Polycystic Ovary Syndrome: A Comprehensive Review of Pathogenesis, Management, and Drug Repurposing

1 Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran 11369, Iran; moc.liamg@9731smansoh (H.M.S.); moc.oohay@adya_ileda (I.A.); ri.ca.smut.tneduts@ivasuoM-ts (T.M.); moc.liamg@57ilainadheizram (M.D.)

2 Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 11369, Iran

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Ida Adeli

2 Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 11369, Iran

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Daniela Calina

3 Department of Clinical Pharmacy, University of Medicine and Pharmacy of Craiova, 200349 Craiova, Romania

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Anca Oana Docea

4 Department of Toxicology, Faculty of Pharmacy, University of Medicine and Pharmacy, Petru Rares, 200349 Craiova, Romania; moc.liamg@00anaoad

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Taraneh Mousavi

2 Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 11369, Iran

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Marzieh Daniali

2 Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 11369, Iran

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Shekoufeh Nikfar

5 Department of Pharmacoeconomics and Pharmaceutical Administration, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 11369, Iran; moc.liamg@rafkin.hefuokehs

6 Personalized Medicine Research Center, Endocrinology and Metabolism Research Institute, Tehran University of Medical Sciences, Tehran 11369, Iran

7 Evidence-Based Evaluation of Cost-Effectiveness and Clinical Outcomes Group, Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran 11369, Iran

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Aristidis Tsatsakis

8 Department of Analytical and Forensic Medical Toxicology, Sechenov University, 119991 Moscow, Russia; moc.liamg@sikastastsira

9 Department of Forensic Sciences and Toxicology, Faculty of Medicine, University of Crete, 71003 Heraklion, Greece

10 Laboratory of Toxicology, Medical School, University of Crete, 70013 Heraklion, Greece

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Mohammad Abdollahi

2 Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 11369, Iran

Find articles by Mohammad Abdollahi Raheela N. Khan, Academic Editor and Saad Amer, Academic Editor

2 Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 11369, Iran

3 Department of Clinical Pharmacy, University of Medicine and Pharmacy of Craiova, 200349 Craiova, Romania

4 Department of Toxicology, Faculty of Pharmacy, University of Medicine and Pharmacy, Petru Rares, 200349 Craiova, Romania; moc.liamg@00anaoad

5 Department of Pharmacoeconomics and Pharmaceutical Administration, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 11369, Iran; moc.liamg@rafkin.hefuokehs

6 Personalized Medicine Research Center, Endocrinology and Metabolism Research Institute, Tehran University of Medical Sciences, Tehran 11369, Iran

8 Department of Analytical and Forensic Medical Toxicology, Sechenov University, 119991 Moscow, Russia; moc.liamg@sikastastsira

9 Department of Forensic Sciences and Toxicology, Faculty of Medicine, University of Crete, 71003 Heraklion, Greece

10 Laboratory of Toxicology, Medical School, University of Crete, 70013 Heraklion, Greece * Correspondence: moc.liamg@aleinadanilac (D.C.); rI.cA.SMUT@dammahoM (M.A.) † These authors contributed equally to this work. Received 2021 Nov 12; Accepted 2021 Dec 31. Copyright © 2022 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Abstract

Polycystic ovary syndrome (PCOS) is an endocrine-gynecology disorder affecting many women of childbearing age. Although a part of the involved mechanism in PCOS occurrence is discovered, the exact etiology and pathophysiology are not comprehensively understood yet. We searched PubMed for PCOS pathogenesis and management in this article and ClinicalTrials.gov for information on repurposed medications. All responsible factors behind PCOS were thoroughly evaluated. Furthermore, the complete information on PCOS commonly prescribed and repurposed medications is summarized through tables. Epigenetics, environmental toxicants, stress, diet as external factors, insulin resistance, hyperandrogenism, inflammation, oxidative stress, and obesity as internal factors were investigated. Lifestyle modifications and complementary and alternative medicines are preferred first-line therapy in many cases. Medications, including 3-hydroxy-3-methyl-3-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors, thiazolidinediones, sodium-glucose cotransporter-2 inhibitors, dipeptidyl peptidase-4 inhibitors, glucose-like peptide-1 receptor agonists, mucolytic agents, and some supplements have supporting data for being repurposed in PCOS. Since there are few completed clinical trials with a low population and mostly without results on PCOS repurposed medications, it would be helpful to do further research and run well-designed clinical trials on this subject. Moreover, understanding more about PCOS would be beneficial to find new medications implying the effect via the novel discovered routes.

