Damaging Effects Of Reactive Oxygen Species Biology Essay

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Antioxidants are compounds that protect cells against the damaging effects of reactive oxygen species such as singlet oxygen, superoxide, peroxyl radicals, hydroxyl radicals and peroxynitrite. Oxidative damage occurs to cells in vivo and in vitro from exposure to free radicals generated by exogenous agents (e.g., radiation, chemicals, hyperoxia) and endogenous processes such as normal cellular metabolism. An imbalance between antioxidants and ROS results in oxidative stress, leading to cellular damage. Oxidative stress (OS) has been linked to cancer, ageing, atherosclerosis, ischemic injury, inflammation, and neurodegenerative diseases. Under extreme oxidative conditions, or if the antioxidant protective mechanisms of cells are compromised, cellular injury and death may occur. Early mammalian embryos are susceptible to damage from reactive oxygen species, and they increase the production of oxygen free radicals when cultured in vitro. The ROS generation can result from oxidative phosphorylation occurring in the mitochondria. Electrons leak from the electron transport chain at the inner mitochondrial membranes, being transferred to the oxygen molecule, resulting in an unpaired electron in the orbit. This leads to the generation of the superoxide molecule. The other points of generation of ROS are the cytoplasmic NADPH-oxidase, cytochrome p450 enzymes and the xanthine oxidoreductase enzymes. Excessive OS can have deleterious effects on the cellular milieu and can result in impaired cellular growth in the embryo or apoptosis resulting in embryo fragmentation. Similarly to what happens in females, oxidative energy production in males is inevitably associated with the generation of ROS excessive concentrations of which can lead to cellular pathology. It has been established that ROS can function as signaling molecules and evidence is emerging that sperm may generate low and controlled concentrations of ROS, specifically O2-H2O2, as well as other species such as nitric oxide (NO), which, in his turn, act to mediate the processes of capacitation, hyperactivation and acrosome reaction crucial to the acquisition of fertilizing ability.

In the present review the most important antioxidants and their mechanisms of action related to animal reproduction, are fully discussed.

Introduction

The term antioxidant, also so called antioxygen, was originally referred specifically to a chemical that prevented the consumption of molecular oxygen. In the 19th and early 20th century, antioxidants were the subject of extensive research as these molecules are compounds that protect cells against the damaging effects of reactive oxygen species (ROS), such as singlet oxygen, superoxide, peroxyl radicals, hydroxyl radicals and peroxynitrite. An imbalance between antioxidants and ROS results in an oxidative stress (OS), leading to cellular damage. OS has been linked to cancer, ageing, atherosclerosis, ischemic injury, inflammation and neurodegenerative diseases [1, 2].

There are two types of antioxidants: enzymatic and non-enzymatic:

Enzymatic antioxidants are also known as natural antioxidants, act by neutralizing excessive ROS, prevent it from damaging the cellular structure. They are composed of superoxide dismutase, catalase, glutathione peroxidase and glutathione redutase, which also causes reduction of hydrogen peroxide to water and alcohol. Superoxide dismutase and glutathione peroxidase are natural antioxidants present in organisms which eliminate some ROS and glutathione peroxidase catalyzes the reduction of peroxide by oxidizing glutathione (GSH) to oxidized glutathione [3].

Non-enzymatic antioxidants are also known as synthetic antioxidants or dietary supplements. The body's complex antioxidants system is influenced by dietary intake of antioxidant vitamins and minerals such as vitamin C, vitamin E, selenium, zinc, taurin, hypotaurin, glutathione, beta carotene and carotene [4]. Vitamins C and E are not produced in the body but must be obtained through food. Glutathione is produced by the body, but levels of this antioxidant decline with age [5]. Research into how vitamin E (α-tocopherol and derivatives) prevents the process of lipid peroxidation, led to the current understanding of antioxidants as reducing agents that break oxidative chain reactions, often by scavenging reactive oxygen species before they can cause damage to the cells [6]. This vitamin, the predominant lipid-soluble antioxidant in animal cells, protects cells from oxygen radicals in vivo and in vitro, and is believed to be the primary free radical scavenger in mammalian cell membranes [2]. Vitamin E has been considered as a natural antioxidant that reacts with soluble free radicals in lipids membranes. Its active place locates in the group OH in the position 6 of the phenol ring. The oxidative attack that greasy acids polyunsaturated suffer by the radical OH• and O2•, produces radical peroxil (ROO•) [7]. This vitamin protects lipids from peroxidation, being oxidized to tocopheryl quinone or into tocopheroxyl free radical. In both cases, it is reduced by ascorbate (Vitamin C), which is afterwards oxidized into dehydroascorbate or ascorbate free radical.

