Fig. 6.1
Glutahtione (GSH) and redox regulation in the oocytes
NO is a vasodilator that mediates function of endothelial cells by regulating the vascular tone, platelet aggregation, leukocyte adhesion, and smooth muscle development [51]. Anion superoxide (·O2 −) is another important free radical in PE. Reperfusion causes oxidative damage after ischemia via conversion of xanthine dehydrogenase (XD) to xanthine oxidase (XO). Also, the metabolism in an ischemic tissue involves ATP breakdown to hypoxanthine (HX). Xanthine and hypoxanthine can be converted to uric acid by XO. XO can also convert oxygen to ∙O− and H2O2 [44]. This scenario becomes more complicated in the placenta where iron needs to be restricted from passing to the syncytiotrophoblast and is stored in the tissue until its concentration increases in the maternal circulation. Metals including iron produce pro-oxidants named hydroxyl radicals (∙OH) by Fenton reaction (Fig. 6.2) [52].
Fig. 6.2
Fenton reaction. Molecular oxygen can be metabolized by different enzymes to form superoxide anion (O−·), which then undergo dismutation forming hydrogen peroxide (H2O2). In the presence of metal (e.g. Fe2+), Fenton reaction occurs leading to the formation of hydroxyl radical
A different source of superoxide is NADPH oxidases. It has been shown that the NADPH oxidase NOX1, triggered by the angiotensin II cascade, is highly expressed in PE placentas and causes inflammation. Lastly, mitochondria are equally essential during pregnancy to maintain the metabolic function of the placenta. But under pathological conditions it turns out to be another source of superoxide. Mitochondrial dysfunction has a potent effect on both fetal and placental growth and function. Noticeably, oxidative stress in PE has a role in the development of endothelial dysfunction [44].
In pregnancy, NO production is stimulated by NO synthase (NOS), which has three different isoforms, namely, neural (nNOS), endothelial (eNOS), and inducible NOS (iNOS). In the vascular endothelium eNOS is expressed to maintain NO synthesis and the vascular tone, but also inhibits the inflammatory state caused by leukocytes and platelets adhesion [45]. Elevated levels of ROS appear to suppress eNOS [53]. Moreover, NO is scavenged by ROS forming peroxynitrite (ONOO−). This peroxynitrite impedes different vascular signaling pathways. Also, it oxidizes DNA, proteins, and lipids. The increase in both ROS and ONOO− are believed to be the cause of the reduction in the availability of NO and a possible cause of endothelial dysfunction. This can be a the mechanistic basis of the underlying etiology of PE [45]. eNOS is important at the cellular level to maintain the vascular function in pregnancy. High levels of free radicals impair eNOS function leading to vascular dysfunction, which characterizes PE.
The treatment strategy for patients with preeclampsia remains experimental and there is no well-known intervention for this disease. Although enzymatic antioxidants might be effective in preventing the development of preeclampsia, studies with the use of vitamins C and E have not yield conclusive results [54].
6.5 Menopause
Maternal age is directly related to oocyte quality. Age related reproductive dysfunctions, including menopause, are associated with decreased oocyte quality, increased mitochondrial DNA (mtDNA) damage, and chromosomal aneuploidy [55, 56]. Typically, as women age her body fat, blood lipids, and iron stores increase [3]. Hormonal changes in menopausal women include a markedly elevation of pituitary gonadotropins and decrease in the levels of anti-Mullerian hormone [57]. The increase in LH and FSH can increase estrogen biosynthesis in the corpus luteum. In post-menopausal women, estrogen exert a concentration dependent either pro-oxidant or anti-oxidant roles [58]. Estrogen deficiency prevents its antioxidant protective effect. Although estrogen helps in the prevention of bone turnover by inhibiting osteoclast formation and function, in menopausal women macrophages produce TNF-alpha increasing osteoblast and osteoclast formation that results in bone turnover [59].
