By-products of O2 metabolism, detoxification mechanisms and the role of oxidative stress

Mitochondrial oxidative phosphorylation is the final stage of cellular respiration, following glycolysis, pyruvate oxidation and the citric acid cycle. During oxidative metabolism, O2 is employed as the terminal electron acceptor for mitochondrial electron transport chain (ETC). In the mitochondrial matrix O2 is reduced into H2O by the respiratory chain. However, one-electron reduction of molecular oxygen (O2) which happens because of electron leakage through respiratory chain complex I and III, will generate highly reactive molecules known as reactive oxygen species (ROS). Therefore, free radicals derived from oxygen (ROS) are a product of aerobic cellular respiration [10, 11].

The most common toxic radical species include superoxide anion, oxygen radical, hydroxyl radical, alkoxy-radical, peroxyl radical, nitric oxide and nitrogen dioxide [12, 13]. The non-radical species such as hydrogen peroxide, hypochlorous acid, hypobromous acid and other species, can induce cellular damage after being converted into more aggressive radical species [13, 14]. The most important ROS derived from enzymatic and by a non-enzymatic reaction is superoxide anion [15]. Superoxide anion is rapidly dismutated to H2O2 either in the matrix by manganese superoxide dismutase (MnSOD) or in the intermembrane space by Cu/ZnSOD. Hydrogen peroxide (H2O2) is further degraded to O2 and H2O by antioxidant enzymes as catalase (CAT) and glutathione peroxidase (GPx). Reduced glutathione (GSH) acts as peroxide scavenger and its oxidised form (GSSG) can be further reduced by glutathione reductase (GR) using NADPH as substrate [16]. H2O2 is a key messenger molecule in redox signaling but at higher concentrations can induce oxidative damage [16, 17]. The mitochondria, peroxisomes, endoplasmic reticulum, endothelial cells, neutrophils, macrophages, xanthine oxidoreductase and myeloperoxidase are endogenous source of ROS generation [18,19,20]. In addition, the exogenous sources of ROS production include air pollution, alcohol, tobacco smoke, heavy metals, industrial solvents, pesticides, gamma and UV radiation as well as certain drugs [21]. Aerobic eukaryotes have developed antioxidant defense mechanisms which act by mitigating damaging effects of excessive ROS production, hence under physiological conditions there is a dynamic balance between the ROS generation and elimination [22]. The oxidative stress is a consequence of excessive ROS production, reduced antioxidant capacity and mitochondrial dysfunction [22, 23]. When oxidative stress (OS) occurs, macromolecular homeostasis can be substantially affected because of lipid peroxidation, protein modifications and DNA oxidation by free radicals [23,24,25].

At physiological levels ROS modulate cell cycle and proliferation, activate angiogenesis, enable phagocytosis, activate antioxidant genes and pro-inflammatory cytokines [22, 24, 26, 27]. Low, physiological levels of ROS are necessary for self-renew of stem cells while increased ROS levels activate proliferation, differentiation, senescence and apoptosis of stem cells in concentration-dependent manner [28]. Detrimental effects on living organisms arise when ROS, exceed basal levels needed for cell signaling and transduction [22]. Therefore, the beneficial effects of ROS are achievable within physiological levels and deviation from redox window (as oxidative or reductive stresses) [29] will be associated with different pathologies including neurodegenerative diseases [30] diabetes [31] cardiovascular disease and atherosclerosis [32] rheumatoid arthritis [33] respiratory disease [34] carcinogenesis [35] ageing [36] female reproductive disease, pregnancy complications and recurrent pregnancy loss [37].

Oxidative stress (OS) and oxidative biomarkers in recurrent pregnancy loss (RPL)

Oxidative biomarkers are important tool in measuring oxidative stress in clinical samples in different pathologies. Common biomarkers measured in oxidative stress research include hydrogen peroxide (H2O2), hydroxyl radicals (OH−), peroxyl radicals (ROO−), protein carbonyl content (PC) as a marker of oxidative modifications of proteins, malondialdehyde (MDA) as a marker of ROS-mediated damage of membrane lipids, 8-hydroxyguanosine (8-OHG) as a RNA damage product and 8-hydroxydeoxyguanosine (8-OHdG) as a biomarker of oxidative DNA damage [38].

Early pregnancy is characterized by increased level of polymorphonuclear leukocyte count, which in turn contribute to the oxidative stress because of superoxide generation from these primed leucocytes [39].