Enzymatic and non-enzymatic antioxidants rapidly scavenge the attacking radical and thereby terminate its destructive pathways. This mechanism presumes that the resulting antioxidant derived radical is a "harmless" one, i.e., the reactivity of the antioxidant radical toward typical biomolecules must be low [8]. Moreover, the antioxidant β-Mercaptoethanol (β-ME) is a low weight thiol compound with a reducing power, interacting directly with some oxidized radicals and can chelate metallic ions. It protects cysteine, a precursor of GSH, from oxidation into cystine and increases its entry into the cell, which is known to trigger GSH synthesis [9].

Nevertheless, there are disadvantages associated to the use of antioxidant. For example, it is important to point out that antioxidant supplements are not always safe. Excessive intake of vitamins can certainly cause trouble, toxicity can occur at very high intake levels for some commonly consumed antioxidants [10, 11].

Oxidative Stress

By definition a free radical is any atom (e.g. oxygen, nitrogen) with at least one unpaired electron in the outermost shell, which is capable of independent existence. A free radical is easily formed when a covalent bond between entities is broken and one electron remains with each newly formed atom [12]. Free radical species are unstable and highly reactive, becoming stables by acquiring electrons from nucleic acids, lipids, proteins, carbohydrates or any nearby molecule causing a cascade of damage and disease [4]. There are two major types of free radical species: ROS and reactive nitrogen species (NOS).

Any free radical involving oxygen can be referred as ROS. Oxygen centred free radicals contain two unpaired electrons in the outer shell. When free radicals steal an electron from a surrounding compound or molecule a new free radical is formed in its place [13]. As above referred, the most common ROS include: the superoxide anion (O2-), the hydroxyl radical (OH•], singlet oxygen (1O2), and hydrogen peroxide (H2O2]. Superoxide anions are formed when oxygen (O2) acquires an additional electron, leaving the molecule with only one unpaired electron. Within the mitochondria O2-• is continuously being formed. The rate of formation depends on the amount of oxygen flowing through the mitochondria at any given time. Hydroxyl radicals are short-lived, but the most damaging radicals within the body. This type of free radical can be formed from O2- and H2O2 via the Harber-Weiss reaction (O2• - + H2O2 → OH• + OH- + O2• -] [14].

Hydrogen peroxide is produced in vivo by many reactions, being converted to the highly damaging hydroxyl radical or catalyzed and excreted harmlessly as water [13]. The principal problem is that H2O2 crosses easily the cellular membranes and while receiving one more electron, normally originating from the iron or copper, gives rise to the radical hydroxyl. This radical are the most reactive oxygen species, as they need only one more electron to be stabilized. Cells living under aerobic conditions constantly face the oxygen (O2) paradox: O2 is required to support life, but its metabolites such as reactive oxygen species (ROS) can modify cell functions, endanger cell survival, or both [15].

ROS have been implicated in more than one hundred diseases [16, 17], playing a particular role in the female reproductive tract as ovaries [18, 19], and even on embryos [20]. ROS is also involved in the modulation of an entire spectrum of physiological reproductive functions such as oocyte maturation, ovarian steroidogenesis, corpus luteal function and luteolysis [18], being thus related with female fertility.