It has been suggested that oxidative stress is exacerbated in menopausal women partially due with impaired antioxidant reserve [59]. A healthy diet and antioxidant supplementation may improve antioxidant levels of post-menopausal women.
6.6 Endometriosis
Endometriosis is a chronic, benign, inflammatory disease characterized by the growth of ectopic endometrial glands and stroma, often implanted in the ovaries and the cul-de-sac, but that can also be found in the lungs, urinary tract and the central nervous system [3, 60, 61]. Patients presenting with endometriosis can be asymptomatic or have a diverse range of symptoms as well as present with infertility.
Infertility and chronic pelvic pain are the two most prevalent clinical symptoms of endometriosis, however, dysmenorrhea, dyspareunia and bladder/bowel symptoms can also be manifested [62]. It is an estrogen-dependent disease that is classified into three types: peritoneal, ovarian and rectovaginal septum endometriosis (deep endometriosis). Depending on the location of the ectopic endometrial tissue in the abdomen, each is a distinct type of disease and differs from the other in the pathogenesis and presentation. Lesions of peritoneal endometriosis appear in the peritoneum whereas those of ovarian endometriosis present in the ovaries. Deep endometriosis is considered the most chronic and invasive form of endometriosis since infiltration of endometriotic tissue is required [63]. Many studies have shown that endometriosis affects 10 % of reproductive aged women, greater than 33 % of women with chronic pelvic pain and 5-50 % of women with infertility [60, 64–66]. The gynecological disorder of endometriosis is common affecting almost seven million of US women [67].
Although many studies report high prevalence of endometriosis, its pathogenesis remains unclear and it is therefore considered a multifactorial disease, which could result from a genetic predisposition. Among the known factors of endometriosis; coelomic metaplasia, metastatic spread, retrograde menstruation and altered immunity are the leading theories behind the causation of endometriosis [62]. The coelomic metaplasia theory, proposed in the 1960s explaining endometriosis at distant sites, is based upon the theory of presence of endometrial stem cells in the peritoneum. These stem cells undergo metaplasia to develop lesions of endometriosis [68]. Also, the presence of endometrial implants in the pelvic cavity could be explained by the theory claiming that menstrual tissue is transported from the uterus to the pelvis through lymphatics and veins [62]. On the other hand, retrograde menstruation is the most accepted theory behind the etiology of endometriosis, clearly explaining the presence of endometrial tissue in the peritoneal cavity as a result of its backflow through the fallopian tubes [3, 62]. Ectopic endometrial tissue then develops its new blood supply and proliferates in the pelvic/peritoneal cavity. Higher volume of refluxed menstrual blood has been reported in women with endometriosis than in control [69]. Nevertheless, retrograde menstruation cannot be considered as a definitive cause of endometriosis because it has been seen that the occurrence of retrograde menstruation is indistinguishable in women with and without endometriosis [62]. Furthermore, women with deficient cell mediated immunity have a higher chance of developing endometriosis as a result of retrograde menstruation. In these women, leukocytes are not capable of differentiating the ectopic endometrial fragments from the reflux therefore failing to clear it out. The cytotoxicity of natural killer cells against endometrial cells was found to be significantly lower in patients with endometriosis compared with controls explaining their contribution to the development of the disease [70]. The other important cause linked to endometriosis is oxidative stress as discussed below.
6.6.1 Biomarkers of Oxidative Stress (OS) in Endometriosis
Oxidative stress (OS) also plays a significant role in the development and progression of endometriosis. Patients suffering from abundant retrograde menstruation and defective immune response are more prone to iron accumulation in the peritoneal cavity as a consequence of the increase number of erythrocytes in the reflux. An abundance of iron decreases the capacity of ferritin to store it, leading to its accumulation. This in turn leads to the production of free radicals, which act as a catalyst in the Fenton reaction [71].
