Polycystic Ovary Syndrome Causes

### **Chapter 3**

## Causes of Polycystic Ovarian Syndrome

*Subrat Panda, Rituparna Das, Lisley Konar and Manasi Singh*

### **Abstract**

PCOS is a multifactorial syndromic disorder—the exact etiology is not known. Genetic, epigenetic, and environmental factors may be the causative factors. It is the most common cause of an-ovulatory infertility, and in adolescents, the young girl may present with irregular periods. Most of the women with PCOS are either overweight or obese. Another variety of PCOS women is lean. Sleep apnea, metabolic syndrome, and endometrial carcinoma are the late consequences of women with PCOS. As new research shows that gut microbiome is one of the attributing factors of PCOS, it will lead to a new horizon in the management of PCOS. Fecal implantation or probiotics may be helpful in PCOS management. Physical and emotional stress is one of the contributing factors to PCOS. Neuroendocrine factors are also an attributive factor for the development of PCOS. Most of the research about neuroendocrine factors is very preliminary and limited to the mice model. The incidence of PCOS varies from region to region as dietary and environmental factors differ. More human research is required to have more knowledge about the etiology of PCOS, which will guide the management of PCOS.

**Keywords:** PCOS, FSH, LH, EDC, SFA, ROS, Genomic variants, hyperandrogenism, NEFAs, SHBG, gut microbiome

### **1. Introduction**

PCOS is a reproductive dysfunction resulting from an interaction between endocrine and metabolic disorders. Hyperandrogenism and insulin resistance complement one another in the development of PCOS. Primarily, it is affected by alteration of the hypothalamus-pituitary-ovarian axis. The cyclic pattern of hormone concentrations, which is the characteristic determinant of normal ovulatory menstrual cycles, is lost in women with PCOS. In these women, there is chronic anovulation resulting in a "steady low state" of gonadotropin and sex steroid concentrations. Daily production of both androgens and oestrogens is increased. Chronic hyperandrogenemia and insulin resistance (usually, though not always, associated with obesity) also result in ovarian dysfunction of PCOS. PCOS women suffer from oligomenorrhoea, infertility, metabolic syndrome, acne and oily skin, hirsutism, and endometrial carcinoma at a later age. The pathophysiology of PCOS is most likely multifactorial, involving endocrine, metabolic, genetic, epigenetic, and environmental factors. Increased

serum luteinising hormone (LH) concentrations, low-normal follicle-stimulating hormone (FSH) levels and increased LH-to-FSH ratios is a feature of women with PCOS. Insulin resistance is usually seen in obese PCOS women and, to a lesser extent, in women with lean PCOS. In general, the prevalence varies from 50 and 75% [1]. About 35% of women with polycystic ovary syndrome exhibit impaired glucose tolerance, and up to 10% meet the criteria for type 2 diabetes mellitus. Insulin and LH act synergistically, resulting hyperandrogenism. Insulin resistance and hyperinsulinemia are the cause, not the result, of hyperandrogenism in PCOS. Women with PCOS have increased visceral fat than subcutaneous fat. Women with lean PCOS have an increased percentage of body fat, a higher waist–hip ratio, and greater intra-abdominal, peritoneal, and visceral fat compared to normal women matched for body mass index (BMI) [2]. Women with PCOS suffer from different psychological disorder. The diagnosis of PCOS is based on the 2003 Rotterdam criteria, where the incidence of two out of the three criteria hyperandrogenism (clinical and/or biochemical), irregular cycles, and polycystic ovary morphology. Evidence-based medicine suggests that the anti-Mullerian hormone in serum can be used as a substitute for the follicular count, and it is thereby emerging as an official polycystic ovarian morphology/PCOS marker. The limitation of this criteria is mainly in the diagnosis of adolescents as the features of PCOS overlap with the normal physiological change in puberty. Therefore, PCOM should not be considered a criterion in the first 8 years of menarche, and the essential criteria are irregular menstrual cycles and clinical and/or biochemical hyperandrogenism.

### **2. Genetic association**

Familial aggregation studies identify PCOS as an inherited disorder. Higher frequency of genetic polymorphism at 2p16.3 (rs13405728), 2p21 (rs13429458), and 9q33.3 (rs2479106) are noted in women with PCOS. In one group of patients, X-linked inheritance was seen. Siblings and parents of patients suffering from PCOS have a high prevalence of hyperinsulinemia and hypertriglyceridemia, PCOS in females, and premature balding in males. Monozygotic twin sisters have twice the risk of developing PCOS. About 50% of sisters of patients with PCOS have increased levels of total or bioavailable testosterone concentrations, and 35% of their mothers also suffer from PCOS [3]. First-degree relatives of women with PCOS have features of metabolic syndrome. PCOS is described as a polygenic disorder that includes the interplay of various genomic variants (**Table 1**).

In pregnancy in PCOS mothers, there is placental dysfunction due to hyperinsulinemia leading to hyperandrogenic foetal micro-environment. The theory of hyperinsulinemia suggests that there are two factors involved: maternal insulin resistance and decreased placental aromatase, which work together to maintain balance in the system. Epigenetic reprogramming can occur with or without alterations or deletions to the existing DNA, potentially affecting chromatin. Methylation of DNA inhibits gene expression, while hydroxymethylation enhances gene expression. The first type of reprogramming involves cytosine at its fifth carbon in the pyrimidine ring, known as CpG islands. This can include methylation, hydroxymethylation, formylation, or carboxylation. The second method is histone modification, which can involve acetylation, methylation, phosphorylation, ubiquitination, or sumoylation. This reprogramming, which occurs in germ cells, has the ability to pass these changes on to *Causes of Polycystic Ovarian Syndrome DOI: http://dx.doi.org/10.5772/intechopen.113877*


