Male Infertility, Oxidative Stress and Antioxidants

Within the male reproductive system, oxidative stress (OS) has been identified as prevailing etiology of male infertility. The effects of reactive oxygen species (ROS) on male fertility depend on the dimensions, “ modus operandi ” of the ROS and the oxido-reduction potential (ORP) of the male reproductive tract. Hereupon, for an adequate response to OS, the cells of our body are endowed with a well-sophisticated system of defense in order to be protected. Various antioxidant enzymes and small molecular free radical scavengers, maintain the delicate balance between oxidants and reductants (antioxidants), crucial to cellular function and fertility. Therapeutic use of antioxidants is an optimal and coherent option in terms of mitigating OS and improving semen parameters. Therefore, recognizing and managing OS through either decreasing ROS levels or by increasing antioxidant force, appear to be a requesting approach in the management of male infertility. However, a clear defined attitude of the experts about the clinical efficacy of antioxidant therapy is still deprived. Prominently, antioxidant such as coenzyme Q10, vitamin C and E, lycopene, carnitine, zinc and selenium have been found useful in controlling the balance between ROS production and scavenging activities. In spite of that, healthy lifestyle, without smoke and alcohol, everyday exercise, reduction of psychological stress and quality well-designed meals, are habits that can overturn male infertility.


