The Novelty of miRNAs as a Clinical Biomarker for the Management of PCOS

Abstract

Polycystic ovary syndrome (PCOS) is a common endocrine disorder that affects around 5–10% of women of reproductive age. The aetiology of PCOS is not fully understood with various genetics, iatrogenic (e.g. chemotherapy) and environmental factors have been proposed. microRNAs (miRNAs) are small non-coding single-stranded RNAs which are known to act as a regulator to gene expression at the post-transcriptional levels. Altered expression of miRNAs has been linked to several disorders including infertility. Recent reports demonstrated the expression of differential levels of miRNAs in the serum, ovarian follicular cells and follicular fluid of PCOS patients when compared with healthy women. Therefore, miRNAs may play important role in the pathogenesis of PCOS. The aim of this chapter is to summarise the current understanding pertaining to miRNAs and PCOS and to expedite its possible role in the diagnosis and management of this disorder.

1. Introduction

Polycystic ovary syndrome (PCOS) is the most common multifactorial endocrinopathy female disorder. It affects approximately 5–10% of women in their reproductive age [1, 2, 3]. It is characterised by oligoanovulatory ovarian dysfunction, polycystic ovarian morphology and clinical and biochemical hyperandrogenism (HR). In addition, other factors such as endocrine dysfunctions, suboptimal follicular environment and oocyte competence make it a leading cause of female infertility. PCOS is also an important risk factor for metabolic disorders such as obesity, hyperlipidaemia and insulin resistance (IR), leading to type-2 diabetes mellitus (T2DM), cardiovascular disease (CVD) and endometrial cancer and other diseases [4, 5]. To date, the aetiology of PCOS remains elusive; however, environmental and genetic variations are proposed as a potential contributing factor. Although recent pedigree and genome-wide association studies have revealed apparent interrelation of several genes with PCOS, none have shown direct cause to the occurrence of the syndrome [6]. Thus, it has been postulated that their effect might be apparent in a dose-dependent or synergistic fashion rather than being a one or none effect [6]. Recent reports have suggested miRNAs’ involvement in PCOS incidence and development, thanks to the differential expression in patients with this disorder and healthy fertile patients [1, 7].

2. miRNAs as a potential novel clinical biomarker for PCOS

miRNAs are a new class of endogenously produced short non-coding single-strand RNAs with 20–25 nucleotides [1, 4, 8]. miRNAs are first transcribed from the genome as a primary miRNA (pri-miRNA) by RNA polymerases to form a hairpin like structure. For the miRNA to exert its biological function, it cleaves into a precursor miRNA (pre-miRNA) by a nuclear protein complex containing a Dorsha enzyme. These small transcripts (60–110 nt) can then leave the nucleus to the cytoplasm and subsequently processed further by the Dicer enzyme and another protein complex into the mature form of the miRNA. The mature form of the miRNA can regulate gene expression post-transcriptionally via binding to the 3’untranslated region (UTR) of the target mRNA, thus preventing their translation and/or causing their destabilisation of molecule [1, 9]. miRNAs have been shown to be widely expressed throughout the body, including organs such as muscles, adipose tissue and ovaries, at the intra- and extra-cellular levels [1, 4]. Recently, miRNAs have been isolated from extracellular fluids with variable expression profile depending on the fluid [1]; urine [10], saliva, plasma/serum [9] and follicular fluid [11]. Furthermore, miRNAs can be encapsulated in microvesicles [12] which are extracellular hetergeneous membraneous vesicles (EVs) originating from the cells. The smallest subpopulation of which are called exosomes, and they are known to act as a mediator to transport and release miRNAs between target cells [12]. It has been reported that miRNAs are more stable in exosomes with greater gene regulatory activity [13]. miRNA’s functions become feasible through participation in processes involved in biological growth, development and disease state. The main impact of miRNAs has been demonstrated during cellular proliferation, differentiation, metabolism, and apoptosis with regulatory roles on RNA processing and transcription, chromatin structure and chromosome segregation [14]. In humans, it has been reported that around 60% of proteins-coding genes serve as target sites of miRNA [14, 15]. miRNAs can also act in an epigenetic manner to regulate the amplification and inhibition of miRNA signals through the feedback mechanism which may lead to a significant modification expression that contributes to different pathological conditions [4]. Moreover, miRNAs have been shown to play an important role in ovarian physiology and pathology such as primordial follicle activation and development, oocyte maturation, ovulation, ovarian cancer and endometriosis [4, 14]. Growing evidence demonstrates a differential expression of miRNAs in patients with and without PCOS [1], potential diagnostic and therapeutic markers for PCOS. Nevertheless, considering the large degree of heterogeneity of PCOS and the complexity of miRNAs regulatory actions, to our knowledge to date is still preliminary. Understanding the role of miRNAs in PCOS pathogenesis is crucial for possible alternative management and treatment approaches to this syndrome.