### **Table 1.**

*Genomic varients related to pcos.*

offspring. Next-generation sequencing (NGS) is used to identify this genetic reprogramming. The various physiological processes related to follicular development, steroidogenesis, glucose metabolism, insulin regulation, and inflammatory mediation are associated with luteinising hormone/choriogonadotropin receptor (LHCGR), FST, LMNA, PPARGC1, and EPHX1 genes. In PCOS, there is decreased methylation and over-expression of LHCGR and PPAR-gamma genes. Obesity also plays an epigenetic modifier in the development of PCOS. These two epigenetic modifications complement each other, resulting in changes in DNA. Two hit theories include the first hit in intrauterine life, and the second one is a provocative factor after delivery in her lifetime. A genome-wide association study identifies a gene that regulates gonadotropin secretion, action, and ovarian function. These genes are FSHB (follicle-stimulating hormone B polypeptide), LHCGR (luteinising hormone/choriogonadotropin receptor), FSHR (follicle-stimulating hormone receptor), anti-Mullerian hormone (AMH), and DENND1A (DENN domain-containing 1A) and genes associated with metabolism, such as THADA (thyroid-adenoma-associated gene) and INSR (insulin receptor).

### **3. Environmental factors**

Endocrine-disrupting chemicals (EDC) are present in whatever we use in our dayto-day life. These are phenols or halogens like chlorine and bromine [4]. They mimic steroid hormone action. Longer and uninterrupted exposure to EDCs from prenatal to adolescence is a causative factor of PCOS [5]. Bisphenol A (BPA), which is used for packaging food and drink and many other purposes, interferes with oogenesis [4, 6]. BPA acts on granulosa cells and reduces the expression of aromatase enzyme and the production of oestrogen, thereby interfering with oocyte development and maturation. BPA is also a potent ligand for sex hormone-binding globulin (SHBG) and replaces testosterone thereby increasing free testosterone concentration. High androgen deactivates the uridine diphosphate-glucuronosyl transferase enzyme and

decreases BPA clearance in the liver. Thereby, it increases the concentration of free BPA in blood, leading to further negative effects on the ovaries [4, 6–8]. BPA upregulates adipogenesis-related genes and activates glucocorticoid receptors, thus acting as an obesogenic. BPA also gives rise to the release of interleukin 6 (IL6) and tumour necrosis factor α (TNF-α), which prompts adiposity and insulin resistance. BPA also affects glucose homeostasis by directly influencing pancreatic cells. Advanced glycation end products (AGEs), also called glycotoxins, are pro-inflammatory molecules affecting women's health. They interact with the surface receptor called RAGE (receptor for AGE) and stimulate proinflammatory pathways and oxidative stress [9, 10]. It prompts adiposeness. Increased body mass index decreases the clearance of glycotoxin and is responsible for its deposition in ovaries. This worsens the inflammatory process and metabolic syndrome of PCOS [9].

### **4. Physical and emotional stress**

Chronic stress causes hypertrophy and hyperplasia of adipocytes, stimulating adipocrine secretion attraction and activation of stromal fat immune cells [11]. Chronic stress also causes increased levels of inflammatory cytokines, like IL-6 and TNF-α, and affects oxidant-antioxidant balance. It has a role in increased insulin resistance. Stress induces hypothalamic-pituitary axis to release cortisol. Cortisol leads to IR by stimulating visceral fat accumulation, gluconeogenesis, lipolysis, and increased glucose production in liver [12]. Stress effect on PCOS may refer to intervention with anti-Mullerian hormone (AMH) and changing sex hormone levels [12, 13]. Recent research studies on animals have shown that AMH stimulates LH release and increases the gonadotropin-releasing hormone, thereby linking AMH to endocrine instability.

Diet with saturated fatty acids (SFAs): it produces an inflammatory status [14] and lowers insulin sensitivity [15]. Vitamin D deficiency aggravates comorbidities associated with PCOS [15, 16]. Vitamin D deficiency increases insulin resistance by causing chronic inflammation [15, 17]. Vitamin D supplementation may have a beneficial effect on AMH and antral follicle count (AFC) in women with PCOS [18].

There is a decreased response of cells to insulin in women with PCOS. It is seen both in lean and obese PCOS women. Insufficient response to insulin is tissuespecific. Insulin response in ovarian tissue and adrenal is not affected [19, 20]. Insulin promotes growth of ovarian follicle and steroidogenesis. Insulin like growth factor coacts with LH. Hyperinsulinemia increases androgen production from ovary [21]. IR independently augment CYP17A1 activity, the secretory enzyme in androstenedione and testosterone production [22]. Hyperinsulinemia decreases hepatic SHBG and elevates free testosterone levels in blood. It also increases IGF-1 production in liver, which increases androgen production by stimulating theca cells. Hyperinsulinemia also stimulates LH receptors in pituitary and increases both frequency and amplitude of LH secretion. Insulin has been found to be associated with enhanced pituitary gonadotropin sensitivity to GnRH. Thus, hyperinsulinemia promotes GnRH neuron activity. It stimulates adiposeness and lipogenesis and inhibits lipolysis. Insulin resistance causes a decrease in omentin level that is independent of the patient's body mass index (BMI). Insulin's indirect effect on PCOS is enhanced by pituitary gonadotropin sensitivity to GnRH, and hyperinsulinemia promotes GnRH neuron activity [23]. Hyperglycaemia causes inflammation by secreting TNF-α from mononuclear cells (MNCs) [24].