Keywords: polycystic ovary syndrome, hyperandrogenism, insulin resistance, molecular mechanisms, management, repurposing drugs

1. Introduction

Polycystic ovary syndrome (PCOS) is a heterogeneous endocrine disorder that impacts many women of the reproductive age worldwide [1]. This syndrome is often associated with enlarged and dysfunctional ovaries, excess androgen levels, resistance to insulin, etc. [2]. It is estimated that approximately every 1 in 10 women face PCOS before menopause and struggle with its complications [3].

Although the high ratio of luteinizing hormone (LH) to follicle-stimulating hormone (FSH) and increased frequency of gonadotropin-releasing hormone (GnRH) is known as the underlying causes of PCOS [4], the exact etiology and pathology have not been comprehensively well-known [4,5]. Evidence suggests the role of different external and internal factors, including insulin resistance (IR), hyperandrogenism (HA), environmental factors, genetic, and epigenetics. In addition, it is worth mentioning that PCOS increases the risk of further complications like cardiovascular diseases [5,6], type 2 diabetes mellitus [5,6], metabolic syndrome [6], depression, and anxiety [7].

To manage this condition, the most crucial step is to lose at least 5% of the weight; therefore, having a regular exercise plan and fat and sugar-free diets are also recommended to every woman with PCOS. Furthermore, in some cases, taking complementary and alternative medicine strategies with or without other treatments is preferable due to their prior beliefs, lower costs, etc.

Physicians tend to use (combined) oral contraceptives, antiandrogen agents, insulin sensitizers, and ovulation inducers [4]. Up until today, there is no United States Food and Drug Administration (USFDA) approved medication specifically for PCOS, and all mentioned medications are used off-label [8]. Apart from the essential need for improvement in the research and development of new drug molecules and new drug discovery, novel medications could be found with drug repurposing methods [9]. On this very spot, there are plenty of medications, previously approved by USFDA for indications rather than PCOS; and, today, there is a desire to implement them as the therapeutic options in the management of PCOS.

These agents vary from anti-diabetic medications such as pioglitazone, empagliflozin, sitagliptin, liraglutide to 3-hydroxy-3-methyl-3-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors like simvastatin and atorvastatin, as well as mucolytic drugs like N-acetyl cysteine.

Given that PCOS is a growing issue that is unfortunately followed by many unwanted complications and that available methods and medications are not 100% effective, it is essential to investigate its pathogenesis and find out new pharmacological targets carefully. This could be done through repositioning approaches, saving time and cost.

This review discusses PCOS’s definition, diagnosis, and etiology, focusing on the pathogenesis and management of this syndrome. Internal and external factors contributing to PCOS have been comprehensively studied, and several commonly prescribed medications with their complete drug information are provided. Subsequently, a couple of repurposed medications are mentioned thoroughly, reviewing the related clinical trials over the past five years.

2. Methods

PubMed, Google Scholar, ScienceDirect, TRIP database, and UpToDate were comprehensively searched for publications including PCOS relevant keywords in different areas, focusing on the new ones (since 2016) and excluding those with a language rather than English or animal studies. In addition, Clinicaltrials.gov was searched to find data about completed or running trials of repurposed drugs in PCOS over the past five years.

3. Diagnosis

PCOS is among the conditions that cannot be diagnosed with basic diagnostic tests, including blood tests, culture, and biopsy; thus, there is no certain test for PCOS diagnosis. Differential diagnosis is called excluding the relevant disorders according to the symptoms and narrowing the choices. In order to establish a differential diagnosis for PCOS, hyperprolactinemia, thyroid disease, Cushing’s syndrome, and hyperplasia of adrenal should be excluded based on the associated investigations [10,11]. Although considering past medical history, weight changes, and symptoms of insulin resistance might be helpful, pelvic examination, a transvaginal ultrasound, and measuring the level of hormones are among the most frequently recommended investigations [12]. According to the National Health Service (NHS), irregular or infrequent periods, high levels of androgenic hormones or symptoms, and scans showing polycystic ovaries are the specified criteria for PCOS [13]. In addition, Rotterdam PCOS diagnostic criteria in adults are the most commonly used method. In an ultrasound, the presence of two clinical or biochemical hyperandrogenism, ovulatory dysfunction, or polycystic ovaries would finalize a PCOS diagnosis [14].

