MicroRNAs (miRNAs) are a class of small, non-coding, and highly conserved RNAs that are ~20-22 nucleotides in length and control many biological functions through the posttranscriptional and translational regulation of mRNA. Binding to the 3′untranslated region (3′UTR) of the target, miRNAs adversely affect their expression, leading to the degradation of target genes’ transcription or translation inhibition [1,2]. miRNAs are transcribed as long primary transcripts from the genome (pri-miRNA) and are then cleaved by the endonuclease enzyme Drosha and the cofactor DGCR8 (DiGeorge syndrome critical region gene 8) in the nucleus [3]. Pre-miRNA, a stem-loop structure of approximately 70-100 nucleotides, is transferred to the cytoplasm by exportin 5 and processed into a double-stranded mature miRNA of approximately 20-22 nucleotides in length through cleavage by Dicer [4,5]. The guide strand of this mature miRNA, which binds to target genes, is associated with the RNA-induced silencing complex (RISC), thus contributing to decreased protein expression [6,7]. This pathway (Drosha/DGCR8) is the canonical pathway for miRNA biosynthesis [8]. In the non-canonical pathway, miRNAs are generated without processing by Drosha/DGCR8 but still require Dicer for maturation, or they are transferred directly from Drosha/DGCR8 to the RISC, and Dicer is not involved [9,10]. miRNAs can regulate multiple target genes simultaneously and are therefore involved in many vital cellular functions, such as the regulation of cell proliferation and differentiation, homeostasis, angiogenesis, development, the immune system, apoptosis, and hormone biosynthesis and secretion [11-15]. miRNA expression is tissue-specific and closely related to tissue function. This feature has also been observed in ovaries [16,17]. The ovary is a complex organ that contains oocytes and somatic cells such as granulosa, theca, and cumulus cells (CCs), which have specific miRNA profiles. Many miRNAs play important roles in the development of follicles, ovulation, and other developmental processes in the ovary [18]. Therefore, miRNAs can be attributed to various types of ovarian disorders and diseases, including polycystic ovary syndrome (PCOS) [19,20]. In patients with PCOS, abnormal expression of miRNAs in serum, follicular fluid, and granulosa cells has been detected, which is reportedly related to anti-apoptotic function and inconclusive testosterone response [17,21].

PCOS is the leading endocrine disorder among women of reproductive age worldwide. The frequency of PCOS is widely dependent on ethnicity, physical characteristics, and diagnostic factors [22,23]. The prevalence of PCOS is calculated as 15% by Rotterdam criteria, reaching 30% in obese and overweight women [24,25]. This disorder is diagnosed based on two of the following three criteria: menstrual irregularity (oligo-ovulation or anovulation), hyperandrogenism (clinical or biochemical), and polycystic ovarian morphology. PCOS is often associated with infertility, obesity, insulin resistance, and dyslipidemia [26-28]. This complex disorder is caused by a combination of environmental and genetic factors [29]. Ethnic diversity in patients with PCOS is strongly associated with their genetic background [30,31]. PCOS is one of the most common endocrine disorders in women and the most common cause of infertility due to lack of ovulation [32]. Moreover, miRNAs play a pivotal role in the posttranscriptional regulation of gene expression and signaling pathways. Therefore, we reviewed recent findings on the expression profiles of exosomal and non-exosomal miRNAs in serum and ovarian follicular fluid and their potential role in ovarian development and PCOS. A better understanding of their functions at different stages of ovarian development can shed new light on women’s reproductive health and the prediction and prevention of various ovarian disorders and infertility.

Similar to other human tissues and organs, miRNAs are expressed in ovarian tissue (Fig. 1). These molecules play important roles in ovarian follicular development, including follicular growth, cell proliferation, atresia, and ovulation [33]. Ovarian development begins in the early embryonic period; however, follicular growth and ovulation, followed by luteal tissue development and cell death, continue throughout life. Targeted knockout of argonaute (AGO2) and Dicer in specific tissues of mouse models has revealed the valuable role of miRNAs in ovarian development and the function of various proteins involved in these pathways [34-36]. Bioinformatic and gene ontology analyses have shown that miRNA-related target genes in the ovary are involved in cell cycle regulation, growth, proliferation, apoptosis, endocrine disorders, and ovarian function [37,38]. miRNAs also play a role in the regulation of ovarian follicle growth and are involved in certain stages of ovarian follicular development, ovulation, luteal function, and period.

miRNAs exhibit tissue-specific expression patterns in various organs and systems. Data from various studies have shown that changes in serum levels of miRNAs are valuable and important tools for the diagnosis, tracking, prognosis, and treatment of numerous diseases. This is mostly due to their unique characteristics, such as stability, resistance to nuclease degradation, simple detection, and the potential to be disease-specific [51]. The mechanism by which miR-NAs are transferred from inside the cell to the extracellular environment, such as serum, is accomplished through two main pathways: 1) active transfer via extracellular vesicles, such as exosomes and 2) transfer as part of a protein-miRNA complex; they can also leak from damaged cells [52]. Table 1 shows the various miRNAs and their potential functions in different parts of ovarian tissue.

