Overview of reactive oxygen and nitrogen species.
Oxidative stress (OS) is a condition caused by an imbalance between reactive oxygen species (ROS) overgeneration and decreased antioxidant defense mechanisms in the cell. OS has become a prominent factor in male reproductive dysfunction as ROS cause damage to sperm DNA, lipids and proteins, alterations to critical sperm structures and signaling pathways, leading to a decreased sperm activity and fertilizing capacity. At the same time, small amounts of ROS play vital roles in events leading to sperm maturation and acquisition of functional activity, which is why a proper oxidative balance is of paramount importance for a proper male fertility. Understanding the physiological and pathological roles of ROS in male reproduction has become an essential pillar of modern andrology; however, numerous questions related to the controversial behavior of ROS in male reproductive cells and tissues still remain unanswered. This chapter aims to summarize current evidence available on the relationships between free radicals, antioxidants and male reproduction and to trigger more scientific interest, particularly with respect to the design of efficient strategies to diagnose or treat male sub- or infertility associated with OS.
- free radicals
- reactive oxygen species
- oxidative stress
- male infertility
Aerobic life inherently depends on oxygen, which is essential for a controlled oxidation of molecules containing carbon, subsequently leading to the release of energy. Nevertheless, aerobic cells, including spermatozoa, are persistently counteracting the so-called Oxygen Paradox: while oxygen is crucial to sustain aerobic life, it is simultaneously toxic to the cell survival . Normal aerobic metabolism leads to the generation of by-products called free radicals (FR) [2, 3], which, under physiological conditions, are necessary for a normal cell function . On the other hand, if FR concentrations become too high, either because of their overgeneration or due to low levels of antioxidant defense mechanisms, oxidative stress (OS) emerges with unpredictable consequences on the cell behavior and survival .
Oxidative stress has been implicated in the pathogenesis of a variety of human diseases such as atherosclerosis, cancer, diabetes, liver damage, AIDS, Parkinson’s disease and health complications associated with premature birth . In the meantime, seminal OS is believed to be one of the main factors in the pathogenesis of sperm dysfunction in male sub- or infertility [7, 8, 9]. Several intrinsic and extrinsic factors have the ability to promote reactive oxygen species (ROS) generation in the testicular as well as post-testicular (e.g. epididymal) environment, resulting in defective spermatogenesis and altered sperm function . As expected, approximately 25% of infertile patients exhibit higher ROS levels in semen as opposed to fertile men [7, 10, 11, 12].
Although the origin of ROS generation in semen and their roles in male reproduction have only recently been uncovered, numerous questions still remain unanswered, thus offering multiple strategies for future research. As such, the role of free radicals and oxidative stress in fertility and subfertility is an area requiring continuous scientific attention.
2. Free radicals: general characteristics
A free radical (FR) is defined as any atom, molecule or a fragment of atoms and molecules with one or more unpaired electrons, capable of short independent existence. The abstraction or gain of one electron by a nonradical molecule may (or may not) convert it to a radical species . Free radicals may have a positive, negative or a neutral charge :
A → minus one electron → A+●.
B → plus one electron → B−●.
It is precisely the presence of an unpaired electron that results in certain common properties shared by most radicals. Free radicals are generally unstable and highly reactive. They can either donate an electron to or accept an electron from other molecules, thus behaving as oxidants or reductants .
The most common and important free radicals related to biological systems are oxygen-derived radicals called reactive oxygen species (ROS) and nitrogen-derived molecules, defined as reactive nitrogen species (RNS) . ROS represent a broad category of molecules including radical and non-radical oxygen derivatives . Reactive nitrogen species are nitrogen-free radicals and commonly accepted as a subclass of ROS [13, 15]. A summary of the most common oxygen- and nitrogen-derived free radicals is provided in Table 1.
|Hydroxyl radical||OH●||Hypochlorous acid||HOCl|
|Peroxyl radical||ROO●||Hypobromous acid||HOBr|
|Hydroperoxyl radical||HO2●||Singlet oxygen||1Δg|
|Lipid peroxyl radical||LOO●||Lipid peroxide||LOOH|
|Nitric oxide||NO●||Nitrous acid||HNO2|
|Nitrogen dioxide||NO2●||Nitrosyl cation||NO+|
|Nitronium (nitryl) cation||NO2+|
3. Sources of ROS in semen
Virtually every ejaculate may contain potential sources of ROS. Leukocytes activated by multiple factors, especially inflammation and infection, are among significant ROS producers in semen . Subpopulations of leukocytes, which may be found in semen, mainly consist of polymorphonuclear (PMN) leukocytes (50–60%) and macrophages (20–30%) . PMN leukocytes represent an important source of ROS due to their abundant presence in semen. Furthermore, external stimuli induce the activation of macrophages, leading to an oxidative burst and ROS overgeneration. Under normal circumstances, these monocytes are of paramount importance in defending male reproductive structures against nearby cells and pathogens .
The Endz test based on myeloperoxidase staining is an efficient technique to quantify seminal leukocytes during semen quality assessment . According to the World Health Organization (WHO), if the leukocyte concentration in the ejaculate exceeds 1 × 106/mL, leukocytospermia is present .
Numerous reports have studied possible relationships between seminal leukocytes and male reproductive dysfunction, resulting in two different directions. On the one hand, some studies failed to reveal any correlation between leukocytospermia and sperm damage , whereas inversely, other studies emphasized on a strong link between the presence of seminal leukocytes and abnormal sperm quality . In particular, Sharma et al.  observed that even small numbers of white blood cells may be responsible for seminal OS, and hence subthreshold levels of leukocytes, as seen in ejaculates collected from otherwise healthy subjects, may not be considered safe as previously believed. Moreover, activated leukocytes may be responsible for a 100-fold increase in ROS production in comparison to non-activated white blood cells .
Leukocytospermia has been furthermore associated with increased ROS production by spermatozoa, most likely triggered by a direct cell-to-cell contact of the leukocyte with the sperm cell or by the release of soluble products acting on the spermatozoon [23, 24].
Spermatozoa have also been reported to generate ROS independently of leukocytes, and this ability primarily depends on the maturation level of the sperm cell. During the epididymal transit, the main morphological change that takes place in the spermatozoon is the migration of the cytoplasmic droplet, a remnant of the cytoplasm associated with testicular sperm. The droplet migrates from the proximal to the distal position during maturation and is normally shed from spermatozoa during or shortly after ejaculation . Failure to extrude excess cytoplasm during sperm differentiation and maturation traps a number of enzymes, including glucose-6-phosphate dehydrogenase (G6PD) and ß-nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which have been associated with ROS generation through the formation of the NADPH intermediate . As such, immature and functionally defective spermatozoa with abnormal head morphology and cytoplasmic retention are another important source of ROS in semen . According to Gil-Guzman et al.  there is a strong positive correlation between immature spermatozoa and ROS production, which in turn is negatively correlated with semen quality. The study revealed that after a density gradient separation of human ejaculates, the layer of immature spermatozoa produced the highest levels of ROS. Furthermore, elevated concentrations of immature spermatozoa were accompanied by increased amounts of mature spermatozoa with damaged DNA .
Sertoli cells have also been revealed to have the ability to generate ROS, which may be inhibited by the addition of scavestrogens (J811 and J861). Scavestrogens are derivates of 17alpha-estradiol and serve as effective FR-quenching molecules that able to inhibit iron-catalyzed cell damage
Varicocele is defined as the excessive dilation of the
3.1. Endogenous ROS production by sperm
Superoxide (O2●−) is considered to be the primary ROS produced by respiring cells, including spermatozoa . It is a regular by-product of oxidative phosphorylation, created between complex I and III of the electron transport chain as a result of a monovalent reduction of oxygen and the addition of a single electron .
In the male gamete, O2●− is predominantly generated through two reduced forms of ß-nicotinamide adenine dinucleotide phosphate (NADPH) oxidases that are similar to those found in phagocytic leukocytes: the NADH-dependent oxidoreductase located in the inner mitochondrial membrane and the NAD(P)H-oxidase found in the plasma membrane . The hypothesis that these enzymes are primarily responsible for low-level generation of O2●− important in cell signaling events in spermatozoa is based essentially on two observations. Firstly, adding pharmacological doses of NADPH to purified sperm suspensions has led to an increase in O2●− production, subsequently leading to a decline in the sperm function [34, 35]. Secondly, such increased O2●− production could be inhibited by superoxide dismutase (SOD), which protects male reproductive cells against the toxic effects of NADPH . Additionally, the cytoplasmic enzyme G6PD controls the rate of glucose flux and intracellular availability of NADPH through the hexose monophosphate shunt. This in turn serves as a source of electrons by spermatozoa to fuel O2●− generation through the NADPH oxidase [35, 36]. Lastly, another relevant source of O2●− in spermatozoa is electron leakage from the mitochondrial electron transport .
Although O2●− is relatively unreactive, in the presence of hydrogen (H+) it undergoes either a spontaneous or SOD-catalyzed dismutation into hydrogen peroxide (H2O2)—a membrane permeable molecule , which is considered to be the major initiator of peroxidative damage in the plasma membrane of spermatozoa . H2O2 can be either scavenged by glutathione peroxidase (GPx) or catalase, catalyzing its dismutation into water and oxygen.
Moreover, O2●− as well as H2O2 can undergo a series of cellular transformations to generate the highly reactive hydroxyl radical (OH●) through the Fenton and Haber-Weiss reactions, comprising a reduction of ferric (Fe3+) to ferrous ion (Fe2+) in the presence of O2●−, followed by the H2O2 conversion to OH●. Furthermore, O2●− has the ability to interact with nitric oxide (NO●) to generate peroxynitrite (ONOO−), subsequent reactions of which may lead to either apoptosis or necrosis .
