Role of Oxidative Stress and Carnitine in PCOS Patients

Abstract

Polycystic ovary syndrome (PCOS) is a common female endocrine and reproductive system disorder which is found in 6–10% of the female population. PCOS is considered a multifactorial metabolic disease characterized by several clinical manifestations, such as hyperandrogenism, polycystic ovaries and ovulatory dysfunctions. PCOS patients have an increase in the oxidative stress with generation of excessive amounts of reactive oxygen species (ROS) and reduction of antioxidant capacity. Oxidative stress is defined as the imbalance between the production of free radicals and the ability of the organism to defend itself from their harmful effects damaging the plasma membrane, DNA and other cell organelles, inducing apoptosis. Oxidative stress markers are circulating significantly higher in PCOS patients than in healthy women, so these can be considered as potential inducers of the PCOS pathology. Therefore, the central role of the oxidative stress may be involved in the pathophysiology of various clinical disorders including the PCOS. This chapter reviewed the role of oxidative stress and carnitine in PCOS patients, indicating the beneficial action of the carnitine pool, and L-carnitine contributes to restore the energy balance to the oocyte during folliculogenesis and maturation, which represent an important strategy to improve the intraovarian environment and increase the probability of pregnancy.

1. Introduction

Polycystic ovary syndrome (PCOS) is a common female endocrine and reproductive system disorder which is found in 6–10% of the female population [1].

In general, it is considered a multifactorial metabolic disease characterized by several clinical manifestations such as hyperandrogenism, polycystic ovaries aspects on ultrasound and ovulatory dysfunctions which makes it the most common cause of anovulation infertility in women, but also from metabolic problems such as obesity, insulin resistance, hyperinsulinemia and type II diabetes which may enhance cardiovascular complications and other neurological and psychological implication such as anxiety and depression [2, 3].

The carnitines are essential in the metabolism of fatty acids and can act to protect from mitochondrial damage and altered energy balance conditions such as those present in polycystic ovary syndrome (PCOS) as also highlighted by the reduced levels of L-carnitine in the serum of patients with this disease.

Restoring the energy balance and adequate energy reserves to the oocyte during folliculogenesis and maturation can represent an important strategy to improve the intraovarian environment and increase the probability of pregnancy. In this context, metabolic compounds, such as carnitines, with positive effects on mitochondrial activity and free radical scavenging, can contribute to mitigate the effects of PCOS.

2. Clinical remarks of PCOS

In the last decade there has been a plenty of discussion regarding the pathogenesis of PCOS, the causes are not yet known, but environmental and genetic factors may be involved [4, 5].

In particular, the genetic abnormalities appear to play a key role in the metabolic complications with a high rate of hyperandrogenism and type II diabetes in first degree relatives of women with PCOS [6, 7].

Recently, some studies have indicated that a defect in insulin action could be the primary cause of PCOS [8, 9].

Other studies have instead observed how important the role of socio-economic status and unhealthy life style, which includes smoking, poor diet, poor exercise and obesity [10, 11]. Furthermore, other studies have suggested that ethnic background may also be associated PCOS probably due to the increased number of insulin resistance and type II diabetes in this population [12, 13]. Polycystic ovary syndrome is the most common cause of menstrual irregularity leads to infertility and it is estimated that 90% of anovolution cases are caused by PCOS [14].

In addition to endocrine and reproductive clinical findings, PCOS also leads to consequences on mental health. Studies showing the correlation between PCOS and reduced quality of life [15, 16] with the increase in anxiety and depression [17]. This is not surprising, since the main phenotypes of this syndrome (obesity, infertility and hirsutism) are major problems that can cause psychological stress. Neuroendocrine dysfunction in gamma-aminobutyric acid (GABA) signaling and neuronal androgen receptors that might alter hypothalamic sensitivity and lead to an impairment of estradiol and progesterone feedback. Elevated concentrations of GABA in the cerebrospinal fluid of women with PCOS, GABA seems to exert an excitatory effect on GnRH neurons and this leads to greater secretion of LH by the pituitary gland, as occurs in PCOS [18, 19].

The metabolic implications of PCOS increase the risk of cardiovascular complications in PCOS patients [20]. Chronic anovulation in PCOS patient may lead to endometrial hyperplasia increasing the risk of endometrial cancer. Obesity, insulin resistance and type 2 diabetes associated to PCOS will enhance the risk of endometrial cancer in PCOS patients [21, 22].