To this end, the aim of this chapter is to summarise and discuss the current knowledge regarding the possible interplay between miRNAs and PCOS and to establish the potential clinical role of some miRNAs that may offer a novel insight for the management and treatment of the syndrome.

2.1 Possible role of miRNAs in ovarian dysfunction in PCOS

Several studies and theories have been proposed in an endeavour to explore the possible cause of the altered follicle growth and development, large number of small and generally immature follicles as well as anovulation associated with PCOS. Hormonal-induced alterations to granulosa cells (GCs) appearance and function, as well as steroidogenesis abnormalities by the theca cells (TC), have also been reported (Figure 1). However, the mechanisms, chronology and the relative criticality in the cascade of events leading to PCOS are still unestablished. Since PCOS is largely influenced by environmental and genetic modifications including miRNAs [16], and their epigenetics alteration can modulate gene expression at the cellular levels, thus miRNAs’ profiles in the ovarian compartments in PCOS have been studied in several animal and human models and are discussed further in the section below.

Figure 1.

The complexity of miRNAs interactions with multiple organs leading to PCOS.

2.1.1 miRNAs in granulosa cells (GCs) and PCOS

During follicle development, GCs govern oocyte growth by modulating nutrient availability and activity of regulatory molecules. GCs have been identified as the primary site of endocrine signalling and oestrogen synthesis [17]. It has been demonstrated that GC may contribute to the abnormal folliculogenesis in PCOS patients [18]. These abnormalities have been further defined by the proliferation inhibition and increased rate of GCs death, thus supporting the link between the functional disorder of GCs and the disease nature of the PCOS [18].

A vast array of miRNAs has been shown to be differentially expressed among various size follicles. They have been also indicated to modulate GCs apoptosis and proliferation [19]. miR-1275, a regulator of GC death, has been reported to upregulate during follicular atresia and induce early porcine GCs apoptosis [20]. miR-23a and miR-27a have been shown to stimulate GC apoptosis, whereas miR-93 and Let-7 family of miRNAs act to promote proliferation [4, 17]. Moreover, Let-7 family of miRNAs has also been reported to induce GC apoptosis via the inhibition of mitogenic activated protein kinase 1 (MAP3K1). Interestingly, let-7c, 23a and 27a were all shown to be highly expressed in patients with premature ovarian failure when compared with a healthy control patient [21]. Furthermore, a high expression of miR-93 [22] and lower miR-23a have been identified in the GCs from PCOS patients. They indicated that miR-23a induces cell cycle arrest via the inhibition of FGD4 signalling [23]. Another group has also demonstrated a significantly lower expression of miR-126-5p and miR-29a in the GCs of PCOS patients when compared with healthy individuals, which were indicated to induce GC apoptosis in PCOS [24]. A recent study proposed that the high expression profile of miR-141 and miR-200 in PCOS may target the Wnt and PI3K signalling pathway to inhibit GC proliferation [25]. Moreover, miR-3940-5P, miR-486-5P, miR-206 and miR-204 are known to modulate ovarian GC proliferation and apoptosis [26, 27]. In PCOs, miR-3940-5p was reported to be markedly upregulated; however, low expression of miR-206, miR-204 and miR-486-5p has been indicated in the GC of PCOS when compared with normal controls [19, 26]. One questionable miRNA, which has been known to be involved in the proliferation and apoptosis of GC, is miR-485-5P. This miRNA has been reported contradictory in PCOS, with one report demonstrating its upregulation [18], and the other showed a lower expression of miR-485-5P in the GC of PCOS [28].

Furthermore, the dysregulation of miRNAs in GC can be a cause of oestrogen difeiciency which is known as a main characteristic of PCOS. Zhang et al. [29] reported a significantly lower expression of miR-320a in the cumulus cells (CCs) of PCOS, which served via 320a/RUNX2/CYP11A1 (CYP19A1) cascade to induce oestrogen deficiency. miR-182 and miR-15a are known as an essential regulator of GC proliferation and apoptosis as well as steroidogenesis. These miRNAs were found to markedly decreased in the GC of PCOS patients [4].

