Polycystic ovarian syndrome (PCOS) is the most common underlying cause of anovulatory infertility in women of reproductive age (1) and is associated with metabolic features that include clinical and biochemical hyperandrogenism, insulin resistance, obesity, type 2 diabetes (T2DM), and hypercholesterolemia (2–5). Epigenetic abnormalities, genetic mutations, and alterations of noncoding RNAs are involved in its etiology (6–9).

microRNAs (miRNAs) are noncoding RNAs of ~22 nucleotides in length that posttranscriptionally regulate gene expression. Their binding to target messenger RNA (mRNA) causes mRNA cleavage, translational repression, and mRNA decay (10–13). Only ~600 of the 2,600 mature human miRNAs described have been confidentially validated as microRNA in the miRBase miRNA registry (14, 15). Due to their multitudinous base pairing, allowing each miRNA sequence to attenuate multiple target mRNA expressions, they affect many different biological/cellular pathways. Their involvement in almost every biological process results in miRNAs being implicated in a number of disease states, including obesity and T2DM, and their associated comorbidities (16, 17). MiRNA have been reported to regulate many biological processes linked with obesity, including lipid metabolism, adipogenesis, insulin secretion, and glucose uptake (18–21).

miRNA expression is highly complex, and therefore, it is not surprising that a number of published studies in PCOS subjects appear discordant (22). MiRNA-21, miRNA-27b, miRNA-103, and miRNA-155 have been reported to be differentially expressed in both PCOS and obesity (23). Serum miRNA-21 (24) and miRNA-6767-5p (25) are elevated, while miRNA-320 is lowered (26), in PCOS and have been proposed as biomarkers for diagnosis of PCOS. In the ovary, miR-92a and miR92b have been described to be differentially expressed in PCOS (27), while in the ovarian cortex of PCOS subjects, miR-93 expression was highest (28). MiR-93 has also been implicated in downregulating the expression of GLUT4, the insulin-sensitive glucose transporter present in adipose tissue (29); miRNA-223 also plays an ill-defined role in the insulin resistance of PCOS. In adipose tissue, miRNA-93 has been reported to be overexpressed in insulin-resistant PCOS women (although, of note, the overexpression was discordant with expression of MCM7, the host gene) (30). A study in Chinese women with and without PCOS reported five upregulated circulating miRNAs (let-7i-3pm, miR-5706, miR-4463, miR-3665, miR-638) and four downregulated miRNAs (miR-124-3p, miR-128, miR-29a-3p, let-7c) in PCOS patients.

In the present study, we utilized serum samples collected by the PCOS Biobank in the follicular phase of the menstrual cycle in control women and compared them to serum samples from anovulatory PCOS subjects. Our objective was to determine whether circulating miRNAs differed between women with and without PCOS when weight and age were matched. Our hypothesis was that there would be no difference in circulating levels of miRNAs between women with and without PCOS when weight and age were matched.

Baseline data for the 58 patients, 29 PCOS and 29 normal controls, are shown in Table 1, where it can be seen that patients were overweight, age and BMI matched, and differed significantly in their insulin levels, insulin resistance (HOMA), testosterone, free FAI, and AMH.

One hundred seventy-six miRNA were detected, of which 15 differed significantly (Table 2, Figure 1) between normal women and PCOS. The top 9 of these (p < 0.02) were miR-486-5p, miR-24-3p, miR-19b-3p, miR-22-3p, miR-19a-3p, miR-339-5p, miR-185-5p, miR-101-3p, and miR-let-7i-5p. There was no association of the top 9 miRNA that differed with BMI, androgen levels, insulin resistance, or AMH in either PCOS or normal women (Supplementary Table 1).