3.2. Endogenous RNS production by sperm
The primary RNS species produced by male gametes is nitric oxide (NO●). Its production is catalyzed by nitric oxide synthase (NOS) in a redox reaction between L-arginine and oxygen, initiated by NADPH, and with L-citrulline as a byproduct. NO● interacts with O2●− to create peroxynitrite (ONOO−), a highly toxic-free radical . Interestingly, both high and low concentrations of NO● may result in significant alterations of the sperm function as a result of the production of ONOO− .
Inversely, physiological NO● levels are reported to have beneficial effects, acting in signal transduction pathways involved in spermatozoa motility, capacitation and acrosome reaction .
3.3. External sources of ROS
ROS generation can be exacerbated by a multitude of environmental, infectious and lifestyle-related etiologies.
A wide range of industrial by-products and waste chemicals (e.g. polychlorinated biphenyls, nonylphenol or dioxins) have been associated with several adverse health effects, many of which are related to male infertility. These chemicals have been shown to increase the production of reactive species such as O2●− and H2O2 in the testes, damage sperm DNA and impair spermatogenesis . Persistent environmental contaminants, such as heavy metals and pesticides, may also lead to OS, particularly among workers exposed to such pollutants. These individuals often present with a decreased semen volume and density, accompanied by increased oxidative damage to the sperm lipids, proteins and DNA .
Radiation is a natural source of energy with significant effects on living organisms. Mobile devices are becoming more accessible to the general population, particularly to adolescent males and men of reproductive age. Cell phones release radiofrequency electromagnetic radiation, exposure to which has shown to increase the risk of oligo-, astheno- or teratozoospermia. Furthermore,
Various components of cigarette smoke have been associated with OS exacerbation. Cigarettes contain a broad array of free radical-inducing agents such as nicotine, cotinine, hydroxycotinine, alkaloids and nitrosamines [41, 42]. The prime component of tobacco is nicotine, which is a well-known ROS producer in spermatozoa with detrimental effects on the sperm count, motility and morphology. Moreover, smokers exhibited a lower hypo-osmotic swelling test percentage, indicating a weaker plasma membrane integrity when compared to non-smokers . Smoking increases ROS production by causing leukocytospermia as shown by Saleh et al. , who also demonstrated that in smokers, the seminal ROS and total antioxidant capacity score was increased—a direct indication of oxidative imbalance in affected ejaculates. A different study showed that levels of seminal plasma antioxidants were diminished in smokers. This was furthermore confirmed by the presence of increased levels of 8-hydroxy-2′-deoxyguanosine .
By directly affecting the liver, alcohol intake increases ROS production while simultaneously decreasing the antioxidant capacity of the body. Although alcohol consumption has been repeatedly associated with systemic OS, its effect on semen parameters has not been explored to a larger extent. In a study comprising 8344 subjects, moderate alcohol consumption did not negatively affect semen parameters . Nevertheless, it was revealed that chronic drinkers had reduced levels of testosterone, possibly due to an impaired hypothalamic-pituitary axis and damage to the Leydig cells . Increased alcohol levels block gonadotropin-releasing hormone, leading to reduced luteinizing hormone and testosterone levels. Furthermore, alcohol has been shown to increase ROS generation when consumed by malnourished individuals .
Lastly, diet may affect semen parameters. In a Danish study, men with the highest saturated fat intake presented with a significantly lower total sperm count and concentration in comparison to those with the lowest saturated fat intake . These observations were supported by a later report focused on studying the link between dairy food intake and male fertility and revealing that a low-fat dairy diet may lead to a higher spermatogenesis . On the other hand, omega-3 fatty acids and omega-6 fatty acids were shown to improve sperm count, motility and morphology . With regard to obesity and its relation to semen parameters, currently available data are conflicting. In a study on Iranian men, it was found that overweight men tend to have lower sperm counts . Inversely, a different study reported that underweight subjects had lower sperm counts than normal and overweight men . Moreover, a study comprising Tunisian men revealed that sperm concentration, motility and morphology did not vary across different BMI values .
4. Physiological roles of ROS
Aerobic metabolism utilizing oxygen is essential for energy requirements of reproductive cells, and free radicals do play a significant role in physiological processes occurring within the male reproductive tract. Spermatozoa themselves produce small amounts of ROS that are essential for a variety of physiological processes such as capacitation, hyperactivation, acrosome reaction and sperm-oocyte fusion .
4.1. Sperm maturation
During transit and storage in the epididymis, spermatozoa undergo membrane, nuclear and enzymatic remodeling, involving the release, attachment and rearrangement of surface proteins [6, 30, 51]. Such changes are based on the assembly of several signal transduction pathways necessary for the subsequent ability of spermatozoa to undergo hyperactivation and capacitation.
ROS are essential for a proper chromatin packing during the maturation of mammalian spermatozoa, leading to a characteristic chromatin stability. This unique chromatin architecture results from an extensive inter- and intra-molecular disulfide bond stabilization between the cysteine residues of protamines—small nuclear proteins that replace histones during spermatogenesis. Oxidation of the thiol groups in protamines takes place during the transport of spermatozoa from the caput to the cauda epididymis . As demonstrated by Aitken et al. , a spontaneous luminol peroxidase signal indicating the presence of ROS was exclusive to mature spermatozoa collected from the cauda region. ROS may act as oxidizing agents in this process, hence facilitating the formation of disulfide bonds, increasing chromatin stability and protecting DNA from possible damage [30, 52]. As spermatozoa possess minimal to none repair mechanisms , chromatin condensation is a crucial protective mechanism, in which ROS actually protect male gametes against future oxidative insults.
Likewise, peroxides have been associated with formation of the mitochondrial capsule—a coat surrounding sperm mitochondria providing protection against possible proteolytic degradation . It is suggested that during spermatogenesis peroxides may oxidize the active form of phospholipid hydroperoxide glutathione peroxidase (PHGPx), creating an intermediate that subsequently interacts with thiol groups to form a seleno-disulfide bond. The resulting mitochondrial capsule is made out of a complex protein network rich in disulfide bonds. Mitochondria require such protection as their proper function is crucial for metabolism, cell cycle control and oxidative balance [51, 53, 54].
Although several studies have reported improved sperm DNA integrity and reduced ROS production as a result of daily antioxidant consumption , an unusual decondensation of sperm DNA has been revealed as well . Hence it may be hypothesized that high antioxidant levels may alter the oxidative conditions necessary for a proper formation of the inter- and intra-molecular disulfide bonds, leading to a lower DNA compaction.
Capacitation is a prominent process of final maturation that spermatozoa undergo in the female reproductive tract, during which sperm motility changes from a progressive state to a highly energetic one. It is hypothesized that capacitation occurs exclusively in mature spermatozoa in order to reach the oocyte taking advantage of hyperactive motility and an increased responsiveness to chemotactic agents. Numerous receptors on the sperm head become activated, providing energy to the sperm to penetrate the zona pellucida. As such, capacitation sets up the path necessary for subsequent hyperactivation and acrosome reaction . Most prominent molecular processes associated with capacitation include Ca2+ and HCO3− influx, cholesterol efflux, increased cAMP activity, ROS generation, pH, protein phosphorylation and membrane hyperpolarization [32, 58].
Numerous of studies on both human and animal spermatozoa indicate that H2O2 is the primary ROS responsible for capacitation to occur. This process is associated with an increase in tyrosine phosphorylation, and it has been shown that the amount and banding pattern of tyrosine phosphorylation by adding exogenous H2O2 was similar to that observed during endogenous ROS production, providing evidence that H2O2 may be responsible for the enhancement of capacitation [32, 57, 58]. This hypothesis was further confirmed by Rivlin et al.  who showed that catalase decreased, while H2O2 increased the tyrosine phosphorylation in a dose-dependent manner, thereby solidifying the involvement of H2O2 in the process of capacitation.
At the same time, de Lamirande and Gagnon  indicated that O2●− may be also involved in this process. Of note is also the role of NO●, which is present in the female genital tract. NO● may initiate the acrosome reaction, the effects of which are likely achieved through a complex mechanism involving H2O2 [59, 60].
Finally, a combination of O2●−and NO● forms ONOO−, which allows oxysterol to be produced. Oxysterol, which removes cholesterol from the lipid bilayer, inhibits tyrosine phosphate and promotes cyclic adenosine 3′,5′-monophosphate (cAMP) production . This process is vital as cAMP must increase in concentration for capacitation to occur. cAMP and its subsequent pathways involve protein kinase A, which phosphorylates MEK (extracellular signal-regulated kinase)-like proteins as well as tyrosine present in fibrous sheath proteins [57, 58].
The results of the above studies show that ROS can positively enhance sperm capacitation, but diverge over the specific ROS involved. Both O2●− and H2O2 may stimulate different molecules in the biochemical pathways, and depending on the
Although physiological ROS levels are necessary for capacitation, their overgeneration may trigger apoptosis. When the levels of oxysterols and lipid aldehydes increase, cell-mediated suicide may occur accompanied by an enhanced mitochondrial O2●− production, lipid peroxidation (LPO), cytochrome c release and subsequent caspase activation [53, 61].
4.3. Motility and hyperactivation
Hyperactivation is an incompletely understood process to be observed in the final maturation stage of spermatozoa and is considered a subcategory of capacitation. Normally spermatozoa exhibit a low amplitude flagellar movement accompanied by low, linear velocity. In the hyperactivated state, spermatozoa movement is of high amplitude, asymmetric flagellar movement, pronounced lateral head displacement and non-linear trajectory, allowing the sperm to penetrate the
Extracellular O2●− is considered nearly essential for hyperactivation in mammalian spermatozoa, as the presence of SOD, but not catalase, reduced the percentage of spermatozoa exhibiting hyperactivity in a variety of culture media .