Concerning Nitric oxide (NO), it is synthesized during the enzymatic conversion of L-arginine to L-citrulline by nitric oxide synthase (NOS) [21-23]. With an unpaired electron, NO, which is a highly reactive free radical, damages proteins, carbohydrates, nucleotides and lipids and, together with other inflammatory mediators, results in cell and tissue damage, low-grade, sterile inflammation and adhesions [22]. NO potently relaxes arterial and venous smooth muscles and, less strongly, inhibits platelet aggregation and adhesion. NO donors, acting as vasodilating agents, are therefore a possible therapeutic approach [24]. NO acts in a variety of tissues to regulate a diverse range of physiological processes, being, however, toxic in excess [4, 25].

Reactive nitrogen species have been associated with asthma, ischemic/reperfusion injury, septic shock and atherosclerosis [26-29]. The two common examples of reactive nitrogen species are the above referred NO and nitrogen dioxide [4, 30]. NO is produced by the enzyme NO synthase. There are tree types of nitric oxide synthase (NOS) isoenzymes in mammals involving endothelial NO synthase (NO synhase 3), neuronal NO synthase (NO synthase1) and inducible NO synthase (NO synthase 2). Neuronal NO synthase (nNOS) and endothelial NO synthase (eNOS) are constructive NO synthases, and responsible for the continuous basal release of NO. Inducible NO synthase (iNOS) is present in mononuclear phagocytes (monocytes and macrophages) and produces a large amount of NO. This is expressed in response to proinflammatory cytokines and lipopolysaccharides [25]. Inducible NO synthase is activated by cytokines such as, interleukin-1, and TNF-α and lipopolysaccharides. NOS is expressed in thecal cells, granulosa cells, and surface of oocyte during the follicular development. In pathological conditions, inducible NO synthase might play a major role in NO production. In most organs, inducible NO synthase is expressed only in response to immunological stimuli [31].

Oxidative Stress in the female reproduction

It is a given fact that perfectly normal and healthy individuals, even under basal conditions, produce ROS through their aerobic metabolism. Therefore cells have developed a wide range of antioxidant systems to limit the production of ROS, inactivate them and repair cell damage [4, 32].

In a healthy body, ROS and antioxidants remain in balance. Nevertheless, when this balance is disrupted towards an overabundance of ROS, oxidative stress occurs. ROS are a double-edged sword: they can serve as key signal molecules in physiological processes but also have a role in pathological processes involving the female reproductive tract.

Recently, OS has also been reported to have an important role in the normal functioning of the female reproductive system and in the pathogenesis of female infertility [32], influencing the entire reproductive span of a females' life. De Bruin and collaborators [33] suggested that the age-related decline in fertility is modulated by this form of stress.

