Introduction

Polycystic ovary syndrome (PCOS) is a common disorder affecting females during the reproductive age. This condition can result in hormonal imbalance, irregular menstruations, excess androgen level and cysts in the ovaries. It is frequently associated with lack of ovulation and subsequent infertility [1]. PCOS is known as a complex metabolic condition that is expressed in genetically vulnerable females after a variety of negative exposures to the dietetic and environmental influences associated with modern lifestyle [2]. A novel evolutionary model has been suggested for description of the pathoetiology of PCOS. This model integrates a wide array of evidence came from the evolutionary theory, genetic investigations, epigenetic programming during fetal period, trans-generational inheritance, and metabolic features [2].

PCOS results from interactions between environmental and genetic factors. However, genetic factors explain less than 10% of PCOS susceptibility [3,4,5]. A large-scale genome wide study has provided evidence for participation of several genetic loci in the pathogenesis of PCOS and comparable genetic characteristics for all PCOS diagnostic standards [6]. Several susceptibility loci have been found for PCOS suggesting that diverse phenotypes and sub-phenotypes result from uncommon genetic variants [7]. Meanwhile, the importance of epigenetic factor should not be ignored in this condition. The observed alterations in the methylation profile of DNA and miRNAs in different biological sources from PCOS subjects indicate that these patients have a different epigenetic regulation. This abnormal epigenetic regulation can be induced by an unfavorable intrauterine milieu or by postnatal environmental factors, including nutritional conditions or obesity [8]. The current review focuses on the role of circular RNAs (circRNAs) as molecular sponges for microRNAs (miRNAs) in the pathoetiology PCOS.

CircRNAs as epigenetic regulators

CircRNAs are a class of non-coding RNAs that are detected in varied range of cells in diverse tissues across species. These non-polyadenylated, single-stranded, covalently enclosed transcripts are resistant to exonuclease digestion [9]. They have some important features, including diversity, stable structure, conservation during evolution, and specific expression signature. These features give circRNAs several vital biological functions. Therefore, circRNAs are regarded as regulator of physiological processes as well as pathological conditions. In addition to the regulation of transcription and splicing, they act as miRNA sponges to affect expression of miRNA targets [10].

The impact of circRNAs in the pathogenesis of PCOS-associated pathologies

CircRNAs contribute to the pathogenesis of several metabolic disorders, including obesity [11], insulin resistance [12] and diabetes [13]. Most importantly, the role of circRNAs in diverse pathologies related with infertility has been recently acknowledged [14, 15]. Certain circRNA signature has been associated with success rate of assisted reproductive technology. To be more specific, circRNA_103829, circRNA_103827 and circRNA_104816 have been up-regulated, whereas circRNA_101889 has been down-regulated in ovarian granulosa cells (OGCs) obtained from females with advanced age [16]. From a mechansitical viewpoint, circRNA_103827 and circRNA_104816 have a role in the regulation of ANKRD20A9P, KCNQ1OT1 and XIST, possibly through regulation of has-miR-4500, has-miR-4458, has-miR-3187-5p and has-miR-98-5p [16]. Since circRNA_103827 and circRNA_104816 have shown an age-related up-regulation in OGCs, they have been suggested as possible indicators of unfavorable follicular microenvironment and predictors of IVF prognosis [16]. Thus, several clues point to the involvement of circRNAs in the pathophysiology of PCOS-related conditions.

CircRNA/miRNA axes in PCOS

A number of studies have validated circRNA/miRNA axes in PCOS using appropriate experiments (Table 1). For instance, Chen et al. have demonstrated up-regulation of circ_0043533 in OGCs and insulin-treated OGCs. They have also examined the interaction between this circRNA and miR-1179 using bioinformatics tools, as well as dual-luciferase and RNA immunoprecipitation assays. Circ_0043533 silencing has significantly decreased viability and proliferation and enhanced apoptosis of insulin-treated OGCs. In the meantime, circ_0043533 silencing could decrease expression levels of Bcl-2, CDK2, and Cyclin D1, and enhance Bax expression. Mechanistically, circ_0043533 acts as a sponge to engage miR-1179. Remarkably, miR-1179 suppression has weakened the effects of circ_0043533 silencing on proliferation and apoptosis of insulin-treated OGCs. Cumulatively, circ_0043533/miR-1179 axis has been found to contribute to the pathogenesis of PCOS [17]. Circ_0030018/miR-136 axis is another molecular axis participating in the progression of PCOS as revealed by Xu et al. [18]. Interactions between these two transcripts have been verified through luciferase reporter and RNA pull-down experiments. Expression of circ_0030018 has been shown to be elevated in PCOS patients and granulosa tumor cells (KGN cells). Circ_0030018 silencing has suppressed proliferation, migration, and invasion of these cells, and promoted their apoptosis. This circRNA could adsorb miR-136, a miRNA that targets MIEN1 [18]. miR-136-5p has also been found to be sponged by another circRNA, namely circ_RANBP9 in the context of PCOS. Notably, circ_RANBP9 silencing has repressed proliferation and increased apoptosis of KGN and COV434 cells, while miR-136-5p silencing has reversed these processes. In addition, XIAP, a target of miR-136-5p could abolish the effects of miR-136-5p on proliferation and apoptosis [19]. hsa_circ_0118530 is another circRNA that sponges miR-136. Expression of this circRNA has been highly elevated in PCOS patients and OGCs compared to controls. hsa_circ_0118530 silencing has led to up-regulation of E-cadherin and depression of N-cadherin. Notably, hsa_circ_0118530 silencing could inhibit ROS accumulation and MDA levels whereas inducing SOD activity. Moreover, hsa_circ_0118530 down-regulation could suppress release of inflammatory cytokines [20].

