively associated with the insulin resistance index of HOMA-IR in PCOS patients. Furthermore, we found that lower ATG7 levels were associated with a higher risk of developing PCOS after adjusting for various confounding factors, but the association no longer existed after further adjustment of the insulin resistance index of HOMA-IR. These findings suggest that insufficient autophagy, caused by decreased ATG7 levels, contributes to the onset of insulin resistance, which mediates the development of PCOS. Another study reported that inhibiting autophagy pathways through siRNA-mediated reduction of ATG7 in granulosa cells intensified insulin resistance [9], as demonstrated by decreased levels of insulin receptor substrate-1, reduced phosphorylation of Akt, impaired activity of GLUT4, and diminished glucose uptake in granulosa cells of the PCOS mouse model [9].

The AUC values from our study indicate that combining ATG7 with the insulin resistance index HOMA-IR achieves good performance in predicting PCOS. Such metrics are particularly valuable in PCOS, where the heterogeneity of symptoms can complicate diagnosis. The identified cutoff value for ATG7 (11.58 ng/mL) could offer clinicians a practical tool to diagnose or rule out PCOS with reasonable confidence, complementing traditional diagnostic criteria and potentially accelerating the decision-making process in clinical practice. They could improve the diagnosis of PCOS by providing objective, quantifiable biomarkers that are potentially less invasive and more cost-effective than current standard practices, such as transvaginal ultrasonography. Incorporating these biomarkers into clinical practice could not only improve diagnostic accuracy but also enhance the personalization of care, ultimately leading to better health outcomes for women with PCOS. However, further studies are necessary to validate these findings across diverse populations and to investigate the pathophysiological links between these biomarkers and PCOS.

Our study has several notable strengths. Firstly, it is the first study to examine the association between serum ATG7 levels and the risk of PCOS, potentially advancing our understanding of the development of PCOS. Secondly, all laboratory and clinical assessments were performed using standard and highly reliable procedures. Lastly, all the patients in the study were newly diagnosed with PCOS and had not received any medical treatment, thereby reducing the impact of confounding factors on the study. However, there are also limitations to consider. Firstly, our study did not explore the exact mechanisms behind the decreased ATG7 levels in women with PCOS. Secondly, the sample size was relatively small and confined to Chinese women, which may limit the generalizability of the results to other populations. Thirdly, we used methods such as stratification, matching, and definition of clear inclusion and exclusion criteria to minimize selection bias, but we could not completely rule out selection bias. Lastly, despite adjusting for various potential confounding factors in our logistic regression analysis, there remains the possibility of confounding factors that were not accounted for, such as stress, genetic predispositions, and lifestyle choices.

Conclusions

Our study found that serum ATG7 levels were significantly lower in women with PCOS, associated with both an increased risk of PCOS onset and a negative correlation with insulin resistance. This suggests that ATG7 could potentially serve as a biomarker for diagnosing and managing PCOS, particularly when insulin resistance is a predominant feature. Additionally, the study has potential implications for understanding the underlying mechanisms of PCOS pathogenesis and developing targeted therapeutic interventions. However, further studies are necessary to validate these findings across diverse populations.