4.4. Acrosome reaction
Acrosome reaction (AR) is related to the release of proteolytic enzymes, primarily acrosin and hyaluronidase, in order to degrade the zona pellucida of the oocyte. Once degraded, hyperactive motility propels the spermatozoa into the perivitelline space, at which point the spermatozoa may eventually fuse with the oocyte . Compared to the slow, reversible process of capacitation, this is a permanent, fast-acting step associated with a respiratory burst (rapid extracellular O2●− production) increasing the tyrosine phosphorylation of specific proteins [57, 64]. O2●− produced
Moreover, ROS act as signal transducers in the AR. Elevated ROS production may occur upon interaction with the
4.5. Sperm-oocyte fusion
A link exists between enhanced ROS levels and increased sperm-oocyte fusion. High rates of sperm-oocyte fusion are correlated with increased expression of phosphorylated tyrosine proteins , suggesting that sperm-oocyte fusion is related to the events of capacitation and AR. Both H2O2 and O2●− contribute to the increase in fertilization rates as revealed by the fact that the addition of catalase or SOD significantly decreased the fusogenicity, whereas the addition of H2O2 or O2●− significantly increased the fusogenicity [53, 64].
Ultimately, ROS are thought to increase membrane fluidity using two mechanisms: (1) de-esterification of membrane phospholipids and (2) activation of phospholipase A2 (PLA2) .
Once the zona pellucida and corona radiata are penetrated by the sperm cell, the oocyte prevents eventual polyspermy by turning the vitelline layer into a hard envelope. o,o-Dityrosine crosslinks catalyzed by ovoperoxidase lead to the formation of a single macromolecular structure acting as the envelope . H2O2 serves as the substrate to ovoperoxidase to provide for the envelope formation. With our understanding of ROS and their spermicidal effect, H2O2 proves to be an effective spermicide agent against polyspermy [66, 67].
5. Oxidative stress (OS)
The term oxidative stress refers to a critical imbalance between ROS production and antioxidant defense mechanisms available to the biological system . According to Sies , it is a disturbance in the prooxidant-antioxidant balance in favor of the former, leading to potential cellular damage.
Essentially, OS may result from:
Diminished antioxidants, e.g. mutations affecting antioxidant defense enzymes or toxic agents that deplete such mechanisms .
Increased ROS or RNS generation either by exposure to increased levels of toxins that act as reactive species themselves or are metabolized to induce further biological oxidation or by excessive activation of ‘natural’ FR-generating systems (e.g. phagocytic oxidative outburst during chronic inflammatory diseases) [5, 15]. This mechanism is normally thought to be more relevant to mammalian diseases and is frequently the target of attempted therapeutic intervention.
OS can result in:
Adaptation: Usually by upregulation of antioxidant defense systems.
Cell and tissue injury: OS can cause damage to all molecular targets: DNA, proteins and lipids. Often it is not clear which is the first point of attack, since injury mechanisms may overlap .
Cell death: This process may occur by two mechanisms, necrosis or apoptosis. During necrotic cell death, the cell swells and ruptures, releasing its contents into surrounding areas and affecting adjacent cells. The intracellular content can include antioxidants such as catalase or glutathione (GSH) as well as prooxidants such as copper and iron. As such, necrosis may lead to further oxidative insults in the internal milieu [3, 4, 5, 15]. During apoptosis, the cell’s own “suicide mechanism” gets activated. As such, apoptotic cells do not release their content into surrounding environment and apoptosis does not cause damage to the neighboring cells .
An intricate cellular architecture of spermatozoa renders them to be particularly sensitive to OS. Sperm plasma membranes contain large quantities of polyunsaturated fatty acids (PUFAs). On the other hand, their cytoplasm contains low concentrations of scavenging enzymes . OS usually results in a decreased sperm motion and viability, accompanied by a rapid loss of ATP, axonemal damage, increased midpiece morphology defects, followed by alterations in the sperm capacitation and acrosome reaction . Lipid peroxidation has been repeatedly postulated to be the key mechanism of ROS-induced sperm damage, possibly leading to male reproductive dysfunction .
5.1. Lipid peroxidation (LPO)
Sperm plasma membranes are largely composed of PUFAs, which are exceptionally susceptible to oxidative damage due to the presence of more than two carbon–carbon double bonds . These fatty acids maintain the fluidity of membranes . ROS attack PUFAs, leading to a cascade of chemical reactions called lipid peroxidation (LPO). As the LPO proceeds, more than 60% of PUFAs may be lost. LPO affects most prominent structural and functional characteristics of the membrane, including fluidity, ion gradients, receptor transduction, transport processes as well as enzymatic activities. As a result, properties that are crucial for a normal fertilization are impaired [68, 69].
LPO is a self-propagating process that may be divided into three phases: the initiation phase, the propagation phase and the termination phase. Before any of these processes takes place, O2●− is generated either intracellularly through the NADPH system or through leukocytes as an extracellular source. O2●− can be directly protonated to create the hydroperoxyl radical (HO2●) or it can be converted into H2O2
Numerous pathological effects of LPO on the sperm function are currently known. Overall, LPO causes DNA and protein damage through oxidation of lipid peroxyl or alkoxyl radicals. DNA fragmentation by LPO can occur
Furthermore, during LPO, ROS initiate a cascade of events involving the xanthine and xanthine oxidase system and deplete the ATP production which may ultimately lead to sperm death .
5.2. DNA damage
The unique sperm chromatin packing alongside antioxidant molecules present in the seminal plasma provide notable protection to sperm DNA against oxidative damage. Nevertheless, spermatozoa lack any specific DNA repair mechanisms and hence depend on the oocyte for eventual DNA repair following fertilization. ROS-associated catalysis and apoptosis are considered to be the primary mechanisms that induce DNA fragmentation in spermatozoa .
DNA bases and phosphodiester backbones are believed to be most susceptible to ROS-associated peroxidative damage. At the same time, sperm mitochondrial DNA is more vulnerable to oxidative insults when compared to the nuclear genome . Furthermore, because of the structure of the Y chromosome as well as its inability to repair double strand breaks, Y-bearing spermatozoa are more susceptible to DNA damage than X-carrying counterparts . Y-bearing spermatogonia can be a target of mutations in the euchromatic Y region (Yq11), known as the azoospermia factor, resulting in infertility .
Various types of DNA abnormalities may occur in sperm that have been exposed to ROS artificially. These include base modifications, production of base-free sites, deletions, frame shifts, DNA crosslinks and chromosomal rearrangements. OS has also been associated with high frequencies of single- and double-strand DNA breaks. ROS can also cause gene mutations, such as point mutation and polymorphism, resulting in decreased semen quality. These changes may be observed especially during the prolonged meiotic prophase, when the spermatocytes are particularly sensitive to damage and widespread degeneration can occur [72, 73, 74]. Also, mutations in the mitochondrial DNA (mtDNA) may cause a defect of mitochondrial energy metabolism and therefore lower levels of mutant mtDNA may compromise sperm motility
Increased DNA damage has become a serious issue during artificial reproduction techniques (ARTs), as it has been correlated with decreased fertilization rates
5.3. Protein oxidation
Proteins are a critical target for oxidation because of their abundance and high rate constants for interactions with diverse ROS. As such, protein damage is a major consequence of both intracellular and extracellular oxidative insults. ROS may attack both the side chains and backbone, and the extent of the insult depends on multiple factors. In some cases, the damage is limited to specific residues, whereas in case of other ROS, the damage is widespread and nonspecific .
Oxidative attacks on proteins generally result in site-specific amino acid modifications, fragmentation of the peptide chain, aggregation of cross-linked reaction products, altered electric charge and increased susceptibility or extreme tolerance to proteolysis .
The resulting products of protein oxidation include reactive hydroperoxides, which may be employed as biomarkers for protein oxidation
The amino acids in a peptide differ in their susceptibility to oxidative insults, while various ROS differ in their potential reactivity. Primary, secondary and tertiary protein structures alter the relative susceptibility of certain amino acids. Sulfur-containing amino acids and particularly thiol (−SH) groups are very susceptible to ROS-associated damage [79, 80].
According to Mammoto et al. , protein oxidation in spermatozoa leads to a blocked sperm-egg fusion, the capacity to penetrate the zona pellucida, as well as sperm-egg binding. Sinha et al.  showed that oligospermia is linked to a quantitative reduction in the SH-groups in spermatozoa. Thus, oxidation of the sperm SH-proteins may be a notable mechanism responsible for the suppressive effects of ROS on sperm functions.
Usually, when cellular components undergo serious damage, apoptosis or programmed cell death is initiated. During spermatogenesis, abnormal spermatozoa are eliminated primarily through apoptosis. The exact mechanism of action is not fully understood yet; however, previous studies have speculated that ROS serve as an activator of the mitochondria to release the signaling cytochrome c [82, 83]. This molecule initiates a cascade of events involving caspases 3 and 9, eventually leading to sperm apoptosis. The Fas-protein may be also an integral component in the apoptotic pathway. When Fas-ligand or anti-Fas antibody binds to Fas, apoptosis is initiated . An additional mechanism involves the inflammatory production of ROS, primarily hypochlorous acid (HOCl), which is a product of H2O2 and chloride ion. This molecule oxidizes a variety of cellular components, thus causing apoptosis . Said et al.  emphasized that HOCl is associated with elevated levels of apoptotic markers in spermatozoa.
Numerous studies have focused to study apoptosis in spermatozoa. Various authors [35, 86] have reported increased ROS levels and apoptotic markers measured by fluorescence in samples of infertile subjects. In deer spermatozoa, it was demonstrated that H2O2 addition stimulates apoptosis, whereas O2●− and OH● do not have this ability . Meanwhile studies in primate, murine and boar spermatozoa indicated that NO● was correlated with apoptosis possibly through caspase activation [87, 88].