During gestation OS plays a role in the initiation of preterm labor [34, 35] and during normal parturition [36, 37], assuring oocyte maturation, ovarian steroidogenesis, ovulation, blastocyst formation, luteolysis and luteal maintenance in pregnancy [19, 38]. Normal concentrations of ROS may also play a major role both in the implantation and fertilization of eggs [39]. OS operates in normal cycling ovaries, follicular development and cyclical endometrial changes. Markers of oxidative stress such as superoxide dismutase, Cu-Zn superoxide dismutase, glutathione peroxidase, γ glutamil synthetase and lipid peroxides have been investigated by immunohistochemical location, m-RNA expression and thiobarbituric acid methods [38, 40]. The expression of various markers of OS has been demonstrated in normal cycling human ovaries [19, 41]. All follicular stages have been examined for superoxide dismutase expression including primordial, primary, preantral, nondominant antral follicles, dominant follicles and athretic follicles [19] founding a delicate balance between ROS and antioxidant enzymes in the ovarian tissues. The presence of superoxide dismutase in the ovary revealed intense staining by immunohistochemistry in the theca interna cells in the antral follicles [41].On the other hand, the pathological effects are exerted by various mechanisms including lipid damage, inhibition of protein synthesis and depletion of ATP [42]. OS is also involved in the etiology of defective embryo development and seems responsible for numerous types of embryo damage. ROS such as O2- are able to diffuse and pass through cell membranes and alter most types of cellular molecules such as lipids, proteins and nucleic acids. The consequences are multiple and include mitochondrial alterations, embryo cell block, ATP depletion and apoptosis. ROS also induce lipid peroxidations with related effects in cell's division, metabolite transport and mitochondrial dysfunction. The 2-cell block observed in mouse embryos is associated with a rise in lipid peroxides [43, 44]. ROS can induce protein sulphydryl oxidation and disulphide formation. With oxidative stress, the rates of disulphide bonds and mixed disulphide formations increase within the cell. As a consequence, inactivation of enzymes such as glyceraldehydes 3-phosphate dehydrogenase (G3PDH) can occur [45]. It was also reported that ROS induce DNA strand breaks [46]: a four-fold increase in the nuclear DNA fragmentation rate is observed after spermatozoa are exposed to ROS [47]. Such nuclear DNA lesions are involved in embryo development arrest observed in vitro. OS induces mitochondrial damage [48, 49]. Mitochondrial DNA (mtDNA) is especially susceptible to mutation because of its lack of histones which normally quench ROS. During OS, mtDNA mutation is four-fold more frequent than nuclear DNA mutations. This way, defective embryo mtDNA may induce metabolic dysfunction and, consequently, disturb embryo development; arrest of embryo development observed in vitro is associated with dysfunction of mitochondria. The consequences of these alterations are multiple, and include embryo development retardation and arrest, metabolic dysfunctions and possibly also apoptosis. ROS have been implicated in the impaired development of mammalian embryos in vitro [50] [51]. The 2-cell embryo block observed in mouse embryos is associated with a rise in ROS [43, 44], which is only observed after culture in vitro. No such effect is observed in embryos collected in vivo [44]. Deleterious effects of ROS during oocyte maturation may alter embryo development [52]. ATP depletion occurs in cells via inactivation of glycerol-3-dehydrogenase (G3PDH) and/or inactivation of glycolitic and mitochondrial pathways [53]. OS induces consumption of reducing equivalents such as GSH. Glutathione reductase (GR) activity allows the GSH endogenous pool to be maintained. GR is NADPH dependent and the main source of NADPH in the monophosphate shunt (pentose phosphate pathway). Consequently, oxidative stress, via competitive consumption of reducing equivalents, can interfere with important metabolic functions and divert glucose from other pathways by inducing the monophosphate shunt.

Accumulation of superoxide radicals and a decline in SOD levels are involved in apoptotic cell death, whereas antioxidants including SOD can inhibit apoptosis. H2O2 is a mediator of apoptosis in blastocysts [54]. The appearance of cytoplasmic fragments in blastocysts seems to be related to apoptosis [55]. A direct relationship was also observed between increased H2O2 concentration and apoptosis in human fragmented embryos [56]. Fragmentation in mouse and human embryos occurs just before the time of in vitro block; such fragmentation may be a mechanism to regulate the nucleocytoplasmic ratio in blastomeres, and/or a protective mechanism against damage induced by oxidative stress.

In assisted reproduction technology (ART), the freeze-thaw process makes cells more sensitive to ROS. DNA instability is observed in mouse oocytes after cryopreservation. Furthermore, the freeze-thaw process reduces GSH concentrations by 78% and SOD activity by 50 % in bovine spermatozoa [57]. The increase in lipid peroxidation observed after sperm cryopreservation is due to a loss of SOD activity [58], which strongly suggests that oxidative stress occurs during and/or after the cycle of freeze-thaw. This way partly explain the observed deleterious effect of cryopreservation on gamete/embryo viability. Modifications of membrane lipids related to ROS and the resulting spatial modifications of membrane structures may clearly lead to cryodamage.

Nevertheless, it should be borne in mind that ROS play a physiological role in gametes and embryos. They are implicated in the control of capacitation, acrosomal reaction [59] and fertilization [60] processes, being probably implicated in regulating the speed of pre-implantation embryo development [61].