4. Etiology and Risk Factors

4.1. External Factors

4.1.1. Epigenetic Mechanism

Epigenetic refers to inheritable alterations in genome and gene expression without any changes in DNA sequence [15,16]. These changes involve adding or omitting chemical components on DNA or histone [17]. Increased LH activity is a seen phenomenon in PCOS women. It may relate to the problems in follicle development and HA, which are common among PCOS patients [18]. LH/choriogonadotropin receptor (LHCGR) is responsible for the steroidogenesis process in theca cells [19]. This receptor hypomethylation leads to higher gene expression and sensitivity to LH [18,20].

A study on PCOS patients approved that hypomethylated sites are related to overexpression of LHCGR [15,19] on theca cells surface [19]. In addition, epoxide hydrolase 1 (EPHX1) is an active enzyme in degrading aromatic compounds [15,19,21]. Its gene promoter hypomethylation [15,19] increases enzyme expression [15]. Overproduction of EPHX1 reduces the transformation of testosterone to estradiol, which can contribute to PCOS [15]. Furthermore, peroxisome proliferator-activated receptor gamma (PPAR-γ) plays a role in ovaries’ function [15,18,19,22]. Hypermethylation of PPARγ, hypomethylation of nuclear co-repressor 1 [19,22], and alteration in acetylation of histone deacetylase 3, for which both are PPARγ co-repressors [15], are observed in PCOS patients showing HA [15,19,22]. These alterations were noticed in PCOS women’s granulosa cells [18,23].

4.1.2. Environmental Toxicants

The United States Environmental Protection Agency (USEPA) defines endocrine-disrupting chemical (EDC) as “an exogenous agent that interferes with the synthesis, secretion, transport, binding, action, or elimination of natural hormones in the body that are responsible for the maintenance of homeostasis, reproduction, development and/or behavior” [24].

EDCs may act as hormones’ agonists or antagonists in binding to their receptors [25]. EDCs are almost parts of everything we use in our daily life [21]. Their structures consist of phenols or halogens like chlorine and bromine, so they imitate steroid hormones’ actions [21]. Studies have approved the higher serum concentration of EDCs in PCOS suffering women [21,26]. Prolonged and continuous exposure to EDCs from prenatal to adolescence can cause susceptibility to PCOS [21,27].

As an example, bisphenol A (BPA) BPA is a synthetic compound used in polycarbonate plastics, epoxy resins [25,28], dental filling, food and drink packages [25], baby bottles, and polyvinyl chloride (PVC) [28], which affects metabolism through different pathways. BPA directly affects oogenesis [29] by interacting with estrogen receptor (ER) α and β, non-classical membrane ER, and G-protein coupled receptor 30 (GPCR30) [21,28,29]. It also triggers androgen secretion and restrains testosterone catabolism in theca cells [21,29].

Another effect of BPA on interstitial theca cells is the overproduction of androgens by dysregulation of 17β-hydroxylase (P450c17) [28,30], cholesterol side-chain cleavage enzyme (P450scc), and steroidogenic acute regulatory protein [30]. BPA’s influence on granulosa cells refers to reducing the expression of aromatase enzyme and production of estrogen [21,29]. Lastly, it disturbs the intrafollicular environment and damages the oocyte development and maturation [21,29]. BPA’s indirect effect on HA involves downregulation of testosterone 2a-hydroxylase and testosterone 6b-hydroxylase enzymes in liver level, and thus a higher concentration of testosterone [30,31].

In addition, BPA is a potent ligand for sex hormone-binding globulin (SHBG) and replaces testosterone; thereby, free testosterone concentration increases. Androgen and BPA have a two-way relation; high androgen inactivates the uridine diphosphate-glucuronosyl transferase enzyme and reduces BPA clearance in the liver. This process causes a high concentration of free BPA in blood and worsens its negative effects on the ovaries [21,29,30,31].

Additionally, it is believed that BPA may act as an obesogen [28,30]. Its obesogenic influence includes upregulation of adipogenesis-related genes [30], stimulation of adipocytes differentiation [28,30], potentiation of the accumulation of lipid in cells incorporated in medical syndrome, and triggering the conversion of target cells to adipocytes via phosphatidylinositol 3-kinase pathway [30].