A more recent study has shown that sets of microRNAs can coordinately act in distinct biological pathways, thereby revealing an emerging concept of regulatory microRNA networks (35). When Ingenuity Pathway Analysis was undertaken to look at the functional relationships, the significant miRs mapped to the pathways of reproductive abnormalities as shown in Figure 2. Notably, all nine microRNAs revealed functional connectivity as part of a regulatory network of genes predominantly associated with central regulators of insulin signaling and glucose metabolism such as the AKT-FOXO1 circuit (36). In addition, the generated pathway also suggested vascular endothelial growth factor (VEGF) to be associated with the altered microRNA functions, which is important because VEGF contributes to the pathogenesis of PCOS (37).

Altered microRNAs in circulation reflect significant changes in peripheral tissues and are considered as potential biomarkers similar to enzymes and cytokines in obesity-associated metabolic diseases (38). The key results of this study were the significant alterations in miRNAs in anovulatory PCOS women compared to their age- and BMI-matched control counterparts, all of whom were documented to be in the follicular phase of their menstrual cycle. In the follicular phase, in PCOS patients compared to controls, there were 15 miRNAs that differed significantly; the 9 most significant were miR-486-5p, miR-24-3p, miR-19b-3p, miR-22-3p, miR-19a-3p, miR-339-5p, miR-185-5p, miR-101-3p, and miR-let-7i-5p.

Given that the subjects were BMI matched, then it is perhaps unsurprising to see that the miRNAs that have been reported to be related to BMI, such as miR-21, miR-27b, miR-103, and miR-155 (23), did not differ in this study between groups. miR-93 has been implicated in downregulating the expression of the insulin-sensitive glucose transporter GLUT4 in adipose tissue (29) and has been shown to be overexpressed in adipose tissue (29). In addition, the discrepancy with the literature may be in part due to the differing unmatched populations that have been studied and the potential confounding influence of miRNA changes through the menstrual cycle that are still undefined.

While some of the miRNAs have been associated with insulin resistance such as miR-93, much of the validation of these miRNAs described have been done in the cancer-related field. Previous studies have shown that miR-486-5p plays a role as a tumor suppressor of nonsmall cell lung cancer (NSCLC) (1), hepatocellular carcinoma cells (2), and leukemia cells, where it induces apoptosis by targeting FOXO1 (3); it also prevents the migration, invasion, and endothelial-to-mesenchymal transition (EMT) by regulating Smad2 in breast cancer (4). MiR-24-3p was reported to suppress cellar proliferation in hepatocellular carcinoma (5) as well as promote cellular invasion and migration in nasopharyngeal carcinoma by targeting TEL2 (6) and in lung cancer by targeting SOX7 (7). Studies on hepatocellular carcinoma have shown that miR-19a-3p contributes to tumor metastasis and regulates cell growth via the PTEN/Akt and PI3KIP1/Akt pathways, respectively (8, 9). MiR-19a-3p downregulation in prostate cancer activates the transforming growth factor beta (TGF-β) signaling pathway, which stimulates migration, invasion, and bone metastasis (10). Reports on miR-19b-3p demonstrated that it inhibits cell proliferation in breast cancer via the PI3K/Akt pathway (11), whereas, by contrast, it promotes cell proliferation in colon cancer via targeting SMAD4 (12). A study in human cervical squamous carcinoma reported that miR-22-3p causes inhibition of cell apoptosis by targeting the eIF4EBP3 gene (13) and suppresses cell proliferation in hepatocellular carcinoma and arterial smooth muscle by regulating SP1 and HMGB1, respectively (14, 15). Studies investigating the role of miR-339-5p indicate that it inhibits tumor invasion in breast cancer (16) and in hepatocellular carcinoma by targeting ZNF689 (17, 18); by regulating EMT, it inhibits metastasis of NSCLC (19). However, it was reported to promote stem cell leukemia syndrome development through the downregulation of the BCL2L11 and BAX proapoptotic genes (20). MiR-185 was reported to induce potent autophagy by AKT signaling in hepatocellular carcinoma (21), whereas in human prostate cancer, it was reported to promote apoptosis (22). Reports indicate that miR-101-3p targets Pim-1 in salivary gland adenoid cystic carcinoma to enhance the sensitivity to chemotherapy and suppress cell proliferation and invasion (23); miR-101-3p was also reported to target MALAT-1 in nonsmall cell lung cancer to inhibit growth and metastasis (24). Recent studies have found that the role of let-7i-5p is to suppress cellular migration and proliferation via HMGA1 gene targeting in bladder cancer (25) and kallikrein-related peptidase 6 in colon cancer (26). Moreover, the inhibition of let-7i was reported to enhance progesterone-induced functional recovery in an ischemic mouse model (27).

