Polycystic ovary syndrome (PCOS) is one of the prevailing gynecological, endocrine and metabolic disorders among women of reproductive age, representing one of the leading causes of anovulatory infertility. According to the criteria in practice, this disease affects 5% to 20% of women worldwide ^[1][2][3][4][1,2,3,4].

According to the Rotterdam Consensus on Diagnostic Criteria for PCOS, this is a multifaceted pathology that encompasses many clinical manifestations. It is necessary to present two out of three of these characteristics in order to be diagnosed with PCOS: oligoanovulation [5]; clinical and/or biochemical hyperandrogenism (HA) [6]; polycystic ovarian morphology that does not encompass other abnormalities, such as Cushing’s syndrome, congenital adrenal hyperplasia, and androgen-secreting tumors [7].

To be considered a woman with polycystic ovary morphology, it is also necessary to present at least one ovary with “12 or more follicles, measuring 2–9 mm in diameter, and/or increased ovarian volume, namely above 10 mL” ^{[8][9][10][11][12]}[8,9,10,11,12]. This definition, however, does not apply to women who use the oral contraceptive pill because its use alters ovarian morphology ^[8][13][8,13]. A scan should be repeated in the next cycle if a corpus luteum or dominant follicle are discovered. Any evidence of ovarian asymmetry or an abnormal cyst requires a more in-depth inquiry, as well as asymptomatic polycystic ovarian morphology women, since they do not present ovulatory disorder or hyperandrogenism ^{[8][14][15][16]}[8,14,15,16].

Metabolic abnormalities are also common, even if they are not considered to classify a PCOS woman. Three out of the following five qualify for metabolic syndrome: blood pressure ^[8][17][18][8,17,18]; abdominal obesity (waist circumference) ^[8][17][19][8,17,19]; high levels of triglycerides (TG) ^[8][17][8,17]; fasting and two-hours glucose from oral glucose tolerance test; low levels of HDL-C. These might further generate an increased risk of type 2 diabetes ^[17][20][21][17,20,21], insulin-resistance (IR) ^[17][22][23][17,22,23], and cardiovascular diseases ^{[17][24][25][26][27][28]}[17,24,25,26,27,28], and they might disturb the body mass index (BMI) ^[8][17][8,17].

Because of the variety of clinical and biochemical manifestations of PCOS, two of the hallmarks of these women, which heavily influence the disease phonotypy, are overweight and obesity, with only 30–50% of PCOS patients exhibiting an average weight. Such conditions may result in IR and metabolic syndrome [2]. Glucose intolerance and an oral glucose tolerance test should be used to screen obese women with PCOS for metabolic syndrome. IR is described as the decrease in the use of glucose mediated by insulin. This metabolic abnormality occurs in more than 50% of PCOS cases and leads to reproductive complications. Improving the lifestyle and recovering with pharmacological intervention can help mitigate further irregularities ^[8][29][8,29]. Insulin can keep lipid metabolism in check by blocking the release of free fatty acids from adipose tissue, and in patients with IR, inhibition of the lipid oxidation rate is weakened, resulting in an increase in the concentration of free fatty acids in the FF ^[4][30][31][4,30,31]. Some criteria for defining a metabolic syndrome were already developed. These include components associated with the IR syndrome, such as centripetal obesity, hypertension, fasting hyperglycemia, and dyslipidemia ^{[17][20][21][32]}[17,20,21,32]. It was proposed to add an oral glucose tolerance test (OGTT) to the fasting blood tests. The 2-h glucose level after a 75-g oral glucose challenge for glucose intolerance could be evaluated in this manner. Within obese PCOS women, impaired glucose tolerance and type 2 diabetes are two common features, both being diagnosed by OGTT ^[8][21][8,21]. As referenced before, IR, is common in PCOS patients and, increases the risk of metabolic syndrome,as well as cardiovascular morbidity, and it generates higher glucose concentrations ^[3][33][3,33]. PCOS women that present obesity, a family history of type 2 diabetes, IR, or beta-cell dysfunction have high probabilities of developing diabetes ^{[20][21][22][34][35]}[20,21,22,34,35]. Apart from these metabolic abnormalities, it is also suggested that these woman present an increased risk of having strokes and cardiovascular diseases ^[8][24][36][8,24,36]. IR is linked to coronary heart disease, and PCOS patients might also present dyslipidemia and abnormal vascular function ^{[37][38][39][40][41][42][43]}[37,38,39,40,41,42,43]. Chronic anovulation with unopposed estrogen exposure to the endometrium has also been linked to an increased risk of endometrial cancer ^[8][44][8,44]. Other aspects may be considered as additional risk factors, such as a family history of diabetes [8]. Clinical studies have revealed that PCOS patients with weight and BMI reduction were often associated with menstrual bleedings, the return of ovulation, and the normalization of other metabolic parameters ^[4][45][4,45].

