Polycystic Ovary Syndrome-Epigenetic Mechanisms and Aberrant MicroRNA

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Polycystic Ovary SyndromeEpigenetic Mechanisms and Aberrant MicroRNA Ioana R. Ilie, Carmen E. Georgescu1 Department of Endocrinology, University of Medicine and Pharmacy “Iuliu-Hatieganu”, Cluj-Napoca, Romania 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. The Epigenetic Landscape 2.1 Follistatin 2.2 PPAR-γ 2.3 CAG Androgen Receptor 2.4 LMNA 2.5 LHCGR 2.6 EPX1 2.7 Other Epigenetically Regulated Genes 3. miRNA 3.1 Biomarkers 3.2 Pathophysiology 4. Conclusions Acknowledgments References

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Abstract Polycystic ovary syndrome (PCOS) is the most common endocrinopathy in women of reproductive age affecting various functions including reproduction and metabolism. This syndrome is associated with increased prevalence of subclinical cardiovascular disease as well as endometrial and ovarian cancer. This syndrome is highly heterogeneous and it is not yet clear which factors are responsible for the development of a particular phenotype. Current research has shown that the interaction of susceptible and protective genomic variants under the influence of environmental factors can modify the clinical presentation via epigenetic modifications. MicroRNA (miRNA) are regulators of gene expression. Altered miRNA expression has been associated with various diseases such as diabetes, insulin resistance, inflammation, and cancer. Several miRNA have been identified in PCOS. This review examines the role of epigenetics and miRNA in the pathophysiology of this complex disease process. Advances in Clinical Chemistry ISSN 0065-2423 http://dx.doi.org/10.1016/bs.acc.2015.06.001

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2015 Elsevier Inc. All rights reserved.

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ABBREVIATIONS AR androgen receptor CpG cytosine–phosphate–guanine CYP 17 cytochrome P450, family 17 CYP11A cholesterol side-chain cleavage cytochrome P450 DENND1A DENN domain-containing protein 1A E2 estradiol EPHX1 gene epoxide hydrolase 1, microsomal (xenobiotic) FSH follicle-stimulating hormone FST follistatin FTO fat mass and obesity-associated gene GC granulosa cells GLUT4 Glucose transporter type 4 GO gene ontology HDAC3 histone deacetylase 3 IVF in vitro fertilization KEGG Kyoto Encyclopedia of Genes and Genomes LH luteinizing hormone LHCGR LH/choriogonadotropin receptor LMN protein coding, lamin A/C NCOR1 nuclear receptor co-repressor 1 PCOS polycystic ovary syndrome PPAR-γ peroxisome proliferator-activated receptor-γ SHBG sex hormone-binding protein THADA thyroid adenoma-associated TNF-alpha tumor necrosis factor-alpha VNTR insulin gene variable number of tandem repeats XCI X chromosome inactivation

1. INTRODUCTION Polycystic ovary syndrome (PCOS) affects approximately 5–7% of reproductive age women placing it among the most common female endocrine disorders [1]. Its cardinal features are hyperandrogenism, chronic anovulation, and polycystic ovaries. Additionally, these women are frequently characterized with insulin resistance and central obesity, possess several cardiovascular risk markers and early subclinical atherosclerosis [2–4]. Genetic factors are widely believed to contribute to PCOS [5]. To date, several candidate genes have been proposed. These include luteinizing hormone (LH)/choriogonadotropin receptor, thyroid adenoma-associated (THADA) and DENND1A (DENN domaincontaining protein 1A), D19S884, CYP 17, CYP11A, androgen receptor (AR), sex hormone-binding protein (SHBG), tumor necrosis factor-alpha