Embryos may also have different sensitivities to ROS at different developmental stages. For example, it has been observed [62] that 9 to 16 cell bovine embryos are more resistant to exogenous H2O2 than zygotes and blastocysts. These different sensitivities are due to variations in defense mechanism thresholds.

Oxidative Stress in the male reproduction

Similarly to what happens in females, oxidative energy production in males is inevitably associated with the generation of reactive oxygen species (ROS) in which excessive concentrations can lead to cellular pathology. It has been established that ROS can function as signaling molecules and evidence is emerging that sperm may generate low and controlled concentrations of ROS, specifically O2-H2O2, as well as other species such as nitric oxide (NO), which act to mediate the processes of capacitation, hyperactivation and acrosome reaction crucial to the acquisition of fertilizing ability [63]. Mild oxidative conditions resulting from low concentrations of ROS may also stimulate sperm-binding to the zona pellucida [64]. Although the precise nature and concentration of ROS varies with experimental conditions, data are converging to describe this event as oxidative or redox regulated. The balance between the timing and location of ROS production and scavenging must be respected if sperm function is not to be compromised.

Although the precise mechanism by which ROS operate as transduction molecules in sperm are yet to be elucidated, a scheme of action can be proposed. Sperm in the basal state generate low net concentrations of ROS. Incubation in capacitating conditions stimulates generation of superoxide anion (O2-) through an oxidase not yet characterized. O2- and hydrogen peroxide (H2O2) fored by dismutation of O22- can induce sperm capacitation, which is also promoted by NO. The targets for this ROS are unknown but there is a redox-regulated increase in tyrosine phosphorylation of specific proteins during capacitation. Induction of the AR in capacitated sperm stimulates O2 production which causes release of unesterified fatty acids from the plasma membrane of these cells. The ROS target for AR induction is unknown but again tyrosine phosphorylation of specific proteins is involved [63] .

The mechanism of ROS production by sperm is still unclear. An NADPH oxidase generating O2-, similar to that found in phagocytic leucocytes has been proposed [65], based on observations of the effect of NADPH addition to purified sperm using a lucigenin-based chemiluminescence assay. However, NADPH was shown not to stimulate extracellular O2- production in sperm as detected by 2-methyl-6-(rho-methoxyphenyl)1-3,7 dihydroimidazo [1,2-a] pyrazin-3-one chemiluminescence whereas addition of biological fluids inducing capacitation (e.g. follicular fluid) did so [66].

McLeod [67] first demonstrated that incubation under oxygen in vitro was detrimental to human spermatozoa, decreasing motility and viability. Since then, many reports have associated ROS with impaired sperm function including decreased motility, abnormal morphology, and decreased sperm-egg penetration [64, 68-71]. Spermatozoa are highly susceptible to damage by excess concentrations of ROS due to the high content of polyunsaturated fatty acids within their plasma membrane and, although conventional basic semen characteristics other than motility are not obviously influenced by the oxidative state of semen [65], such damage may underlie several aspects of male infertility. Increased lipid peroxidation and altered membrane function can render sperm dysfunctional through impaired metabolism, motility, acrosome reaction reactivity and fusogenic capacity as well as oxidative damage to sperm DNA [72]. Patients with asthenozoospermia (impaired sperm motility) have increased concentrations of ROS in seminal plasma and increased ROS-mediated damage of sperm membranes, although whether oxidative damage occurs in the testes, epididymis or semen is still uncertain. Abnormal sperm, characterized by retention of excess residual cytoplasm as a result of defective spermatogenesis, are a source of ROS in semen [73], but t is generally accepted thet contaminating leukocytes are the primary source of ROS in unpurified sperm suspensions [74-77].