Introduction
The World Health Organization (WHO) defines infertility as the inability (failure) to attain clinical pregnancy after one year or more of regular unprotected sexual intercourse [1]. Since infertility presents a certain disability (impaired reproductive function), medical assessment and treatment falls under the umbrella of the United Nations Convention on the Rights of Persons with Disabilities -UNCRPD, which is formally accepted by many countries. The article 1 of this Convention summarizes the overall objective as: "to promote, protect and ensure the full and equal enjoyment of all human rights and fundamental freedoms by all persons with disabilities, and to promote respect for their inherent dignity" [2]. Due to its health, cultural and socio-economic impact, infertility is a major globally underestimated public health concern [3, 4]. Therefore, proper evaluation of male infertility is a substantial stride in qualifying, quantifying and configuring necessary laboratory assessment, credential treatment strategies as well as policies to diminish the burden of this global sensitive health issue.
There are approximately 186 million infertile people [5] or 15% of couples globally, 50% due to male factor infertility which experience problems in conceiving [6,7]. In male dominated societies, generally, the female partner is blamed for barrenness, even though ancient Greeks were aware that male factor is a contributor to the reproductive success [8].
In fertile couples, spontaneous conception is most likely to occur in 30% of cases during the first month, 75% after 6 months, 90% after 12 months and 95% between 18 to 24 months [9]. Also, there are studies which consider that 80% of couples having unprotected sexual intercourse will achieve pregnancy in the 6-month [10] or 12-month interval [11].
In addition, male fertility reaches its maximum potential at ages of about 25 to 30 years and declines sharply in the beginning of fifties [12], however, there are men reported to give life to offspring into their eighties [13]. Paternal age of >40 years is associated with more than 20% higher chance of congenital defects in the offspring [14]. Over the past decades, an age-related decline in semen quality resulting in declined fertility was observed [15].
Oxidative stress (OS) has been identified as one of the major contributors affecting male fertility potential [16] and has thus been extensively studied in the last three decades. Although cells of the human body have efficient mechanisms to cope with factors disturbing the normal cell homeostasis, OS may arise due to an imbalance between generation of oxidants and antioxidants mechanisms, resulting in cell damage.
Reactive oxygen species (ROS) are important mediators of OS status, because of their capacity to oxidize proteins, lipids, and DNA, resulting in cellular dysfunction [17]. ROS are oxygen-based molecules that have unpaired electrons on their most outlier spin-orbit, derived from the reaction of carbon-centered radical with oxygen (except hydrogen peroxide), which makes them highly reactive [18]. The most common ROS are hydroxyl radical (OH•), hydrogen peroxide (H 2 O 2 ) and the superoxide anion (O 2 •-). ROS are generated not only by leukocytes (neutrophils and macrophages mostly) [19], but also by any aerobe living cell including spermatozoa [20]. Moreover, another subclass of free radicals deriving from nitrogenbased molecules are called reactive nitrogen species (RNS) [21,22]. At physiologic amount, RNS are important for various functions within the male reproductive tract such as: (1) signal transduction, (2) regulation and assembly of tight junction within the blood-testis barrier, (3) mediation of cytotoxic and pathological events, (4) production of hormones, (5) inflammation and (6) other important physiological changes of spermatozoa [23].
Some of the most common ROS and RNS are listed in Table 1. Effects, consequences, mode of formation and action of these molecules are presented in details in Table 2.
Under physiological conditions, high levels of ROS are counterbalanced by antioxidants, which competently maintain a delicate redox balance by donating their electrons to the ROS and thus interrupting further intake of electrons from surrounding compartments [37]. The seminal antioxidant system comprises a network of enzymatic and non-enzymatic molecules, dispersed mostly within seminal plasma and spermatozoa [38]. The three major antioxidant enzymes are glutathione peroxidase (GPx), catalase (CAT) and the superoxide dismutase (SOD) [39].
With an increasing knowledge on the role of OS in the clinical manifestation of male infertility, antioxidant prescription and its implementation in treating male infertility may be helpful. Several antioxidant compounds are currently prescribed without any scientific rationale, ensuing neither semen parameters improvement, nor fertilization outcomes. Contrary, some other studies even showed a worsening of semen parameters [40][41][42], because an excess intake of antioxidants can contribute in the establishment of reductive stress (RS), a condition which has been reported being as harmful as OS [43]. Therefore, there still lack of conclusive consensus regarding the clinical advantages of antioxidants -based therapy in male infertility.
Hydrogen peroxide is not a free radical, because it does not contain an unpaired electron, but it is classified as ROS because it participates in the generation of highly reactive hydroxyl free radicals through interactions with iron and copper, based on the Fenton reaction. [22,[24][25][26] It is generated by electron transport leaks from several reaction in cytosol. It does not spread easily and faraway its origin. It is responsible for cell injury, by deconstructing iron-sulphur clusters in proteins through the inactivation of iron regulatory protein-1. Superoxide is insoluble for the cell membrane.
This represents the neutral form of the hydroxide ion, deriving from the reaction between Fe 2+ and H 2 O 2 (Fenton reaction). It is the most reactive free radical. The hydroxyl radicals and hydroxide ions can be generated also by the reaction of H 2 O 2 and O 2 •catalyzed by iron (Haber-Weiss reaction). The hydroxyl radical has the potential of reacting fast and nonspecifically. [30,31] Peroxynitrite (ONOO -) Ref.
It is generated during reaction of nitric oxide (NO) with O 2 -, it can react with thio groups of structural proteins, resulting in the formation of nitrosotioles, which can disunite metal-protein interactions and result in the formation of metal-derived free radicals. [32] Peroxyl radical (ROO • ) Ref.
Peroxyl radicals remove electrons from lipids during the process of lipid peroxidation. During this process, intermediates are generated that participate in further reactions with oxygen to form lipid peroxyl (LOO•) and lipid hydroperoxide (LOOH) which are responsible for sperm DNA and protein damage. [33][34][35].
Hypochloric acid is produced by macrophages and neutrophils during respiratory burning that accompanies phagocytosis. This radical is generated in the reaction between H 2 O 2 and chloride ion (Cl À ). [36]

Oxidative stress and male infertility
OS is a condition characterized by an elevated generation of ROS and a reduced response of biological mechanisms to promptly neutralize the reactive intermediates or to repair the damage [44]. An increased quantity of ROS and RNS has now been established with strict evidence to be a prominent attribute of many acute and chronic pathologies [45].
Nearly eight decades after the Macleods discovery in 1943, highlighting ROS as key players in cell physiology and sperm motility [46], scientists all over the world turned their attention toward the association between free radicals and the male infertility.