Adipogenesis due to BPA happens because of the activation of the glucocorticoid receptor. Activation of the receptor upregulates the enzyme involved in the conversion of cortisone to cortisol, thus inducing adipogenesis [28]. Moreover, BPA prompts the release of interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) [30,31], both involving adiposity and IR [30]. In addition, it restrains the release of adiponectin [28,29,30,31] and the beneficial compound in protecting against IR [28,30,31].

It can also change glucose homeostasis [28,29,31] by directly influencing the pancreatic cells [29]. BPA causes a chronic increase in insulin and further IR in long exposure [30] by affecting the mitochondrial activity and metabolic pathways of β-pancreatic cells [28]. BPA reduces glucagon secretion by inhibiting the intracellular calcium ion fluctuating pattern with a lack of glucose condition [30].

Advanced glycation end products (AGEs), also called glycotoxins, are another chemical group affecting body health. AGEs are pro-inflammatory molecules [21,23,29,32] that interact with their surface receptor called RAGE (receptor for AGE) [21,23,29] and stimulate pro-inflammatory pathways and oxidative stress [21,23,29,32]. AGEs can be absorbed into the body as exogenous compounds or derived from nonenzymatic glycation and oxidation of proteins and lipids [21]. Increased concentration of AGEs in serum has been detected in PCOS patients [21]. AGEs interrupt pre-ovulatory follicles growth via ERK1/MAPK pathway and damage follicles by oxidative stress caused by interaction with RAGEs [21]. This interaction increases intracellular inflammatory molecules [21].

In vitro studies on 3T3-L1 cell lines showed that glycotoxins are likely to trigger adipogenesis [21]. On the other hand, a higher body mass index corresponds to a lower extent of soluble RAGEs, which is responsible for glycotoxin clearance and deposition of AGEs in the reproductive system, especially in ovaries [21,29]. This bilateral relation worsens inflammatory processes and metabolic syndrome in PCOS [21]. AGEs also play a role in IR [21,29]. These compounds disrupted glucose transport in the human granulosa KGN cell line [21] and reduced glucose uptake by adipocytes in previous research [21,29]. They also involve IR by causing oxidative stress, inflammation, and glycation of proteins, which considerably diminishes insulin sensitivity [21]. Moreover, increased concentration of AGEs changes the insulin signaling pathway and interferes with glucose transporter 4 (GLUT-4) translocation [23].

4.1.3. Physical and Emotional Stress

Although there is minimal information on the role of stress in PCOS, it is known that PCOS possesses adverse effects on self-esteem and mental health. Chronic stress results in hypertrophy and hyperplasia of adipocytes. This phenomenon happens as a result of glucocorticoids’ effect on pre-adipocytes maturation. Chronic stress is also associated with adipokine secretion, attraction, and activation of stromal fat immune cells [33].

In addition, it is responsible for making an inflammatory condition by leading to high levels of inflammatory cytokines like IL-6 and TNF-α, along with disrupting oxidant-antioxidant balance [33]. In addition, chronic stress plays a vital role in IR.

Stress triggers the hypothalamic-pituitary-adrenal (HPA) axis to release cortisol [34,35]. Cortisol leads to IR by stimulating visceral fat accumulation, gluconeogenesis, and lipolysis [35]. Moreover, cortisol arouses glucose production in the liver [35]. Stress is also involved in enhancing insulin levels [34]. Other stress influences on PCOS may refer to inference with anti-mullerian hormone (AMH) and changing sex hormone levels [34,35].

4.1.4. Diet

Although nutrition contributions to PCOS is unclear, studies showed a relationship between some nutrient levels and PCOS indices.

Saturated fatty acids (SFAs) intake plays a role in PCOS by producing an inflammatory status [36] and reducing insulin sensitivity [37]. Taking SFAs induces inflammation by triggering an increase in TNF-α level in circulation and expressing a specific cytokine suppressor [36].

Vitamin D deficiency may exacerbate PCOS [37,38] or the comorbidities induced by PCOS [38]. Calcitriol upregulates insulin receptors at mRNA and protein levels. It also increases insulin sensitivity directly and indirectly. The direct effect occurs by activating PPAR-δ, the involved receptor in fatty acids metabolism in adipose tissue and skeletal muscle. The indirect impact is the regulation of intracellular calcium, which is vital for insulin-mediated signaling in fat and muscle [38]. On the other hand, vitamin D deficiency may result in insulin resistance by causing an inflammatory response [37,39]. Furthermore, vitamin D downregulates the AMH promoter [39].