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(TNF-alpha), insulin receptor gene (INSR), insulin gene variable number of tandem repeats (VNTR), calpain-10, peroxisome proliferator-activated receptor-γ (PPAR-γ), or fat mass and obesity-associated gene (FTO) (Fig. 1). Despite their apparent association with PCOS, none were identified as a major factor in its etiology [6–9]. There is extensive evidence supporting the premise that environmental factors also influence clinical presentation via epigenetic modifications [10], thereby contributing to the origin, pathophysiology, and development of PCOS [11]. Epigenetic changes have been widely connected to common diseases such as type 2 diabetes, various cancers, and mental disorders including schizophrenia and depression [12,13]. Epigenetic modifications are changes in gene expression with no change in DNA sequence inheritable through mitosis or meiosis leading to phenotypic changes [14]. Epigenetic regulation usually includes DNA hypo- or hypermethylation as well as histone modifications. They can cause irregular gene expression, thereby predisposing individuals to developing PCOS [15]. However, to date, little is known about epigenomics, especially the DNA methylation profiles in the pathophysiology of PCOS [16]. Candidate genes Genes involved in androgen steroidogenesis

INSR

FTO CYP17

? Calpain-10 PPAR-g

Genes involved in the secretion and action of insulin, in obesity and insulin resistance and chronic inflammation

Insulin gene VNTR CYP11A TNF-alpha AR SHBG

Fibrillin-3 (D19S884)/ DENND1A/ LHCGR/ THADA/ loci-susceptibility to PCOS

Genes involved in gonadotrophin action and regulation

Figure 1 Candidate genes identified in association with PCOS. (AR, androgen receptor; DENND1A, DENN domain-containing protein 1A; FTO, fat mass and obesity-associated gene; INS, insulin receptor gene; LHCGR, luteinizing hormone/choriogonadotropin receptor; PPAR-γ, peroxisome proliferator-activated receptor-γ; SHBG, sex hormonebinding protein; THADA, thyroid adenoma-associated protein; TNF-alpha, tumor necrosis factor-alpha; VNTR, insulin gene variable number of tandem repeats; ?, other, yet unknown candidate genes).

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Understanding the complex network of gene expression and epigenetic regulation has been further complicated by the recent discovery of microRNA (miRNA). These unique molecules are intimately involved in the regulation of gene expression and play a critical role in a wide number of cellular processes including development, differentiation, cell-cycle regulation, senescence, and metabolism. In fact, aberrant miRNA expression is now linked to several human diseases [17]. MiRNA directly modulate target molecules and are connected with the epigenetic machinery. They are involved in the feedback regulatory loop, the purpose of which is to finely tune gene expression. It is now known that miRNA expression can be affected by the same mechanisms modulating protein-coding genes (PCGs), including epigenetic regulation [18,19]. The interplay of miRNA and epigenetics is very complicated. For example, miRNA itself can modulate the expression of epigenetic components such as DNA methyltransferases, histone deacetylases, and polycomb repressive complex genes, thereby producing a highly controlled feedback mechanism. Their aberrant expression, i.e., “epi-miRNA,” has often been associated with the development or progression of human cancer [18,19]. An aberrant miRNome, i.e., the full spectrum of miRNA for a specific genome [19], is likely representative of underlying pathophysiology. This premise led to an increased number of studies that investigated the potential regulatory mechanisms responsible for this dysregulated expression. In PCOS, a number of miRNA (miR-146a, miR-22, miR-132, miR-200c, miR-141, and miR-21) were differentially expressed in ovarian tissue [20,21]. Serum miRNA may also be of potential use in different diseases including PCOS [21]. However, it is far from clear as to the possible modes of miRNA action in epigenetic pathophysiology of PCOS. Their interplay has only recently been investigated and much needs to be done with understanding their complex interrelationships with respect to altered gene expression in PCOS. It is likely that a more comprehensive understanding of underlying molecular mechanisms will facilitate diagnosis and improve treatment of this complicated syndrome.