On the other hand, in certain males, abortive apoptosis appears to fail in the clearance of spermatozoa that are marked for elimination by apoptosis. As such, the subsequent population of ejaculated spermatozoa may exhibit an array of anomalies consistent with characteristics typical for cells that are in the process of apoptosis. Apoptotic failures may lead to a decreased sperm count resulting in subfertility [82, 83].
5.5. Effects on sperm motility
Spermatozoa motility is an important prerequisite to secure their distribution in the female sexual system, followed by an effective passage through the cervical mucus and penetration into the egg . Increased ROS levels have been repeatedly correlated with a decreased sperm motility [10, 11, 12, 90], although the exact mechanism involved is still not completely understood. One hypothesis suggests that H2O2 diffuses across the membranes into the cells and inhibits the activity of vital enzymes such as NADPH oxidase . At the same time, a decreased G6PDH leads to a reduced availability of NADPH accompanied by a build-up of oxidized glutathione. Such changes may lead to a decline in the intracellular antioxidant levels and a subsequent peroxidation of membrane phospholipids .
Another hypothesis presents a series of interrelated events leading to a decreased phosphorylation of axonemal proteins, followed by sperm immobilization, both of which are linked to a reduced membrane fluidity crucial for sperm-oocyte fusion [10, 32]. When spermatozoa are incubated with selected ROS overnight, loss of motion characteristics observed is highly correlated with sperm LPO. Furthermore, the ability of antioxidants to revive sperm motility is evidence that LPO is a major cause for motility loss in spermatozoa [68, 69].
6. The role of antioxidants in male reproduction
Because ROS have both physiological and pathological functions, biological systems have developed defense systems to maintain ROS levels within a certain range. Whenever ROS levels become pathologically elevated, antioxidants scavenge them to minimize any potential oxidative damage .
Antioxidants are defined as molecules that dispose, scavenge and inhibit the formation of ROS or oppose their actions. According to Ďuračková , antioxidants can protect cells against OS
Antioxidants may be divided into two dominant categories:
Enzymatic (e.g. superoxide dismutases, catalase and glutathione peroxidases).
Non-enzymatic (e.g. vitamin C, vitamin E, vitamin A, carotenoids, albumin, glutathione, uric acid, pyruvate, etc.) .
Due to the size and small volume of cytoplasm, as well as the low concentrations of scavenging enzymes, spermatozoa have limited antioxidant defense possibilities. Mammalian spermatozoa predominantly contain enzymatic antioxidants, including SOD and glutathione peroxidases (GPx), which are mainly located in the midpiece. A few non-enzymatic antioxidants, such as vitamins C and E, transferrin and ceruloplasmin, are present in the plasma membrane of spermatozoa and act as preventive antioxidants .
Under normal circumstances, the seminal plasma is an important protectant of spermatozoa against any possible ROS formation and distribution. Seminal plasma contains both enzymatic antioxidants, as well as an array of non-enzymatic antioxidants (e.g. ascorbate, urate, vitamin E, pyruvate, glutathione, albumin, taurine and hypotaurine) .
Studies have shown that antioxidants protect spermatozoa from ROS generating abnormal spermatozoa, scavenge ROS produced by leukocytes, prevent DNA fragmentation, improve semen quality, reduce cryodamage to spermatozoa, block premature sperm maturation and generally stimulate sperm vitality [91, 92].
6.1. Superoxide dismutases (SOD)
where M = Cu (n = 1); Mn (n = 2); Fe (n = 2); Ni (n = 2).
The enzymes are present in both intracellular and extracellular forms. The first intracellular form is the dimeric copper-zinc SOD, localized primarily in the cytosol and/or intermembrane space and containing copper and zinc (Cu/ZnSOD, SOD-1) in its active center. The second form is manganese SOD, which is found predominantly in the mitochondrial matrix and has manganese in its active center (MnSOD, SOD-2) .
The secretory tetrameric SOD (EC-SOD, SOD-3) may be detected in the extracellular space. The enzyme is associated with surface polysaccharides although it may also be found as a free molecule. Structurally, SOD-3 is similar to SOD-2; however, it has zinc and copper in its active center instead of manganese [1, 5, 15]. The cytosolic Cu/Zn-SOD is the dominant SOD isoenzyme found in the seminal plasma and spermatozoa .
Numerous studies have suggested a significant role for SOD in sperm motility both
6.2. Catalase (CAT)
Catalase catalyzes the decomposition of hydrogen peroxide to molecular oxygen and water, thereby completing the detoxifying reaction started by SOD. A characteristic feature of its structure is a heme system with centrally located iron [1, 13]:
Fe()-E represents the iron center of the heme group attached to the enzyme.
CAT has been found in peroxisomes, mitochondria, endoplasmic reticulum and the cytosol in a variety of cells . In semen, the enzyme was detected in human, bovine and rat spermatozoa, as well as seminal plasma, with the prostate as its source [97, 98].
Catalase activates sperm capacitation induced by nitric oxide [59, 60]. Furthermore, it plays an important role in decreasing lipid peroxidation and protecting spermatozoa during genitourinary inflammation .
Numerous studies have revealed a positive relationship between sperm motility and the presence of CAT in mammalian ejaculates. Also, positive correlations were observed between sperm morphology and protein expression of CAT in seminal plasma [98, 99]. Furthermore, CAT supplementation to fresh, processed and cryopreserved semen resulted in a higher sperm vitality, progressive motility and DNA integrity .
6.3. Glutathione peroxidase (GPx)
Glutathione peroxidases are a family of selenium-containing enzymes, which catalyze the reduction of H2O2 and organic peroxides, including phospholipid peroxides . In their active site, the enzymes contain selenium in the form of selenocysteine.
The net reaction catalyzed by glutathione peroxidase may be represented as:
where GSH symbolizes reduced glutathione and GS-SG represents glutathione disulfide. The reaction is based on the oxidation of selenol of a selenocysteine residue by H2O2. This process leads to its derivation with selenic acid (RSeOH). This by-product is subsequently converted back to selenol through a two-step process that starts with a reaction comprising GSH to generate GS-SeR and water. A second GSH molecule then reduces the GS-SeR intermediate back to selenol, releasing GS-SG as a by-product [1, 5, 13]:
Glutathione reductase then reduces the oxidized glutathione to complete the cycle:
The classic intracellular GPx1 is expressed in sperm nucleus, mitochondria and cytosol, as well as in the testes, prostate, seminal vesicles, vas deferens, epididymis, and has a significant relationship with sperm motility [101, 102].
More importantly, a direct relationship has been reported between male fertility and phospholipid hydroperoxide glutathione peroxidase (PHGPx; GPx4), a selenoprotein that is highly expressed in testicular tissue and has a prominent role in the formation of the mitochondrial capsule [51, 53, 54]. Glutathione peroxidases remove peroxyl (ROO●) radicals from various peroxides, including hydrogen peroxide .
6.4. Other enzymes
Other enzymes, such as glutathione reductase, ceruloplasmin or heme oxygenases, may also participate in the enzymatic control of oxygen radicals and their products. A short overview of minor antioxidant enzymes is provided in Table 2.
|Glutathione reductase (GR)|
|Glutathione S-transferase (GST)|
|Heme oxygenase (HO)|
6.5. Non-enzymatic antioxidants
Non-enzymatic antioxidants are also known as synthetic antioxidants or dietary supplements. The body’s complex antioxidant system is affected by dietary intake of antioxidants, vitamins and minerals, such as vitamin C, vitamin E, zinc, selenium, taurine and glutathione.
6.5.1. Glutathione (GSH)
Glutathione is the most abundant thiol protein in mammalian cells . Being an endogenous source, it is synthesized by the liver but it can also be derived from dietary sources such as fresh meat, fruits and vegetables. This molecule has three precursors: cysteine, glutamic acid and glycine. Its cysteine subunit provides and exposes -SH that directly scavenges free radicals. Once oxidized, GS-SG is then regenerated/reduced by glutathione reductase to complete the cycle .
High levels are found especially in the testis of rats  and the reproductive tract fluids and epididymal sperm of bulls . GSH protects the cell membranes from lipid oxidation and prevents further formation of free radicals. Its deficit leads to instability of the sperm midpiece, which results in motility disorders . Glutathione supplementation in infertile subjects has led to a significant improvement in sperm parameters and prevents oxidative damage to sperm DNA. A factor increasing the level of GSH is pantothenic acid, which by doing so also protects tissues against oxidative stress [117, 118].
6.5.2. Vitamin C
Vitamin C or ascorbic acid (AA) may be found in its reduced (ascorbate) as well as oxidized form (dehydroascorbic acid), both of which are easily interconvertible and biologically active. Vitamin C is found in citrus fruits, peppers, strawberries, tomatoes, broccoli, brussels sprouts and other leafy vegetables. AA is a water-soluble vitamin, and because of its hydrophilic nature, it has more effective scavenging properties at the plasma level than in the lipid bilayer .
Vitamin C has been used in the management of male infertility on empirical grounds, particularly in the presence of non-specific seminal infections . Its presence in the seminal plasma of healthy males has been reported by various authors [121, 122, 123]. Chinoy et al.  stated that AA was essential for the structural and functional integrity of androgen-dependent reproductive organs. Low concentration of vitamin C showed significant degenerative changes in the testes, epididymis and vas deferens of scorbutic guinea pigs. On the other hand, excessive intake of vitamin C has been reported to cause reproductive failure in the men .
AA deficiency may lead to an increase in oxidative damage induced by ROS and a disturbed oxidative balance was observed in ejaculates of 25–45% of infertile men . This was further corroborated by the association of decreased AA followed by an increase in the seminal plasma LPO as observed in a human trial [126, 127]. Moreover, it has been reported that AA supplementation leads to a significant reduction in the ROS concentration, sperm membrane LPO and DNA oxidation together with an increased sperm quality. The results of a recent animal experimental study indicate that vitamin C improves the activity of antioxidant enzymes and significantly reduces malondialdehyde (MDA) concentration in testicular structures .