4.2. Internal Factors

4.2.1. Insulin Resistance

IR means an insufficient cells response to insulin [40]. IR is independent of patients’ adiposity, body fat topography, and androgen levels [18,41]; i.e., it has been reported in lean patients as well [18,42]. It should be mentioned that IR is tissue-selective in PCOS women [18,43], although skeletal muscles [18,43,44], adipose tissue, and liver lose their sensitivity to insulin, adrenal glands [18,43], and ovaries remain sensitive [18,28,43,45].

Insulin directly triggers androgens production in ovarian theca cells [32,44,46,47,48] and grow [48]. Insulin effectively stimulates ovarian follicle growth and hormone secretion by stimulating its receptors in the follicle membrane cells [49]. It also triggers ovarian P450c17 [18,23,50] and P450scc enzyme activity to promote ovarian steroidogenesis [18,51] and increases them with the synergistic effect of chorionic gonadotropin [52]. This hormone, as well as insulin-like growth factor 1 (IGF-1) [18], synergizes with luteinizing hormone [18,45]. Hyperinsulinemia increases LH-binding sites and androgen-producing response to LH [44]. LH and insulin interaction enhance steroidogenic acute regulatory enzyme and CYP450c17 mRNA expression [52,53]. CYP450c17 is involved in androgen production [23,44]. Likewise, IR independently enhances CYP17A1 activity, the productive enzyme in androstenedione and testosterone production [52].

On the other hand, hyperinsulinemia reduces hepatic SHBG [18,32,40,49,52,54,55,56], increasing free testosterone levels in blood [18,32,52,54,56]. In addition, hyperinsulinemia inhibits IGF-1 binding protein production in the liver. IGF-1 is responsible for triggering the production of androgens in thecal cells. Inhibition of the production of IGF-1 binding proteins leads to a higher concentration of this substance in blood circulation and then higher production of androgens in thecal cells [18,46]. Moreover, IGF-1 upregulation decreases a specific miRNA and thus accelerates granulosa cells apoptosis and inhibits folliculogenesis [52]. HA [46] and hyperinsulinemia [45,46,57] both play a role in stopping follicles growth [45,46]. This stoppage is attributed to menstrual irregularity, anovulatory sub-fertility, and amassing of immature follicles [46].

Furthermore, hyperinsulinemia contributes to PCOS by affecting the pituitary gland. Excessive insulin stimulates its receptors in the pituitary gland to release LH [49]. Accumulation of insulin stimulates GnRH and LH pulse secretion via influencing both amplitude and frequency [23]. Insulin’s indirect effect on PCOS is augmented by pituitary gonadotropin sensitivity to GnRH [18], and hyperinsulinemia increases GnRH neuron activity [58].

The insulin’s influence on adipose tissue and inflammation is another essential PCOS pathogenesis topic. Insulin stimulates adipogenesis and lipogenesis and inhibits lipolysis [42], resulting in fat accumulation [44]. IR leads to enhanced plasma levels of free fatty acids (FFAs), affecting the liver and adipose tissue [32]. Moreover, IR causes a reduction in omentin level independent of the patient’s body mass index (BMI). In addition, hyperglycemia can lead to inflammation by producing TNF-α from mononuclear cells (MNCs) [50].

4.2.2. Hyperandrogenism

Generally, hyperandrogenism (HA) reduces the SHBG level, leading to a higher concentration of free testosterone [18,59]. It was observed that PCOS women have higher concentrations of testosterone in plasma which can convert to estrone in adipose tissue. Increased alteration of estrone to estradiol affect follicle growth and increases the LH to FSH ratio causing ovulatory dysfunction [23].