Sperm possesses a variety of antioxidant scavenger defence mechanisms including catalase, uric acid, taurine, thiols, ascorbic acid and alpha-tocopherol but principally superoxide dismutase (SOD) and the glutathione-peroxidase-reductase system [78, 79]. Extracellular SOD binds to the neck region of a subgroup of sperm which retain motility longer than those without bound SOD and both the proportion of sperm binding SOD and total SOD activity vary widely among samples; however, any reproductive significance of this remains unknown. Despite this range of defences apparently available, it has been suggested that mature sperm may yet be inadequately protected due to their high concentration of membrane unsaturated lipids together with a relative paucity of enzymes such as SOD due to the virtual absence of cytoplasm [80]. This is partly compensated for by the powerful antioxidant system present in seminal plasma which, in contrast to other biological fluids, contains significant concentrations of SOD, xanthine oxidase, nitric oxide, catalase, glutathione peroxidase, plus ascorbic acid, thiols, uric acid, alpha-tocopherol and a high level of glutathione [81-83]. Moreover, sperm do retain functional concentrations of antioxidant enzymes despite their sparsecytoplasm [84, 85]. Sperm defences need only ensure their survival until they achieve fertilization; they are the removed fro the female reproductive tract. However, it is crucial that this balance be struck as inadequate defences lead to premature loss of function and thus impair fertility.

Oxidative stress in Assisted Reproductive Techniques

Assisted reproductive techniques (ART) involve the direct manipulation of the oocytes, sperm or embryos outside the body, to establish a pregnancy.

The follicular fluid microenvironment has a crucial role in determining the quality of the oocyte. This, in turn, impacts the fertilization rate and the embryo quality. Oxidative stress markers have been localized in the follicular fluid in patients undergoing IVF/embryo transfer (ET) [40, 86, 87]. Low intrafollicular oxygenation has been associated with decreased oocyte developmental potential as reflected by increasing frequency of oocyte cytoplasmic defects, impaired cleavage and abnormal chromosomal segregation in oocytes from poorly vascularised follicles [88]. ROS may be responsible for causing increased embryo fragmentation, resulting from increasing apoptosis [56]. Thus increasing ROS levels are not favourable to embryo growth and result in impaired development.

Increase in the total antioxidant capacity (TAC) was observed in follicular fluid of oocytes that later were successfully fertilized. Therefore, lower total antioxidant capacity is predictive of decreased fertilization potential [87]. Lower levels were associated with increased viability of the embryos until the time of transfer, and the fertilization potential decreased with decreasing concentrations of total antioxidants. Similarly mean glutathione peroxidase levels were increased, in follicles yielding oocytes that were subsequently fertilized [89]. Attaran and collaborators (2000) reported that levels of ROS are significantly lower in women who does not became pregnant when compared with those who became pregnant, indicating that intrafollicular ROS levels can be used as a potential marker for predicting success with IVF. On the other hand, high levels of superoxide dismutase activity were present in fluid from follicles whose oocytes did not fertilize as compared with those in which fertilization has been achieved [90]. Patients who became pregnant following IVF or ICSI had higher lipid peroxidation levels and TAC. Besides both markers were unable to predict embryo quality, pregnancy rates, levels of lipid peroxidation and TAC demonstrated a positive correlation. OS in follicular fluid from women undergoing IVF was inversely correlated with the women's age [91] in which the slope was found to positively correlate with maximal serum estradiol levels, number of mature oocytes and number of cleaved embryos and inversely with the number of gonadotropin ampoules used. The pregnancy rate achieved was 28% and all pregnancies occurred when the thermochemiluminescence amplitude was small. This is in agreement with another study that reported minimal levels of OS were necessary for achieving pregnancy [86]. Follicular fluid ROS and lipid peroxidation levels may be markers for success with IVF.

Oocyte quality is a very important determining factor in the outcome of IVF/ET. 8-hydroxy-2-deoxyguanosine is a reliable indicator of DNA damage caused by oxidative stress. This compound is an indicator of OS in various disease processes and high levels of this compound are associated with low oocytes's quality and consequently lower fertilization rates and poor embryo quality [92].