One of the key features of PCOS is the presence of clinical and/or biochemical androgen excess, aside from other diseases ^[8][46][8,46]. Some clinical features of hyperandrogenism encompass the presence of hirsutism, acne, and androgenic alopecia when coupled with oligoovulation ^{[8][46][47][48]}[8,46,47,48]. In addition, some studies pointed out that the circulating androgen levels, corresponding to the biochemical fraction, might represent an inherited marker for androgen excess, even though this aspect may not be clear in all cases ^{[49][50][51][52][53][54]}[49,50,51,52,53,54]. However, the quantification of the different androgens is challenging, since most of the commercially available kits are inaccurate ^{[8][55][56][57]}[8,55,56,57], and not all chromatographic methods allow the precise detection of these hormones ^[58][59][60][58,59,60]. Additionally, multiple androgens that are characterized through these methods cannot be considered in the final evaluation ^[8][61][8,61]. Due to the wide variety of the population, the values of the hormones may differ more than expected, and, therefore, control limits have not yet been set ^[8][28][62][8,28,62]. Another pertinent point is that androgen suppression is extremely difficult to reverse, even after hormonal treatment has been discontinued [8]. The evaluation of free androgens and their index are sensitive markers for assessing hyperandrogenemia, with the second one being more accurate ^{[8][57][63][64][65]}[8,57,63,64,65].

Due to an increase in the amplitude and frequency of LH pulses, LH concentrations and their relationship to FSH levels are immensely elevated in PCOS women ^{[8][66][67][68]}[8,66,67,68]. These levels may be influenced by the timing of ovulation, BMI (being lower in PCOS women with a higher BMI), and the analytical method used. The effects of LH on human reproduction are highly debated, with some studies suggesting that high levels of this hormone may reduce oocyte maturation and fertilization, resulting in higher miscarriage rates ^{[8][67][69][70]}[8,67,69,70]. Others concluded that abnormal LH concentrations did not affect oocyte and embryo quality, implantation, fertilization, or pregnancy outcomes ^[8][71][72][8,71,72]. Many studies resort to GnRH in order to reduce endogenous LH. However, while some studies suggested that this practice reduced miscarriage rates ^[8][73][8,73], others questioned such a therapeutic effect ^[8][74][75][8,74,75]. Nonetheless, LH can be used as a secondary parameter, particularly in women who are not overweight [8]. Until today, both the subsequent etiology and pathophysiology of PCOS remain unclear. To reduce the psychological and social pressures that come with this pathology, several studies were conducted to facilitate the diagnosis and treatments ^[1][76][1,76]. Patients with PCOS that undergo assisted reproductive techniques (ART) might present a poor to exaggerated response, low oocyte quality, ovarian hyperstimulation syndrome, as well as changes in the FF metabolites pattern. These abnormalities originate a decrease of MII oocytes and decreased rates for fertilization, cleavage, implantation, blastocyst conversion, poor egg to follicle ratio, and increased miscarriages ^[1][2][1,2]. Over the years, the focus of research shifted from embryo to oocyte quality to optimize IVF outcomes and to improve pregnancy rates. However, since PCOS is considered a heterogenous disease, obtaining high-quality embryos is taken into more consideration ^[1][33][1,33], where every patient should undergo custom tests to receive the most accurate guidance.