Exosomes are a heterogeneous group of extracellular microspheres with diameters of approximately 4-100 nm [96]. Exosomes exist in different types of mammalian cells in the form of multivesicular bodies in the intracellular space and are secreted out of the cell or into biological fluids such as joint fluid, amniotic fluid, plasma, cerebrospinal fluid, urine, saliva, breast milk, alveolar fluid, follicular fluid, and bile through integration with the plasma membrane [97,98]. Exosomes may have different surface markers depending on their cellular origin and physiological state. They contain a variety of cell/tissue-specific proteins, RNAs, lipids, and biological biomolecules that facilitate cell communication with the surrounding environment and have a significant impact on biological and pathological events [99]. AGO2 and some RNA-binding proteins such as hnRNPA2B [100] and Y-box1 [101] are involved in the regulation of miRNA loading into exosomes. During the formation of multivesicular bodies in the cytoplasm, AGO2 binds to the exosome marker protein CD63, leading to the loading of miRNAs into exosomes [102]. Table 2 displays the exosomal miRNAs compared to non-exosomal miRNAs in patients with PCOS.

miRNAs are among the most significant RNA groups in clinical research. Because a specific miRNA may be involved in different biological pathways disrupted in a disease, it can be very useful from a therapeutic perspective, especially if the disease is not associated with a genetic defect. In this regard, bioinformatics software used to identify miRNA binding sites, target genes, and the biological pathways involved, along with laboratory in vitro and in vivo research models, have greatly assisted in accelerating the identification of miR-NAs in clinical medicine [116]. In healthy individuals, a large proportion of ovarian follicles undergo apoptosis and die during their reproductive life through a vital process known as follicular atresia [117]. Follicular atresia begins with the apoptosis of follicular granulosa cells (granulosa cells), followed by the degeneration of other follicular components. Factors such as IGF-1 are follicle viability factors that control atresia [118]. Das et al. [119] showed that there was a significant variation in the apoptosis rate and proliferation of granulosa cells in patients with PCOS. They were the first to find that the level of active caspase-3, which is functionally required to initiate cell death, was significantly lower in the granulosa cells of anovulatory PCOS follicles than in those of normal ovulatory follicles. The expression of Bcl-XLong, an anti-apoptotic factor, increased approximately two-fold, while that of Bax, an apoptosis inducer, slightly decreased in the PCOS group. The expression of Ki-67 (an indicator of cell proliferation) was significantly higher in the PCOS group, indicating a significant imbalance between the rates of apoptosis and proliferation in patients with PCOS [119]. Studies have shown that miRNAs are involved in this pathway, and their regulation can be useful in disease treatment. miR-99a is one of the miRNAs found in granulosa cells, and its expression is significantly decreased in PCOS. This decrease was associated with an increase in the IGF-1R protein levels. IGF-1R belongs to the transmembrane receptor tyrosine kinase family and is activated by IGF-1 [120]. During follicular development, the increase in IGF-1R through the stimulation of tyrosine kinase pathways leads to an increase in proliferation and a decrease in apoptosis of granulosa cells in the first stage of folliculogenesis, which contributes to the pathogenesis of PCOS. Therefore, the re-regulation of miR-99a may be an effective method for reducing the adverse effects of IGF-1/IGF-1R system overactivation in PCOS [121]. miR-323-3p is another miRNA involved in the pathogenesis of PCOS that regulates cell apoptosis and steroidogenesis by targeting IGF-1. Studies have shown that the expression of miR-323 declines in CCs of PCOS patients [122]. CCs, a subclass of granulosa cells that surround the oocyte in the antral follicle, are the source of ovarian steroid metabolism and growth hormones and play an important role in oocyte maturation [123]. Apoptosis is one of the primary causes of PCOS [124]. Increased miR-323-3p expression in CCs stimulates cell proliferation and inhibits apoptosis. As previously mentioned, CYP19A is a critical enzyme in the production of ovarian estradiol, which increases in CCs in PCOS [125]. The expression level of this enzyme is indirectly controlled by miR-323; thus, the reduction of miR-323 in PCOS CCs leads to an increase in the level of IGF-1, followed by an increase in CYP19A, and a disruption in the completion of oocyte development. Thus, miR-323-3p is a novel and promising molecular target for ameliorating CC dysfunction in PCOS [105]. Programmed cell death 4 (PDCD4) is a key factor in apoptosis that suppresses protein translation by binding to eIF4A and inhibiting RNA helicase activity [126]. Available evidence demonstrates that the expression of PDCD4 increases in patients with PCOS and is involved in the pathogenesis of the disease [127]. In an investigation of 36 female C57BL/6 mice, Zhao et al. [128] observed high PDCD4 expression in mice with PCOS compared to that in controls. Delivery of exogenous miR-323-3p by adipose mesenchymal stem cell derived exosomes can suppress the expression of PDCD4. As the apoptosis of CCs increases in PCOS to induce premature follicular atresia, studies have suggested that PDCD4 contributes to premature apoptosis in PCOS CCs, and its pro-apoptotic effect can be eliminated by miR-323-3p. Therefore, miR-323-3p upregulation suppresses CCs apoptosis by targeting PDCD4 in PCOS, and the delivery of miR-323-3p via adipose-derived mesenchymal stem cell exosomes is an effective approach for regulating PCOS progression [128]. miR-135 is another miRNA found in granulosa cells and its expression is highly upregulated in PCOS. miR-135a is regulated by androgens at the transcriptional level. Increased androgen levels in PCOS lead to an increase in miR-135a, and miR-135a can suppress proliferation, enhance the DNA damage response, and induce apoptosis by directly binding to the vascular endothelial growth factor C gene. Consequently, the rate of apoptosis increases during the middle stage of the antrum, leading to cyst formation. Therefore, regulating miR-135a expression can be considered a new therapeutic goal for PCOS patients [129]. miR-335-5p expression is inversely correlated with the number of antral follicles, AMH and testosterone levels. Research has shown that miR-335 levels in the granulosa cells and follicular fluid are significantly decreased in patients with PCOS. Bioinformatics analyses have shown that SGK3 is the direct target of this miRNA, a member of the family of kinases involved in cell proliferation through the PI3K/AKT-mTOR signaling pathway [130,131]. In granulosa cells, SGK is localized in the cell nucleus and regulates cell proliferation and differentiation. In the early stages of folliculogenesis, a reduction in miR-335 levels leads to an increase in AMH levels and granulosa cell proliferation. AMHs prevent the premature depletion of ovarian follicles and regulate steroidogenesis in granulosa cells [132]. In contrast, the reduction of miR-335 expression in an SGK-dependent pathway leads to increased proliferation of granulosa cells, and delayed follicular atresia, and contributes to PCOS pathogenesis. These observations provide a new perspective on the function of granulosa cells in PCOS and suggest that miR-335-5p can serve as a new molecular target for improving dysfunctional granulosa cells [131]. Díaz et al. [133] investigated circulatory concentrations of miR-451a, miR-652-miR-451a, miR-652-3p, miR-106b-5p, and miR-206 in girls with PCOS treated with oral contraceptive or spironolactone-pioglitazone-metformin. They observed that the concentration of the studied miRNAs decreased significantly compared with that in the control group. These target genes are involved in energy homeostasis and cell-cycle regulation. This study demonstrated the capability of miR-451a to diagnose PCOS with 100% sensitivity [133].