Studies on miRNAs expression in GC and its relation to PCOS are intensive and generally linked to the increased GC proliferation and apoptosis rates in GCs. Although it may sound contradicting, but it does make sense when thinking about the progression features of the disease in relation to follicular development. The increased proliferation rate of GCs can contribute to a more follicles progressing to the primary stage, which is reflected in the polycystic countenance of PCOS [30]. Whereas the increased apoptosis rate is indicated by the anovulatory feature of the disease [4, 31].

Overall, to date, all reports indicate that GCs miRNAs are clearly involved in the regulation of folliculogenesis and steroidogenesis, and any dysregulation or alteration may subsequently contribute to the pathogenicity of PCOS.

2.1.2 miRNAs in theca cells (TCs) and PCOS

Theca cells (TCs) are the primary site of androgen synthesis in the ovaries. Under the influence of LH, theca cells express the mRNA of the three important steroidogenic enzymes CYP11A, CYP17 and 3ß-HSD, which are involved in the androgen de novo synthesis pathway [32, 33]. Androgen excess is one of the most important key features that is used to diagnose PCOS [14, 34]. It can negatively impact ovarian follicle growth and maturation and therefore female infertility. It has been reported that the increased expression or activity of CYP17 and P450scc enzyme is associated with the abnormal high androgen production in the theca cells [2, 35, 36]. Another key androgen producing-related gene GATA6 and an insulin receptor gene IRS-2 were demonstrated to be markedly upregulated in the theca cells of PCOS patients. Lin et al. [2] documented that miR-92a and miR-92b are involved in the regulation of GATA6 and IRS-2, and their expressions were significantly lower in theca tissues of women with PCOS. The results of miRNA microarray analysis have demonstrated that miR-200a, miR-200c, miR-141 and miR-502-3p were significantly increased in the theca interna in women with PCOS [14].

Limited data are available on the theca cells miRNAs in PCOS; however, it indicates that it may play a pivotal role in the dysregulation of androgen synthesis pathways in women with PCOS.

2.1.3 miRNA in follicular fluid and PCOS

Follicular fluid (FF) provides a perfect microenvironment for oocyte development and maturation. It allows for an efficient cross talk between the blood, granulosa and TCs [9]. FF comprises various hormones, multiple proteins, metabolites, anti-apoptotic factors and regulatory nucleotides including miRNAs [13]. Therefore, FF composition reflects the secretory activity of oocyte, GC and TCs. In addition, it may serve as a relatively easy and less invasive method for the collection and the analysis of miRNA during oocytes retrieval for assisted reproduction [4].

miRNAs have been reported abundant in FF, and some researchers focused on investigating their association to PCOS [1, 13]. A recent study found 29 differentially expressed miRNAs in the FF of subjects with and without PCOS, with 12 involved in reproductive pathways [37]. Of these, miR-382-5p was found to positively associate with free androgen index (FAI) and age, miR-199b-5p allied with AMH, and miR-127-3p was linked to insulin resistance [37]. A study by Roth et al. [38] reported a significantly increased expression of miR-9, miR-18b, miR-32, miR-34c and miR-135a in the FF of women with PCOS. In addition, the study demonstrated that synaptogamin 1, insulin receptor 2 and interleukin 8 were the target genes for these miRNAs [38]. The study by Scalici et al. showed an increased expression of miR-30a and a significantly decreased levels of let7b and miR-140 in the FF of PCOS individuals. FOXL-2, essential gene for ovarian development, was identified as the target gene for miR-30a, and the inhibition of this miRNA in mouse resulted into disrupted GC morphology and androgen production by the TCs [39]. Furthermore, two members of the TGF-ß family, Smad 2/3 and activin receptor I, were possible target genes for let-7b. The dysregulation of TGF-ß was reported to be a potential cause of PCOS [1, 40]. Scalici et al. [39] also concluded that the combination of Let-7b, miR-30a and miR-140 can be used as a possible novel biomarker, with a sensitivity of 70% and specificity of 80%, to distinguish between normal controls and PCOS. miR-93 has been described as a novel diagnostic marker for PCOS due to the significantly consistent increased expression when compared with healthy individuals [41]. Moreover, a study by Lin et al. reported a significant downregulation of miR-92a and miR-92b in PCOS [2].