In women with recurrent abortions increased generation of radical species from leukocytes was demonstrated via increased granulocyte spontaneous chemiluminescence when compared to the reference group [40].

NADPH-oxidase found in polymorphonuclear leukocytes, present a major source of superoxide generation in early and term pregnancy [41, 42]. NADPH oxidases (Nox) are a family of isoenzymes found in neutrophils, vascular smooth muscle cells and placenta [43, 44]. Placental isoform or human placental NADPH oxidase has different properties compared to neutrophils and macrophages [43].

Evidence suggest that NADPH oxidase is the main source of superoxide generation before 10 weeks of gestation when chorionic villi are exposed to relative hypoxia [41, 45].

A study investigating NADPH oxidase activity and antioxidant capacity in placental tissues from early and term pregnancies showed corresponding increase of antioxidant capacity and Nox activity emphasizing the role of superoxide production in modulation of antioxidant defense in early pregnancy [42].

Altered balance between oxidants and antioxidants can trigger oxidative stress in human placenta and has important implications in the etiology of RPL.

Studies investigating oxidants, consistently reported increased levels in plasma and placental tissues of recurrent miscarriage patients. Superoxide anion radical (SOA) is the most widespread ROS and an imbalance in the homeostatic concentrations of superoxide anion and H2O2 can lead to the production of hydroxyl ions. Significance of this highly reactive free radical is that can cause severe cell injury by reacting with organic and non-organic molecules [16]. A recent study reported significant increase of SOA in plasma samples of RPL patients compared to the control samples (45.2 ± 6.10 nmol/ml vs 35.3 ± 5.45 nmol/ml). Similar difference was observed for the placental tissue SOA between the two groups (5.93 ± 0.78 µmol/min/mg in RPL patients and 4.67 ± 0.62 µmol/min/mg in healthy pregnant women) [8].

Another parameter of oxidative stress assayed in plasma and placental tissue is H2O2. Two studies assessing this biomarker yielded similar results. Ghneim et al., reported increased levels of plasma H2O2 in RPL group compared to healthy women and non-pregnant women (7.4 ± 0.70 pM; 6.70 ± 0.70 pM; 6.20 ± 0.70 pM; respectively) [9]. Comparably, Al-Sheikh and coauthors found increased levels of this marker in placental tissues of recurrent miscarriage patients 3.38 ± 0.46 nmol/min/mg in RM group and 2.41 ± 0.35 nmol/min/mg in healthy pregnant subjects [8].

Importantly, increased levels of SOA and H2O2 in both studies were associated with depletion of enzymatic antioxidants such as superoxide dismutase (SOD) catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPx) as well as decreased expression of all examined antioxidant genes (GPx, GR, SOD and CAT) [8, 9].

The superoxide dismutase (SOD) is one of the main antioxidant enzymes and its activity is regarded as a first line of defense in preventing oxidative stress (OS). There are three isoforms of SOD including Cu/ZnSOD defined as SOD1, MnSOD or SOD2 and extracellular enzyme, or SOD3. A study conducted to examine SOD1 and SOD2 activities in samples of RPL patients and controls found interesting results. SOD1 plasma activity was 5.43 ± 0.69 nmol/min/ml in recurrent miscarriage group and 6.11 ± 0.73 nmol/min/ml in healthy controls. Similar results were observed for SOD2 plasma activity in study and control group (4.50 ± 0.55 nmol/min/mg vs 5.12 ± 0.66 55 nmol/min/mg) [46]. Furthermore, intracellular hsSOD1 transcripts were downregulated by 54% in placental tissue of women with RPL. In this study increased levels of SOA were associated with concurrent decrease of SOD1 and SOD2 activity in plasma and placental tissues of women with RPL [46].

Yiyenoglu et al., conducted a prospective controlled study to evaluate oxidative stress markers and observed increased oxidative stress index levels and decreased antioxidant capacity in women with a history of RPL [47].

Therefore, oxidative stress associated with deficient antioxidant defense is regarded as one of the key factors in the etiopathogenesis of RPL.