### **5. Hyperandrogenism**

It reduces sex hormone-binding globulin and increases serum-free testosterone. In women with PCOS, higher testosterone is converted to estrone in adipose tissue. Estrone is converted to estradiol, which affects follicular growth and alters LH and FSH ratio, which affects ovulation [10]. Hyperandrogenism upregulates AMH level, which inhibits follicular growth and ovulation. IGF-II level, which is positively related to follicular diameter, decreased in follicular fluid caused by increased androgen level. Hyperandrogenism increases LH level. Hyperandrogenism adds to PCOS-related inflammatory factors, insulin resistance, and oxidative stress. PCOS women have adipose tissue similar to men because of hyperandrogenism. Increased levels of white blood cell, C-reactive protein (CRP), and other inflammatory biomarkers in peripheral blood are associated with PCOS [25]. Inflammation induces hyperandrogenism. TNF-αexacerbates insulin resistance. IL-1 affects the FSH and LH receptors, which interfere with follicular development and ovulation.

### **6. Oxidative stress**

It denotes an imbalance between pro-oxidants and antioxidants. Oxidative stress molecules include reactive oxygen species (ROS) (e.g., O2−, H2O2, and OH− ) and reactive nitrogen species (RNS). Excess production of oxidative chemicals causes various damage to vital molecules such as lipids, proteins, and DNA. Oxidative stress inhibits the insulin signalling pathway and adds to IR. Oxidative stress leads to obesity [26].

### **7. Obesity**

Obesity can lead to insulin resistance, hyperinsulinemia, and hyperandrogenism. Visceral obesity can cause an increase in non-esterified fatty acids (NEFAs) in the bloodstream. Skeletal muscles utilise NEFAs as an energy source instead of glucose. This can cause hyperglycaemia, triggering a rapid response in the pancreas and resulting in hyperinsulinemia. Visceral fat adipocytes undergo necrosis in response to catecholamines. This leads to release of cytokines like TNF and IL6, which are further responsible for adrenal steroidogenesis and hyperandrogenemia. This obesityinduced inflammatory process is also responsible for impairing insulin clearance, hence also contributing to hyperinsulinemia. Adipocytes produce high levels of leptin, which inhibits the expression of aromatase mRNA in granulosa cells and prevents the conversion of androgens to oestrogen and thus hampers ovarian folliculogenesis. Adiponectin, secreted by adipocytes, is insulin-sensitising, anti-diabetic, and has anti-inflammatory properties. Visceral fat secretes lower levels of adiponectin compared to subcutaneous fat. Omentin-1, a chemical secreted by adipose tissue, enhances IGF-1-induced progesterone and estradiol secretion, stimulates the expression of steroidogenic acute regulatory protein and CYP450 aromatase, and improves IGF-1 receptor signalling in adipose tissue. The accumulation of lipids in non-adipose tissues, known as lipotoxicity, can lead to oxidative/endoplasmic reticulum stress associated with inflammation and insulin resistance. Excessive fatty acids in muscles and liver can induce insulin resistance through serine phosphorylation of the insulin receptor by diacylglycerol.

**Figure 1.** *Gut microbiome and PCOS.*

### **8. Gut microbiome**

The clinical manifestations of PCOS women are also associated with the brain-gut axis [27]. The release of endotoxin by the gut microbiome as a response to gut inflammation is found to be a contributing factor in PCOS. Various gut microbiota play a role in maintaining physiological balance and preserving structural integrity and histological homeostasis. Additionally, the gut microbiome is involved in the production of vitamins, short-chain free fatty acids, and conjugated linoleic acid. It also plays a role in the biotransformation of bile acids, ammonia synthesis, and detoxification. Short-chain fatty acids such as acetate, butyrate, and propionate are produced by the gut microbiome and have anti-inflammatory, anticarcinogenic, and immunomodulatory effects [28]. The metabolism of butyrate and propionate is important for energy metabolism, as it regulates the gluconeogenesis process and cholesterol metabolism. Furthermore, the gut microbiome modulates the immune system [29]. The diversity of the microbiome is crucial for restoring the host's health. Obese individuals with PCOS have lower alpha and beta diversity compared to those without PCOS [30]. The main species of firmicutes in the human gut are *Lactobacillus*, *Clostridium*, and *Ruminococcus*. Among these, *Lactobacillus* has a direct association with PCOS and its impact on human health [31]. Liu et al. observed a decrease in Ruminococcaceae and *Clostridium* in obese individuals with PCOS [32]. The presence of the Proteobacteria phylum, specifically *Salmonella*, is associated with PCOS. Bacteroidetes, on the other hand, were significantly lower in the stools of PCOS patients and were associated with reproductive hormones such as thyroid-stimulating hormone (TSH) and luteinising hormone (LH) [33]. There is an immune neurological network formed by the central nervous system, intestinal neural network, and pituitary adrenal axis. Correlation between gut microbiome and PCOS will lead to a newer management option for women suffering from PCOS (**Figure 1**).

### **9. Neuroendocrine correlation**

Polycystic ovary syndrome is symbolised by excess androgen and ovulatory dysfunction. There is a rapid pulse frequency of gonadotrophin-releasing hormone due to the decreased sensitivity to the progesterone and oestrogen feedback inhibition at the level of hypothalamus [34]. The pulsatile release of GnRH is from the median

**Figure 2.** *Causes of PCOS.*

eminence, which controls the pulsatile LH secretion from the anterior pituitary. This hormonal imbalance leads to increased LH and FSH ratio which accounts for excess androgen secretion from ovary and arrest of follicular growth and oligo ovulation [35]. The various neuronal modulators associated with PCOS are KDNy neurons, kisspeptin released from hypothalamus, γ-aminobutyric acid (GABA), AMH, and others. There is an increased level of kisspeptin in women with PCOS. Thus, the blending effect of kisspeptin and LH is lost in PCOS women, resulting in dysregulated gonadotrophin-releasing hormone pulsatility. γ-Aminobutyric acid (GABA) and anti-Mullerian hormone also stimulate gonadotrophin-releasing hormone neurons directly. GABA has an excitatory effect on gonadotrophin-releasing hormone neurons. Thus, an increased level of γ-aminobutyric acid (GABA) is found in cerebrospinal fluid in women with PCOS. Direct action of hyperandrogenism in brain might have a role in the development of PCOS traits [36]. Many clinical studies have found a positive correlation between PCOS and psychiatric disorders. A higher incidence of PCOS in bipolar disorder might be due to neurotransmitter imbalance on the neuroendocrine axis executing reproductive dysfunction [37]. Women suffering from depression have a 35% of occurrence of PCOS [38]. Studies show there is also an increased incidence of PCOS in women with epilepsy. Women using valproate have an increased incidence of PCOS due to increased GABA levels. A clinical trial has shown a favourable result by targeting KDNy neuron with Ank3 receptor antagonist in women with PCOS to decrease LH pulsatility [39]. Early response of antral follicle to dysregulated LH reaches the incipient end-stage resulting in ovarian morphology in women with PCOS (**Figure 2**).