Additionally, alterations in the circadian rhythm can trigger PCOS through oxidative stress and inflammation. Circadian rhythms can alter circulating exosomes and related miRNAs, leading to alterations in physiological conditions [134]. miR-NAs play an important role in maintaining homeostasis of the circadian system, and miR-132 and miR-219 are involved in regulating circadian rhythms in mammals by targeting clock genes. Furthermore, miR-494, miR-27b-3p, miR-155, and miR-142-3p are involved in regulating the clock gene, brain, and muscle Arnt-like protein 1 (BAML1) [135,136]. Changes in the expression of miR-192 and miR-194 not only affect the rhythm fluctuation of BMAL1 mRNA but also suppress the expression of the clock gene period (Per) [137]. Therefore, circadian rhythms can be modulated by targeting miRNAs to investigate their potential in PCOS treatment [134].

miRNAs that are free or enclosed in extracellular vesicles (exosomes) can affect various signaling pathways. Abnormal expression of miRNAs occurs in the granulosa cells, theca cells, adipose tissue, follicular fluid, serum, and peripheral blood leukocytes of women with PCOS. They regulate follicular growth and maturation, insulin signaling, glucose and lipid metabolism, and steroid hormone synthesis. Additionally, exosomes can protect miRNAs from degradation, making them stable and efficient diagnostic tools.

The use of miRNAs in the clinical management of PCOS is a dynamic part of research, and there are several potential functions of miRNAs in this context. miRNAs can be used clinically in various pathways, such as diagnostic and prognostic biomarkers, therapeutic targets, and drug delivery vehicles; therefore, as diagnostic and prognostic biomarkers of PCOS. Many studies have identified specific miRNAs that are dysregulated in individuals with PCOS, and their detection in the blood or other fluids may be useful for early diagnosis and monitoring of disease progression. Also, the expression of certain miRNAs has been found to be associated with insulin resistance, a common feature of PCOS. By measuring the levels of these miRNAs, it may be possible to predict the probability of increased IR-related complications, such as type 2 diabetes.

Although miRNAs are potentially promising diagnostic and therapeutic factors, their clinical applications in diagnosis and treatment are still under development. Concerning diagnosis, many of the identified miRNAs are still at the experimental level and require further investigation. Additionally, only a limited number of miRNAs have been approved by the Food and Drug Administration as therapeutic agents. Therefore, the application of miRNAs as diagnostic or therapeutic factors in medicine is in its early stages, and there is still a long way to go.