Differing types of microRNA can coordinate in biological pathways, indicating their regulation of microRNA networks (35). The Ingenuity Pathway Analysis revealed that all of the top miRNAs reported had functional connectivity as part of a regulatory network of genes predominantly associated with central regulators of insulin signaling and glucose metabolism such as the AKT-FOXO1 circuit (36). Furthermore, the microRNAs reported here were related to the pathway associated with the vascular endothelial growth factor A (VEGFA) gene, which is associated with genetic alterations of vascular endothelial growth factor (VEGF), and suggests that VEGF has a role in the pathogenesis of PCOS as a candidate locus (37). Genetic variations in the VEGFA gene contribute to VEGF secretion and PCOS.

For VEGFA, the single nucleotide polymorphism (SNP) rs3025020 was significantly higher in PCOS cases than in control women, while increased fasting insulin and HOMA-IR and bioactive testosterone were linked with rs3025020 (37). This has been recently repeated in a Chinese population (39). A systematic review suggested that VEGF was involved in the pathophysiology of PCOS and, in particular, the development of ovarian hyperstimulation syndrome (40). Our data indicate that both miR-339-5p and miR-424-5p can directly influence VEGF expression and potentially contribute to abnormal VEGF level and function in PCOS. Further studies, particularly the overexpression of the mRNA in animal models of PCOS, would yield greater insights into the function of these miRNAs and their role in the pathogenesis of PCOS.

Quantitative polymerase chain reaction (qPCR) is the preferred method for determining miRNA expression. It is considered to be more accurate, simpler to perform, and delivers superior reproducibility when compared to other methods (hybridization or sequencing-based technologies) (41). It should be noted that most reported changes in circulating microRNAs in metabolic disorders fall within the same range of 1.5- to a maximum of 4-fold changes (42) as found here, unless a much more severe pathophysiological condition is present, resulting in significant tissue damage and cell death (43). Thereby, owing to their high abundance and stability, a twofold difference in body fluid microRNAs is considered as significant (38). The microRNA levels in this study were analyzed using the most reliable PCR-based platform developed by Exiqon, which relatively performs at least as well as in RNA sequencing (44). A deeper analysis of the reflected changes in identified microRNAs would provide a better understanding of the cellular mechanisms associated with PCOS, and this would require intense functional cell-based studies.

The strengths of this study were that the subjects were age and sex matched and that the PCOS population was homogeneous, demonstrating all three diagnostic features. It has been reported that PCOS subjects with all three diagnostic features have a more metabolic phenotype independent of obesity (45); therefore, with this exaggerated phenotype, if a relationship existed between the metabolic parameters and miRNA, it would be apparent. As a pilot study, one limitation was the relatively small number of subjects available. Despite this, the results clearly showed that miRNA appeared to differ between control women in the follicular phase of the menstrual cycle and anovulatory PCOS women.

In conclusion, these data indicate that both miR-339-5p and miR-424-5p can directly influence VEGF expression and potentially contribute to abnormal VEGF level and function in PCOS; miRNAs were found to differ between women without PCOS in the follicular phase of the menstrual cycle and anovulatory PCOS women, but when age and BMI were matched, there were no associations with AMH and the metabolic parameters.

The data that support the findings of this study are available from the corresponding author upon request.

AB contributed to data analysis and wrote the manuscript. SHN-S performed the miR measurements. VR, TS, RD, NG, MB, SD, and SH contributed to data analysis. TS supervised sample collection. SA designed the studies, supervised the work, contributed to data analysis, and was involved in preparation of the manuscript. SA is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

RD is currently employed by the company AstraZeneca. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.