PCOS patients have an increased risk of type II mellitus [23], in addition, insulin resistance plays a central role in the pathogenesis of PCOS [24] as it provokes hyper-insulinemia and accelerates the over-production of androgens in the ovary. Hyper-insulinemia which, in turn, contributes to the development of diabetes and dyslipidemia [25].

3. Oxidative stress in PCOS

Central role of the oxidative stress may be involved in the pathophysiology of various clinical disorders including the PCOS.

PCOS patients have an increase in the oxidative stress with generation of excessive amounts of reactive oxygen species (ROS) and reduction of antioxidant capacity [26].

Oxidative stress is defined as the imbalance between the production of free radicals and the ability of the organism to defend itself from their harmful effects damaging the plasma membrane, DNA and other cell organelles, inducing apoptosis [27]

Oxidative stress markers are circulating significantly higher in PCOS patients than in healthy women, so these can be considered as potential inducers of the PCOS pathology [28].

4. Advanced glycation end products (AGEs)

Among the most important post-translational modifications is the non-enzymatic modification of proteins, lipids and nucleic acids with glucose and their consequent conversion into AGEs.

AGEs (advanced glycation end products) therefore represent the final products of a chemical process known as the Maillard reaction, in which carbonyls of glucose or other reactive sugars react non-enzymatically with amino groups of proteins. The further reorganization of which leads to the formation of the Amadori product: the proteins containing this product are known as glycated proteins and the process of formation is known as glycation. Depending on the nature of these glycation products, protein adducts or protein cross-linking are formed, giving rise to the AGEs [29].

The end product of this reaction (AGE), in turn, induce oxidative stress and accelerate the Maillard reactions ultimately leading to inflammation and the propagation of tissue damage [30, 31, 32].

Advanced glycation end-products (AGEs) such as glycated hemoglobin commonly used in clinical practice as a marker of hyperglycemia is an Amadori product implicated in the development diabetes mellitus [32].

AGEs can be taken exogenously, through the consumption of food and smoke, or produced endogenously. In fact, in physiological conditions, AGEs are formed very slowly while, in particular conditions like hyperglycemia, insulin resistance, obesity, aging, oxidative stress and hypoxia, their formation process is accelerated [33].

Any accumulation of AGE is associated with various diseases, such as diabetes mellitus type 2, metabolic syndrome, cardiovascular diseases, ovarian aging, neurodegenerative disorders, obesity and PCOS [33, 34, 35].

Once formed, AGEs can damage cellular structures through a number of mechanisms, including the formation of cross-links between key molecules of the basement membrane of the extracellular matrix and interaction with receptors on cell surfaces, leading to this way to alteration of cellular function [36, 37].

However, the AGE content in the body is not defined only by their rate of formation, but also from rate of removal. The body cells in fact have developed pathways of detoxification against the accumulation of AGE [38].

The interaction between circulating AGEs and RAGE (receptor for AGEs) will trigger and enhance the pro-inflammatory state, cell toxicity cell and damage Figure 1 [40].

Figure 1.

Advanced glycation end products and their relevance in female reproduction [39].

RAGE is a transmembrane receptor and is expressed in numerous tissues including ovaries, heart, lung and skeletal muscle, but also in monocytes, macrophages and lymphocytes [41]. In physiological conditions this receptor is down-regulated while with aging its expression increases, probably due to the accumulation of ligands which, through positive feedback, regulate the expression of receptor itself [40, 41, 42].

In conditions like diabetes, inflammation, atherosclerosis and PCOS, there is a marked induction of RAGE due to the action of ligands and the numerous mediators activated by inflammatory cells Figure 2 [44].

Figure 2.

The role of advanced glycation end products in human infertility [43].

5. Factors that induce the production of advanced glycation end products (AGEs)

AGE levels in blood and tissues depend on endogenous sources (chemical reactions) and exogenous sources (diet and smoking). In particular foods rich in protein and fat, like meat, cheese and egg yolk, they are in fact rich in AGE, moreover, cooking methods (such as high temperatures) also increase their concentration drastically [45].

Smoke is another exogenous source of AGE and it has in fact been seen that the serum levels of AGE in smokers are significantly higher compared to non-smokers [46].