2. Follicular Fluid (FF)

Follicular fluid (FF) serves as a complex microenvironment for germ cell–somatic cell communication. It encompasses a variety of metabolites and enables different reactions to take place that are crucial to oocyte growth [2]. It is derived from the diffusion of serum, transudate of plasma, and metabolites synthesized in the follicle wall that will later be altered by granulosa cells (GCs) and theca cells. In addition, compounds that derive from local follicular metabolic processes and the biological activities of ovarian cells are also present. This biological matrix is the only one directly associated with the oocyte since it is where its growth and differentiation occurs in vivo. It contains a variety of bioactive molecules that change in quantity and quality during follicle development, as well as specific changes in the follicular microenvironment that lead to follicle and oocyte maturation and development. The biosynthesis and transport of these metabolites are crucial to multiple metabolic reactions; they regulate meiosis, are involved in the synthesis of steroid hormones and glycoproteins by the dominant follicle, and promote follicle and oocyte maturation and development, fertilization, and implantation ^[77][78][79][77,78,79]. The follicle wall acts as a highly rough molecular sieve that allows passage to small metabolites while restringing the access to molecules over 100 kDa. There is bidirectional signal regulation and metabolite transport between GCs and oocytes, such as steroid hormone biosynthesis, oocyte gene transcription, and protein synthesis regulation, showing a deep connection between the oocyte and GCs [78]. Some research also points to the role of oxidative stress (OS) in follicular fluid as a causative factor of female infertility. FF contains reactive oxygen species (ROS) and antioxidant enzymes, with ROS being physiologically produced during the ovulatory process. Under physiological conditions, antioxidant defense systems prevent ROS production and scavenge existing free radicals. It is also proposed that ROS significantly speed up ovarian aging. Proteins are among the most affected metabolites by ROX, and carbonyl groups (aldehydes and ketones) are produced on protein side chains when oxidized. Carbonylated proteins are formed early in OS conditions and are relatively stable. They induce modifications at the protein level (identified as oxidative damage to the proteins), leading to the proteolytic breakdown of the peptide bond, crosslinking and/or modifications of amino acids such as carbonylation, or the formation of disulfide bridges intra and intermolecularly. These changes may alter protein function and antigenicity, triggering immunological processes associated with inflammation and autoimmune damage. The faulty immune system activation and regulation, coupled with the unbalanced production of ROS, may result in oxidative damage. Therefore, many pathological conditions affecting female fertility may derive from the synergistic action of OS and immunity ^{[77][80][81][82][83][84][85][86][87][88][89][90][91][92][93][94][95][96][97][98][99][100][101][102][103][104][105]}[77,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105]. A more detailed understanding of FF and its metabolic profile is crucial for further analysis of several pathologies, such as PCOS, endometriosis, and early ovarian failure. FF has become an essential source of information since it is a non-invasive matrix that gathers biological insights about fertility, reflecting the alterations of the patient’s microenvironment. Recently, the molecular and biomolecular signature of FF have aroused many interests, leading to several studies that aim to identify new targets that allow for evaluation of the development of the oocyte. Consequently, an exhaustive characterization and comprehension of FF may then help the recognition of metabolites that could potentially disturb normal female function and promote infertility ^{[78][79][106]}[78,79,106].