Table 1 CircRNA/miRNA axes in PCOS.
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Figure 1A shows the mechanistic process underlying contribution of miR-136 in PCOS.

Fig. 1
figure 1

(A) The mechanistic process underlying contribution of miR-136 in PCOS. hsa_circ_0118530 and circ_0030018 are two circRNAs that adsorb miR-136 in the context of PCOS. (B) The mechanism of involvement of circ_FURIN in the pathogenesis of PCOS

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Circ_FURIN is another circRNA that affects PCOS pathogenesis through sponging at least two miRNAs. Expression of circ_FURIN has been found to be up-regulated in the ovarian cortex tissue of PCOS cases and TTR-treated KGN cells, parallel with up-regulation of MTM1 and down-regulation of miR-423-5p. Circ_FURIN silencing has alleviated TTR-induced proliferation suppression and apoptosis enhancement. Furthermore, silencing of miR-423-5p, a target of circ_FURIN, has decreased circ_FURIN silencing-mediated effect under TTR exposure. Thus, circ_FURIN/ miR-423-5p/MTM1 axis has a prominent role in the pathoetiology of PCOS [20]. This circRNA can also regulate apoptotic signaling pathways, since its up-regulation in PCOS cases has been associated with up-regulation of BCL2 via adsorbing miR-195-5p [21]. Figure 1B shows the mechanism of involvement of circ_FURIN in the pathogenesis of PCOS.

Circ_0043532 is another up-regulated circRNA in PCOS OGCs and KGN cells. Circ_0043532 knock-down has inhibited proliferation and cell cycle transition and stimulated cell apoptosis in the mentioned cells. Mechanistic analyses have shown that circ_0043532 sponges miR-182, a miRNA that targets SGK3. Therefore, circ_0043532/miR-182/SGK3 axis is another molecular axis contributing to the pathogenesis of PCOS [22].

Another study has used high-throughput chips to assess expression of circRNAs in PCOS cases. This study has shown up-regulation of circRHBG in OGCs of these patients. Knockdown of this circRNA has inhibited proliferation of KGN and SVOG cells. Further studies have shown that circRHBG contests with SLC7A11 to bind to miR-515-5p, indicating the function of circRHBG/miR-515-5p/SLC7A11 axis in PCOS. Moreover, circRHBG silencing could promote ferroptosis in PCOS [23].

circMTO1 is another circRNA participating in the PCOS pathogenesis. Its knockdown has promoted proliferation of KGN and SVOG cells and suppressed their apoptosis rate. The effects of circMTO1 on these cells are mediated through miR-320b/MCL1 axis. Besides, circMTO1 expression in these cells has been stimulated by Snail family transcriptional repressor 2 (SNAI2) [24].

Expression of circASPH has also been found to be increased in PCOS patients, parallel with up-regulation of MAP2K6 and down-regulation of miR-375. This circRNA directly targets miR-375, which targets MAP2K6. Additionally, circASPH silencing represses proliferation of KGN cells and enhances their apoptosis [25]. circPUM1 is another circRNA that contributes to the development of PCOS via adsorbing miR-760 [26].

On the other hand, circPSMC3 expression has been shown to be abnormally reduced in the ovarian tissue of PCOS patients. Notably, up-regulation of this circRNA in PCOS model mice has relieved the symptoms. Moreover, circPSMC3 could inhibit cell proliferation and enhance apoptosis of OGCs through hindering cell cycle. These effects are mediated through miR‐296‐3p/PTEN axis [27].