### **10. Conclusion**

The exact aetiology of PCOS is not known, and it may be multifactorial in origin. Genetic factors, metabolic factors, and environmental factors interplay in the pathophysiology of PCOS. Genomic studies have identified the various genes involved in gonadotropin secretion, gonadotropin action, ovarian follicular development, and insulin sensitivity. This genetic association, with their epigenetic regulators, are still under constant research, which will further improve the understanding of disease, thereby improving its diagnosis and treatment modalities. Newer research on neuroendocrine factors and gut microbiome will facilitate innovative approach for the

management of PCOS and its consequences. Neuroendocrine and gut microbiome topics of research for the aetiology of PCOS are in early stage and mostly based on the mice model. Further research, specifically on humans, is required to establish these factors as the causative factors of PCOS, and that will open the innovative window for the management of this endocrine and metabolic disorder.

### **Author details**

Subrat Panda1 \*, Rituparna Das2 , Lisley Konar1 and Manasi Singh1

1 AIIMS, Kalyani, India

2 AIIMS, Guwahati, India

\*Address all correspondence to: pandadrsubrat@rediffmail.com

© 2024 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### **Chapter 4**

## Would Non-coding RNA Resolve the Polycystic Ovary Syndrome (PCOS) Puzzle?

*Rana Alhamdan*

### **Abstract**

Polycystic ovary syndrome (PCOS) is the most common endocrine heterogeneous reproductive disorder. This metabolic disease affects around 5–10% of women and accounts for 75% of anovulatory infertility all over the world. The complexity of the disease as manifested by the involvement of multiple underlying mechanisms and the lack of specific and sensitive biomarkers, make it difficult to timely manage and treat the disease. Remarkably, genetic, epigenetics, and environmental variations may contribute considerably to the pathogenicity of PCOS. Recent investigations indicated that non-coding RNAs (ncRNA) were involved in the occurrence and development of PCOS. Thus, this chapter aimed to summarize the current knowledge around the expression and dysregulation of ncRNA in human PCOS.

**Keywords:** polycystic ovarian syndrome (PCOS), infertility, non-coding RNA (ncRNA), micro RNAs (miRNAs), Long non-coding RNAs (lncRNAs)

### **1. Introduction**

PCOS is the most common endocrine heterogeneous reproductive disorder. This metabolic disease affects around 5–10% of women and accounts for 75% of anovulatory infertility all over the world [1]. The fundamental features of this condition are oligo- or anovulation, hyperandrogenism, and polycystic ovaries. Women with PCOS may also develop other disorders, including type II diabetes, obesity, dyslipidemia, endometrial cancer, and insulin resistance (IR), in addition to being at risk of cardiovascular diseases [2]. Considering the complexity and diversity of this disorder, a deeper thinking and wider study of its etiology is certainly needed. In clinical settings, diagnosis is mainly based on the presentation of no less than two out of the three Rotterdam agreed main symptoms. These include polycystic ovaries, irregular periods, and high androgens. Therefore, the expert diagnostic opinion is now based on pelvic ultrasound examination and hormonal blood tests. This can emphasize the growing need for a specific and sensitive biomarker for the diagnosis of this condition [3].

Over the past two decades, a striking finding has indicated the presence of large amounts of non-protein-coding sequences in the genome of complex organisms [4, 5]. These previously named 'transcriptional noise" sequences have now been shown to play multiple crucial biological roles and are referred to as non-coding RNAs (ncRNA). The involvement of those ncRNAs in the occurrence and development of PCOS has recently been indicated. A differential significant expression of ncRNAs has been found in the granulosa cells (GCs), follicular fluid (FF), Theca cells (TCs), serum, and plasma of patients with PCOS when compared to non-PCOS women [6, 7]. Therefore, this chapter aimed to provide an updated narrative review summarizing the current knowledge around the presence and dysregulated expression of ncRNAs. The chapter will review two main ncRNA regulators (microRNA (miRNAs), and long ncRNAs (lncRNAs)) and frame the discussion in the context of linking ncRNAs to the pathogenicity of PCOS as well as the possibility of using them as a biomarker to resolve the puzzle of PCOS.

### **1.1 Overview of ncRNAs**

A significant amount of the human genome encodes functional transcripts that are destined for translation into proteins. Only recently, new classes of ncRNAs have been identified and were classified by length into small ncRNAs (sncRNAs) transcripts of small RNA sequences of <200 nucleotides and transcripts >200 nucleotides that lack any defined open frames and called lncRNA [5, 8, 9]. sncRNAs are further classified into miRNAs, small interfering RNA (siRNA), piwi-interacting RNA (piRNA), and transfer RNA (tRNA), small nuclear RNA (snRNA) [8]. Advanced sequencing technology led to the discovery of some of the underlying molecular mechanisms linking those ncRNAs with PCOS. It highlighted some precise and feasible biomarkers, of which miRNA and lncRNA have been the most well-characterized as functional ncRNAs in PCOS [5, 9, 10].