2. THE EPIGENETIC LANDSCAPE Genetic and early-life environmental factors acting in utero might determine whether or not PCOS ultimately develops in later life [22]. For example, recent studies have suggested that early inappropriate epigenetic reprogramming is carried forward [15,23–25]. It is important to note

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that these epigenetic changes not only affect somatic cells but also gametes thus influencing future generations [22,26]. Animal model studies point to a fetal origin. For example, androgen exposure of rhesus monkeys, sheep, mice, and rats in early or late gestation results in the subsequent development of PCOS phenotypes in adulthood [15,27]. Excessive environmental exposure to endogenous such as those in PCOS or exogenous androgens such as androgenic endocrine-disrupting chemicals (EDC) during early development trigger persistent abnormalities in the reproductive and metabolic systems in both the exposed and subsequent generations [22]. Although clearly established in animals, underlying molecular mechanisms associated with this phenomenon remain unclear. Interestingly, no significant differences were noted in global DNA methylation patterns in 20 PCOS patients versus 20 body mass index- and age-matched controls [28]. The authors did, however, indicate that further investigation on methylation in specific gene regions was warranted. Using visceral adipose tissue, a genome-wide site-specific methylation analysis was performed in infant and adult rhesus monkeys following androgen exposure [25]. The array identified numerous differentially methylated loci in treated infant (n ¼ 163) and adult (n ¼ 325) monkeys versus controls. Several significant pathways were identified including the antiproliferative role of TOB in T cell and transforming growth factor-β signaling. Despite these remarkable findings, direct association of these candidate gene methylation profiles with PCOS remains elusive [29].

2.1 Follistatin Follistatin (FST) is an activin-binding protein that antagonizes activin in vitro. Although this ubiquitous protein is present at the highest concentration in ovarian tissue, its role in PCOS is controversial [30–32]. For example, circulating FST is increased in PCOS, but its mRNA expression is absent in granulosa cells (GCs). Attempts to correlate endometrial tissue (vs. peripheral blood) methylation patterns with the promoter region of the FST gene in PCOS were largely equivocal [29].

2.2 PPAR-γ PPAR-γ appears greatly involved in reproduction and metabolism through its different isoforms. For example, PPAR-γ1 specifically regulates ovarian function [33]. Transcription of PPAR-γ is regulated by co-repressors,

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i.e., nuclear co-repressor 1 (NCOR1) and histone deacetylase 3 (HDAC3), and co-activators [34]. Increased testosterone was noted in the serum and follicular fluid of hyperandrogenic PCOS women [24]. Significant genetic alterations of methylated PPAR-γ1 and NCOR1 and acetylated HDAC3 were also noted. There were differences between the failed and successful pregnancy groups. For example, decreased PPAR-γ1 and increased NCOR1 and HDAC3 expression was reported for the former. Studies in PCOS rat models demonstrated that PPAR-γ1 hyper- and NCOR1 hypomethylated promoters was associated with decreased PPAR-γ1 and increased NCOR1 gene transcription during ovarian maturation and development. Ovarian dysfunction induced by excessive androgen exposure may be greatly influenced by significantly altered methylation of PPAR-γ1 and NCOR1 genes and markedly altered HDAC3 acetylation. As such, follicular hyperandrogenism may induce these epigenetic alterations in GCs which could play a substantial role in the underlying mechanism of the ovarian dysfunction and the difference in pregnancy outcomes. More studies are clearly needed to explore over other potential epigenetic mechanisms involved in this process.

2.3 CAG Androgen Receptor Some studies investigating the relationship between DNA methylation and PCOS focused on the CAG repeat sequence in exon 1 of the AR gene [35]. Because the AR gene is located on the X chromosome, the epigenetic modification leading to preferential activation of either the short or long allele may account for the differential AR activity that triggers clinical heterogeneity, i.e., androgenic features. For example, Dasgupta et al. [35] demonstrated that the CAG allele distribution profile was homogeneous in PCOS versus controls. This finding indicates that CAG repeat polymorphism cannot, by itself, differentiate PCOS. Interesting, preferential activation of the short allele was noted among PCOS cases with non-random X chromosome inactivation (XCI) pattern. Differences in localization and DNA methylation pattern of CAG repeat sequence in AR gene might influence signal transduction and lead to development of PCOS [36]. In fact, AR gene polymorphism, i.e., CAG microsatellite repeat sequence and XCI, was shown to affect follicle-stimulating hormone (FSH) and LH expression and might be associated with PCOS development [37]. In other studies, the assessment of XCI revealed that