During normal cellular metabolism, reactive oxygen species (ROS) are generated and antioxidants present in the body prevent their toxicity. However, an imbalance between the generation of ROS and antioxidants can lead to oxidative stress, a factor contributing to the pathogenesis of endometriosis. Pathological levels of ROS have been reviewed by Defrere et al. and Agarwal et al. and were found to contribute to the regulation of Nuclear Factor-κβ (NF-κβ) -a transcriptional factor involved in the progression of the disease. NF-κβ stimulates the release of pro-inflammatory cytokines like IL-6, TNF-α, and IL-β, which helps in the recruitment of phagocytes (Fig. 6.3). It is an established fact that phagocytes are the major contributors in the production of ROS [3, 71]. The link between high levels of ROS and NF-kB has been reviewed by Gupta et al. [72].
Fig. 6.3
Productions of ROS in the peritoneal environment. Increase in peritoneal fluid, leukocytes lead to release of growth factors and cytokines (including regulated on activation, normal T-cell expressed and secreted (RANTES)); which then induce an inflammatory response (by activating different cells e.g. monocytes, T-cells), resulting in ROS production
Furthermore, higher levels of 8-iso-prostaglandin F2- alpha (8-iso-PGF2-alpha) were identified in the urine and peritoneal fluid of women with endometriosis. This study, conducted by Sharma et al., found that an increase in levels of 8-iso-PGF2-alpha resulted in lipid peroxidation and thus, oxidative stress. The measurement of 8-iso-PGF2-alpha can be used as a biomarker for oxidative stress in patients with endometriosis [73].
6.6.2 Proteomics and Oxidative Stress in Endometriosis
The role of proteomics has been explored to provide a linkage between oxidative stress and endometriosis [74]. Prieto et al. measured the levels of superoxide dismutase (SOD), an antioxidant enzyme, in the follicular fluid of women with endometriosis, and found a decrease in SOD leading to oxidative stress [75]. Higher levels of Afamin, a vitamin E binding protein, were detected in the peritoneal fluid of patients with endometriosis, which caused a decrease in the scavenging role of antioxidants leading to oxidative stress [76]. While this study indicated an increase in a protein contributing to oxidative stress in patients with endometriosis, another study noted a decrease in hemopexin, a protein responsible for inhibiting the production of reactive oxygen species [77]. These two findings show a clear correlation between oxidative stress and endometriosis. Heme binding protein was also found to be lower than normal further underlining the relevance of oxidative stress in relation to endometriosis [63]. Thioredoxin binding protein (TBP2) plays a role in the regulation of thioredoxin (TRX), an antioxidant responsible for cell proliferation and apoptosis. TBP2 stimulates apoptosis in cases of increased levels of ROS. Decreased levels of TRX and TBP2 were noted in a study that measured their levels in the peritoneal fluid of women with endometriosis. The following results marked a decrease in antioxidant activity as well as cell apoptosis explaining the development of oxidative stress related endometriosis [78].
Heat shock proteins (HSP) are chaperones stimulating cell proliferation by inhibiting apoptosis. They are found in low concentration under normal physiological states, but accumulate in conditions where cells undergo physiological stress [3]. Elevated levels of HSP70, a member of the HSP family, were measured and found to lead to increased proliferation of ectopic endometrial cells present outside the pelvic cavities [79]. In a systematic review, it has been suggested that these oxidants released as a result of an increase in HSP70 influence the progression of ectopic endometrial cells by stimulating pro-inflammatory cytokines [3]. Regardless of its increase in the aforementioned study, the role HSP contributes to oxidative stress related endometriosis is still unclear [74]. Contradictory reports in the literature have shown that levels of HSP90, another member of the HSP family, were evaluated in ectopic endometrial tissue and showed an increase in one study [80] but a decrease in another [81].