HA can result in AMH upregulation, which inhibits ovulation and the development of follicles by a different mechanism. Furthermore, the IGF-II level is negatively related to androgen levels, and HA reduces IGF-II in follicular fluid. IGF-II positively relates to follicle diameters and estradiol concentration in follicular fluid [23]. In addition, HA increases LH indirectly [58,60]. Estradiol and progesterone are responsible for GnRH and LH secretion via negative feedback [58,61,62]. HA disrupts the negative feedback on secretion [18,23,61,62] resulting in increased LH levels [18,62]. Interaction of androgen and its receptor interferes with progesterone receptor transcription. Moreover, this receptor is involved in converting high levels of androgens to compounds that modulate the gamma-aminobutyric acid A (GABA_A). Modulation of the GABA_A receptor triggers GnRH neurons and weakens the response to negative progesterone feedback [58]. In addition, it is assumed that androgens might decrease hepatic nuclear factor-4α (HNF-4α) levels by inhibiting lipid synthesis. HNF-4α stimulates SHBG expression by binding to its promoter [63].

HA contributes to other influential factors of PCOS, including IR, inflammation, and oxidative stress.

HA aggravates IR via different routes; it reduces the insulin sensitivity, expression of GLUT-4 and inhibits insulin degradation in the liver [23,32]. Moreover, HA increases a type of skeletal muscle fibers that have low insulin sensitivity [32]. On the other hand, HA worsens central adiposity, which is involved in IR [23,32]. Additionally, it was observed that testosterone increases inflammatory chemicals such as lipopolysaccharide-induced IL-6 in 3T3-L1 adipocytes by activating some signaling pathways [64]. One way androgen results in oxidative stress is by increasing MNC sensitivity to glucose and aggravating glucose-stimulated oxidative stress [65]. It is worth mentioning that dehydroepiandrosterone as an androgen decreases interferon-γ (IFN-γ), an essential regulator in normal ovarian physiology and cell function [64].

In addition, it should be mentioned that studies on PCOS women approved the resemblance of their fatty tissue to men, and hence the effect of HA on adipose tissue dysfunction [8]. In addition, HA is a cause of adipocyte hypertrophy and consequential damages to adipokine secretion [55].

4.2.3. Inflammation

Appropriate inflammation is a vital cause of oocyte growth and ovulation [66]. However, high levels of white blood cell [46,66], C-reactive protein (CRP) [4,46,50,66,67], and other inflammatory biomarkers in peripheral blood are associated with PCOS [4,46,66,67,68]. Inflammation is a cause of HA [44,69]. TNF-α is a pro-inflammatory chemical that can worsen IR. Contribution to IR happens due to interference of pro-inflammatory molecules with insulin signaling pathways [32,67] and reduction of GLUT-4 expression [23]. Some studies showed that the insulin receptor substrate (IRS) serine residue phosphorylation inhibits insulin receptor signaling [32,70]. This phenomenon results in the prevention of GLUT-4 translocation and glucose reuptake [70]. In addition, TNF-α showed the ability to prompt theca cells proliferation in vitro [71]. Furthermore, IL-1 hinders the FSH and LH receptors. Inhibition of these receptors leads to inhibition of follicular development and ovulation [66]. Both TNF-α and IL-1β inhibit activation of HNF-4α by different mechanisms [23]. In addition, NLRP3 inflammasomes induce follicular pyroptosis, ovarian fibrosis, and disturbance of follicular formation [66]. An increase in CRP level is another cause of IR in insulin-sensitive tissues. IR occurs because of increased pro-inflammatory factors secreted by the liver and monocytes. CRP stimulates this increase in secretion [72]. Moreover, another study approved the higher-than-normal level of IL-6 mRNA in granulosa cells [66].

4.2.4. Oxidative Stress

Oxidative stress (OS) is an imbalance between pro-oxidants and antioxidants [71,72,73]. Oxidative molecules include different chemicals such as reactive oxygen species (ROS) [73,74,75] (e.g., O 2− , H₂O₂, and OH − ) [76] and reactive nitrogen species (RNS) [74,75]. ROS plays a role in different mechanisms like signaling pathways [71,73,76], cell growth [71,73], and differentiation, as well as RNS [73]. RONS also acts on ovaries functions like steroidogenesis [67,77] and affects neurons responsible for feeding behavior to induce hunger [71]. Overproductions of oxidative chemicals cause various damage to vital molecules such as lipids, proteins, and DNA [73,74,75,77].