### *1.1.1 miRNAs and lncRNAs*

ncRNAs are key regulators of gene expression and chromatin structures. They have particularly been shown to be relevant since the early stages of germline development.

miRNAs are small single-strand RNAs (22–23 nucleotide (nt) long). It plays a crucial biological role in regulating protein expression via either gene degradation or silencing. It acts as a core element to mediate gene and protein interactions at a multiple intra- and inter-cellular site to regulate their functions [11]. Hence, they are heavily available in biological fluids, a feature that facilitates their use as a biomarker research target.

Their biosynthesis is orchestrated at multiple events by which miRNAs are transcribed in the nucleus into a large primary form (pri-miRNAs). The immature primiRNAs are then methylated and subsequently processed by the DORSHA-DGCR8 complex and transferred to the cytoplasm. The cytoplasm is where pri-miRNAs are cleaved by DICER to form the mature double-stranded miRNA (22 nt), which are incorporated into a ribonuclear vesicle and form the RNA-induced silencing complex (RISC) [8, 11, 12].

lncRNAs, on the other hand, are typically longer than 200 nt. They are abundantly generated from 80–90% non-coding modulatory elements in the human genome and account for 60,000 lncRNA. Functional studies have recently classified the molecular mechanisms of lncRNAs into four types: guide, signal, decoy, and scaffold. They possess their regulatory role at the transcriptional, post-transcriptional processing levels, chromatin modification, and epigenetic modulation. Like miRNAs, they are

*Would Non-coding RNA Resolve the Polycystic Ovary Syndrome (PCOS) Puzzle? DOI: http://dx.doi.org/10.5772/intechopen.114387*

radially available in body fluids and present in exosomes. A fact that makes them a good candidate as a non-invasive biomarker research tool.

LncRNAs, like other ncRNA, exhibit a complex three-dimensional structure and share a similar biosynthesis pathway. Based on their positional properties, they can be easily transcribed by polymerase II, polyadenylated, and spliced from different genomic sites. Owing to this property, lncRNAs are grouped into (1) natural antisense transcripts, (2) stand-alone, (3) pseudogenes, (4) intronic transcripts, and (5) enhancer RNA, promotor-associated, and divergent transcripts. LncRNAs lack open reading frames (PRFs); however, they exhibit a high tendency for cell-specific expression and possess a 3-terminal process specificity.

Both miRNAs and lncRNAs are present in abundance in body fluids and various cell types, and they have become a hot research topic for investigations related to PCOS diagnostic and therapeutic biomarker targets.

### **2. ncRNA and PCOS**

To explore the cause, we need to critically assess the problem. This will lead us to explore the available knowledge associated with this disorder and further link that to how ncRNAs may contribute to the physiology and pathophysiology of PCOS.

Gonadotropin-releasing hormone (GnRH) is typically high in PCOS, leading to increased secretion of pituitary luteinizing hormone (LH). Consequently, ovarian androgen levels increase, leading to lower follicle-stimulating hormone (FSH) production by the pituitary gland. This would result in egg maturation issues and, therefore, anovulation. Moreover, numerous small follicles are observed in the ovary. In addition, altered steroidogenesis will result in abnormal production of estrogen (E2), progesterone (P4), and androgens [9]. Insulin resistance (IR) has also been described as a momentous cause of PCOS [6].

A vast number of ncRNAs have been observed altered in various reproductive tissues and body fluids from PCOS women. Their aberrant expression has been associated with abnormal ovarian cell apoptosis and/or proliferation, steroidogenesis, IRs, as well as adipocyte dysfunction among PCOS patients compared to healthy controls.

### **2.1 The role of ncRNAs in PCOS-associated ovarian steroidogenesis**

Androgen production is vital for estrogen synthesis. Thus, the malfunction and/ or incomplete conversion of androgens to estrogens cause hyperandrogenism (HA). PCOS is characterized by the dysregulation and increased production of the six main androgens: free testosterone (fT), testosterone (TT), androstenedione (A), sex hormone binding globulin (SHBG), 17-hydroxy progesterone (17-OHP), and dehydroepiandrosterone sulfate (DHEA [13]). Hyperandrogenism is substantially attributed to the ovaries and adrenals and with minimal contribution from fatty tissues. The imbalance in these hormones negatively impacts follicle development and oocyte maturation, leading to infertility. The etiologies associated with androgen excess are not solely uncovered and are still controversial. One possible reason is the increased P450 steroidogenic enzymatic activity, and another might be androgen receptor (AR) malfunction, cortisol metabolism defects, and/or the overproduction of androgen by TCs in response to LH overstimulation [14, 15]. It has been suggested that increased testosterone bioavailability in women with PCOS is the result of decreased SHBG [14]. AR has been proposed implicated and was reported to be highly expressed in patients with PCOS [16]. A growing body of evidence has indicated a regulatory role of ncRNAs in the sex steroids-associated physiological mechanisms [17].

### *2.1.1 miRNAs*

The statistical significance of several **miRNAs** has already been documented as clinical biomarkers for PCOS [7, 18]. However, the complexity of the mechanisms involved in sex hormone production makes it harder to define those guilty miRNAs. A positive relationship has been indicated between miR-592 and LH/chorionic gonadotropin receptor (LH/CGH), a vital element in the HA-PCOS-involved mechanism [19]. In PCOS women, LH overstimulation has been shown to stimulate the expression of Pak3, thus down-regulating miR-125b-5p expression and leading to ERK1/2 signaling trigger, consequently, the increase expression of androgen synthesis-related enzymes and testosterone production [20]. The dysregulation of miR-23a and miR-146a has been found to inversely correlate with the significance of PCOS development. Both miRNAs were identified as negative regulators of serum testosterone [21, 22]. The overexpression of miR-278 and miR-181a has been shown to negatively regulate aromatase activity, thus leading to lower estrogen synthesis [23, 24]. Adding to those players modulating steroid synthesis, miR-34C, miR-9, miR-135a, miR-18b, and miR-32 have been found implicated [25]. miR-96-5P has also been shown to modulate E2 synthesis via the inhibition of serum, FF, and/or GC forkhead transcription factor (FOXO1) expression and its downstream genes [26]. miR-200b has also been proposed as a downstream target for AR via the hypothalamic-pituitary-ovarian axis (HPO-A) and may act as an actress in the processes leading to HA-PCOS [27]. Moreover, miR-320 is a controversial miRNA that has been once reported to be upregulated and next to be downregulated. The first group studied the expression of miR-320 in the FF of PCOS. They suggested the involvement of this miRNA in the inhibition of E2F1/ SF1 proteins, thus leading to the downregulation of estradiol release into the FF [28]. However, the second study reported a significantly lower expression of miR-320 in PCOS when compared to controls and proposed a role of this miRNA in regulating steroidogenesis [29]. Despite the discrepancies between the studies, which might be due to the differences in the experimental settings and patients' population, miR-320 appears to have a direct relevance to the HA-PCOS phenotype.