6.6.3 Metabolomics and Oxidative Stress in Endometriosis
Metabolomics is the study of metabolites in relation to the etiology and pathophysiology of disease. Proton Nuclear Magnetic Resonance Spectroscopy Based Targeted Metabolite Profiling (NMR) was used to measure the levels of OS biomarkers and detect metabolite imbalances in patients with endometriosis to further comprehend the causation of their disease. Among the 135 women recruited for the study, 55 % were diagnosed with endometriosis and comprised the study group whereas 45 % with tubal factor infertility were used as controls. The inclusion criteria strictly allowed only those who did not receive any medical or hormonal treatment for the past three months to participate in the study. The exclusion criteria was any history of chocolate cysts being removed, pelvic tuberculosis and gynecological surgeries like abdominal or lower pelvic surgeries. Patients were asked to present fasting and in their early follicular phase, and venous blood was drawn and the following parameters were measured in the serum: levels of reactive oxygen species (ROS), total antioxidant capacity (TAC), lipid peroxidation (LPO), superoxide dismutase (SOD), glutathione (GSH), and catalase. Metabolic profiling using NMR was then performed on 26 samples from the study group and 24 from the controls. An increase in ROS and LPO was noted while a decrease in the antioxidant levels of SOD, catalase and GSH were observed in the serum of the study group in comparison to the controls. A decline in the levels of GSH contributes to the biosynthesis of ophthalamate, also releasing 2-hydroxybutyrate as a by-product. Metabolic profiling displayed elevated levels of both opthalamate and 2-hydroxyvutyrate indicating oxidative stress in patients with endometriosis. Advanced oxidation protein products (AOPP), albumin aggregations impaired by OS, were also measured in the study [67]. The presence of ROS in the plasma induces oxidation of amino acids through their reaction with chlorinated oxidants finally forming AOPP [82]. These findings determine the contribution of oxidative stress to the pathophysiology of endometriosis. This study also provides a more comprehensive assessment of free radicals as well as their end products such as AOPP and how it translates into changing metabolic profile and its relationship with OS.
6.6.4 Treatment of Oxidative Stress Associated with Endometriosis
Being an estrogen dependent disease, it has been suggested that endometriosis could be approached using gonadotropin-releasing hormone agonists (GnRHa) as well as contraceptive pills, both of which generate an estrogen-deficient state. Consequently, growth of ectopic endometrial tissue is impeded and symptoms of endometriosis are relieved [61]. Furthermore, treating infertile women with endometriosis using GnRHa has proved to improve embryo implantation and to increase pregnancy rates [83]. Aromatase inhibitors have also shown to alleviate symptoms of endometriosis by hindering the activity of Aromatase P450, an enzyme responsible for the biosynthesis of estrogen in the ovaries [60]. Aromatase activity is significantly higher in patients with endometriosis in comparison to those without the condition (Fig. 6.4). Increased estrogen levels caused by aromatase P450 stimulate prostaglandin E2 further activating aromatase and progressing the development of ectopic endometrial deposits [84].