Sources of ROS
Semen comprises a variety of cells including spermatozoa, germ cells, leukocytes and epithelial cells [47], whereby leukocytes produce about 1000-times more ROS than immature sperm cells [48].
ROS originate from a different countless endogenous and exogenous sources. Endogenous sources of ROS can be generated extracellularly and intracellularly. Intracellular ROS include O 2 -, H 2 O 2 and OH À , generated mainly in the mitochondria [49]. In the mitochondria, about 5% of the consumed oxygen is physiologically converted into ROS. The ROS production is increased when the electron transporting chain (ETC) derails as a result of mitochondrial dysfunction [50].
Exogenous sources of ROS include smoking, alcohol and drugs abuse, environmental pollutants, heavy metals, ionizing radiation, diets rich in energy-yielding nutrients like carbohydrates, saturated fats and proteins [51].

Mechanism of ROS production within human sperm
ROS are generated in two pathways: the extrinsic and the intrinsic pathway, described in Figure 1.
Leukocytes are responsible for the extrinsic pathway of generating ROS, while spermatozoa for the intrinsic pathway of ROS generation [52]. Granulocytes are the white blood cells (WBC) in seminal fluid which are predominantly responsible for demolishing pathogens by ROS production [53,54].
An association between OS and the elevated leukocyte numbers has been found [19]. On the other hand, the relationship between the seminal leukocyte concentration and male infertility is not clear. In fact, leukocytospermia, i.e. the presence of more than 1Â10 6 WBC/mL, is not predictive of male infertility [55,56]. However, the significance of WBC activation in ROS generation and its impact on elevated OS levels cannot be left unnoticed. Various studies reported high levels of proinflammatory chemokines in human semen along with high ROS quantity [57,58]. Recently, in the seminal plasma of oligozoospermic and azoospermic men it was observed a negative correlation between levels of interleukin-6 (IL-6), interferon alpha (IFN-α) and interferon gamma (IFN-γ) and sperm parameters such as concentration, motility and morphology [59,60].
Among spermatozoa, it has been shown that morphologically abnormal spermatozoa are the main source of ROS generation [61]. Excess residual cytoplasm (ERC) around the mid-piece of spermatozoa (observed in teratozoospermic sperm) contains high levels of cytoplasmic enzymes responsible for generating ROS [62].
ERC has a considerable amount of enzymes to regulate glucose metabolism, specifically glucose-6-phosphate dehydrogenase (G6PD) [63], which induces increased ROS levels by activating (1) the NADPH -nicotinamide adenine dinucleotide phosphate located in the plasma membrane of spermatozoa, and (2) NADPHdependent oxide-reductase, known as diphorase, detected in the middle piece of mitochondrial level [64][65][66]. In a study by Sabeur et al., calcium-dependent NADPH oxidase 5 (NOX5) of spermatozoa plays a considerable role in ROS generation [67]. However, there is a difference between NOX5 found in spermatozoa, which does not require protein kinase C for expressing its activity, and in leukocytes, where protein kinase C is essential [68].

Physiological role of ROS
ROS are very important molecules as they act as cellular mediators essential for (1) normal spermatogenesis, (2) activation of steroidogenic pathway, (3) modulation of mitochondrial and death receptor-apoptotic pathways. These fundamental cascades are required for the process of: maturation, hyperactivation, capacitation, acrosome reaction as well as sperm-oocyte fusion, crucial for the fertilization process, all presented in Figure 2.