Studies focusing on miRNAs’ involvement in androgen metabolism and PCOS have identified a specific group of miRNAs in the FF of PCOS patients; however, there is no specific consensus on the effect of these miRNAs in steroidogenesis in PCOS patients. Of these miRNAs, miR-151 was shown to negatively associate with free testosterone; while miR-29a and miR-518 were positively correlated to testosterone; miR-155, miR-9 and miR-18b inhibited testosterone and miR-146a, miR-132 and miR-135 inhibited both testosterone and progesterone secretion [42]. Other contradicting miRNAs, miR-132 and miR-320, which are known to be involved in oestradiol release, showed significant lower expression in PCOS patients [43], while another author reported increased expression of miR-320 in PCOS individuals [39, 44]. A third report demonstrated no change in 320a expression in PCOS subjects when compared with healthy women [3, 38]. miR-518-3p was shown to highly expressed in androgenic PCOS phenotype. Further analysis demonstrated the reduction in miR-24-3p, miR-29a, miR-151-3p and miR-574-3p levels in PCOS subjects when compared with healthy controls [3].

All above, data indicates that miRNAs in PCOS can serve as a potential novel biomarker for the diagnosis and maybe further for the classification of different PCOS phenotypes. Furthermore, it may provide insights into the molecular changes related to the cells from which the fluid is derived from and thus support therapeutic decisions. Ovarian cells and circulating miRNAs that have been proposed to be dysregulated and involved in the pathogenicity of PCOS are summarised in the Table 1.

miRNA	Place of detection	Proposed function
miRNA-376	GC [4]	Modulate ovarian GC proliferation and/or apoptosis
miRNA-155	GC/FF [42]	Modulate androgens
miRNA-33b-5p	GC [4]	Promote IR
miRNA-483-5p	GC [1]	Modulate ovarian GC proliferation and/or apoptosis
miRNA-145 [1]	GC	Modulate ovarian GC proliferation and/or apoptosis
miRNA-126-5p	GC [1]	Modulate ovarian GC proliferation and/or apoptosis
miRNA-29a-5p [1]	GC	Modulate ovarian GC proliferation and/or apoptosis
miR-29a	GC [1, 42]	Modulate ovarian GC proliferation and/or apoptosis, androgens
miRNA-224	GC [42]	Modulate ovarian GC proliferation and/or apoptosis, androgens
miR-485-5P [18]	GC	Modulate ovarian GC proliferation and/or apoptosis
miRNA-15a	GC [4]	Modulate ovarian GC proliferation and/or apoptosis, androgens
miR-1275	GC [20]	Modulate ovarian GC proliferation and/or apoptosis, androgens
miR-23a	GC [4]	Modulate ovarian GC proliferation and/or apoptosis
miR-27a	GC [4]	Modulate ovarian GC proliferation and/or apoptosis
miR-182	GC [4]	Modulate ovarian GC proliferation and/or apoptosis, steroidogenesis
miR-200 [14]	GC	Modulate ovarian GC proliferation and/or apoptosis
miR-3940-5P [26, 27]	GC	Modulate ovarian GC proliferation and/or apoptosis
miR-486-5P [26, 27]	GC	Modulate ovarian GC proliferation and/or apoptosis
miR-206	GC [19, 26]	Modulate ovarian GC proliferation and/or apoptosis
miR-204	GC [19, 26]	Modulate ovarian GC proliferation and/or apoptosis
miRNA-320a [1]	Cumulus cells	Inhibit steroidogenesis
miRNA-509-3p	Cumulus cells [1]	Modulate androgens
miR-141 [14]	GC/TC	Modulate ovarian GC proliferation and/or apoptosis, steroidogenesis
miRNA-92b	GC/TC [1]	Modulate androgens
miRNA-222 [42]	TC/serum	Modulate androgens
miRNA-92a [1]	TC	Modulate androgens
miR-502-3p	TC [14]	Modulate androgens
miR-200a [14]	TC	Modulate androgens
miR-200c [14]	TC	Modulate androgens
miRNA-233	Adipose tissues [1]	IR
miRNA-93 [1]	Adipose tissues/GC	Modulate ovarian GC proliferation and/or apoptosis
miRNA-320	Adipocytes/FF/GC [1, 4, 42]	Modulate androgens
miRNA-132 [1, 42]	GC/ FF	Modulate androgens
Let7-b [1, 11]	FF/GC	Modulate ovarian GC proliferation and/or apoptosis
miR-382-5p [37]	FF	Modulate androgens
miR-24 [42]	FF	Modulate androgens
miRNA-32 [38]	FF	Modulate androgens
miRNA-34c [38]	FF	Modulate androgens
miRNA-135a	FF/GC [38]	Modulate androgens
miRNA-18b [42]	FF/GC	Modulate androgens
miR-21 [42]	FF/GC	Modulate androgens
miRNA-30a [42]	FF	Modulate androgens
miRNA-140 [42]	FF	Related to ovarian follicle development
miR-9 [42]	FF/GC	Modulate androgens
miR-127-3p	FF [37]	IRs
miR-151 [42]	FF	Modulate androgens
miR-151-3p [3]	FF	Modulate androgens
miR-146a [42]	FF/serum	Modulate androgens
miR-518-3p [3]	FF	Modulate androgens
574-3p [3]	FF	Modulate androgens
miR-518	FF	Modulate androgens
miRNA-103	Blood/GC [42]	Modulate androgens
miRNA-27b [1]	Blood	Modulate androgens