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shorter CAG repeats could change the sensitivity of the receptor to androgen and thus trigger PCOS [14,38,39]. XCI, associated with early embryogenesis, is thought to occur randomly. In contrast, non-random inactivation triggered by environmental exposure or allelic differences may play a significant role in PCOS due to the location of the AR gene that resides on the X chromosome. In a study by EscobarMorreale et al. [40], no significant differences were reported in XCI in women with hyperandrogenic hirsutism or idiopathic hirsutism. Similar findings were reported by Shah et al. [38]. However, in a subset of patients with non-random inactivation, the chromosome with the shorter CAG allele was preferentially active. This finding suggests that genetic and epigenetic changes may be involved in its pathogenesis.

2.4 LMNA There seems to be a connection with variation of the lamin A/C (LMNA) gene and the clinical manifestations of fat metabolism disorders, i.e., hyperandrogenemia, insulin resistance, polycystic ovary, etc. However, previous studies [41,42] that examined the relationship between LMNA gene variation and PCOS were largely inconclusive. Using MassARRAY DNA methylation, the relationship between LMNA gene methylation status and insulin resistance was examined in PCOS. Significant differences were noted in 12/20 cytosine–phosphate– guanine (CpG) sites. Hypermethylation was associated with insulin resistance in PCOS. As such, this gene may play an important role in the regulation of PCOS-associated insulin resistance [14].

2.5 LHCGR Other reports have supported the role of epigenetic mechanisms in PCOS. These include demethylation of the LH receptor (LHR) gene in a mouse model [10] and skewed XCI in humans [40,43]. The relationship of the LH/choriogonadotropin receptor (LHCGR) with PCOS has been investigated using a genome-wide association study. Wang et al. [44] sequenced the exons and flanking regions of LHCGR in 192 women with PCOS and reported no novel somatic mutations. Methylation status of six CpG sites in the LHCGR promoter region was assessed by pyrosequencing peripheral blood from 85 PCOS individuals versus 88 controls. This approach identified two hypomethylated sites at 174 and 111 CpG. Subsequent bisulfite sequencing detected additional CpG

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sites in the promoter including significant hypomethylation of +17 CpG. Methylation status was further examined using GCs. Eight significantly hypomethylated CPG sites (174, 148, 61, 43, 8, +10, +17, and +20) were noted resulting in increased LHCGR transcription. Hypomethylation across different tissues and corresponding expression supports its role in PCOS. It is clear that additional research is required to more fully substantiate this relationship [44].

2.6 EPX1 Another study examined three genes involved in steroid synthesis and metabolism [45]. Using peripheral blood from PCOS patients, decreased methylation of a subset of CpG clusters in the promoter region of the epoxide hydrolase 1 (EPHX1) gene was demonstrated. This gene encodes EPHX1, a critical enzyme that transforms reactive epoxides (resulting from degradation of endogenous and exogenous aromatic compounds) into transdihydrodiols that can be conjugated and excreted. Interestingly, this protein also plays a key role in the female reproductive system [46–48] and two exon single-nucleotide polymorphisms in this gene appear associated with PCOS [49]. As such, reduced methylation of the EPHX1 promoter region might activate its expression in PCOS. Moreover, the latter could suppress estradiol (E2) production from testosterone and further increase PCOS risk [13].