Maturation
After spermiation, spermatozoa are transported into the epididymis where they undergo a maturation process, leading to significant chemical and physiological modifications including recombination of cell-surface proteins, and enzymatic and nuclear modifications [69,70]. These result in the assembly of the signal transduction machinery that is crucial for the sperm capacity to undergo hyperactivation and capacitation [69,71]. The nuclear DNA of mammalian spermatozoa is densely packed, as histones are substituted by smaller-sized (arginine-rich) protamine [72]. Protamines substitute histones during spermiogenesis [73] and compact DNA tightly through inter/intramolecular disulphide bonds between cysteine residues [74]. The oxidizing process of thiol groups on protamines and the formation of disulfide bonds increase chromatin stability and DNA protection from any physical or chemical damage [75], which is fundamental because human spermatozoa have 6 limited capability to repair DNA damage [76]. Protamination occurs when spermatozoa pass through the caput and caudal part of the epididymis [77].
Another important event is the formation of "mitochondrial capsule" made by a complex protein material, which is necessary to abolish proteolytic degradation [78].

Hyperactivation
Hyperactivation is a particular state of sperm motility characterized by vigorous, large asymmetric flagellar (whiplash-type) beat and head sperm shifting (large lateral head displacement) [79]. Hyperactivation is reported to facilitate the capacitation process and is indispensable for successful accomplishment of acrosomal reaction, sperm-egg fusion, and fecundation [74].
Undoubtedly, ROS play an inclusive role in the regulation of these processes, by triggering hyperactivation and capacitation. This occurs by induction of Ca 2+ and HCO 3 À influx, probably through the deactivation of the enzyme Ca 2+ -ATPase and further basification of the cytosol [80]. ROS (especially O 2 •-) upregulate the Ca 2+ mediate adenylate cyclase (AC) enzymatic activity, increasing cAMP (cyclic adenosine monophosphate) generation by activating protein kinase A (PKA). Further, this triggers NADPH oxidase activation and thereby promotes the upregulation of ROS production [81]. PKA-mediated phosphorylation leads to protein tyrosine kinase (PTK) activation, phosphorylating consecutive tyrosine residues in the axonemal fibrous sheath and the cytoskeleton of sperm tail [69,82].

Capacitation
Capacitation has been documented in 1951 by Austin and Chang [83,84]. Capacitation involves cholesterol outflow from the sperm membrane and a global intensification of tyrosine phosphorylation [85]. The signal transduction pathway is guided by the cAMP and modulated by the oxido-reductive state [86]. During capacitation, spermatozoa undergo molecular modifications such as alkalization of inner cell pH, activation of cAMP-dependent pathways, cholesterol efflux from cell-membrane and phosphorylation of surface proteins by cAMP-dependent kinase [87]. Researchers have emphasized the impact of free radicals in modulating the cAMP pathway, which involves PKA activation and phosphorylation of its substrates [88]. A correlation between elevated protein phosphorylation rate, increased presence of the second messengers and ROS synthesis have been observed during capacitation [69]. The cholesterol oxidation and its consequent discharge from the sperm membrane is necessary in tuning-up spermatozoa for the next step, resulting in greater bicarbonate and Ca 2+ ion permeability via activation of sodium/bicarbonate cotransporter (NBC) and ion channels [89].

Acrosome reaction (AR)
The hyperactivated spermatozoon tends to penetrate over the cumulus-oocytecomplex and attach to the zona pellucida of the egg, whereas acrosome reaction (AR) is a well-regulated exocytotic reaction in response to coordinated stimuli [90]. These changes are triggered by tyrosine phosphorylation of sperm-membrane proteins regulated by ROS signaling [63,88]. NO is implicated in AR by activating the second messenger cyclic guanosine-mono-phosphate (cGMP), PKC and protein kinase G (PKG) [91]. Physiological levels of H 2 O 2 , O 2 À and NO are needed for AR [88]. In the oocyte, the release of Ca 2+ is followed by cleavage of phosphatydilinositol-4,5-bisphosphonate (PIP 2 ) into inositol tri-phosphate (IP 3 ) and diacylglycerol (DAG), which are responsible for acrosomal exocytosis and activation of PKC. This further results in Ca 2+ inflow and activation of PLA2 (phospholipase A2), which play a key role in the cleavage of secondary fatty and consequently increasing the membrane fluidity, necessary sperm-oocyte fusion [92].