It has been suggested that miRNA-423-3P may act guilty for the dysregulation of progesterone production by the TCs. A significant reduction of AdipoR2 (adiponectin receptor 2), a receptor to adiponectin, has been found in the TCs of women with PCOS. AdipoR2 has been shown to directly interact with miRNA-423-3P and thus may play an important role in the pathogenesis of PCOS [7].

### *2.1.2 lncRNAs*

**LncRNAs** have been implicated in divergent indications of PCOS via various interaction networks including miRNAs and ceRNAs [10]. Chen et al. suggested that the lncRNA H19 can modulate steroidogenesis at the post-transcriptional levels. The group utilized an animal and a human model to demonstrate that H19 disturbance may alter androgen production via a Cyp17-related mechanism and can be an important player in the pathogenicity of PCOS-associated HA [17]. Another group demonstrated an inhibitory effect of lncRNA HUPCOS on CYP11 activity through the interaction with RNA-binding protein with multiple splicing (RBPMS). Via which,

### *Would Non-coding RNA Resolve the Polycystic Ovary Syndrome (PCOS) Puzzle? DOI: http://dx.doi.org/10.5772/intechopen.114387*

this lncRNA was positively correlated to FF testosterone in PCOS individuals [30]. Similarly, lncRNA OC1 silencing experiments have indicated the role of this lncRNA to suppress aromatase activity in the GCs of women with PCOS [31]. Furthermore, lncRNA terminal binding protein 1 anti-sense (CTBP1-AS) is thought to be an important modulator of AR and can tightly correspond to TT [10]. Moreover, lncRNA Neat1 has been previously described as implicated in the regulation of follicle development and P4 function. LncRNA Neat1 knockout (KO) mice exhibited low serum P4 expression levels and CL malfunction and, thus, pregnancy failure [7]. Interestingly, lncRNA zinc finger anti-sense 1 (ZFAS1), exhibiting a chromosome 20q13 location, was found to play a role in stimulating the secretion of E2 and P4 in the GCs of PCOS women. This study explored the mechanistic network between lncRNA ZFAS1/miR-129/and high-mobility group box protein 1(HMGB1) in PCOS. Their work demonstrated that both ZFAS1 and HMGB1 were upregulated and miR-129 was downregulated in the GCs of PCOS patients and that the silencing or reversing the action of either may repress the endocrine disturbance leading to this pathological condition [32].

These insights can indicate the complex interaction network between both miRNAs and lncRNAs to modulate the endocrine disturbance associated with the PCOS phenotype. Thus, further work is required to highlight the possible use of these ncRNAs as a potential therapeutic target. A summary of miRNAs and lncRNAs included in this section is provided in **Table 1**.


### **Table 1.**

*The role of ncRNAs in PCOS-associated ovarian steroidogenesis.*

### **2.2 The role of ncRNAs in PCOS-associated IR**

It sounds like there is a strong link between IR, HA, and PCOS-related infertility. IR in PCOS is caused by impaired insulin action and is marked by compensatory hyperinsulinemia (HI) and reduced insulin response to glucose overload [33]. It is because insulin plays a significant role in facilitating androgen secretion from the pituitary, ovaries, liver, and adrenal glands, and it acts as a co-gonadotropin to regulate ovarian steroidogenesis. Additionally, it can arrest ovarian follicular development, modulate LH pulsatility, and negatively regulate SHBG production [34]. Androgen excess can lead to a chain reaction with IR and HI. It is prevalent in 65–95% of women diagnosed with PCOS, and it has been found that obesity can exacerbate these conditions. IR can stimulate an increase in CYP17 enzymatic activity, which in turn inhibits SHBG levels, leading to increased serum fT levels [8, 14]. Interesting to note that HA in the ovaries can be attributed to the insulin growth factor-1 (ICF-1) receptor, which can play a role in mediating LH-induced TCs androgen overproduction [35].

### *2.2.1 miRNAs*

IGF-1 signaling pathway in synergy with peroxidase proliferator receptor (PPAR) and angiopoietin, has been shown to contribute to PCOS. Interestingly, miR-223 has been indicated to have the potential to modulate this pathway [36]. The expression miR-222 has been reported to positively correlate with serum insulin from patients with PCOS [21, 37]. It is apparent that miR-146 is an important factor in IR and is believed to have a significant role in its pathogenesis. The expression of miR-146 triggers a series of proinflammatory signals that activate nuclear factor kappa B (NFkB), leading to the upregulation of miR-146a, which in turn initiates negative feedback and regulates immune response. However, during hyperglycemia, this miRNA is downregulated despite NFkB activation and proinflammatory response [33, 38]. It is interesting to note that according to Sang et al. [29], miR-320 and miR-132 have been suggested to play a role in modulating IR. The author predicted that RABSB and HMGA2 are the target genes for miR-320 and miR-132, respectively, and that both gene expression are altered by reduced levels of these miRNAs in the FF of women with PCOS [29]. One of the reported miRNAs to be elevated in PCOS individuals is miR-93. The overexpression of this miRNA has been associated with IR and was shown to correspond strongly with GLUT4 receptor suppression in adipose tissues in women with PCOS [39]. It has been found that miR-133a-3p is highly expressed in patients with PCOS as compared to controls. This miRNA inhibits the PI3K/AKT (PKB) signaling pathway, which plays a crucial role in insulin action. The insulin receptor substrate-1 activates PI3K, which activates AKT, the main downstream protein. However, in PCOS, IR leads to inhibition of the PI3K/AKT signaling pathway. Nevertheless, the mechanism is not yet fully understood and requires further investigation [38].