The presence of AGE in ovarian tissue, together with an altered metabolic profile and elevated testosterone levels therefore provides evidence for a double effect of the AGE taken with the diet on reproductive and metabolic function [39].

6. Correlation between AGEs and feature of PCOS

PCOS has been defined as a disorder due to an excess of androgens and insulin resistance, about 50–70% of women with PCOS have a certain degree of resistance to insulin, which is defined as a state in which more insulin is required than the normal to obtain an appropriate response [47]. Besides contributing to PCOS-associated hyperandrogenism, insulin resistance is also linked the development of impaired glucose tolerance and type 2 diabetes mellitus [48], in both obese and non-obese women with PCOS [49]. It is unclear whether hyperandrogenism is the result of hyperinsulinemia or vice versa [50]. Both insulin-like growth factor 1 (IGF-1) and insulin are potent stimulators of production of ovarian androgens, an action probably mediated by the insulin receptor [50, 51], furthermore, it is possible that the increase of circulating insulin levels potentiate the effect of luteinizing hormone (LH) on cells of the ovarian theca. Another mechanism of possible hyperandrogenism observed in the PCOS is the insulin-mediated inhibition of sex hormone binding globulin, which results in an increase of free androgens [52].

Since oxidative stress and inflammation are closely associated with insulin resistance, it is conceivable that the AGE-RAGE system may play a role in pathogenesis of insulin resistance observed in PCOS [30], regardless of circulating glucose levels, weight and obesity. A study conducted by Cai et al. has in fact identified the AGEs as a risk factor for insulin resistance independent of over-nutrition in non-obese mice [53], with such insulin resistance that occurred before changes in blood glucose levels. Additionally, recent work on overweight women reported that a low AGE diet improves insulin sensitivity [54].

About 30–75% of women with PCOS are obese [55] and such patients are likely at more risk to suffer from severe consequences than PCOS, such as hyperandrogenism and metabolic syndrome, compared to patients with a normal BMI [1, 56]. Moreover, it has been shown that modest weight loss regulates menstruation, improves reproductive performance and hirsutism, reduces serum androgen and insulin levels and improves the index of insulin sensitivity in women with PCOS [1].

In addition, the distribution and morphology of adipose tissue appear to contribute significant to the pathophysiology underlying PCOS: most affected women in fact, it presents an abdominal distribution of adipose tissue (central obesity) independent of BMI, which is an effect probably associated with the high amount of circulating androgens [56].

Circulating AGEs correlate with indicators inflammation, such as C reactive protein (CRP), and with oxidative stress [57]. In addition, the accumulation of AGE in the tissues induces cellular oxidative stress and promotes inflammation, thus increasing the vulnerability of the target tissues [58]. The dietary restriction of AGE, in fact, is associated with a significant reduction in inflammatory markers, such as plasma CRP, TNF-α (tumor necrosis factor-α) and VCAM-1 (vascular cell adhesion molecule-1) [59]. Furthermore, AGEs are directly correlated with the physiology of adipocytes as AGEs may also stimulate adipogenesis [60].

Patients with classic PCOS phenotype show alterations in the follicular fluid intermediate metabolites and the cumulus cells have an increase in oxidative stress, which causes the alteration of processes of follicular growth and oocyte development, causing the reduction in the pregnancy rate [61].

7. AGE-RAGE system in serum and ovarian PCOS

Insulin resistant women with PCOS without hyperglycemia have elevated levels serum of AGE and the expression of RAGE in circulating monocytes [44]. Furthermore, serum AGE levels are positively correlated with levels of testosterone, free androgens and insulin [50]. An increase in the serum AGE levels suggesting that serum AGEs are high in PCOS regardless of the presence of insulin resistance [62]. Recent studies have also shown that RAGE and AGE-modified proteins are expressed in human ovarian tissue [35, 63]. Specifically, women with PCOS have an increase in the expression of AGE and RAGE in the theca and granulosa cell layers, compared to healthy women [34, 64].

The AGE-RAGE system may be responsible for the failure of ovulation characteristic of PCOS: in a model of human cell lines of granulosa, observed that the AGE interfere in vitro with the action of LH leading to altered follicular development and therefore the dysfunction ovulatory associated with PCOS [65]. The AGEs within the ovary alter glucose metabolism and the folliculogenesis, the AGE could be responsible for the reduction of glucose uptake by granulosa cells, with consequent alteration of follicular growth [66].