6.5.3. Vitamin E
Vitamin E is a term that encompasses a group of potent, lipid-soluble tocol (tocopherol) and tocotrienol derivatives qualitatively exhibiting the biological activity of RRR-α-tocopherol. Structural analyses have revealed that molecules having vitamin E antioxidant activity include four tocopherols (α-, β-, γ- and δ-) and four tocotrienols (α-, β-, γ- and δ-) with α-tocopherol being the most abundant form in nature and mostly available in food, having the highest biological activity and reversing vitamin E deficiency symptoms. The molecular functions fulfilled specifically by α-tocopherol have yet to be fully described; however, the antioxidant feature is the flagship of the biological activity related to vitamin E .
Vitamin E is present within the seminal plasma and plasma membrane. It is a lipid soluble, chain-breaking antioxidant that able to terminate free radical chain reactions, particularly the peroxidation of PUFAs [129, 130].
Numerous reports emphasize on the role of α-tocopherol in the management of male infertility. A positive association was found between α-tocopherol in sperm plasma membranes and the percentage of motile, living and morphologically intact spermatozoa . At the same time, α-tocopherol levels were decreased significantly in oligo- and azoospermic patients in comparison to normospermic controls .
A significant improvement in the
6.5.4. Other non-enzymatic antioxidants
There are other substances which may contribute to the maintenance of oxidative homeostasis. The prime function of these compounds is not to combat the production or action of ROS; however, their presence may decrease the risk of OS development. Albumin, cysteine, taurine, zinc and selenium are the most known representatives. Furthermore, antioxidant substances isolated from natural resources, such as resveratrol, curcumin or lycopene, have recently emerged as suitable dietary supplements or therapeutics due to their chemical diversity, structural complexity, availability, lack of significant toxic effects and intrinsic biologic activity. A short overview of secondary non-enzymatic antioxidants is provided in Table 3.
7. Strategies to reduce oxidative stress in male reproduction
Antioxidant supplementation has proven to be effective against male reproductive dysfunction
|Vitamin C and vitamin E|
|Vitamins A, C, E, N-acetyl-cysteine and zinc|
|Selenium and Vitamins|
|Zinc sulfate (ZnSO4)|
ROS-induced damage may have significant clinical implications in the context of ARTs. Numerous reports have indicated that significantly increased ROS levels may occur in response to repeated cycles of centrifugation involved in conventional sperm preparation techniques used for ARTs . Spermatozoa selected for ART often face OS and a high risk for DNA damage. When intrauterine insemination or
Selection of an effective sperm preparation technique is important to minimize ROS overgeneration and eventual oxidative insults to the male gamete. The density gradient technique is able to separate leukocytes and immature or damaged spermatozoa from normal spermatozoa, which may be subsequently used in ARTs [77, 179, 180].
Assisted reproduction techniques may benefit from
In cases of IVF, incubation times of more than 16–20 hours have been correlated with increased oxidative damage. Shortening the insemination timeframes (up to 1–2 hours or less) may reduce ROS overgeneration in culture media and possibly improve fertilization, embryogenesis and pregnancy rates [77, 179].
8. Methods for detecting reactive oxygen species
Because high levels of ROS have been associated with a decreased male infertility, measuring ROS levels in semen is an important part of the initial evaluation as well as follow-up of men with reproductive dysfunction [10, 11, 12]. Chemiluminescence and flow cytometry are currently the most common techniques in clinical andrology to assess and study seminal OS.
Chemiluminescence measures light emitted following administration of specific reagents to a semen sample. Two major probes currently used to assess ROS generation by spermatozoa are luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) and lucigenin (10,10′-dimethyl-9,9′-biacridinium dinitrate). Lucigenin is membrane-impermeable and responsive to ROS, particularly O2●−, in the extracellular space. Inversely, luminol is relatively membrane-permeable and reacts with a variety of ROS, including O2●−, H2O2 and OH● intracellularly as well as extracellularly. Chemiluminescent assays are sensitive, convenient for diagnostic purposes and have relatively well-established normal ranges [11, 12]. Nevertheless, significant set up costs have to be taken into consideration, and the data generated by chemiluminescence must be interpreted carefully because a variety of factors can affect the signals obtained .
A possible solution to the disadvantages associated with the chemiluminescence approach can be found in a variety of redox-sensitive fluorescence probes that can be loaded into spermatozoa and subsequently monitored by flow cytometry . Two probes can be used. Dihydroethidium or hydroethidine is a non-fluorescent probe that is oxidized by the superoxide to become ethidium bromide, which will stain the mitochondrial and nuclear DNA [183, 184]. The other fluorescent probe is 2,7-dichlorofluorescein diacetate, a stable non-fluorescent cell-permeable probe that de-esterifies in the presence of intracellular H2O2 to form 2,7-dichlorofluorescein . Other ROS such as peroxynitrite, HOCl, and OH● can also oxidize this probe . Flow cytometry has a higher specificity, accuracy, sensitivity and reproducibility than fluorescent microscopy or chemiluminescence. A large number of cells can easily be analyzed, leading to high specificity and sensitivity . One major disadvantage is that sophisticated and expensive hardware is needed. Also, the results do not quantify the target ROS but simply indicate the percentage of cells exhibiting a high level of activity .
Other methods to assess the oxidative balance in semen include indirect measurements such as the total antioxidant assay. This protocol is based on the ability of all antioxidants present in the sample to cease the oxidation of 2,20-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) to ABTS+ by metmyoglobin. Hence, the antioxidants suppress oxidative processes to a degree that is proportional to their final concentration, which may be detected at 750 nm . Another option is to assess the activity of antioxidant enzymes (SOD, CAT, GPx) or the redox potential defined by the ratio of oxidized and reduced glutathione using commercially available assay kits. A popular option is the measurement of oxidative end-products, including protein carbonyls , lipid hydroperoxides , MDA  and oxidative DNA adduct 8-hydroxy 2-deoxyguanosine .
Despite a remarkable progress in the evolution and design of new techniques to evaluate seminal OS, more straight-forward and accessible assays with well-defined and clinically significant physiological ranges reflecting normal sperm functions have yet to be introduced in order for oxidative stress to become a standard sub- or infertility marker in andrology laboratories.
Oxygen toxicity is an inherent double-edged sword to aerobic life. Increased oxidative insults to sperm lipids, proteins and DNA are associated with alterations of signal transduction mechanisms crucial for fertility. The origin of ROS generation and the etiologies of increased ROS in men with low sperm quality are becoming increasingly clear, offering multiple management and/or treatment options. Recent evidence suggests that spermatozoa possess an inherent ability to generate ROS essential for the fertilization process. A variety of defense mechanisms against ROS overproduction encompassing antioxidant enzymes, vitamins and other biologically active molecules are involved in biological systems. A balance of the benefits and risks from free radical production seems to be crucial for the sperm survival and function. As male infertility continues to play an increasing role in contributing to the inability to conceive in couples of reproductive age, it is pivotal for andrologists to fully comprehend the importance of thoroughly evaluating seminal oxidative profiles in order to provide a better care for male patients with reproductive dysfunction. Although the therapeutic use of antioxidants appears attractive, clinicians need to be aware of exaggerated claims of antioxidant benefits by various commercial supplements for fertility purposes until proper multicenter trials have been completed. However, initial data emphasizing on the potential of antioxidant supplementation in improving semen quality and conception rates are indeed encouraging.
This study was supported by the Slovak Research and Development Agency grants APVV-15-0543 and APVV-15-0544.