Moreover, a recent investigation on glucagon-like peptide 1 agonist receptor agonist (GLP-1RA) and dipeptidyl peptidase-4 (DPP-4) inhibitors have shown a relationship with altered expression of miRNAs such as mIR-222, miR-221, miR-33, miR-155-5, miR-6763, miR-75-5p, miR-197, miR-1197-3p, and miR-6356, which may have an impact on insulin sensitivity and contribute to the management of symptoms related to PCOS by increasing glucose metabolism and transport [33, 40–42].

### *Would Non-coding RNA Resolve the Polycystic Ovary Syndrome (PCOS) Puzzle? DOI: http://dx.doi.org/10.5772/intechopen.114387*

Furthermore, an antagonistic glycolytic regulatory activity has been observed to be exhibited by miR-155-5p and miR-143-2p in PCOS-related follicular dysplasia [43]. miR-29, miR-19a, miR-1, and miR126 have also been suggested to modulate glucose uptake via PI3K signaling pathways [44].

### *2.2.2 lncRNAs*

The role of **lncRNAs** in association with IR has been addressed in a few studies. Intriguingly, around 20 lncRNA were found to co-expressed with the neuropeptide Y1 receptor (NPY1R), a candidate gene for type 2 diabetes mellitus, and these coexpressed lncRNAs may have a significant role in the development of PCOS [5]. According to a study by Zhao et al. [34], using WGCNA analysis, six hub lncRNAs (RP11-151A6.4, LNC00475, RP11-54A4.2, TTTY14, RP1-93H18.1, and RP3-439F8.1) were found to be significantly associated with IRs in patients with PCOS. Of all six lncRNAs, lncRNA RP11-151A6.4 was identified to express at a significantly higher level in both subcutaneous and omental adipose tissues of patients with IR compared to healthy controls. Moreover, the expression levels of RP11-151A6.4 in ovarian GCs were increased in patients with PCOS compared to control patients with HI. These findings may suggest a significant role of this lncRNA in IR-related PCOS [34]. A limited number of studies have explored the relationship between lncRNAs and PCOS with IRs. However, the model used by Zhao and his colleagues, and the potential molecular candidates explored by the group could be crucial for future investigations and may help to understand the mechanisms of IRs. Thus, further investigation is urged to explore the possible pathogenic role of miRNAs and lncRNAs in patients with PCOS and IRs. A summary of miRNAs and lncRNAs included in this section is provided in **Table 2**.

### **2.3 The role of ncRNAs in PCOS-associated GCs proliferation and apoptosis**

GCs play a crucial role in the growth and maturation of oocytes, and any dysfunction in these cells can contribute to abnormal folliculogenesis and hormone production. Thus, aberrant GC proliferation and apoptosis are believed to be a significant factor in the development of PCOS.

### *2.3.1 miRNAs*

Several studies have profiled ncRNAs in GCs taken from women with and without PCOS. It has been shown that women with PCOS tend to have lower levels of miR-145 in their GCs. Interestingly, the upregulation of this miRNA not only inhibited cell proliferation but also promoted cell apoptosis in human GCs. Further investigations demonstrated that miR-145 can suppress the activation of the MAPK/ERK signaling pathway via binding to insulin receptor substrate 1(IRS1). Based on these findings, the author suggested that the inhibition of miR-145 levels may promote GC proliferation via IRS1/MAPK/ERK regulatory network in women with PCOS [45]. Mao et al. [46] have also reported lower expression of miR-29a-5p, 92b, and miR-126-5p in GCs of PCOS women, which could induce GC apoptosis [46]. The role of miR-486-5p, miR-3940-5p, miR-182, miR-206, miR-15a, and miR-204 in modulating ovarian GC proliferation and apoptosis have been reported. While miR-3940-5p was found to be upregulated, low expression of miR-182, miR-15a, miR-486-5p, miR-206, and


### **Table 2.**

*The role of ncRNAs in PCOS-associated IR.*

miR-204 has been observed in the GC of PCOS individuals when compared to healthy controls [14, 47, 48]. However, the profile of miR-486-5 in PCOS has been reported with contradicting results [49, 50].

It is well established that GCs are the primary site of endocrine signaling and estrogen synthesis in the ovaries. Thus, it is expected that the dysregulation of miRNAs in GC may contribute to the dearth of E2, a major feature of PCOS. Zhang et al. [51] reported a low expression of miR-320a in the CCs in females with PCOS when compared to healthy controls. The group proposed that miR-320a induced E2 deficiency via 320a/RUNX2/CYP11A1 (CYP19A1) regulatory pathway [51].

The overexpression of miR-93 has been reported in the GCs of PCOS patients. Jiang et al., further indicated that cdkn1a is the target gene of this miRNA and that the absence of this gene supported the stimulating proliferation role of miR-93, whereas the reintroduction of cdkn1a reversed this effect [52]. He et al. [53] have suggested that miR-141 and miR-200 expression in the GC of PCOS patients may target the PI3K/Wnt signaling pathway to inhibit their proliferation [53]. The significant upregulation of miR-513b, miR-509-3p, and miR-423-3p has been reported by several studies in the CCs of women with PCOS [50, 54, 55].