It should be emphasized that reactive oxygen species (ROS) and reactive nitrogen species (RNS) can act together to induce cellular injury. Nitric oxide (NO) is nitrogen based-reactive species and as membrane permeable radical can react with superoxide to form peroxynitrite, an agent with oxidizing and nitrating properties [48]. Therefore, NO can mediate ROS-induced lipid peroxidation (LPO). In particular polyunsaturated fatty acids (PUFA) are less resistant to free-radical attack because of the abundance of double bonds in their structure [49]. Malondialdehyde (MDA) is toxic end-product of autoxidation of polyunsaturated fatty acids (PUFA) and important index of oxidative damages. As a result of lipid peroxidation several membrane functions are disturbed including membrane permeability, fluidity and enzyme activity [48].

Studies evaluating the level of oxidative stress by assessing malondialdehyde (MDA) as a product of lipid peroxidation, have found elevated plasma and placental levels of this marker in patients with recurrent pregnancy loss (RPL). Reported plasma MDA levels were 5.95 ± 0.91 pM for recurrent miscarriage patients and 5.12 ± 0.81 pM for healthy controls [9]. Similarly in another study, placental MDA levels were highly significantly increased in recurrent miscarriage subjects compared to the controls (334 ± 45.8 nmol/g wet weight versus 258 ± 35.7 nmol/g wet weight) [8].

A case–control study investigating the MDA levels in RPL patients, healthy pregnant and non-pregnant women found a significant elevation of this marker in the study group. Additionally a highly significant difference was observed in the MDA/vitamin E ratio between recurrent miscarriage group and healthy controls [50].

El-Far et al., investigated MDA levels in women with idiopathic recurrent abortions. They reported significantly increased MDA levels and NO production in the study group compared to the control groups (p < 0.05 for each comparison). The authors conclude that possible oxidative damage due to the increased generation of oxidative species and diminished antioxidative defense may be responsible for recurrent abortions [51].

Although, mechanism by which oxidative stress (OS) contribute to anticardiolipin antibodies (aCL) formation is not fully clarified, a study conducted by Ferro et al., demonstrated a mean decrease in aCL titer and reduced rate of thrombin generation in patients treated with non-enzymatic antioxidants. In this study all patients treated with antioxidants showed a significantly increased plasma levels of vitamin C and vitamin E as well as a mean decrease in aCL titer by 61% (range 16–84%) [52].

Another study evaluating extent of somatic DNA damages and oxidative stress conditions found a statistically significant increase in the mean micronuclei frequency (MN). MDA levels were significantly increased in women with recurrent pregnancy loss when compared to the control group (1.63 and 0.66 respectively) [53]. Collectively, these studies highlight the role of oxidative stress (OS) in recurrent pregnancy loss (RPL).

Antioxidant defense mechanisms and recurrent pregnancy loss (RPL)