3. Biomarkers in Follicular Fluid

3.1. Biomarkers of Oxidative Stress (OS)

The antioxidant defense system has many components ^[107][124]. The imbalance between antioxidant and pro-oxidant molecules characterizes OS [80]. Healthy women present higher total antioxidant status (TAS) concentrations in the FF. These present a positive association with clinical pregnancy rates. The TAS in FF samples can be determined using available immunoassays that measure the total antioxidant capacity of a sample ^[107][124]. The presence of various OS markers in human FF has been reported, and these appear to be involved in the pathogenesis of female infertility. An imbalance in the production of ROS could also be harmful. The effects of OS on female fertility are a topic of great interest, both scientifically and clinically. Several studies have found that follicular fluid ROS play a role in ovarian aging and, as a result, oocyte quality ^{[80][98][108][109][110][111]}[80,98,167,168,169,170]. When accompanied by hyperglycemia, IR causes OS and lipid peroxidation, affecting steroidogenesis and follicular development. OS may be one of the causative factors of female infertility, originating many degenerative changes to the oocytes during aging ^[107][124]. There is a delicate balance of oxidants and antioxidants in an oocyte and its surrounding environment, and any disruption can compromise its competence. FF forms the immediate microenvironment of the developing oocyte and is the best medium to assess OS marks. Some studies showed that antioxidants increase dominant follicle selection and the cytoplasmic maturation of MII oocytes, impring embryo development. However, the correlation between ROS and the total antioxidant capacity (TAC) concentrations in the FF and embryo quality is not yet clear. It is possible to measure the levels of ROS, TAC, and lipid peroxidation [33]. 8-Isoprostane (8-IP) (Figure 1a) is a highly sensitive, chemically stable, and quantifiable marker of OS in PCOS. It is a lipid peroxidation marker that can be measured using immunoassays. In addition, 8-IP is a prostaglandin F2-like compound formed by the free radical-catalyzed peroxidation of phospholipid-bound arachidonic acid, a pathway not involving cyclooxygenase. Its formation is modulated by antioxidant status, which increases in response to oxidant injury. Lipid peroxidation is caused by the free radical attack. Lipid peroxidation is a self-propagating phenomenon that is terminated by antioxidants, and the measurement of lipid peroxidation products is commonly used to assess OS. Women with PCOS have higher median 8-IP values in their FF. Pregnant women with PCOS who had carried abortions had significantly higher levels of 8-IP. As a result, 8-IP may predict a higher risk of miscarriage in PCOS women ^{[33][107][112]}[33,124,171]. Fabjan and co-workers ^[107][124] found that 8-hydroxy-2’-deoxyguanosine (8-OHdG) (Figure 1b) in the FF is a good predictor of oocyte fertilization and maturation in PCOS patients. This OS biomarker concentration is significantly lower in these women. In addition, 8-OHdG is an oxidized deoxyguanosine derivative, one of the most common oxidative modifications in mutagenic damage. Guanosines are easily oxidized, and this reaction can result in G:C→T:A mutations. These mutations have the potential to cause serious consequences. Special DNA repair machinery typically recognizes and excises oxidized bases. High ROS levels stimulate the expression of antioxidant enzymes, reducing the extent of oxidative stress and, as a result, preventing ROS interactions with DNA and decreasing 8-OHdG formation. Several studies found that major antioxidant enzymes are significantly increased in PCOS patients ^{[107][113][114][115]}[124,172,173,174]. The concentration of 8-OHdG in the PCOS group was linked to a mature egg and its successful fertilization. However, more research is required before 8-OHdG can be considered a PCOS biomarker ^[107][124].