### *2.3.2 lncRNAs*

It is interesting to note that Huang et al. [54] demonstrated that there was a significant upregulation of the expression of 620 **lncRNAs** in PCOS cumulus cells, with only three lncRNAs being downregulated [54]. Similarly, Liu et al. [55, 56] observed that 692 lncRNAs were upregulated and 170 lncRNAs were downregulated in PCOS GCs [56]. Chen et al., discovered that lncRNA hcp5 may play a role in promoting cell proliferation and apoptosis via the miR-27a-3p/IGF-1 axis in the human KGN cell line model [57]. Utilizing a knockout model, the Zhu group has demonstrated that ZFAS1 can potentially increase GC proliferation and inhibit apoptosis in PCOS [32]. Moreover, LINC00477 has been recently reported to be significantly highly expressed in PCOS women, and it is postulated that may promote PCOS by inhibiting the GCs proliferation and increasing apoptosis through sponging miR-128. The study demonstrated that the expression levels of this lncRNA in the serum can be used to distinguish PCOS individuals from healthy counterparts [58]. Furthermore, another study found that the lncRNA PVT1/miR-17-5p/PTEN axis may have a role in regulating E2


**Table 3.** *The role of ncRNAs in PCOS-associated GCs proliferation and apoptosis.* and P4 secretion, as well as GC proliferation and apoptosis in PCOS [58]. It is interesting to note that Taurine upregulated 1 (TUG1) was found to be highly expressed in PCOS GCs and that was directly correlated to an increased AFC. Furthermore, TUG1 was primarily localized in the nuclei of human GCs. The findings that TUG1 silencing inhibited cell proliferation and regulated apoptosis and autophagy in MAPKs pathway-dependent and P21-dependent manners are also noteworthy. Moreover, the TUG1 knockdown model increased aromatase expression and E2 biosynthesis in GCs [59]. The results of a recent independent microarray analysis performed by Liu et al. [60] have demonstrated an increased expression of lncRNA human leukocyte antigen complex group 26 (HCG26) in PCOS patients when compared to their matched controls. Further functional analysis revealed that the knockdown of HCG26 inhibits cell proliferation while promoting aromatase gene expression and E2 production. These findings suggest that HCG26 plays a role in GC proliferation and steroidogenesis and thus contributes to the pathogenicity of PCOS [60]. On the whole, these data indicate the presence of different profiles of miRNAs and lncRNAs in GCs in individuals with PCOS compared to healthy controls. These ncRNAs (**Table 3**) may play various roles in cellular proliferation and apoptosis, hormone regulation, and angiogenesis.

### **3. Did ncRNA resolve the puzzle for PCOS?**

Efforts to gain a better understanding of the molecular endogenous RNA networks affecting PCOS are vast. These studies successfully shed some light on the complicated interactions and functions in association with the endocrine and female reproductive system overall. The research demonstrated the crucial role of ncRNAs in forming a mutual functional endogenous network that can significantly develop and modulate a disease state.

Sequencing technology and transcriptomics analysis have been used by various groups to investigate potential ncRNAs as biomarkers for PCOS with varying biological significance. However, to agree on the statistical significance of any of the found biomarkers, it is important to take into consideration several factors. Among the most important are the p-value, the specificity and sensitivity of the fold change in PCOS and related symptoms, and how relevant the indicated role of the biomarker is [3]. The data available to date is contradictory, and very few ncRNAs can be defined as significant to the condition of PCOS. The reason for that can be related to the various types of samples and methods used by the different groups. Adding to the complexity of testing is the fact that PCOS is a multi-organ disorder, which means it can be affected by the various ncRNAs expressed in association with extracellular signaling. Therefore, it is important to study these functional biomolecules by utilizing more experimental investigations and large data build-up and analysis. Several models have been shown to be useful, such as the bioinformatics approach used by Zeng et al. The group was able to develop an RNA-drug network based on differentially expressed miRNAs and lncRNAs. This approach allows for the identification of molecular correlations among drugs and can guide future work in locating the underlying mechanisms and functional roles of these biomolecules in PCOS [3, 61]. Furthermore, studies on human cell lines such as KGN cells can also be promising, especially in studying intracellular signaling networks [60, 62]. This approach in combination with tissue and cell analysis is prominent to investigate biomolecular interactions and molecular mechanisms involved in follicular development and PCOS. Gene Ontology (GO) and pathway analysis with the Kyoto Encyclopedia of Genes and

*Would Non-coding RNA Resolve the Polycystic Ovary Syndrome (PCOS) Puzzle? DOI: http://dx.doi.org/10.5772/intechopen.114387*

Genomes (KEGG) has been used by several groups to discover significant correlations between ncRNAs biomarkers for PCOS in association with functional biological significance [3, 5]. The statistical significance and true causation analysis to point and define effective biomarkers for the diagnosis and treatment of PCOS require further clarity of the underlying mechanisms. Thus, the combination of these approaches mentioned above would be a good way to move forward in discovering the PCOSassociated therapeutic complexes.

### **4. Conclusion**

In conclusion, PCOS is a condition associated with hormonal imbalances manifesting into infertility and other serious long-term health problems. Early and accurate diagnosis is crucial, as it can help prevent complications like diabetes. The fact that PCOS affects endocrine cellular functions is certainly concerning and urges more work to clearly define the accurate causation of the condition. The advancement in sequencing technology is promising and can help us expect reliable ncRNA biomarkers that can be used in clinical settings worldwide soon. Further research is needed to explore complex networks of ncRNAs/RNAs and protein combinations and useful functional signaling pathways. This could help unveil and resolve the PCOS puzzle.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Rana Alhamdan King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia

\*Address all correspondence to: amsf199705@hotmail.co.uk

© 2024 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Section 3