Sperm-oocyte fusion
ROS are also necessary in the finalization of the fertilization process. This final step is due to enhanced membrane fluidity, which is controlled and directed by ROS in inhibiting the protein tyrosine phosphatase activity, which prevents deactivation of PLA 2 , a necessary step for accomplishing sperm-oocyte fusion [93]. When the spermatozoon penetrates the zona pellucida and the corona radiata, the oocyte changes the composition of the vitelline layer [24]. This envelope is catalyzed by ovoperoxidase making o,o-dityrosine crosslinks to prevent polyspermy [94].

Pathological repercussions of oxidative stress
High levels of ROS have the potential to damage cellular components by mediating lipid peroxidation, apoptosis, DNA damage, mitochondrial dysfunction and protein oxidation.

Lipid peroxidation (LPO)
Sperm membranes are mostly constituted by poly-unsaturated fatty acid (PUFAs), which represents a disadvantage in terms of OS susceptibility [95].
Lipid peroxidation (LPO) is as a chemical reaction by which oxidants assault carbon double bond(s) in lipid compounds, especially PUFAs, by detaching hydrogen and adding oxygen to carbon, by generating LOO• and LOOH [96]. In vitro research highlighted a negative correlation between malondialdehyde (MDA -end product of LPO) concentration, and sperm morphology and motility [97][98][99]. LPO is a self-propagating process passing through three phases: (1) initiation; (2) propagation; (3) termination. Through all three phases free radicals enter in a radical-chain reaction [32].
The propagation of the oxidative wave can also result in DNA fragmentation and protein damage, affecting particularly sperm motility, morphology and fertilizing capacity.

Apoptosis
The programmed cell death, known as apoptosis, is a physiological phenomenon. In the male reproductive tract, apoptosis is responsible for supervising the excess production of male gametes, a process being regulated by extrinsic and intrinsic stimuli [80]. The intrinsic stimuli include apoptosis-including genes like p53, Bax and Fas, but also Bcl-2 and c-kit genes which act as apoptosis suppressors [100], while extrinsic stimuli consist of varicocele, infection, heat stress, environmental toxins, advanced male age lifestyle factors, ionizing and nonionizing radiations, defective protamination and idiopathic causes [101,102] . During the process of spermatogenesis, spontaneous germ cell apoptosis in all developing stages of spermatozoa has been seen in the testis of normozoospermic and non-obstructive azoospermic men [20]. This guarantees that only functionally and genetically competent germ cells become mature spermatozoa [103]. Prolactin and insulin are considered as pro-survival hormones which bind to specific receptors on sperm membrane [104]. The inhibition of this cascade will result in increased ROS generation by mitochondria, followed by the release of cytochrome C, which in turn activates the apoptotic caspases, triggering the apoptosis [74,82,105]. High levels of cytochrome C have been found in seminal plasma of infertile men [82,106].

DNA damage
It is reported that infertile males with high seminal OS levels present high fragmentation of sperm DNA [107]. Numerous contributors can include lifestyle factors, radiation, advanced male age, varicocele, infection and idiopathic causes [108,109]. Guanine base (G) is the most common DNA's organic base exposed to OS assault and converts into 8-hydroxy-deoxyguanosine (8-OHdG) by free radicals [110]. Mechanisms by which OS cause DNA damage involve warping single and double-stranded DNA crosslinks, direct oxidation of DNA bases and DNA mutations [111]. Comparing to nuclear DNA, mitochondrial DNA is more susceptible to DNA damage, due to the lack of histones and protamines, and nucleotide excision repair mechanisms [112].
In addition, mitochondrial damage affects the interior mitochondrial membrane, causing electron outflow from the transporting chain, inducing a further increase of OS status [113].