Table 1.

Circulating and ovarian miRNAs that been proposed altered in PCOS.

2.2 miRNAs altered androgenic and metabolic consequences of the PCOS

HA and metabolic disorders are inevitably associated in women with PCOS. It is always manifested by hyperandrogenism (HA), insulin resistance (IR) and compensatory hyperinsulinemia, which is known as an essential contributor to the pathogenicity of PCOS. Therefore, the mechanism behind this connection could shed lights on key markers in the diagnosis of PCOS.

2.2.1 miRNA and androgens dysregulation in PCOS

HA is a common characteristic of PCOS that is detected and used for diagnosis in both serum and ovarian compartment. The source of androgen excess is not exclusively resolved, it might be due to increased steroidogenic enzyme activity, increased androgen synthesis by the TCs as a response to LH overstimulation, androgen receptor (AR) defects at the target organs level, cortisol metabolism defects and/or increased adrenal gland androgen production [3, 4, 45]. Evidence indicates that testosterone, androstenedione (A4) and dihydrotestosterone (DHT) are all involved in the pathogenicity of PCOS. In normal subjects, a major fraction of free testosterone is bound to sex hormone binding globulin (SHBG) and albumin. In women with PCOS, SHBG levels are decreased resulting into an increased level of bioavailable testosterone [4]. Furthermore, increased expression of AR has been reported in patients with PCOS [46].

The complexity of abnormal sex hormone production makes it difficult to define the specific miRNAs involved in this process. Several studies have highlighted the role played by miRNAs in androgens and steroid synthesis in the ovarian cells and body fluids. miR-592 was found to positively correlate with LH/chorionic gonadotropin receptor (LH/CGH), a key factor in the mechanism involved in HA in PCOS [47]. A recent study showed a negative association between serum testosterone and miR-146a, an miRNA that has been found to inhibit the secretory activity of steroid hormones [1, 48]. Oestradiol was noted to positively linked with miR-222, miR-132, miR-320 and miR-520-3p, whereas a negative relationship has been observed between progesterone concentration and miR-193b, miR-24 and miR483-5p [43, 49]. Furthermore, miR-320, miR-518, miR107 and miR-29a were also found to positively correlated with high levels of serum testosterone [4, 9]. On the other hand, expression profile of miR-151 was found to negatively relate to serum testosterone [9]. Another study demonstrated a strong positive association between levels of free testosterone and miR-155, miR-27b, miR-103 and miR-21 [1]. Interestingly, Xiong et al. reported that the chance of PCOS development has been reduced by 0.01-fold for every elevated fold expression of miR-23a [50]. Lower oestrogen synthesis has been linked to the over-expression of miR-181a and miR-378 via the downregulation of aromatase enzyme [51, 52]. It is intriguing to note that miR-200b is involved in ovulation at the hypothalamo-pituitary ovarian axis level and is a downstream target for AR; such information may indicate that miR-200 plays a role in HA and PCOS [14].

HA is associated with an array of pathological changes that interact to promote the development of PCOS. Of these changes, hyperinsulinemia, IR and dyslipidaemia are well characterised.