Increased OS has been seen in PCOS patients in different studies [74,78,79]. Increased levels of OS activate the nuclear factor-kappa B (NF-κB) [72,75]. NF-κB is involved in inflammatory pathways [75] and affects the production of pro-inflammatory cytokines like TNF-α and IL-6 [72,80]; the effect in IR and PCOS was explained above. A high level of OS also increases the release of TNF-α [77]. On the other hand, increased OS actuates some protein kinases that trigger serine/threonine phosphorylation instead of normal tyrosine phosphorylation of IRS. Thus, the insulin signaling pathway is inhibited, and OS leads to IR [67]. OS also plays a role in obesity. It increases mature adipocyte size and consequently stimulates pre-adipocyte proliferation and adipocyte differentiation. OS also imposes a major effect on obesity [71].

4.2.5. Obesity

Obesity is a key in low-grade chronic inflammation [72]. Accumulation of adipocytes in visceral fat leads to hypoxia and consequent necrosis, which causes inflammatory cytokines production [66]. Adipocyte death due to hypertrophy causes an inflammatory state [44,69]. The mononuclear cells of adipose tissue produce pro-inflammatory cytokines [6,44,81]. Excess abdominal fat is also responsible for the inflammatory condition [6,44,81].

Obesity also plays a role in hyperinsulinemia, IR, and HA occurrence. Visceral obesity arouses an increase in non-esterified fatty acids (NEFAs) levels in the blood. Skeletal muscles uptake NEFAs as the energy source instead of glucose. This hyperglycemia leads to a pancreas rapid reaction and hyperinsulinemia [55]. In addition, the lipolytic response of visceral fat to catecholamines causes lipotoxicity [44] and impairment of insulin clearance and activity [81].

FFA stimulates IRS-1 serine/threonine phosphorylation and reduces tyrosine phosphorylation. Increased FFAs reduce insulin and glucose uptake sensitivity in intramyocellular lipids [52]. Notably, that visceral fat is weightier in IR than abdominal [44] and subcutaneous fat [81] as the visceral fat lipolytic response to catecholamines is more severe [44,81]. The reason is the increased function of the β3 and higher expression of β1 and β2 receptors [81]. Moreover, the type 1 isoenzyme of 11β-hydroxysteroid dehydrogenase (11β-HSD) is involved in converting cortisone to active cortisol, which is highly expressed in adipose tissue, especially in adipose tissue visceral ones. Glucocorticoids reduce glucose uptake and insulin signaling in omental adipocytes [81]. In addition, visceral fat’s adiponectin secretion is less than subcutaneous fats, and this phenomenon leads to decreased adiponectin secretion in obesity [46].

In addition to all adipose tissue’s functions mentioned above, this tissue has endocrine function and secretes chemicals called adipokines or adipocytokines. Adipocytes produce leptin, a high concentration of which inhibits the expression of aromatase mRNA in granulosa cells—thus interrupting androgens to estrogen conversion [52]. In addition, it is suggested that increased leptin levels are related to the absence of folliculogenesis [81]. Moreover, adiponectin, secreted by adipocytes [52], has insulin-sensitizing, anti-diabetic, and anti-inflammatory effects [46]. The adiponectin insulin-sensitizing effect causes a reduction in FFA uptake and gluconeogenesis. It also plays a role in progesterone and estrogen production, ovulation, and decreased GnRH secretion [52]. Furthermore, adiponectin reduces LH secretion from the pituitary, triggers estradiol secretion in granulosa, and is associated with androgen production in ovaries [81]. Omentin-1, another adipose tissue secreted chemical, improves IGF-1-induced progesterone and estradiol secretion in different ways, including increasing the steroidogenic acute regulatory protein and CYP450 aromatase expression and enhancing IGF-1 receptor signaling [82].

Adipose tissue also has several enzymes responsible for converting androstenedione to testosterone and testosterone to dihydrotestosterone [45]. 17β-HSD converts androstenedione to testosterone [44,81] and estrone to estradiol [81]. This enzyme is expressed in adipose tissue [44,81]. As a result of this process, excess adiposity exacerbates HA [45].

Furthermore, the accumulation of lipid in non-adipose tissues, called lipotoxicity, causes oxidative/endoplasmic reticulum stress linked with inflammation and IR. Excess fatty acids in muscles and liver induce IR via serine phosphorylation of insulin receptor by diacylglycerol [83]. In addition, lipid accumulation in the liver diminishes HNF-4α levels leading to reduced SHBG production [63].

A summary of the most representative molecular mechanisms of PCOS pathogenesis is presented in Figure 1 .