Mitochondrial dysfunction
Mitochondria represent the most important place in generating ATPs, which serves as a fuel for sperm to move. This is why its proper function represents a fundamental key point in the mosaic of male infertility problems. Defects in the pathway for ATP production correlate with low sperm motility, known as asthenozoospermia [114]. There is an inactivation of genes which encode constituting proteins of the electron transport chain, mainly those that are involved in ATP formation [115]. When the extent of such injury overwhelms DNA repair capacity mechanisms, the subsequent alterations in mitochondrial biology stimulate the activation of the genes responsible for stress-response, hereby inducing apoptosis [116].

Protein oxidation
Formation of radical amino acids is of the result of protein oxidation (PO), especially of the alpha-central carbon, causing disintegration of peptide skeletons [117]. Moreover, the SH-rich lateral chains of methionine and cysteine are inclined to be oxidised with propagation of methionine sulphoxide and disulphides, respectively [87]. Similarly, arginine, proline, threonine and lysine are oxidised, resulting in the formation of carbonylated proteins (aldehyde and ketones), markers of PO status [117]. These alterations impact the protein morphology and physiology, with a wide impact on spermatogenesis and fertility potential.

Antioxidants in male infertility treatment
Antioxidants are defined as chemicals compounds with the ability to donate electrons and thereby neutralize an excessive production of ROS [118]. Humans possess a well-sophisticated antioxidant system to shelter the body's cells and tissues against oxidation [119].
As a physiological response to OS, seminal plasma is endowed with various scavengers acting enzymes indexed as total antioxidant capacity (TAC) measured to be 10x higher comparing to blood plasma [120].
The anti-oxidant defense system implicates a co-action of different endo/exogenous players to scavenge the potential oxidative damage of ROS [121]. These consist of CAT, SOD, glutathione peroxidase (GPx), peroxiredoxins and glutathione-Stransferase [122], and water-soluble and fat-soluble vitamins [123]. The role and effect of endogenous and exogenous antioxidants are discussed below.

Endogenous antioxidants
The major endogenous antioxidant enzymes are: (1) CAT, (2) SOD and (3) GPx. Studies about their efficacy in clinical trials are presented in Table 3.

Catalase
Activity of catalase (tetrameric protein) is consisted in dissolving hydrogen peroxide into water and oxygen, through the oxidation of hydrogen ion donors, such as methanol (CH 3 OH), ethanol (CH 3 CH 2 OH), with the consumption of 1 mol of H 2 O 2 [128]. In addition, CAT has an important role in terms of physiological effects during sperm capacitation, inducing NO activity and the removal of ROS [129].

Superoxide dismutase (SOD)
SOD is known as metallo-enzyme, as it has the catalytic metal in the active site [130]. The SOD enzyme consists of three different classes existing in both extraand intracellular compartments. SOD-1 or CuZnSOD is the first intracellular enzyme, with Cu and Zn in the active center; it is usually localized in the cytosol [131]. SOD-2 or MnSOD is the second intracellular isoform, localizing in mitochondria and showing Mn in the active center [132]. The extracellular form of SOD (EC-SOD or SOD-3) is a glycosylated homotetramer mainly secreted into the extracellular area. It is upregulated by cytokines, downregulated by TNF-α, and anchored to the extracellular matrix [133]. CuZnSOD is highly active (75%) in comparison with SOD-3 (25%) [119,130].

Glutathione peroxidase (GPX)
GPx is a cytosolic antioxidant seleno-enzyme mainly expressed in the epididymis and testis [134]. GPx catalyzes the reduction of detrimental hydroperoxides with thiol cofactors [119]. A "catalytic triad" is formed by the selenocysteine in the active site with tryptophan and glutamine: this activates the selenium portion and Enzyme Study findings Ref.