List of abbreviations
|ARTs||Artificial reproduction techniques|
|cAMP||Cyclic adenosine monophosphate|
|ICSI||Intracytoplasmic sperm injection|
|IVF||In vitro fertilization|
|NADPH||ß-nicotinamide adenine dinucleotide phosphate|
|PHGPx||Phospholipid hydroperoxide glutathione peroxidase|
|PUFAs||Polyunsaturated fatty acids|
|RNS||Reactive nitrogen species|
|ROS||Reactive oxygen species|
|WHO||World Health Organization|
Sies H. Strategies of antioxidant defense. European Journal of Biochemistry. 1993; 215:213-219
Boveris A, Chance B. The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen. Biochemical Journal. 1973; 134:707-716
Chance B, Sies H, Boveris H. Hydroperoxide metabolism in mammalian organs. Physiological Reviews. 1979; 59:527-605
Makker K, Agarwal A, Sharma R. Oxidative stress & male infertility. Indian Journal of Medical Research. 2009; 129:357-367
Sies H. Oxidative stress: Oxidants and antioxidants. Experimental Physiology. 1997; 82:291-295
Aitken RJ. Molecular mechanisms regulating human sperm function. Molecular Human Reproduction. 1997; 3:169-173
Chen H, Zhao HX, Huang XF, Chen GW, Yang ZX, Sun WJ, Tao MH, Yuan Y, Wu JQ, Sun F, Dai Q, Shi HJ. Does high load of oxidants in human semen contribute to male factor infertility? Antioxidants & Redox Signaling. 2012; 16:754-759. DOI: 10.1089/ars.2011
Aitken RJ, De Iuliis GN, Finnie JM, Hedges A, McLachlan RI. Analysis of the relationships between oxidative stress, DNA damage and sperm vitality in a patient population: Development of diagnostic criteria. Human Reproduction 2010; 25:2415-2426. DOI: 10.1093/humrep/deq214
Aitken RJ, Roman SD. Antioxidant systems and oxidative stress in the testes. Oxidative Medicine and Cellular Longevinity. 2008; 1:15-24
Zini A, de Lamirande E, Gagnon C. Reactive oxygen species in the semen of infertile patients: Levels of superoxide dismutase- and catalase-like activities in seminal plasma and spermatozoa. International Journal of Andrology. 1993; 16:183-188
Agarwal A, Mulgund A, Sharma R, Sabanegh E. Mechanisms of oligozoospermia: An oxidative stress perspective. Systems Biology in Reproductive Medicine. 2014a; 60:206-126. DOI: 10.3109/19396368.2014.918675
Agarwal A, Tvrda E, Sharma R. Relationship amongst teratozoospermia, seminal oxidative stress and male infertility. Reproductive Biology and Endocrinology. 2014b; 12:45. DOI: 10.1186/1477-7827-12-45
Ďuračková Z. Free radicals and antioxidants for non-experts. In: Laher I, editor. Systems Biology of Free Radicals and Antioxidants. 1st ed. Berlin Heidelberg: Springer Verlag; 2014. p. 3-38. DOI: 10.1007/978-3-642-30018-9_2
Hermes-Lima M. Oxygen in biology and biochemistry: Role of free radicals. In: Storey KB, editor. Functional Metabolism: Regulation and Adaptation. Hoboken, New Jersey, USA: John Wiley & Sons, 2004, pp. 319-368. ISBN: 0-471-410909-X
Halliwell B. Free radicals and other reactive species in disease. Encyclopedia of Life Sciences. 2005; 1:1-9
Agarwal A, Prabakaran SA. Mechanism, measurement, and prevention of oxidative stress in male reproductive physiology. Indian Journal of Experimental Biology. 2005; 43:963-974
Whittington K, Ford WC. Relative contribution of leukocytes and of spermatozoa to reactive oxygen species production in human sperm suspensions. International Journal of Andrology. 1999; 22:229-235
Wolff H. The biologic significance of white blood cells in semen. Fertility and Sterility. 1995; 63:1143-1157
Ochsendorf FR. Infections in the male genital tract and reactive oxygen species. Human Reproduction Update. 1999; 5:399-420
Shekarriz M, Sharma RK, Thomas Jr AJ, Agarwal A. Positive myeloperoxidase staining (Endtz test) as an indicator of excessive reactive oxygen species formation in semen. Journal of Assisted Reproduction and Genetics. 1995; 12:70-74
Cooper TG, Noonan E, von Eckardstein S, Auger J, Baker HW, Behre HM, Haugen TB, Kruger T, Wang C, Mbizvo MT, Vogelsong KM. World Health Organization reference values for human semen characteristics. Human Reproduction Update. 2010; 16:231-245. DOI: 10.1093/humupd/dmp048
Henkel R, Kierspel E, Stalf T, Mehnert C, Menkveld R, Tinneberg HR, Schill WB, Kruger TF. Effect of reactive oxygen species produced by spermato – zoa and leukocytes on sperm functions in non-leukocytospermic patients. Fertility and Sterility. 2005; 83:635-642
Wolff H, Politch JA, Martinez A, Haimovici F, Hill JA, Anderson DJ. Leukocytospermia is associated with poor semen quality. Fertility and Sterility. 1990; 53:528-536
Sharma RK, Pasqualotto AE, Nelson DR, Thomas Jr AJ, Agarwal A. Relationship between seminal white blood cell counts and oxidative stress in men treated at an infertility clinic. Journal of Andrology. 2001; 22:575-283
Plante M, de Lamirande E, Gagnon C. Reactive oxygen species released by activated neutrophils, but not by deficient spermatozoa, are sufficient to affect normal sperm motility. Fertility and Sterility. 1994; 62:387-393
Gatti JL, Castella S, Dacheux F, Ecroyd H, Métayer S, Thimon V, Dacheux JL. Post-testicular sperm environment and fertility. Animal Reproduction Science. 2004; 82-83:321-339
Aitken J, Krausz C, Buckingham D. Relationships between biochemical markers for residual sperm cytoplasm, reactive oxygen species generation, and the presence of leukocytes and precursor germ cells in human sperm suspensions. Molecular Reproduction and Development. 1994; 39:268-279
Gil-Guzman E, Ollero M, Lopez MC, Sharma RK, Alvarez JG, Thomas AJ Jr, Agarwal A. Differential production of reactive oxygen species by subsets of human spermatozoa at different stages of maturation. Human Reproduction. 2001; 16:1922-1930
Cho ChL, Esteves SC, Agarwal A. Novel insights into the pathophysiology of varicocele and its association with reactive oxygen species and sperm DNA fragmentation. Asian Journal of Andrology. 2016; 18:186-193. DOI: 10.4103/1008-682X.170441
Du Plessis SS, Agarwal A, Halabi J, Tvrda E. Contemporary evidence on the physiological role of reactive oxygen species in human sperm function. Journal of Assisted Reproduction and Genetics. 2015; 32:509-520. DOI: 10.1007/s10815-014-0425-7
Shiraishi K, Matsuyama H, Takihara H. Pathophysiology of varicocele in male infertility in the era of assisted reproductive technology. International Journal of Urology. 2012; 19:538-550. DOI: 10.1111/j.1442-2042.2012.02982.x
de Lamirande E, Jiang H, Zini A, Kodama H, Gagnon C. Reactive oxygen species and sperm physiology. Reviews of Reproduction. 1997; 2:48-54
Koppers AJ, De Iuliis GN, Finnie JM. Significance of mitochondrial reactive oxygen species in the generation of oxidative stress in spermatozoa. Journal of Clinical Endocrinology & Metabolism. 2008; 93:3199-3207. DOI: 10.1210/jc.2007-2616
Armstrong JS, Bivalacqua TJ, Chamulitrat W, Sikka S, Hellstrom WJ. A comparison of the NADPH oxidase in human sperm and white blood cells. International Journal of Andrology. 2002; 25:223-229
Richer S, Ford W. A critical investigation of NADPH oxidase activity in human spermatozoa. Molecular Human Reproduction. 2001; 7:237-244
Said TM, Agarwal A, Sharma RK, Mascha E, Sikka SC, Thomas AJ Jr. Human sperm superoxide anion generation and correlation with semen quality in patients with male infertility. Fertility and Sterility. 2004; 82:871-877
Herrero MB, Gagnon C. Nitric oxide: A novel mediator of sperm function. Journal of Andrology. 2001; 22:349-356
Marques-Pinto A, Carvalho D. Human infertility: Are endocrine disruptors to blame? Endocrine Connections. 2013; 2:R15-R29
Wirth JJ, Mijal RS. Adverse effects of low level heavy metal exposure on male reproductive function. Systems Biology in Reproductive Medicine. 2010; 56:147-167. DOI: 10.3109/19396360903582216
La Vignera S, Condorelli RA, Vicari E, D’Agata R, Calogero AE. Effects of the exposure to mobile phones on male reproduction: A review of the literature. Journal of Andrology. 2012; 33:350-356. DOI: 10.2164/jandrol.111.014373
Taha EA, Ez-Aldin AM, Sayed SK, Ghandour NM, Mostafa T. Effect of smoking on sperm vitality, DNA integrity, seminal oxidative stress, zinc in fertile men. Urology. 2012; 80:822-825. DOI: 10.1016/j.urology.2012.07.002
Saleh RA, Agarwal A, Sharma RK, Nelson DR, Thomas AJ Jr. Effect of cigarette smoking on levels of seminal oxidative stress in infertile men: A prospective study. Fertillity and Sterility. 2002; 78:491-499
Gholinezhad CM, Colagar AH. Seminal plasma lipid peroxidation, total antioxidant capacity, and cigarette smoking in asthenoteratospermic men. Journal of Men’s Health. 2011; 8:43-49. DOI: 10.1016/j.jomh.2010.09.230
Jensen TK, Swan S, Jørgensen N, Toppari J, Redmon B, Punab M, Drobnis EZ, Haugen TB, Zilaitiene B, Sparks AE, Irvine DS, Wang C, Jouannet P, Brazil C, Paasch U, Salzbrunn A, Skakkebæk NE, Andersson AM. Alcohol and male reproductive health: A cross-sectional study of 8344 healthy men from Europe and the USA. Human Reproduction. 2014; 29:1801-1809. DOI: 10.1093/humrep/deu118