Other OS markers such as thiobarbituric acid-reactive substances, conjugated dienes and lipid hydroperoxides have been studied in the preovulatory follicular fluid [93], but no correlation was observed between these markers and IVF success [93]. For the above described it becomes clear that overproduction of ROS is detrimental for the embryo, resulting from impaired intracellular milieu and disturbed metabolism resulting in dramatic effects on the fetus [20, 94]. Early embryo development in mammals occurs from fertilization through differentiation of principal organ systems in a low oxygen environment [95] A marginal improvement in pre-implantation embryonic viability has been reported under low oxygen concentrations in patients undergoing IVF and ICSI [96]. Lower concentrations of oxygen in in-vitro culture of porcine embryos decreased the H2O2 content and resulted in reduced DNA fragmentation, which thereby improved developmental ability [97]. The higher oxygen concentration of 20% has been associated with lower developmental competence. Accelerated development was observed under low (5%) oxygen concentrations.

ROS may be generated endogenously or exogenously, but either way it can affect the oocyte and embryo. IVF culture media may be the exogenous site of ROS generation affecting the oocytes and the pre-implantation embryo. There are some specific events in embryo development associated with a change in the redox state. It has been suggested that redox may have a causative role in sperm mediated oocyte activation, embryonic genome activation and embryonic hatching from the zona pellucida [94]. Higher day 1 ROS levels in culture media were associated with delayed embryonic development, high fragmentation and development of morphologically abnormal blastocysts after prolonged culture. A significant correlation was reported between increased ROS levels in Day1 culture media and lower fertilization rates in patients undergoing Intracytoplasmic Sperm Injection (ICSI) [98]. Lower ROS levels were associated with higher fertilization rates, indicating the physiological relevance of low levels of ROS. Incubation of poor quality embryos was associated with a decline in TAC in the pre-implantation embryo culture medium after 24 hours incubation. Poor quality embryos may be associated with increased generation of ROS [99]. In vivo, fertilization and embryo development occurs in an environment of low oxygen tension [95]. During ART, it is thus important to avoid conditions that promote ROS generation and expose gametes and embryos to ROS. During culture, low oxygen tension is more effective at improving the implantation and pregnancy rate than higher oxygen tension [100]. Similarly, higher implantation and clinical pregnancy rates are reported when antioxidant supplemented media is used rather than standard media without antioxidants. Metal ions can sometimes result in the production of oxidants. Metal ions can also increase the production of ROS directly through the Haber-Weiss reaction. It may be useful to add metal ion chelating agents to the culture media to decrease the production of oxidants [100].

As known, amino acids added to the IVF media also have antioxidant properties. The benefits of adding such as ascorbate during cryopreservation reduces hydrogen peroxide levels and thus the oxidative distress in mammalian embryos [101], resulting in a better embryo development improving blastocyst development rates. Follicular vascularity determines the intrafollicular oxygen content and the developmental potential of the oocyte [88, 102]. Intrafollicular hypoxia results in chromosomal segregation disorders and deleterious mosaicisms in the embryo. Sildenafil, an inhibitor of phosphodiesterase enzyme, prevents the breakdown of cGMP and potentiates the effects of NO on vascular smooth muscle. Vaginal Sildenafil and L-arginine have been investigated for their potential to improve intrafollicular blood flow by potentiating the actions of NO on vascular smooth muscle. It augments the effect of NO in inducing vasodilatation and thus improving uterine blood flow. A recent study in humans reported that Sildenafil, administered on day 3 of the menstrual cycle, appeared effective in improving uterine artery blood flow and endometrial development [103].

Conclusion

A comprehensive review of the published literature reveals that the role of oxidative stress is yet quite controversial. It can be concluded that the number of articles on oxidative stress in the last 5 years have significantly increased compared to the previous 5 years indicating that more studies are being conducted to understand the role of oxidative stress in female and male reproduction. The effects of ROS studied and its ability to influence reproduction have been studied on various endpoints in terms of the oocyte, spermatozoa, fertilization, embryo development and gestation. Different markers of oxidative stress are reported in various studies and the sensitivity and specificity of the various biomarkers are yet unknown. While some research is focused on studying the antioxidant capacity, others focus on studying and determining the levels of oxidative stress markers. Further studies need to be designed to validate the results of the earlier studies, with elimination of various factors leading to bias.

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