CAT
• ↑ CAT activity in the group that received antioxidant therapy, comparing to control samples that received placebo. • Positive correlation between levels of CAT and fertilization rates.
• Studies are limited in this field. [124,125] SOD • Its levels are positively associated with sperm concentration (p<0.001) and motility (p=0.008). • Negative relationship was found with DNA fragmentation (p=0.014). [126] GPx • 10x greater GPx activity in the fertile group comparing with the GPx activity in infertile men. • Statistically significant (p<0.001).
[127] neutralizes peroxides [135]. It is mainly expressed in the mitochondrial sperm matrix, while nuclear isoform of GPx has been correlated with sperm DNA preservation from oxidative detrimental impact and chromatin condensation [136]. GPx reduces fat hydroperoxides into alcohols and free H 2 O 2 to H 2 O, it is fundamental for protecting lipid integrity and maintaining sperm viability and membrane integrity [134].

Exogenous antioxidants
Most common exogenous antioxidants refer to carnitines, α-tocopherol, ascorbic acid, carotenoids, zinc and selenium. Spermatozoa carry with them minimal endogenous antioxidant amounts, thus during the entire process of spermatogenesis, sperm rely on exogenous antioxidants [137]. Studies about their efficacy in clinical trials are presented in Table 4.

Carnitines
L-carnitine (LC) and L-acetyl carnitine (LAC), a water-soluble antioxidant, are implicated in sperm metabolism, motility and viability [147]. It helps in preventing lipid peroxidation, sperm DNA protection and apoptosis [148]. The highest concentration of carnitine is found in the epididymis and spermatozoa [132]. Studies of the semen samples of infertile men, especially oligoasthenoteratozoospermic (OAT) men, have shown lower carnitine levels compared to fertile men [133].

Vitamin C (L-ascorbic acid)
This is a water-soluble vitamin. Humans and other vertebrates lack the enzyme L-glucono-gamma lactone oxidase (LGGLO), which is essential for in vivo synthesis. Hence, its intake with diet or as a supplement is fundamental. Vitamin C concentration is 10-times higher in seminal plasma comparing to serum [149]. It nullifies the activity of •OH, O 2 •and H 2 O 2 radicals, thereby protecting against oxidative damage [150].

Carotenoids
Carotenoids can be found naturally in fruits and vegetables. Carotenoid cannot be synthesized by humans, by introduced by the diet. Lycopene, a fat-soluble aromatic carotenoid, is reported to be strong neutralizer of À1 O 2 , but a combination of carotenoids seem to be more effective [151]. It can alter the levels of antioxidant enzymes by modification of the levels of ROS, making great contribution to the human antioxidant system [43,119]. There are studies on fertile men that show high concentration of Lycopene, and reduced levels in seminal plasma of infertile men [152].

Coenzyme Q-10 (CoQ10)
CoQ10 is an intermediate of the mitochondrial electron transport chain [153,154]. Low seminal plasma/sperm concentrations of CoQ10 have been associated with reduced sperm motility [155].

Zinc (Zn)
Zn is one of the most abundant elements in human [156]. It acts as metalloprotein cofactor in the metabolism of nucleic acids transcription, signal transduction, protein synthesis and cell death regulation [157]. Moreover, Zn is fundamental for optimal sustain of spermatogenesis and adequate function of the male reproductive organs [158]. It also plays a key role in preventing LPO and preserves sperm structure, by reducing generation of H 2 O 2 and •OH, through separating active redox transition metals, such as Fe and Cu [144].

Selenium (Se)
Se is an important trace mineral, implicated in many biological processes. Se is the constituent of enzymes such as GPx and seleno-proteins, it shows a major impact in redox defense system, spermatogenesis and increased fertility capacity in both males and females [159]. It protects sperm DNA against OS damage, although the mechanism is still unclear [160].