Jang MH, Shin MC, Shin HS, Kim KH, Park HJ, Kim EH, Kim CJ. Alcohol induces apoptosis in TM3 mouse Leydig cells via bax-dependent caspase-3 activation. European Journal of Pharmacology. 2002; 449:39-45
Jensen TK, Heitmann BL, Jensen MB, Halldorsson TI, Andersson AM, Skakkebaek NE, Joensen UN, Lauritsen MP, Christiansen P, Dalgard C, Lassen TH, Jorgensen N. High dietary intake of saturated fat is associated with reduced semen quality among 701 young Danish men from the general population. American Journal of Clinical Nutrition. 2013; 97:411-418. DOI: 10.3945/ajcn.112.042432
Afeiche MC, Bridges ND, Williams PL, Gaskins AJ, Tanrikut C, Petrozza JC, Hauser R, Chavarro JE. Dairy intake and semen quality among men attending a fertility clinic. Fertility and Sterility. 2014; 101:1280-1287. DOI: 10.1016/j.fertnstert.2014.02.003
Qin DD, Yuan W, Zhou WJ, Cui YQ, JQ W, Gao ES. Do reproductive hormones explain the association between body mass index and semen quality? Asian Journal of Andrology. 2017; 9:827-834
Dadkhah H, Kazemi A, Nasr-Isfahani MH, Ehsanpour S. The relationship between the amount of saturated fat intake and semen quality in men. Iranian Journal of Nursing and Midwifery Research. 2017; 22:46-50. DOI: 10.4103/1735-9066.202067
Hadjkacem Loukil L, Hadjkacem H, Bahloul A, Ayadi H. Relation between male obesity and male infertility in a Tunisian population. Andrologia. 2014; 47:282-285. DOI: 10.1111/and.12257
Ford WC. Regulation of sperm function by reactive oxygen species. Human Reproduction Update. 2004; 10:87-399
Erenpreiss J, Spano M, Erenpreisa J, Bungum M, Giwercman A. Sperm chromatin structure and male fertility: Biological and clinical aspects. Asian Journal of Andrology. 2006; 8:11-29
Aitken RJ, Ryan AL, Baker MA, McLaughlin EA. Redox activity associated with the maturation and capacitation of mammalian spermatozoa. Free Radical Biology and Medicine. 2004; 36:994-1010
Amaral A, Lourenço B, Marques M, Ramalho-Santos J. Mitochondria functionality and sperm quality. Reproduction. 2013; 146:R163-R174. DOI: 10.1530/REP-13-0178
Zini A, San Gabriel M, Baazeem A. Antioxidants and sperm DNA damage: A clinical perspective. Journal of Assisted Reproduction and Genetics. 2009; 26:427-432. DOI: 10.1007/s10815-009-9343-5
Menezo Y. 2007. Antioxidants to reduce sperm DNA fragmentation: An unexpected adverse effect. Reproductive Biomedicine Online. 2007; 14:418-421
de Lamirande E, Leclerc P, Gagnon C. Capacitation as a regulatory event that primes spermatozoa for the acrosome reaction and fertilization. Molecular Human Reproduction. 1997; 3:175-194
de Lamirande E, Gagnon C. Human sperm hyperactivation and capacitation as parts of an oxidative process. Free Radicals in Biology and Medicine. 1993; 14:157-166
Rivlin J, Mendel J, Rubinstein S, Etkovitz N, Breitbart H. Role of hydrogen peroxide in sperm capacitation and acrosome reaction. Biology of Reproduction. 2004; 70:518-522
Zini A, de Lamirande E, Gagnon C. Low levels of nitric oxide promote human sperm capacitation in vitro. Journal of Andrology. 1995; 16:424-431
Aitken RJ. The capacitation-apoptosis highway: Oxysterols and mammalian sperm function. Biology of Reproduction. 2011; 85:9-12. DOI: 10.1095/biolreprod.111.092528
Suarez SS. Control of hyperactivation in sperm. Human Reproduction Update. 2008; 14:647-657. DOI: 10.1093/humupd/dmn029
Baldi E, Luconi M, Bonaccorsi L, Muratori M, Forti G. Intracellular events and signaling pathways involved in sperm acquisition of fertilizing capacity and acrosome reaction. Frontiers in Bioscience. 2000; 5:110-123
de Lamirande E, Tsai C, Harakat A, Gagnon C. Involvement of reactive oxygen species in human sperm acrosome reaction induced by A23187, lysophosphatidylcholine, and biological fluid ultrafiltrates. Journal of Andrology. 1998; 19:585-594
Griveau JF, Le Lannou DL. Reactive oxygen species and human spermatozoa: Physiology and pathology. International Journal of Andrology. 1997; 20:61-69
Min L. The biology and dynamics of mammalian cortical granules. Reproductive Biology and Endocrinology. 2011; 9:149. DOI: 10.1186/1477-7827-9-149
Gadella BM. Interaction of sperm with the zona pellucida during fertilization. Society for Reproduction and Fertility. 2010; 67:267-287
Aitken RJ, Clarkson JS, Fishel S. Generation of reactive oxygen species, lipid peroxidation and human sperm function. Biology of Reproduction. 1989; 40:183-197
Alvarez JG, Touchstone JC, Blasco L, Storey BT. Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. Superoxide dismutase as major enzyme protectant against oxygen toxicity. Journal of Andrology. 1987; 8:338-348
Ayala A, Muñoz MF, Argüelles S. Lipid peroxidation: Production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxidative Medicine and Cellular Longevity. 2014; 2014:1-31. DOI: 10.1155/2014/360438
Aitken RJ, Wingate JK, De Iuliis GN, Kopper AJ, McLaughlin EA. Cis-unsaturated fatty acids stimulate reactive oxygen species generation and lipid peroxidation in human spermatozoa. In Journal of Clinical Endocrinology & Metabolism. 2006; 91, 14154-14163. DOI: 10.1210/jc.2006-1309
Agarwal A, Said TM. Oxidative stress, DNA damage and apoptosis in male infertility: A clinical approach. BJU International. 2005; 95:503-507. DOI: 10.1111/j.1464-410X.2005.05328.x
Wright C, Milne S, Leeson H. Sperm DNA damage caused by oxidative stress: Modifiable clinical, lifestyle and nutritional factors in male infertility. Reproductive Biomedicine Online. 2014; 28:684-703. DOI: 10.1016/j.rbmo.2014.02.004
Hosen B, Islam R, Begum F, Kabir Y, Howlader ZH. Oxidative stress induced sperm DNA damage, a possible reason for male infertility. Iranian Journal of Reproductive Medicine. 2015; 13:525-532
Shirakawa T, Fujisawa M, Kanzaki M, Okada H, Arakawa S, Kamidono S. Y chromosome (Yq11) microdeletions in idiopathic azoospermia. International Journal of Urology. 1997; 4:198-201
Kumar DP, Sangeetha N. Mitochondrial DNA mutations and male infertility. Indian Journal of Human Genetics. 2009; 15:93-97. DOI: 10.4103/0971-6866.60183
Bach PV, Schlegel PN, Sperm DNA. Damage and its role in IVF and ICSI. Basic and Clinical Andrology. 2016; 26:15. DOI: 10.1186/s12610-016-0043-6
Davies MJ. Oxidative damage to proteins. In: Chatgilialoglu C, Studer A, editors. Encyclopedia of Radicals in Chemistry, Biology and Materials. 1st ed. New York, USA: Wiley; 2012. p. 93-109. 2324 p. DOI: 10.1002/9781119953678.rad045
Berlett BS, Stadtman ER. Protein oxidation in aging, disease, and oxidative stress. The Journal of Biological Chemistry. 1997; 272:20313-20316. DOI: 10.1074/jbc.272.33.20313
Sinha S, Pradeep KG, Laloraya M, Warikoo D. Over-expression of superoxide dismutase and lack of surface-thiols in spermatozoa: Inherent defects in oligospermia. Biochemical and Biophysical Research Communications. 1991; 174:510-517
Mammoto A, Masumoto N, Tahara M, Ikebuchi Y, Ohmichi M, Tasaka K, Miyake A. Reactive oxygen species block sperm-egg fusion via oxidation of sperm sulfhydryl proteins in mice. Biology of Reproduction. 1996; 55:1063-1068
Shaha C, Tripathi R, Mishra DP. Male germ cell apoptosis: Regulation and biology. Philosophical Transactions of the Royal Society of London. 2010; 365:1501-1515. DOI: 10.1098/rstb.2009.0124
Shukla KK, Mahdi AA, Rajender S. Apoptosis, spermatogenesis and male infertility. Frontiers in Bioscience. 2012; 4:746-754
Vissers MC, Pullar JM, Hampton MB. Hypochlorous acid causes caspase activation and apoptosis or growth arrest in human endothelial cells. Biochemical Journal. 1999; 344:443-449
Said TM, Paasch U, Glander HJ, Agarwal A. Role of caspases in male infertility. Human Reproduction Update. 2004; 10:39-51
Martínez-Pastor F, Aisen E, Fernández-Santos MR, Esteso MC, Maroto-Morales A, García-Alvarez O, Garde JJ. Reactive oxygen species generators affect quality parameters and apoptosis markers differently in red deer spermatozoa. Reproduction. 2009; 137:225-235. DOI: 10.1530/REP-08-0357
Moran JM, Madejón L, Ortega Ferrusola C, Peña FJ. Nitric oxide induces caspase activity in boar spermatozoa. Theriogenology. 2008; 70:91-96. DOI: 10.1016/j.theriogenology.2008.02.010
Johnson C, Jia Y, Wang C, Lue Y, Swerdloff RS, Zhang X, Hu Z, Li Y, Sinha Hikim AP. Role of caspase 2 in apoptotic signaling in primate and murine germ cells. Biology of Reproduction. 2008; 79:806-814. DOI: 10.1095/biolreprod.108.068833
Elia J, Imbrogno N, Delfino M, Mazzilli R, Rossi T, Mazzilli F. The importance of the sperm motility classes-future directions. The Open Andrology Journal. 2010; 2:42-43
Armstrong JS, Rajasekaran M, Chamulitrat W, Gatti P, Hellstrom WJ, Sikka SC. Characterization of reactive oxygen species induced effects on human spermatozoa movement and energy metabolism. Free Radical Biology & Medicine. 1999; 26:869-880
Twigg J, Irvine DS, Houston P, Fulton N, Michael L, Aitken RJ. Iatrogenic DNA damage induced in human spermatozoa during sperm preparation: Protective significance of seminal plasma. Molecular Human Reproduction. 1998; 4:439-445