2.7 Other Epigenetically Regulated Genes Using genome-wide methylated DNA immunoprecipitation, Shen et al. [23] reported that a total of 79 genes were differentially methylated in insulin-resistant versus noninsulin-resistant PCOS. Forty genes were also identified as having different methylation patterns in PCOS versus controls. Subsequent examination via gene ontology (GO) and pathway enrichment analysis demonstrated that immune response categories were differentially methylated. It was noteworthy that cancer pathway genes were also differentially methylated in these patients as well. Using DNA methylation profiling with transcriptome analysis, Wang et al. [16] first reported that there were at least 3% differentially methylated sites and 650 differential transcripts in PCOS ovaries versus normal ovaries. Furthermore, methylated status of 54 genes was associated with transcription level in PCOS.

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These results open new avenues to identify novel epigenetically regulated genes that may be involved in PCOS. Furthermore, these aberrantly methylated genes and expressed transcription products may also be potential use as PCOS biomarkers. Several important changes in DNA methylation were located in gene-coding regions. Although no direct regulation of gene transcription was apparent, these changes may influence gene expression via alternative splicing pathways [50]. The chemokine (C–C motif ) ligand 2 (CCL2) [50,51], which was hypermethylated and downregulated, and fibrillin 1 (FBN1) [52], which was hypermethylated and upregulated, have been associated with PCOS. Because of this finding, it is likely that other genes are worthy of further investigation. As such, there is evidence suggesting that epigenetic events may partly be responsible for the development of PCOS. Although type I diabetes mellitus, p53, and NOD-like receptor signaling as well as immune and inflammatory diseases have been implicated in this disease process, more research is clearly needed to elucidate the exact epigenetic changes. Unfortunately, few studies have examined genome-wide DNA methylation in combination with expression profiling for PCOS.

3. miRNA MiRNA are defined as endogenous, small, noncoding, singlestranded, regulatory ribonucleic acid (RNA) molecules, 20–24 nucleotides in length [53]. They are processed from larger stem-loop precursor transcripts and regulate gene expression post-transcriptionally by binding to the 30 untranslated region of target mRNA [54]. During the binding process, the miRNA starts a pathway that degrades the transcripts, suppressing or enhancing mRNA translation. Microvesicles have been recently shown to encapsulate miRNA [55–57]. In addition, miRNA circulate freely [58] and can be found in serum [59], plasma [60], urine [61], saliva [55], and semen [61]. miRNA expression can be regulated by several mechanisms including chromosomal abnormalities, mutations, single-nucleotide polymorphisms, transcriptional deregulation, defects in the miRNA biogenesis machinery, and epigenetic changes (Fig. 2). Epigenetic mechanisms, i.e., promoter methylation or histone acetylation, can modulate transcription thereby altering miRNA expression [18] at least for a subset of miRNA-encoding genes. Small RNA itself can have an epigenetic effect causing changes in gene expression [62].

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PCOS phenotype, origin, pathophysiology

Candidate genes miRNAs miRNA genes

SNPs

Transcriptional deregulation

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n tio yla eth Am DN s( ) s ge an ion t a ch tic odific inery ne ach ige s m sis m Ep tone ene biog his RNA

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? “Epigenetic” manner

DNA hypomethylation DNA hypermethylation Histone modificaitons

Directly

Target effectors of the epigenetic machinery

Indirectly

Epigenetically controlled gene expression

Irregular gene expression

Environment

Figure 2 Possible roles of genetics, epigenetics, and miRNA in the pathophysiology and origin of PCOS and its subphenotypes. MicroRNA expression can be affected by chromosomal abnormalities, mutations, single-nucleotide polymorphisms, transcriptional deregulation, defects in the microRNA biogenesis machinery, and epigenetic changes [18]. On the other hand, a specific group of miRNAs (defined as epi-miRNA) can directly target effectors of the epigenetic machinery (such as DNA methyltransferases, histone deacetylases, and polycomb repressive complex genes) and indirectly might affect the expression of those genes, whose expression is controlled by epigenetic factors.

PCOS susceptibility genes, i.e., DENND1A [63], which encodes miR601 [64], could result in the overlap of genetic and epigenetic factors, and thus influencing miRNA target specificity [57]. On the other hand, miRNA themselves can regulate the expression of components of the epigenetic machinery thus creating highly controlled feedback. Aberrant expression of these miRNA, i.e., “epi-miRNA,” has often been associated with cancer development and progression in humans. For example, the miR-29 family has been shown to directly target de novo DNA methyltransferases DNMT3A and -3B in lung cancer [62].