3.2. Lipids

The FF’s composition includes diverse lipids. Abnormalities in these may generate diseases such as hyperandrogenemia, obesity, and IR. Meanwhile, obesity has a significant impact on lipid metabolism. Several biomarkers have shown that lipids are associated with molecular processes in normal-weight PCOS patients, such as inflammatory processes and endoplasmic reticulum stress in the FF. These will endanger oocyte nuclear maturation [2]. Free fatty acids are also considered critical molecular indicators, and several spectroscopy-based studies have found that the concentrations of these molecules in the FF of women with PCOS are also altered. Free fatty acids might derive from abnormal lipid metabolism, and by modulating gene expression, they influence cell growth, differentiation, and metabolism. The composition of fatty acids in the oocyte and their concentration in the surrounding environment may impact oocyte developmental competence and subsequent embryo implantation in mammals. It has been proposed that abnormal levels of these metabolites induce multiple endoplasmic reticulum stress markers that are harmful to mammalian oocytes [116]. As referenced before, lipid abnormalities can also be associated with IR. This specific condition might regulate the expression of glucose transporters in GCs, reducing glucose uptake in oocytes and delimiting the resources available for energy metabolism ^[117][118][112,175]. Defective glucose transportation and IR in PCOS patients induce alternative energy pathways that produce altered FF concentrations bioproducts such as lipids, amino acids, and ketone bodies [116]. Related to lipid metabolism, Chen et al. ^[119][122] studied the concentrations of 7β-Hydroxycholesterol (Supplementary Material Figure S1a, supplementary could be found in https://www.mdpi.com/2227-9059/10/6/1254#supplementary) through LC-MS. 7-Hydroxycholesterol is an oxysterol, a bioactive metabolic intermediate and a product of cholesterol metabolism. Changes in 7-Hydroxycholesterol levels may cause OS, leading to fatty acid metabolism dysfunction. As a result, low levels of 7-Hydroxycholesterol in the FF of PCOS women may relate to a disrupted microenvironment for the growth of the oocytes. In addition, Ban and co-workers detected many species of phosphatidylethanolamines (PE) (Supplementary Material Figure S1b) in the FF of the PCOS patients using LC-MS [2]. PE is a major phospholipid class in the membranes of eukaryotic cells, creating a non-lamellar structure and modulating the membrane fluidity ^[2][120][2,176]. Cordeiro and their research team analyzed FF samples from PCOS patients who underwent IVF and had a hyper response to gonadotropins. Higher levels of PE were also characteristic of the PCOS group. This presence may be involved in the final process of cell division due to high proliferation in response to ovarian stimulation. The lipidomic analysis was performed by electrospray ionization mass spectrometry, and the biomarkers were analyzed by Electrospray Ionization MS/MS ^[120][176]. Phosphatidylinositol (PI) (Supplementary Material Figure S1c) is another subclass of lipids that is highly present in PCOS patients with average weight. PI presents a glycerol backbone, two esterified acyl chains, and an inositol ring linked by a phosphate that can also be detected by LC-MS. Although PI constitutes only 5–10% of total cellular lipids in mammalian cells, it is the source of seven phosphorylated derivatives that play vital roles in many cellular functions, such as signaling, membrane trafficking, ion channel regulation, and actin dynamics ^[2][121][2,177]. Thus, the detection of PE and PI may come from membrane structures of sub organelles or vesicles who suffered cell apoptosis [2]. Another study discovered low levels of 1H-Indol-3-ylacetyl-myo-inositol (Supplementary Material Figure S1d) through UHPLC-MS, another derivative from Indole [84]. Multiple reaction monitoring (MRM) metabolomic analysis of FF revealed that PCOS patients have a different lipid profile ^[122][123][123,166]. The presence of phosphatidylcholine (PC) (Supplementary Material Figure S1e) in human cumulus cells (hCC) was linked to LH from IVF cycles, implying that PC might relate to optimal oocyte development and proper LH response ^[123][124][166,178]. As a result, PCOS women have higher levels of LH in their FF ^[66][123][66,166]. Sphingolipids are cellular membrane structural components. They can function as signaling