Role and effect of vitamin E in male reproduction
Vitamin E is the major lipophilic antioxidant [156] and it has been recognized as an essential nutrient for reproduction since its discovery in 1922 [161]. It neutralizes •OH and O 2 •by lessening lipid per-oxidation commenced by ROS, thus protecting cell membranes from oxidation [160]. Vitamin E ameliorates other scavenging oxidants manners and helps maintaining sperm morphology and motility (which Antioxidants Study findings Ref.

LC & LAC
• Analyzed in certain systematic reviews and meta-analysis.
• Intake (two times daily, not more than 30 weeks) is associated with a remarkable increase in sperm motility and morphology. [138,139] Vit. C • Studies suggest positive association between levels of ascorbic acid in seminal plasma and sperm morphology and viability. • Very effective in controlling sperm agglutination. • Kobori et al. treated 169 males for 6 months with vitamin C, E and CoQ10, and reported a noteworthy improvement of sperm concentration and sperm motility. depends on the integrity of the mitochondrial sheath) [162]. Effects and the roles of vitamin E are presented in Figure 3.
Various vitamin e isoforms have been found, but their role and importance remains enigmatic, and of the eight naturally occurring forms, only α-tocopherol is maintained in the plasma [163]. Therefore, vitamin E is crucial in maintaining all the necessary functions of healthy sperm and protecting it from detrimental effects of OS. Studies show lower levels of vitamin E in infertile men compared to fertile men [135], allowing somehow to increase concentration of the peroxidation byproduct MDA in the seminal fluid [164]. It is mainly used in combination with other vitamins and minerals. In vitro and in vivo studies which show improvements exclusively in the sperm motility and other semen parameters, successful pregnancies and mitigation of oxidative stress markers, presented in Table 5.
Vitamin E intake and its dosage should exclusively be determined by a healthcare professional because of adverse events due to vitamin toxicity. The recommended daily dose of vitamin E is 15 mg (30 IU) for adults [173], a dose of 200-800 mg/day may cause gastrointestinal distress, while a daily dose greater than 1000 mg (1500 IU) is associated with increased risk of hemorrhage (antiplatelet effects), thrombophlebitis,, elevated creatinine, gonadal dysfunction and death [163,174].
Infertile patients which want to increase concentrations vitamin E, its sources can be found in nuts, seeds, vegetable oils, leafy vegetables and fortified cereals.
It needs proper and critical analysis for establishing the correct dosage and duration of antioxidants administration. In case of raised OS status, remedy must be administered at least for 12 weeks, according to the proper minimal period for spermatogonia (72 AE 3 days), or for three to six months [175,176].
Referring to the studies analyzed above, vitamin E consumption has its obvious beneficial effects. But, the question here is whether vitamin E is more effective solely or in a combination? If used solely, is the efficacy more accentuated in in vivo   [171] or in vitro studies? Data presented above from different studies demonstrate the complexity and the unpredictability of vitamin E or antioxidant supplementation, even though there are studies that suggest improvements in sperm parameters, decrease of oxidative stress status, improvements in zona pellucida binding test and higher pregnancy rates. Vitamin E doesn't work only as an antioxidant, but it is also involved in the modulation of cellular responses by modulating enzymes or by regulating the activity of specific transcription factors [173,177].

Conclusion
ROS are very important in certain physiological processes; however they can be very dangerous for male fertility potential if the levels overcome a physiological threshold.
Therefore, normal fine redox equilibrium between ROS and antioxidants is extremely important. The understanding of this fine balance will facilitate steps towards proper diagnosis and treatment in ideal dosages of antioxidant treatment.
The most widely utilized antioxidants either as single therapy or combined are: vitamin C, E, NAC, carnitines, CoQ10, zinc, selenium, and lycopene.
According to current literature we can conclude that vitamin E used alone is more effective when used for in vitro procedures, and very effective used in a dual, triple or more combinations in terms of sperm parameters and oxidative stress status.
Further augmentative clinical trials are needful to ascertain the right and effective antioxidant combination, for reliable and appropriate guiding of this sensitive medical issue.