3.1 Biomarkers miRNA could serve as noninvasive biomarkers for PCOS because they exist in large quantities and are relatively stable in serum, resist nuclease activity, and are easily identified [65]. Unfortunately, there is a paucity of data on the

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precise mechanisms by which miRNA enter the circulation and whether these miRNA are disease-specific. As can be appreciated, serum is composed of many components released from a variety of tissues and organs, thus making it extremely difficult to identify specific cellular origin. miRNA-21, -27b, -103, and -155 have been associated with several mechanisms specific to PCOS pathogenesis [66,67]. These include inflammation, hormone metabolism, adipogenesis, and insulin signaling [68–72] (Fig. 3). For example, a recent case-controlled BMI-adjusted study that included PCOS patients, healthy females, and males (12 each) demonstrated that four miRNA in whole blood were significantly influenced by obesity. miR-21, -27b, -103, and -155 were reduced in controls (women and men), but demonstrated increased expression in PCOS. Subsequent, hormone analysis revealed that free testosterone was positively correlated to three of these mRNA (miR-21, -27b, and -155). This new finding may shed light on why the expression of these miRNA does not decrease in obese women with PCOS, but do decrease in obese men in whom testosterone concentration may be inherently lower. As such, these miRNA that may play an important role in metabolic processes influenced by obesity and circulating androgen [57,72]. Studies have linked increased miR-222 expression in type 2 [73] and gestational diabetes [74]. In contrast, decreased miR-146 expression was

Insulin signaling

miR-222 miR-93

Inflammation, adipogenesis miR-132 miR-103 miR-27b miR-9 miR-18b miR-21 miR-135a miR-320

Androgens/ hyperandrogenemia

miR-155 miR-146a miR-222

Figure 3 miRNA in PCOS. miRNA differently expressed in PCOS that have been associated with several mechanisms specific to the pathogenesis of PCOS, such as insulin signaling, inflammation, adipogenesis, and androgens/hyperandrogenemia, respectively. Some miRNA (as those eight miRNA illustrated in the central circle) target several genes and serve several different functions, as described so far, thus having a potential influence on both inflammation, adipogenesis, and insulin signaling as well as on androgen hormones.

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associated with inflammation and insulin resistance in type 2 diabetes [75]. Using microarrays, Long et al. [21] analyzed the serum miRNA expression profiles in PCOS. This study found that increased expression for eight miRNA (miR-222, -16, -19a, -106b, -30c, -146a, -24, and -186). miR320 expression was, however, decreased in these patients relative to controls. Q-PCR validation revealed that miR-222, -146a, and -30c were the only ones that remained significantly increased in PCOS. Multiple logistic regression analysis indicated that three miRNA were useful as PCOS biomarkers. When adjusted for age and BMI, miR-222 positively correlated to serum insulin whereas miR-146a negatively correlated to serum testosterone in PCOS [21] (Fig. 3). Examination of circulating miRNA in PCOS patients using microarray and qRT-PCR revealed that five circulating miRNA were upregulated (let7i-3pm, miR-5706, -4463, -3665, and -638) and four were downregulated (miR-124-3p, -128, -29a-3p, and let-7c). Hierarchical clustering suggested that PCOS and healthy controls were highly differentiated. Joint prediction by different statistical methods revealed that 34 targeted genes were upregulated in PCOS whereas 41 genes were downregulated in controls. Analyses by GO and Kyoto encyclopedia of genes and genomes (KEGG) demonstrated that the immune system, ATP binding, MAPK signaling, apoptosis, angiogenesis, response to reactive oxygen species, and p53 signaling were involved in PCOS [76]. Follicular fluid represents an important microenvironment for oocyte maturation and development. During oocyte retrieval, follicular fluid may be easily collected and used as a source for miRNA. Two reports using follicular fluid miRNA profiling in PCOS have been published [58,77]. Using the Rotterdam criteria, PCOS patients undergoing in vitro fertilization (IVF) were selected [77]. This study reported that 29 miRNA were differentially expressed when compared to healthy fertile oocyte donors. Of these, hsa-miR-9, -18b, -32, -34c, and -135a were significantly upregulated in PCOS. miRNA expression of interleukin 8, synaptotagmin I, and insulin receptor substrate 2 was further examined using in silico target site prediction with PCOS phenotypes. An inhibitory effect was supported by the fact that all five miRNA negatively correlated with mRNA expression. In a murine study, upregulated miR-143 inhibited GC proliferation thus hindering the formation of primordial follicles [78]. Interestingly, miR-143 was among the 29 miRNA whose follicular fluid expression was significantly different in PCOS as reported earlier [57,77].