Because generation of reactive oxygen species (ROS) is the result of the aerobic cellular respiration and ATP production, living systems have developed necessary defense mechanisms against oxygen toxicity to maintain a delicate homeostatic balance between oxidant and antioxidant state. Antioxidant defense system involves enzymatic and non-enzymatic agents. Endogenous enzymatic antioxidants are the first line of defense and include matrix manganese superoxide dismutase (MnSOD), intermembrane cooper/zinc superoxide dismutase (Cu/ZnSOD), glutathione reductase (GR), glutathione peroxidase (GPx), and catalase (CAT). Ceruloplasmin, transferrin, ferritin and albumin are non-enzymatic antioxidants in the blood plasma [54]. Natural non-enzymatic antioxidants are represented by vitamin A, vitamin E, vitamin C, polyphenols, uric acid, flavonoids, carotenoids, glutathione, bilirubin and melatonin [55]. Metallothionein has antioxidant properties because of the presence of the thiol groups (–SH) and melatonin is effective hydroxyl radical scavenger [54]. Authors investigating antioxidant enzyme activity in the placenta, demonstrated strong correlation of antioxidant activity with gestational age and oxygen concentration within the placental tissues [56]. Data from this study revealed increased activity and increased expression in the mRNA concentration of the antioxidant enzymes CAT, GSH and Cu/ZnSOD with gestational age and with the oxygen tension in the intervillous space [56]. Thus the ensuing conclusion was that at the end of first trimester, establishment of maternal circulation is associated with burst of oxidative stress even in normal pregnancy and has important role in normal placentation [56]. Increased oxygen concentration and diminished antioxidant capacity will result in impaired or abnormal placentation and early pregnancy failure. In normal pregnancies intervillous circulation is initiated in the peripheral regions of the placenta and is fully established during the second trimester. In abnormal pregnancies, premature onset of the maternal placental circulation secondary to reduced trophoblastic invasion, induces oxidative damage to the villous trophoblast and is a key factor in early pregnancy loss [57]. Association of miscarriage and pregnancy with increased oxidative stress was further supported with the results reported by Jenkins et al. In this study normal term pregnancies were associated with increased superoxide dismutase (SOD) levels early in the first trimester while miscarriage group had significantly reduced levels of SOD [58]. Glutathione (GSH) is hydrosoluble antioxidant and comprises three amino acids: glycine, cysteine and glutamic acid. It functions in conjunction with glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione oxidase (GOx) [55]. Glutathione (GSH) and glutathione peroxidase (GPx) are efficient protective antioxidants responsible to maintain redox homeostasis [59]. The activity of GPx depends on the presence of reduced glutathione (GSH), which is major cellular redox buffer in cells and it is oxidized by GPx. Oxidized glutathione (GSSG) is reduced back to GSH by glutathione reductase (GR). Thus, the reducing intracellular environment is maintained by the GSH/GSSG redox couple to ensure low physiological ROS levels [59]. A study evaluating enzymatic antioxidant levels in healthy pregnant women, non-pregnant controls and women with recurrent pregnancy loss (RPL) demonstrated significantly decreased glutathione (GSH) and glutathione reductase (GR) levels, low GSH/GSSG ratio and increased oxidized glutathione (GSSG) levels in plasma and placental tissues of women with RPL compared to healthy controls. Reported plasma glutathione reductase (GR) activity was 1.59 ± 0.20 U/l for RPL patients and 1.83 ± 0.26 U/l for healthy pregnant controls. Authors reported a similar reduction of placental GR activity in the RPL group when compared to the healthy controls (0.36 ± 0.05 U/mg protein against 0.75 ± 0.10 U/mg protein). Placental tissue glutathione (GSH) levels were also significantly decreased in the recurrent miscarriage group compared to the control group (5.80 ± 0.77 nmol/mg tissue vs 6.70 ± 0.71 nmol/mg tissue). Additionally, in this study plasma GSH/GSSG ratio was 52.5 ± 6.77 in non-pregnant controls, 48.3 ± 5.99 in healthy pregnant women and 42.1 ± 5.86 in recurrent pregnancy loss patients [9]. Considering the role of GSH as a ROS scavenger GSH/GSSG ratio is important biomarker of oxidative damage [60]. Al sheik et al., examined levels of enzymatic antioxidants in non-pregnant, healthy pregnant and RPL women. Plasma glutathione peroxidase (GPx) levels in RPL patients and healthy pregnant women (HP) were 0.86 ± 0.10 nmol/min/ml and 1.06 ± 0.13 nmol/min/ml, respectively). Similar pattern was observed for placental tissue GPx in RPL group and healthy controls (0.28 ± 0.04 µmol/min/mg protein and 0.36 ± 0.04 µmol/min/mg protein, respectively). Placental tissue of RPL patients revealed highly significant decreased levels of catalase (CAT) (0.70 ± 0.10 µmol/min/mg protein in RPL group and 0.91 ± 0.12 µmol/min/mg protein in healthy pregnant group). Decreased activity of enzymatic antioxidants GPx and CAT was associated with low GSH/GSSG ratio in the study group [8]. The glutathione S-transferase (GST) families are believed to exert a critical role in protection against OS by detoxifying DNA, catechol products and oxidized lipids, generated as a result of oxidative damage [61]. GST izoenzymes catalyze conjugation of reduced glutathione and act as Selenium-independent GSH peroxidases against organic hydroperoxides [62]. Studies investigating the relation between the RPL and gene polymorphisms of glutathione S-transferase (GST) M1 and T1 suggest increased risk of RPL in women with GSTM1 null polymorphism [63]. Therefore, administration of N-acetylcysteine (NAC), a source of sulfhydryl (SH) groups and acetylated precursor of reduced glutathione, appears beneficial in oxidative stress conditions associated with decreased GSH. Evidence suggest clinical benefits of NAC use, in management of idiopathic RPL [64]. Micronutrients selenium (Se), zinc (Zn), cooper (Cu) and manganese (Mn) are cofactors for the enzymatic antioxidants and have a crucial role in antioxidant defense and ROS scavenge. Zinc, magnesium, cooper and selenium deficiency are associated with pregnancy complications, preeclampsia, premature delivery, fetal growth retardation and low birth weight [65]. Selenium is incorporated into the glutathione peroxidase, thioredoxin reductases and selenoprotein-P [