Fig. 6.4
Role of aromatase in a persistent endometriotic state
Oxidative stress, which is triggered by chronic inflammation in patients with endometriosis, also contributes to the progression of endometriosis. The increase in ROS production affects the quality of the oocytes of these patients as well as implantation explaining infertility in women with endometriosis. In a study conducted by Tamura et al., GnRHa was administered to twenty-three infertile women diagnosed with endometriosis to test its effect on oxidative stress related endometriosis and infertility. The ultralong group, consisting of eleven patients, received three courses of gonadotropin releasing hormone agonist (GnRHa), followed by a standard controlled ovarian hyperstimulation. The control group on the other hand, which consisted of the twelve patients, received one course of GnRHa in the mid luteal phase, followed by a standard controlled ovarian hyperstimulation as well. During oocyte retrieval, follicular fluid aspirated from each woman of the two groups was analyzed. Fertilization, implantation and pregnancy rates were compared between the ultralong and control groups as well. Oxidative stress markers, antioxidants and the numbers of matured follicles and retrieved oocytes were also measured and compared. Implantation rates in the ultralong group (21.4 %) as well as pregnancy rates (27.3 %) were significantly higher than those in the control group (8.3 and 8.3 % respectively). Levels of Tumor necrosis factor (TNF) α, a key oxidative stress marker in patients with endometriosis, showed a remarkable decrease in the ultralong group (5.8 ± 3.2 pg/ml) in comparison to the control group (10.6 ± 3.2 pg/ml). Melatonin, an antioxidant hormone released from the pineal gland, notably increased in the follicular fluid of the ultralong group (139.2 ± 45.7 pg/ml) when comparing it to the control group (85.6 ± 27.4 pg/ml). The study strongly indicates that the administration of GnRHa alleviates the negative impacts of oxidative stress markers in infertile women with endometriosis, leading to an increase their fertility and pregnancy outcomes [83]. Desferoxamine (DFO), an iron chelator could also be used as an effective treatment for oxidative stress related endometriosis. Iron chelators decrease the detrimental effects of oxidative stress caused by iron overload in the peritoneal cavities as a result of retrograde menstruation in patients with endometriosis. In these women, DFO must be administered directly into the peritoneal cavity using intrapelvic implants because of the heavy menstrual cycles these women suffer from. This ensures that DFO will absorb the iron overload present in the peritoneal cavity exclusively, and not further decrease the total body iron, which has already been reduced as a result of the heavy menstrual cycles in these women [71].
An antioxidant rich diet has also shown to play a role in endometriosis regression [3]. A study conducted by Mier-Cabrera et al. showed the correlation between administering vitamin E and C to patients with endometriosis and its effect on certain OS markers. After 4 months of daily supplementation with bars containing vitamins E and C, the levels of malondialdehyde (MDA) and lipid hydroperoxides in the plasma and peritoneal fluids of the study group decreased significantly in comparison to the control group. However, no improvement in pregnancy rates was reported, suggesting that despite the positive impact of antioxidants on OS biomarkers in these women, no definitive treatment has been standardized [85]. Future adequate powered randomized controlled trials are needed to study the alleviation of OS in endometriosis as well as pregnancy outcomes. Treatment needs to be individualized for patients targeted towards the type of endometriosis that each woman is suffering from.
6.7 Gestational Diabetes Mellitus (GDM)
Gestational diabetes mellitus (GDM) is a common pregnancy complication characterized by a state of carbohydrate intolerance. Due to the hyperglycemic situation these women suffer from, the pancreatic beta cells have to release more insulin to compensate, sustaining high insulin levels and developing insulin resistance [86, 87]. The prevalence of GDM has been increasing, especially in women who are obese and in those of advanced age [88, 89]. During routine screening, 2–13 % of pregnant women are diagnosed with GDM [90]. GDM is a risk factor for developing maternal conditions such as gestational hypertension, preeclampsia and cardiovascular diseases [91, 92]. These women are also prone to developing type 2 diabetes mellitus later in life. A study conducted by Teede et al. demonstrated that 26-70 % of GDM women developed type 2 diabetes mellitus 10–15 years after delivery [89]. GDM is also associated with adverse fetal and neonatal outcomes such as macrosomia, shoulder dystocia and development of obesity later in life [90, 91].
The pathophysiology explaining insulin resistance and changes in glucose tolerance in these women is still unclear; however OS was found to play a crucial role in the development of GDM [93, 94]. A study conducted by Zhu et al. reported elevated levels of plasma C-reactive proteins (CRP) in patients with GDM compared to controls. An increase in this inflammatory biomarker was found to be correlated to a decrease in the efficacy of the glutathione antioxidant system as well as an increase in lipid peroxidation [88]. On the other hand, red blood cells (RBC) possess antioxidant characteristics under normal conditions, therefore contributing to OS when the RBCs develop defects. For example, it has been noted that increased levels of physiologically modified RBCs result in lack of scavenging activity in patients with GDM, further underlining the relationship between oxidative state and the development of the disease [88, 95].