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Another study investigated over 100 different miRNA in follicular fluid from PCOS patients diagnosed according to the Androgen Excess Society criteria [58]. The control group was composed of healthy females undergoing intracytoplasmic sperm injection. The 11 most highly expressed miRNA, i.e., hsa-miR-483-5p, -674-3p, -191, -193b, -320, -520c-3p, -24, -132, -146a, -222, and -1290, were similar to those observed previously [79]. These miRNA were found free in the solution and within microvesicles. Only miR-132 and -320 were significantly downregulated in PCOS. In contrast, another study on PCOS follicular fluid and GCs reported upregulated miR-320 expression [80]. Discrepant results regarding regulation of miR-132 and -320 may be explained by the heterogenic nature of PCOS as well as differences in the studied populations including the criteria used to define PCOS, control differences, and method variation. A recent PCOS genome-wide association study identified a singlenucleotide polymorphism in the intron of high mobility group AT-hook 2 and in the exon near Ras-related protein Rab-5B [81] which appears to be targeted by miR-132 and -32, respectively [58]. Further investigation is clearly warranted to clarify these findings and their relationship to PCOS. The findings of Sang [58] and Roth [77] implicate miRNA expression profiles in a number of pathways involving reproduction, reproductive aging, carbohydrate metabolism, steroid synthesis, cellular growth, beta cell function, insulin signaling, and cell communication. Although miR-186, -21, -155, -103, -19a, and -16 are common to follicular fluid and blood, they are differentially expressed in PCOS. These variations, especially those between studies, clearly suggest that PCOS is complex and heterogeneous. While some (miR-146, -222 and -24) are consistently upregulated, others (miR-320) show more variable levels of expression.

3.2 Pathophysiology Animal models have been useful for studying miRNA profiles in PCOS. A number of miRNA have been identified in human, mouse, and cow ovary [82–89]. Altered expression of these miRNA may influence GCs proliferation and differentiation as well as apoptosis due to hyperproliferation, i.e., PCOS. One study found variable miRNA expression patterns in ovarian tissue from rats chronically exposed to dihydrotestosterone [90]. Microarray analysis revealed that 24% (79/346) of miRNA were differentially expressed, the majority of which were upregulated. Interestingly, miR-222, which is