2.2.2 miRNAs and insulin resistance (IR) and/or hyperinsulinemia in PCOS

IR plays a major role in the pathogenicity of PCOS with a pervasiveness of 70% among patients. It is known to be associated with impaired glucose metabolism, T2DM, metabolic syndrome, dyslipidaemia and increased risk of CVD. Hyperinsulinemia contributes to the increased ovarian steroidogenesis and androgen production via insulin growth factor-1 (IGF-1) receptor to initiate LH-induced TCs excess androgen secretion [53]. IR synergistically stimulated androgen via increasing CYP17 enzyme activity leading to lower SHBG levels and thus increase free testosterone levels [4, 54]. IGF-1 signalling, peroxidase proliferator receptor (PPAR) and angiopoietin, have been associated with PCOS and are potentially regulated by miR-223 [41]. miR-483-5p has been shown to lower IRs in women with PCOS [28]. It has been suggested that miR-320 and miR-132 can play a role in modulating insulin resistance [43]. The author predicted that RABSB and HMGA2 are the target genes for miR-320 and miR-132, respectively, and that both gene expressions are altered by the reduced levels of miR-320 and miR-132 in the FF of PCOS individuals [43]. Increased expressions of miR-194, miR-193b and miR-122 have been noted in PCOS patients when compared with controls. The upregulation of these three miRNAs was found to target several pathways including insulin signalling pathway [22]. A strong association has been reported between the expression of miR-93 and miR-33b-5p and the development of IR in women with PCOS. They have been shown to exert their effect through the downregulation of glucose transporter 4 (GLUT4) expression [10, 55]. A significant positive correlation has been found between miR-222 expression and serum insulin [48]. Lin et al. [2] reported that 200a expression is associated with IR and T2DM. miR-1, miR-29, miR126 and miR-19a have been postulated to regulate glucose uptake via modulating PI3K [56].

All these findings indicate a cross talk between HA and hyperinsulinemia in PCOS. miRNAs involved in the regulation of these two dominant features are key for the development of specific markers for the diagnosis and treatment of the syndrome.

2.2.3 miRNA and dyslipidaemia in PCOS

The prevalence of hyperlipidaemia is 70% in PCOS. It is generally manifested by lipid profile dysfunctions, including increased levels of low-density lipoprotein cholesterol (LDL-C), reduced levels of high-density lipoproteins cholesterol (HDL-C) and high triglycerides [57]. Even though obesity is not indicative of PCOS, however, visceral adipose tissues (VATs) were found higher in patients with PCOS, whether they are obese or not, when compared with a control group [33]. To date, studies have found a close correlation between androgen excess, obesity and PCOS [5]. Androgen excess promotes the deposition of abdominal visceral fat, which in turn drives the secretion of adrenal and ovarian androgens via several pathways induced by adipose tissue dysfunction [58].

Current data on the interaction between miRNAs, dyslipidaemia and PCOS is sparse. In animal models, miR-33 has been shown to exert some control on LDL-C secretion and modulation of cholesterol biosynthesis [4, 59]. In addition, miR-128-1, miR-185 and miR-148a were reported to significantly decrease LDL-C levels, whereas miR-148a expression increased HDL-C [60]. The target genes for these miRNAs were adenosine triphosphate (ATP) binding cascade transporter A1 (ABCA1) and ABCG1 [60]. Furthermore, miR-34c-5p, miR-760, miR-597 and miR-1468 were found to be positively linked to hirsutism score. miR-597 and miR-1468 were shown to target the androgen receptor pathway [58]. Murri et al. [58] identified a group of miRNAs that are differentially expressed among PCOS patients. Of which, miR-30c-5p, miR-34c5p, miR-151a-5p, miR-193a-5p, miR-199a-3p, miR-1539, miR-26a-5p, miR-107, miR-142-3p, miR-126-5p and miR-598 were shown to correlate either positively or negatively with abdominal adiposity, obesity and metabolic dysfunction. Among all, miR-107, miR-30c-5p, miR-199a-3p and miR-26a-5p were noted to significantly associate with fatty acid biosynthesis and metabolism, and their expressions were found reduced in PCOS. The same group demonstrated further an increased expression of miR-338-3p, miR-365, miR-223-3p and miR-197-3 in obese PCOS patients when compared with the control subjects. All the reported miRNAs were correlated with androgen excess, glucose metabolic index and BMI [58]. Furthermore, miR-548-3p and miR-34c-5p expressions were also found to increase in PCOS individuals when compared with controls. Both miR-548-3p and miR-34c-5p markedly correlate with fatty acid biosynthesis and metabolisms [58].