The relationship between the AGE-RAGE system and infertility was also documented: AGEs have a negative effects on the reproductive outcome in women undergoing ART (assisted reproduction technology), AGE high levels in NON PCOS women appear to be related to the decrease in ovarian reserve and abnormal folliculogenesis. The pathological significance of these inflammatory AGE molecules, which are harmful to the follicles, clearly requires further investigation, but the identification of specific AGEs could offer potential therapeutic options for treating the decreased response ovarian Figure 3.

Figure 3.

Relationship of the AGE-RAGE system with PCOS and infertility [39].

Intra ovarian dyslipidemia is probably a consequence of the changes associated with the metabolism in the follicles [67]. In addition, the exposure of cumulus-oocyte complex to a high lipid concentrations are known to have negative influences on oocyte maturation [68].

8. Role of Carnitine in PCOS and female infertility

Levocarnitine (L-carnitine) plays a central role in the cellular energy metabolism as it is an essential molecule for the transport of long-chain fatty acids across the internal mitochondrial membrane. It was first isolated in 1905 in bovine muscle [69] and only the L isomer is bioactive. The carnitines as a whole they belong to a special class of nutrients called “quasi-vitamins” or “Conditionally essential” nutrients [70]. L-carnitine can be synthesized endogenously or taken with the diet, in particularly through meat and dairy products [71], hence its homeostasis reflects the balance between endogenous biosynthesis, absorption from the diet and renal reabsorption [72].

Numerous clinical studies have reported that the administration of L-carnitine (LC) and/or acetyl-L-carnitine (ALC) alleviates some effects of PCOS resulting in an increase reproductive outcome [73, 74, 75, 76, 77].

Both LC and ALC are commonly used in reproductive biology to improve mitochondrial function in the treatment of female infertility [78, 79]. Specifically, ALC is predominantly used for its antioxidant and anti-aging effect, while the use of LC to promote capacity of the body to oxidize fat cells to produce energy and burning fat [80]. LC also prevents DNA fragmentation induced by the harmful actions of free radicals [81].

Numerous studies have indicated that administration of L-carnitine (LC) and its acetylated form, acetyl L-carnitine (ALC) improves conditions such as PCOS [73], endometriosis [82] and amenorrhea [83]. In addition, carnitines increase levels of gonadotropins and sex hormones, as well as improve oocyte health Figure 4 [83].

Figure 4.

(a) Molecular structures of L-carnitine and acetyl-L-carnitine, (b) systemic and reproductive functions of L-carnitine. CoA, coenzyme A; ER, endoplasmic reticulum; FFA, free fatty acid; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor [84].

The administration of ALC instead increases the serum levels of other reproductive hormones such as estradiol, progesterone and luteinizing hormone (LH) and decreases prolactin [83, 85]. Hence, through their indirect endocrine effect, carnitines can prevent PCOS, amenorrhea and other pathological conditions related to the reproductive cycle female.

LC and ALC also affect the hypothalamic-pituitary-gonadal (HPG) axis, inducing secretion of reproductive hormones [83, 85, 86]. Among the neural centers, the concentration of LC is higher in the hypothalamus [87]. LC reduces the death rate of nerve cells and the damage associated with aging [88], thanks to its cholinomimetic activity [89]. It also increases the secretion of gonadotropin-releasing hormone (GnRH) from part of the hypothalamus, inducing the depolarization of hypothalamic nerve cells to increase its secretory activity Figure 5 [90, 91].

Figure 5.

Mechanism of L-carnitine action on female fertility [84].

Regarding PCOS, Samimi et al. observed that supplementation with LC (250 mg oral L-carnitine supplementation for 12 weeks) leads to a significant reduction in body weight, body mass index and waist and hip circumference decreasing blood glucose levels and favors the contrast of insulin resistance [73], which may be attributed to the increase in β-oxidation of fatty acids and the metabolic rate base line induced by LC [74].