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present in serum and follicular fluid, was also found in theca cells, but not other ovarian compartments, i.e., GCs of mature follicles [90]. This presence of miR-222 may modulate AR expression and thus paracrine regulation. In support, Xu et al. [91] found decreased miR-222 expression in PCOS cumulus GCs. Others have reported that ovarian follicle miR-221/miR222 expression was repressed by androgens thus regulating cell proliferation via p27/kip targeting [92]. Decreased miR-221/miR-222 in cumulus GCs was most probably due to the high level of androgen in these patients. GO and KEGG pathway analysis was performed for the differentially expressed miRNA to predict targets [91]. Notch3 and MAPK3 proved to be targeted by miR-483-5p based on quantitative real-time PCR, Western blot analysis, and luciferase activity assay. It is important to note that one miRNA could target mRNA expression of different genes. Moreover, a given target may be regulated by multiple miRNA (Fig. 3). However, further studies are required to assess if these miRNA play a role in dysregulation of hormone receptors (androgen, FSH, and E2) and as intraovarian regulators of follicle growth and function in PCOS [57,90]. MiRNA studies performed on GCs are much more extensive than other ovarian cell types. For example, miRNA appear to be intimately involved with steroidogenesis in cultured GCs [93]. This study investigated the production of progesterone, testosterone, and E2 following transfection of cultured primary ovarian GCs with 80 gene constructs encoding human premiRNA. Thirty-six constructs inhibited progesterone release whereas 10 stimulated release. Fifty-seven inhibited testosterone release and only one stimulated release. Fifty-one inhibited E2 release and none were inhibitory. It was also noted that miRNA also influenced cell proliferation and apoptosis. Those involved were subsequently identified by genome-wide miRNA screen. Apoptosis was induced by constructs mimicking endogenous precursor miRNA. Eleven constructs were associated with accumulation of apoptosis marker Bax and its induction [83]. Another 11 were associated increased expression of the proliferation marker PCNA. The finding that miR-383 upregulation was associated with PCOS [83] stimulated research as to their in pathologically increased estrogen in these women. In murine GCs, E2 release was shown to be dose dependent of miRNA-383 via increased CYP19A1 expression [94]. This phenomenon was controlled via inhibition of the transcription factor RBMS1 gene and its downstream target c-Myc, i.e., E2 release. Progesterone production and cell proliferation and apoptosis were not influenced.

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Pathogenesis, abnormal follicular development, and infertility characteristic to PCOS have been associated with altered expression of estrogen receptor expression and signaling [95]. Several miRNA (miR-222, -520c-3p, and 193b) appear involved and worthy of further investigation. Blastocysts from women referred for IVF had downregulated expression for six miRNA (hsa-let-7a, hsa-miR-19a, hsa-miR-19b, hsa-miR-24, hsamiR-92, and hsa-miR-93) [96]. Interestingly, expression of the miR-19a target gene ariadne RBR E3 ubiquitin protein ligase 2 which is important for cell differentiation was decreased in PCOS. The KH-type splicing regulatory protein and nuclear factor of activated T-cells 5 tonicity-responsive genes were upregulated. Although, most miRNA were detected in the human follicular fluid (except for hsa-let-7a), all demonstrated highly variable expression. Last, a positive correlation was also identified with glucose transporter type 4 (GLUT4), the major insulin-dependent glucose transporter and insulin sensitivity, in primary adipocytes from women with PCOS [97]. miR-93 expression was inversely correlated with its suspected target, GLUT4.

4. CONCLUSIONS PCOS remains a problematic pathophysiologic condition with no single criterion sufficient for diagnosis. This issue is exacerbated by its large degree of heterogeneity and is further confounded by environmental and genetic factors including miRNA that likely influence the disease process. Although there appears to be a role for epigenetics in linking genotype and phenotype, a detailed mechanism of action is lacking. Although numerous miRNA have been implicated, miR-222 is perhaps the most consistently associated with PCOS. Despite this, all miRNA should be thoroughly examined to more fully characterize their potential role in PCOS. Future research should focus on elucidating the genetic background including epigenetic mechanisms and aberrant miRNA profiling in PCOS. A more comprehensive and systematic examination of all factors is required in order to clearly understanding this complex and high heterogeneous disease.

ACKNOWLEDGMENTS This work was supported by academic grants—UMF Iuliu—Hatieganu Cluj-Napoca Internal Grant NR. 1494/2/28.01.2014 and Ministry of Education and Research grant PN-II-ID-PCE-2011-3-0879. The authors thank Anca Naiman for providing linguistic technical support with manuscript preparation.

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Conflict of Interest: There is no conflict of interest that would prejudice the impartiality of this scientific work. The authors alone are responsible for the content and writing of the chapter.

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