molecules, second messengers, or paracrine regulators of genetic transcription. These molecules also have the potential to regulate cell growth, proliferation, metastasis, apoptosis, senescence, immune responses, and chemo/radio-resistance ^{[84][123][125]}[84,166,179]. The decrease of these compounds in PCOS patients’ FF might indicate changes in the proper process of steroidogenesis ^[123][166]. As the center lipid, ceramide (Supplementary Material Figure S1f) can be hydrolyzed from glucosylceramide (Supplementary Material Figure S1g) and synthesized from sphingomyelins (SM) (Supplementary Material Figure S1h), sphingosine, or galactosylceramide. Glycerophospholipid metabolism interconnects with the sphingolipid metabolism by the synthesis of SM from PC and ceramide ^[84][126][84,180]. Glucosylceramide was transferable to lactosylceramide, an interconnecting compound linked to glycosphingolipid biosynthesis ^[84][127][84,181]. Regarding the sphingolipid’s pathway, Liu and co-workers detected a decrease in ceramides, galabiosylceramide (Supplementary Material Figure S1i), glucosylceramide, SM, lactosylceramides, and tetrahexosylceramide (Supplementary Material Figure S1j) levels in the FF, using UHPLC-MS [84]. In the glycerophospholipid pathway, using UHPLC-MS and LC-MS, differences were observed between controls and PCOS patients. Lysophosphatidylcholines (lysoPCs) (Supplementary Material Figure S1k), lysophosphatidyl ethanolamines (lysoPE), and glycerophosphocholine were found to be up regulated in PCOS FF ^{[84][106][116][119][127][128][129]}[84,106,116,122,181,182,183]. LysoPCs have been correlated with apoptosis, inflammation, and glucose regulation ^{[84][130][131]}[84,184,185]. The reduced levels of PCs, phosphatidylglycerolphosphate (PGP) (Supplementary Material Figure S1l), lysophosphatidic acid (LPA) (Supplementary Material Figure S1m), and triglyceride (TG) (Supplementary Material Figure S1n), also detected by UHPLC-MS, lead to lower fertilization rates. TG acts as an energy supplier and tends to be present in high concentrations due to the oocyte maturation process [84]. PGP is an important precursor of cardiolipin, which is present in the mitochondrial membrane. Glycerol biosynthesis may arise from high lipolysis that occurs due to oocyte maturation, later originating TG and PGP ^[84][132][84,186]. Liu and their research team observed that LysoPE, lysoPC, and PC were highly associated with age and BMI. There was a positive correlation between lysoPE and the internal secretion parameter LH/FSH in all FF samples, while there was a significantly positive correlation of PC and LH/FSH [84]. Increased concentrations of glycerolipids containing stearic acid residues (Supplementary Material Figure S1o) in the FF of PCOS patients, on the other hand, might connect to the IVF clinical findings. The ovaries of IVF patients synthesize a large amount of estradiol. Its esterification by the E2-acyl-CoA acyltransferase produces stearates, which are then metabolized into fatty acid esters of estradiol ^[122][133][123,187]. According to studies, fertilization failed human oocytes contain more stearic acid than palmitic, oleic, linoleic, and eicosapentaenoic acids (Supplementary Material Figure S1p–s, respectively). The concentrations of oleic and stearic acid are associated with oocyte developmental competence, which may account for the decreased pregnancy rate in women with PCOS ^{[116][117][122][134][135][136]}[112,116,123,188,189,190]. Previously, a GC/MS metabolomics approach involving PCOS and IVF demonstrated the presence of different fatty acids in the FF. An increase in palmitoleic and oleic acids was correlated with embryo fragmentation, deficient developmental competence of embryos, and consequent poor IVF pregnancy outcomes [30]. However, through multiple reaction monitoring (MRM)-profiling, Cordeiro and co-workers did not find oleic acid as a compound of increased abundance, and palmitoleic acid was related to better outcomes ^[122][123]. Sun and collaborators resorted to LC-MS to study the concentrations of both acids, finding higher quantities in the FF of obese PCOS. They also found low FF levels of lysoPCs and phytosphingosine (Supplementary Material Figure S1t), and high levels of eicosapentaenoic acid [116]. Liu and colleagues discovered high levels of pyruvic, citric, isocitric, stearic, and palmitic acid using GC/MS ^[137][115]. The levels of lithocholic and sinapinic acid, on the other hand, were