As women with PCOS present also an imbalance between male and female hormones as their ovaries tend to produce androgens in excessive quantities, such phenomena of hyperandrogenism and/or insulin resistance in non-obese women with PCOS may be associated with the lowering of serum levels of LC [75]. Recent studies based on mass spectrometry confirmed altered fatty acid levels and carnitine in the serum of PCOS patients [92].

9. Carnitine and human assisted reproduction

Due to their beneficial effects on female fertility, carnitines have been used in numerous in vitro studies focused on improving the health and maturation of oocytes, embryonic development in assisted reproduction, in fact they allow to reduce the delay in embryonic development due to ROS, the fragmentation of the DNA and the development of an abnormal blastocyst due to prolonged culture [93, 94].

It has been observed that during oocyte development the cumulus-oocyte complex (COC) plays an essential role in lipid metabolism and therefore in energy production: therefore, in the oocyte, the maintenance of a correct lipid metabolism without or with the minimum generation of free radicals is necessary to preserve its quality [95].

LC is essential for maintaining cellular energy balance and to reduce oxidative stress [96] and to minimize cell death by apoptosis [97], which is necessary for adequate growth of the oocyte and for the maturation of the blastocyst. LC promotes the lipid metabolism of the cumulus-oocyte complex (COC), which is one of the main regulators of oocyte maturation, by transferring the fatty acids in the mitochondria and facilitating their β-oxidation [95].

Carnitine also has an anti-inflammatory effect as the integration of diet with LC decreases the anti-proliferative effect induced by the presence of interleukins such as TNF-α and its detrimental action reducing the consumption of glucose of the embryo in its early development [98] and reducing growth of the inner cell mass and trophoectoderm in the blastocyst, which leads to delayed embryonic development and reduction of vitality of the embryo [99, 100].

It has been observed that the integration of the culture medium with ALC stabilized the mitochondrial membrane, increased the energy supply to the organelles and protected the developing embryo preventing its fragmentation [101]. Furthermore, the integration of the culture medium with LC in addition to showing anti-apoptotic effects, increases the rate of development of blastocysts [97].

Supplementation of the culture medium with LC during the in vitro maturation of the oocytes favored the acquisition of competence for development, as it improved cytoplasmic and nuclear maturation and reduced ROS levels in the culture medium, showing an antioxidant effect [102].

Furthermore, women with endometriosis have a marked increase in TNF-α concentration in the granulosa cells [103, 104, 105, 106], which leads to the reduction in the size of the inner cell mass and in the proliferation of the trophoectoderm in the blastocyst, it was observed that the integration of the culture medium with LC allowed to neutralize the antiproliferative effect of TNF-α and to limit DNA damage during embryo development [93]. LC also had a protective effect against oocytes and embryos against the toxic effects of peritoneal fluid in women with endometriosis, reducing apoptosis levels in embryos and enhancing the microtubular structure [107].

Another typical feature of PCOS is chronic anovulation and the standard approach for the treatment of women with anovulation infertility consists of administration of clomiphene citrate to induce ovulation, however, some women fail to ovulate despite taking increasing doses of clomiphene citrate and, therefore, defined as clomiphene citrate-resistant PCOS. The administration of clomiphene citrate together with LC increases both the ovulation and pregnancy rate in women with clomiphene citrate-resistant PCOS. In addition, the integration with L-carnitine increases the number of follicles capable of ovulating (diameter ≥17 mm), and oocyte maturation, as well as serum levels of estradiol and progesterone [76].

An alternative treatment to induce ovulation in patients with citrate-resistant clomiphene PCOS consists of therapeutic treatment with gonadotropins; however, some of these women do not respond to both treatments, the addition of LC to therapy stimulates the growth of dominant follicles, favoring the pregnancy rate, it also increases the average thickness of the endometrium and the size of ovarian follicles [77].

10. Conclusion

The beneficial effects of carnitines on the reproductive system and ovarian function as well the differential action of the carnitine pool. The carnitines are essential in the metabolism of fatty acids and can act to protect from mitochondrial damage and altered energy balance conditions such as those present in polycystic ovary syndrome (PCOS). The L-carnitine contributes to restore the energy balance and provide adequate energy reserves to the oocyte during folliculogenesis and maturation and can represent an important strategy to improve the intraovarian environment and increase the probability of pregnancy. In this context carnitines, with positive effects on mitochondrial activity and free radical scavenging, can contribute to mitigate the effects of PCOS.