Expression profile of circRNAs has also been assessed in exosomes extracted from follicular fluid of PCOS patients versus controls [28]. Differentially expressed circRNAs have been predicted to contribute to the ovarian steroidogenesis, aldosterone biosynthesis and excretion, and Jak/STAT signaling. One of these circRNAs, namely hsa_circ_0006877 (circLDLR) has a prominent role in ovarian steroidogenesis. This circRNA has also been verified to be depleted in follicular fluid exosomes of additional PCOS cohort. The circLDLR/miR-1294/CYP19A1 ceRNA has been confirmed by functional studies in the cumulus cells of PCOS patients [28].

In addition, in silico studies have predicted some circRNA/miRNA axes in this condition. An example of this type of studies has been conducted by Huang et al. on three datasets of circRNAs, mRNAs and miRNAs, respectively [30]. Differentially expressed genes have been used to construct a network of cirRNA-related competing endogenous RNAs (ceRNAs). This study has validated dysregulation of three circRNAs, namely hsa_circ_0075691, hsa_circ_0075692 and hsa_circ_0085997 in PCOS. The constructed network included mentioned circRNAs as well as three miRNAs (hsa-miR-320c, hsa-miR-455-5p and hsa-miR-346) [30].

In addition to studies that have addressed function of circRNA/miRNA axes in PCOS, some other studies have evaluated expression levels of circRNAs in different tissues of PCOS patients. Most notably, maternal PCOS has been shown to affect expression of circRNAs in the fetus [31]. Zhao et al. have shown up-regulation of 14 circRNAs and down-regulation of 101 circRNAs in the fetal side of placenta from maternal PCOS compared with healthy puerperal through microarray analyses. Among differentially expressed circRNAs, expression profiles of circ_0023942, circ_0002151, circ_0001274, and circ_0008514 have been verified by qRT-PCR. Functional assay have shown that circ_0023942 represses proliferation of human OGCs and suppresses CDK-4 expression. Moreover, this circRNA has been predicted to participate in the regulation of developmental processes and MAPK signaling. Besides, circ_0023942 is predicted to target a number of miRNAs, namely hsa-miR-632, hsa-miR-574-3p, hsa-miR-362-5p, hsa-miR-429, and hsa-miR-502-5p [31].

An expression assay in cumulus cells of PCOS and non-PCOS subjects has shown up-regulation of 167 and down-regulation of 119 circRNAs. qRT-PCR experiments have validated up-regulation of hsa_circ_0043533 and hsa_circ_0043532; and down-regulation of hsa_circ_0097636 in the PCOS cases compared with non-PCOS ones. Levels of hsa_circ_0043533 and hsa_circ_0097636 in the PCOS cases have been correlated with testosterone levels. Most notably, differentially expressed circRNAs have been enriched in cell cycle, oocytes meiosis, progesterone-mediated oocytes maturation, FOXO and phosphatidylinositol signaling pathways as well as glycerophospholipids metabolism [32].

Bioinformatic analyses

In order to gain insight into the ceRNA network implicated in PCOS and offer a basis for further investigations, a number of in-silico analyses were conducted. Initially, we utilized the miRWalk database (http://mirwalk.umm.uni-heidelberg.de/) to identify the potential genes that are controlled by 13 miRNAs that have been thoroughly examined [33]. To ensure accuracy, we implemented a stringent criterion where the target genes must have a score higher than 0.95 and be predicted as target genes in all three datasets: TargetScan, miRDB, and miRTarbase. Our analysis revealed that a total of 80 unique genes have the potential to be regulated by 5 distinct miRNAs (Fig. 2A). Notably, previous research has focused solely on the investigation of BCL2 which is targeted by at least two miRNAs, namely has-miR-195-5p and has-miR-162-5p, as two important miRNAs in the pathoetiology of PCOS. The remaining 79 genes present an intriguing avenue for future studies. The Cytoscape version 3.10.1 was utilized to visualize the network of these interactions [34].

Fig. 2
figure 2

(A) The possible miRNAs-mRNAs interaction network in PCOS, retrieved from the miRWalk database and visualized by the Cytoscape version 3.10.1. (B) The potential circRNAs-miRNAs interaction network in PCOS, obtained from the miRNet version 2.0 database and illustrated by the Cytoscape version 3.10.1

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Subsequently, we utilized the miRNet version 2.0 database (https://www.mirnet.ca/miRNet/home.xhtml) to discover the upstream circRNAs that potentially control the expression or function of 13 miRNAs [35]. As illustrated in Fig. 2B, we observed that among them, only 11 miRNAs could be regulated by 6449 different circRNAs. Moreover, among these 11 miRNAs, five miRNAs were found in the previous step. These five miRNAs could be regulated by 5269 different circRNAs. Considering the published data and our in-silico analyses, it can be inferred that a ceRNA network comprising of 5269 circRNAs, 5 miRNAs, and 80 mRNAs potentially plays a role in the pathobiology of PCOS.