Taken together, miRNAs play a pivotal role in the interaction of HA, hyperinsulinemia, dyslipidaemia and PCOS. Therefore, targeting miRNAs involved in the regulation of these features can shed light on the clinically relevant diagnostics and therapeutics markers of PCOS.

3. Considering miRNAs as a potential therapeutic target in PCOS

Based on the current knowledge presented in previous sections, miRNAs seem to be associated to the development of PCOS. However, no reports are available postulating the use of drugs to target miRNAs as a potential treatment option. Current guidance on disease management options is only focusing on improving prognosis and mitigating the symptoms. Of interest, PCOS patients are mostly undergoing in vitro fertilisation and thus involved in pharmacological protocols to support their fertility treatment. Available treatment options include the combined Letrozole and clomiphene citrate treatment, which is typically used for ovulation induction. Oral contraceptives (OCPs) control ovulation and prevent cyst formation. Anti-androgen drugs such as flutamide and spironolactone are used to manage increased androgens-related problems. Metformin is an insulin sensitivity agent that can improve the life quality for these patients by reducing serum insulin and androgen levels and elevating SHBG [4, 9, 61]. Polyphenols are a natural compound that has been used traditionally as a treatment option for several conditions; however, their limited bioavailability restricted their use. It has recently dragged some attention as a therapeutic target to ameliorate PCOS symptoms and reverse expression of crucial genes; nevertheless, more data are required before their administration into clinical use [61].

Studies focusing on therapeutics that may target small molecules such as miRNAs are rarely reported. Recently, metformin administration has been found to either positively or negatively target some miRNAs. Bao et al. demonstrated that metformin can inhibit markers for pancreatic cancer stem cells via the upregulation of miR-26a [62]. In addition, another study reported that metformin decreased the expression of miR-221 and miR-222 in T2DM patients [4, 9, 63]. Even more, metformin with sitagliptin combined therapy has been proposed as a treatment strategy to ameliorate PCOS in patients with IR [64]. This strategy was found to reduce GC apoptosis via mediating IncRNA H19 expression [33, 64].

Incretins-based therapies have been recently discovered to influence miRNAs expression and its potential for the management of PCOS. Glucagon-like peptide 1 agonist receptor agonist (GLP-1RA) decreased blood sugars via the downregulation of miR-375 and miR-23 and the increased expression of miR-192, miR-132 and miR-27a [65].

Treatment approaches targeting miRNAs associated with the metabolic feature of the disease may improve the clinical outcomes. miRNAs regulating androgen hormones are worthy at the top of the list. However, it is still a rich field of scientific research and validation.

4. Clinical application of miRNAs is still limited

Despite the hope, great effort and the scientific work done to clarify the role of miRNAs in PCOS, it is still a long way to go before it is possible to utilise miRNAs as a diagnostic and therapeutic tool. It is perhaps due to the complexity of both miRNAs and PCOS.

The pathogenicity of PCOS involves several phenotypes described by the Rotterdam criteria and other less sever criterion, thus providing difficult and ununited diagnosis. In addition, the fact that one mRNA maybe regulated by multiple miRNAs and that miRNAs can modulate each other complicates the matter even further. Moreover, most of the studies performed are limited by the sample size and sometimes with contradicting results. Furthermore, metformin is mostly taken by PCOS individuals during their infertility treatment and has been shown to influence miRNA expression, thus may false mask facts and lead to contradicting results. Therefore, it is imperative to deepen the current research to develop consistent and reliable miRNA-based diagnostics and therapeutics.

5. Conclusion and future direction

Taken together, miRNAs play a key role to fine-tune events leading to ovarian cell apoptosis, proliferation, follicular development, HA, altered insulin and dyslipidaemia in PCOS. Yet, it is not possible to determine whether miRNAs-altered expression is a cause or is an aftereffect event of the syndrome. Furthermore, the dynamic expression and action of miRNAs complicate facts further. Thus, further functional studies on mRNA-miRNA and the interplay between epigenetic regulation and altered miRNA expression are required to highlight the pathogenicity of the disease. It is, however, possible to use miRNAs as biomarker to categorise and identify PCOS sub-phenotypes. miRNAs are a promising candidate to modulate pathways involved in the pathogenicity of this syndrome.