significantly lower. Pyruvate and isocitric acid are crucial intermediates in the tricarboxylic acid cycle, a metabolic pathway that involves sugar, lipid, and amino acid metabolism ^{[4][137][138][139]}[4,115,191,192]. Sinapinic acid is a cinnamic acid derivative with strong anti-diabetic properties that can also prevent the formation of hydroperoxides by preventing lipid oxidation. As a result, the significant decrease in sinapinic acid may be related to IR, as well as abnormal lipid metabolism, the tricarboxylic acid cycle, amino acid biosynthesis, the glucagon metabolic pathway, and fatty acid biosynthesis, all of which have a significant impact on metabolic changes in PCOS patients with IR ^[4][59][140][4,59,193]. Reduced triglyceride (TG) levels in the FF are strongly linked to lower fertility rates in PCOS. Increased BMI, on the other hand, is correlated with FF presenting higher TG levels. So far, TGs are the lipid subclass that presents the largest discrepancies between healthy and PCOS women. TGs are composed of three fatty acids and glycerol, a crucial power supply. Patients with PCOS frequently have dyslipidemia, presenting high LDL and TG and low HDL levels ^[2][141][2,194]. TGs in the FF were also associated with high levels of adipokines and proinflammatory cytokines, implying inflammatory processes. As a result, increased TG levels may correlate to poor oocyte quality in PCOS patients. TG accumulation in the FF was also correlated with high adipokines and proinflammatory cytokines, implying inflammatory processes. Therefore, increased TG levels might be associated with the low quality of oocytes in PCOS patients. These were determined by LC-MS [2]. Li and co-workers performed LC-MS to study the concentrations of another fatty acid, arachidonic acid (AA) (Supplementary Material Figure S1u), and its metabolites. The aim of this study was to decipher the role of local AA metabolism in the FF of non-obese PCOS patients that underwent IVF ^[142][117]. Some studies related to non-PCOS patients had already stated that high levels of AA in the FF were detrimental for oocytes ^[142][143][117,195]. The levels of AA metabolites generated via cyclooxygenase-2 (COX-2) (PGI2, PGE2, PGD2, PGF2α, TXB2, PGJ2, and 15d-PGJ2) and cytochrome P450 epoxygenase (8,9-DHET and 11,12-DHET) pathways, but not lipoxygenases, were significantly higher. The metabolites generated via the COX-2 network were significantly correlated with the levels of testosterone and fasting insulin in serum. Insulin played a crucial role in the increased AA metabolites generated via COX-2, which could be interpreted as another novel molecular pathophysiological mechanism of PCOS ^[142][117]. AA-derived metabolites, especially prostaglandins (PGs), play key roles in female fertility. PGE2, PGF2α, and PGJ2 were found to be elevated in the FF of PCOS women. PGE2, an autocrine and paracrine mediator, boosts the release of luteinizing hormone-releasing hormone (LHRH). It affects oocyte maturation, cumulus expansion, and cumulus-oocyte coupling. When present in high concentrations, it might also be damaging, delaying follicle maturation. PGF2α is critical for ovulation once it increases collagenolysis and ovarian contractility. The increase in the PGF2α level in the ovary may serve to overcome the inability to ovulate properly in patients with PCOS ^{[142][144][145][146]}[117,196,197,198]. PGJ2 levels are directly correlated with serum insulin and testosterone. Since PGJ2 is not stable in vivo, it is converted to cyclopentenone PGs such as 9-deoxy-Δ9,12,13,14- dihydro PGD2 (Δ12-PGJ2), and 15d-PGJ2 ^[142][147][117,199]. The last one is an endogenous ligand of peroxisome proliferator-activated receptor gamma that acts as an inflammatory regulator and controls GCs proliferation, steroid hormone biosynthesis, and fibrosis ^{[142][148][149][150][151][152]}[117,200,201,202,203,204]. Many studies have tried to find a correlation between the role of insulin on COX-2, but so far without success. Insulin can increase COX-2, IL-1β-induced COX-2, and PGE2 production ^[153][154][205,206]. However, some studies stated opposite relations, with insulin decreasing COX-2 expression ^[155][156][207,208]. Therefore, the AA metabolites in the FF specifically reflect local ovarian state. It is of notice that gonadotropin stimulation also upregulates PGs levels. Hyperinsulinemia could also exaggerate the induction role of inflammation, stimulating GCs to produce more PGs ^[142][117].