Finally, to identify the relevant pathway for these miRNAs, we employed the miRAnno module of the miRDIP version 5.3.0.1 database (https://ophid.utoronto.ca/mirDIP/miRAnno.jsp), a tool commonly utilized by researchers to investigate miRNA-associated pathways in Homo sapiens [36, 37]. As listed in Table 2, By importing the miRNA list, we discovered that out of the 13 miRNAs examined, 11 miRNAs were associated with a total of 35 distinct human pathways (P-value < 0.001).

Table 2 The possible miRNA-related pathways in PCOS.
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Discussion

Epigenetic factors are involved in the pathogenesis of PCOS as well as the observed heterogeneity in the clinical manifestation and metabolic complications related with PCOS. Moreover, epigenetic inheritance of PCOS provides prospects for novel treatment options in this disorder [38]. CircRNAs have been shown to be involved in the pathogenesis of PCOS and related pathologies. The main mechanism through which they exert their functions is modulation of biological availability of miRNAs. However, the miRNAs sponged by dysregulated circRNAs in PCOS have not been identified completely. We reported several circRNA/miRNA axes that contributed to the pathoetiology of PCOS. Among the reported miRNAs, miR-136 has been shown to be sponged by three circRNAs, namely circ_0030018 [18], circ_RANBP9 [19] and hsa_circ_0118530 [20] in the PCOS context. Thus, this miRNA has a prominent role in this process. The role of this miRNA in the pathogenesis of PCOS has also been evaluated by other studies. For instance, expression of miR-136-3p has been found to be elevated in rat ovaries and isolated OGCs following an ovulatory dose of LH-human chorionic gonadotropin (hCG). At the same time, transcript levels of the hCG receptor (LHR) have been decreased suggesting the effect of miR-136-3p in targeting LHR [39].

Besides, circ_FURIN has been shown to sponge both miR-423-5p [29] and miR-195-5p [21]. Taken together, a certain circRNA can sponge several miRNAs. Meanwhile, a miRNA can be targeted by several circRNAs. Thus, a complex network exists between these two types of transcripts in the context of PCOS.

In addition to circRNAs that regulate function of OGCs, exosomal circRNA can modulate follicular microenvironment and participate in the etiology of PCOS. Thus, future studies can use this biological source to find novel biomarkers for PCOS.

ceRNA axes that contribute to the pathogenesis of PCOS can affect a number of cellular processes, particularly cell apoptosis. Notably, OGCs apoptosis is involved in all phenotypes of PCOS; and suppression of OGCs apoptosis can assuage several clinical manifestations associated with PCOS [40]. Thus, modulation of activity of these molecular axes can be considered as a treatment modality for PCOS. In addition to apoptosis, another route of cell death, i.e. ferroptosis has been found to be affected by a circRNA, namely circRHBG in PCOS [23]. In line with this finding, involvement of ferroptosis-related genes in the pathophysiology of PCOS has been recently highlighted [41].

The mechanism by which expression of cicRNAs is induced in PCOS is not elucidated completely, However, a number of transcription factors such as SNAI2 might be involved in this process [24]. Additional underlying mechanisms and the effects of environmental factors should be investigated in future studies.

To understand the ceRNA network in PCOS, in-silico analyses were conducted using the miRWalk, miRNet, and miRDIP databases. We focused on understanding the role of circRNA/miRNA interactions in PCOS, but we started from dysregulated genes and miRNAs. The miRWalk database identified 80 unique genes potentially regulated by five miRNAs, while miRNet revealed 6449 circRNAs that could control the expression or function of 11 miRNAs. The miRAnno module of miRDIP identified 11 miRNAs associated with 35 human pathways. Among thee pathways are those related with protein translation, adipogenesis, mTOR signaling, WNT signaling, PI3K cascade, cellular responses to stress, chromatin organization and DNA damage response. These findings provide a foundation for further investigations into the role of the ceRNA network in PCOS.

Conclusion and future directions

Cumulatively, evidence discussed in the current literature review supports involvement of circRNAs in the pathophysiology of PCOS and its related pathologies. Of particular note, circRNA signature and the interaction between circRNAs and miRNAs can explain the pathological changes occurred in this condition and predict the response to treatment options for infertility. Moreover, these networks are components of epigenetic machinery that controls expression of genes and chromatin structure.

Finally, the epigenetic model consisted of ceRNA network is in favor of the proposed evolutionary model for PCOS pathogenesis. This synthesis of the available evidence is predicted to evolve over time. Thus, future studies would enhance the understanding of the pathobiology of PCOS and the relevance of environmental versus genetic factors in the modulation of this condition.