Oxidants and antioxidants are produced abundantly in the placenta and the GDM placenta was found to have increased levels of OS markers such as xanthine oxidase and malondialdehyde and decreased capacity to scavenge the free radicals [94, 96]. Furthermore, elevated levels of placental 8-isoprostane have been documented in patients with GDM compared to controls. An elevation in this lipid peroxidation marker additionally correlates OS to GDM [94].
It has been suggested that GDM develops as a result of increased intake of heme-iron supplementation, a common prescription among pregnant women [97, 98]. It was reported that the risk of developing GDM increases by 51 % with every 1 mg intake of heme-iron [99]. OS related GDM induced by heme-iron intake was found to increase lipid peroxidation, DNA damage and enhance insulin resistance [100]. Contradictory findings have been reported concerning the presence of OS biomarkers in GDM patients caused by the induction of the oxidative stress as a result of excess iron [100].
An accumulation of catalytic iron inducing OS, caused by compromised fetuses explains the variability in the results. It has been reported that enzymes such as peroxidases can be used to diminish the detrimental effects of ROS in GDM patients. Furthermore, the DASH diet has shown to decrease ROS production and treat insulin resistance in GDM [101]. Those studies demonstrate that women with GDM have decreased ability to counterbalance the oxidative state they suffer from, which is reflected by their insulin resistance. Nevertheless, further research is needed to determine effective therapeutic interventions to reduce OS, alleviate insulin resistance and treat GDM in these women.
6.8 Unexplained Infertility
A couple is diagnosed with unexplained infertility when all other reasons of infertility such as ovulatory disorders and hormonal imbalances as well as the male factor have been ruled out. Hence, it is considered an exclusion diagnosis and is more prevalent in females with advanced age [102]. Among the couples who consult for infertility treatment, approximately 15 % are diagnosed with unexplained infertility [103]. The pathophysiology behind unexplained infertility is still unclear, however OS can possibly contribute to this condition [104]. The imbalance between ROS and antioxidant levels in the peritoneal fluid of these patients is mainly due to defects in their scavenging systems. Decreased antioxidant activity of both glutathione and vitamin E could very likely be the basis of OS related unexplained infertility [105].
Furthermore, homocysteine, an amino acid triggering OS and apoptosis [106], was found to accumulate in cases of folate deficiency. Folate, a B vitamin, plays a crucial role in female reproduction [107]. Polymorphisms in folate metabolism pathways have been investigated to determine their role in the etiology of unexplained infertility. It has been reported that women with unexplained infertility have polymorphisms in the 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C/T gene [108]. This gene is required for the conversion of homocysteine to methionine through the folate methylation cycle, a process involved in the methylation of DNA, lipids and proteins [109]. Defects in this gene lead to reduced and unstable enzymatic activity resulting in hyperhomocysteinemia [110, 111]. This condition induces ROS production by inhibiting antioxidant enzymes such as glutathione peroxidase and SOD leading to elevated levels of peroxyl and superoxide radicals [112]. Hyperhomocysteinemia was also found to be associated with decreased pregnancy and implantation rates in IVF patients as well as increased abortion rates [113]. Hence, defects in the metabolism pathways of folate lead to distorted levels of homocysteine explaining the development of unexplained infertility [108].
ROS levels were also investigated in the peritoneal fluid of women with unexplained infertility. In one study, Wang et al. detected higher levels of ROS in the peritoneal fluid of patients with unexplained infertility in comparison to those with endometriosis and tubal ligation. Moreover, this elevation did not affect fertility in the other two groups, but played a significant role in those with unexplained infertility further suggesting an association between OS and unexplained infertility [105]. Increased OS has also been documented in the follicular fluid of patients with unexplained infertility undergoing IVF [114]. It has been observed that increased OS damages the oocyte’s cell membrane through lipid peroxidation. This damage leads to poor oocyte quality, which in turn impacts embryo quality [20, 115].
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