Zareba P, Colaci DS, Afeiche M, Gaskins AJ, Jørgensen N, Mendiola J, Swan SH, Chavarro JE. Semen quality in relation to antioxidant intake in a healthy male population. Fertility and Sterility. 2013; 100:1572-1579. DOI: 10.1016/j.fertnstert.2013.08.032
Walczak-Jedrzejowska R, Wolski JK, Slowikowska-Hilczer J. The role of oxidative stress and antioxidants in male fertility. Central European Journal of Urology. 2013; 66:60-67. DOI: 10.5173/ceju.2013.01.art19
Asadpour R, Jafari R, Tayefi-Nasrabadi H. The effect of antioxidant supplementation in semen extenders on semen quality and lipid peroxidation of chilled bull spermatozoa. Iranian Journal of Veterinary Research. 2012; 13:246-249
Perumal P. Effect of superoxide dismutase on semen parameters and antioxidant enzyme activities of liquid stored (5°C) mithun (Bos Frontalis) semen. Journal of Animals. 2014; 2014:1-9. DOI: 10.1155/2014/821954
Buffone MG, Calamera JC, Brugo-Olmedo S, De Vincentiis S, Calamera MM, Storey BT, Doncel GF, Alvarez JG. Superoxide dismutase content in sperm correlates with motility recovery after thawing of cryopreserved human spermatozoa. Fertility and Sterility. 2012; 97:293-298. DOI: 10.1016/j.fertnstert.2011.11.012
Jeulin C, Soufir JC, Weber P, Laval-Martin D, Calvayrac R. Catalase activity in human spermatozoa and seminal plasma. Gamete Research. 1989; :185-196 24
Tvrdá E, Kňažická Z, Lukáčová J, Schneidgenová M, Goc Z, Greń A, Szabó C, Massányi P, Lukáč N. The impact of lead and cadmium on selected motility, prooxidant and antioxidant parameters of bovine seminal plasma and spermatozoa. Journal of Environmental Science and Health Part A. 2013; 48:1292-1300. DOI: 10.1080/10934529.2013.777243
Macanovic B, Vucetic M, Jankovic A, Stancic A, Buzadzic B, Garalejic E, Korac A, Korac B, Otasevic V. Correlation between sperm parameters and protein expression of antioxidative defense enzymes in seminal plasma: A pilot study. Disease Markers. 2015; 2015:1-5. DOI: 10.1155/2015/436236
Moubasher AE, El Din AME, Ali ME, El-Sherif WT, Gaber HD. Catalase improves motility, vitality and DNA integrity of cryopreserved human spermatozoa. Andrologia. 2013; 45:135-139. DOI: 10.1111/j.1439-0272.2012.01310.x
Imai H, Nakagawa Y. Biological significance of phospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) in mammalian cells. Free Radical Biology and Medicine. 2003; 34:145-169
Jelezarsky L, Vaisberg C, Chaushev T, Sapundjiev E. Localization and characterization of glutathione peroxidase (GPx) in boar accessory sex glands, seminal plasma, and spermatozoa and activity of GPx in boar semen. Theriogenology. 2008; 69:139-145
Fujii J, Ito JI, Zhang X, Kurahashi T. Unveiling the roles of the glutathione redox system in vivo by analyzing genetically modified mice. Journal of Clinical Biochemistry and Nutrition. 2011; 49:70-78. DOI: 10.3164/jcbn.10-138SR
Garrido N, Meseguer M, Alvarez J, Simón C, Pellicer A, Remohí J. Relationship among standard semen parameters, glutathione peroxidase/glutathione reductase activity, and mRNA expression and reduced glutathione content in ejaculated spermatozoa from fertile and infertile men. Fertility and Sterility. 2004; 82:1059-1066
Kaneko T, Iuchi Y, Kobayashi T, Fujii T, Saito H, Kurachi H, Fujii J. The expression of glutathione reductase in the male reproductive system of rats supports the enzymatic basis of glutathione function in spermatogenesis. European Journal of Biochemistry. 2002; 269:1570-1578
Gopalakrishnan B, Aravinda S, Pawshe CH, Totey SM, Nagpal S, Salunke DM, Shaha C. Studies on glutathione S-transferases important for sperm function: Evidence of catalytic activity-independent functions. Biochemical Journal. 1998; 329:231-241
Kumar R, Singh VK, Atreja SK. Glutathione-S-transferase: Role in buffalo ( Bubalus bubalis) sperm capacitation and cryopreservation. Theriogenology. 2014; 81:587-598. DOI: 10.1016/j.theriogenology.2013.11.012
Hemachand T. Functional role of sperm surface glutathione S-transferases and extracellular glutathione in the haploid spermatozoa under oxidative stress. FEBS Letters. 2003; 538:14-18
Orlando C, Caldini AL, Barni T, Wood WG, Strasburger CJ, Natali A, Maver A, Forti G, Serio M. Ceruloplasmin and transferrin in human seminal plasma: Are they an index of seminiferous tubular function? Fertility and Sterility. 1985; 43:290-294
Roeser HP, Lee GR, Nacht S, Cartwright GE. The role of ceruloplasmin in iron metabolism. Journal of Clinical Investigation. 1970; 49:2408-2417
Galdston M, Feldman JG, Levytska V, Magnůsson B. Antioxidant activity of serum ceruloplasmin and transferrin available iron-binding capacity in smokers and nonsmokers. American Review of Respiratory Disease. 1987; 135:783-787
Akalın PP, Bülbül B, Çoyan K, Başpınar N, Kırbaş M, Bucak MN, Güngör S, Öztürk C. Relationship of blood and seminal plasma ceruloplasmin, copper, iron and cadmium concentrations with sperm quality in merino rams. Small Ruminant Research. 2015; 133:135-139. DOI: 10.1016/j.smallrumres.2015.08.019
Wojtczak M, Dietrich GJ, Irnazarow I, Jurecka P, Słowińska M, Ciereszko A. Polymorphism of transferrin of carp seminal plasma: Relationship to blood transferrin and sperm motility characteristics. Comparative Biochemistry and Physiology – Part B. 2007; 148:426-431
Abdel Aziz MT, Mostafa T, Roshdy N, Hosni H, Rashed L, Sabry D, Abdel Nasser T, Abdel Azim O, Abdel Gawad O. Heme oxygenase enzyme activity in human seminal plasma of fertile and infertile males. Andrologia. 2008; 40:292-297. DOI: 10.1111/j.1439-0272.2008.00856.x
Abdel Aziz MT, Mostafa T, Atta H, Kamal O, Kamel M, Hosni H, Rashed L, Sabry D, Waheed F. Heme oxygenase enzyme activity in seminal plasma of oligoasthenoteratozoospermic males with varicocele. Andrologia. 2010; 42:236-241. DOI: 10.1111/j.1439-0272.2009.00983.x
Turkseven S, Kruger A, Mingone CJ, Kaminski P, Inaba M, Rodella LF, Ikehara S, Wolin MS, Abraham NG. Antioxidant mechanism of heme oxygenase-1 involves an increase in superoxide dismutase and catalase in experimental diabetes. American Journal of Physiology – Heart and Circulatory Physiology. 2005; 289:H701-H707
Irvine DS. Glutathione as a treatment for male infertility. Reviews of Reproduction. 196; 1:6-12
Meseguer M, Martínez-Conejero JA, Muriel L, Pellicer A, Remohí J, Garrido N. The human sperm glutathione system: A key role in male fertility and successful cryopreservation. Drug Metabolism Letters. 2007; 1:121-126
Chambial S, Dwivedi S, Shukla KK, John PJ, Sharma P. Vitamin C in disease prevention and cure: An overview. Indian Journal of Clinical Biochemistry. 2013; 28:314-328. DOI: 10.1007/s12291-013-0375-3
Mathur V, Murdia A, Hakim AA, Suhalka ML, Shaktawat GS, Kothari LK. Male infertility and the present status of its management by drugs. Journal of Postgraduate Medicine. 1979; 25:90-96
Colagar AH, Marzony ET. Ascorbic acid in human seminal plasma: Determination and its relationship to sperm quality. Journal of Clinical Biochemistry and Nutrition. 2009; 45:144-149. DOI: 10.3164/jcbn.08-251
Das P, Choudhari AR, Dhawan A, Singh R. Role of ascorbic acid in human seminal plasma against the oxidative damage to the sperms. Indian Journal of Clinical Biochemistry. 2009; 24:312-315. DOI: 10.1007/s12291-009-0058-2
Song GJ, Norkus EP, Lewis V. Relationship between seminal ascorbic acid and sperm DNA integrity in infertile men. International Journal of Andrology. 2006; 29:569-575
Chinoy MR, Sharma JD, Sanjeevan AG, Chinoy NJ. Structural changes in male reproductive organs and spermatozoa of scorbutic guinea pigs. Proceedings of the National Academy of Sciences, India. 1983: B49628-B49635
Paul PK, Datta-Gupta PN. Beneficial or harmful effects of a large dose of vitamin C on the reproductive organs of the male rat depending upon the level of food intake. Indian Journal of Experimental Biology. 1978; 16:18-21
Akmal M, Qadri JQ, Al-Waili NS, Thangal S, Haq A, Saloom KY. Improvement in human semen quality after oral supplementation of vitamin C. Journal of Medicinal Food. 2006; 9:440-442
Jelodar G, Nazifi S, Akbari A. The prophylactic effect of vitamin C on induced oxidative stress in rat testis following exposure to 900 MHz radio frequency wave generated by a BTS antenna model. Electromagnetic Biology and Medicine. 2013; 32:4094-4016. DOI: 10.3109/ 15368378.2012.735208
Chow CK, Chow-Johnson HS. Antioxidant function and health implications of vitamin E. The Open Nutrition Journal. 2013; 7:1-6
Bolle P, Evandri MG, Saso L. The controversial efficacy of vitamin E for human male infertility. Contraception. 2002; 65:313-315
Moilanen J, Hovatta O, Lindroth L. Vitamin E levels in seminal plasma can be elevated by oral administration of vitamin E in infertile men. International Journal of Andrology. 1993; 16:165-166
Kessopoulou E, Powers HJ, Sharma KK, Pearson MJ, Russell JM, Cooke ID, Barratt CLA. Double-blind randomized placebo cross-over controlled trial using the antioxidant vitamin E to treat reactive oxygen species associated male infertility. Fertility and Sterility. 1995; 64:825-831
Suleiman SA, Ali ME, Zaki ZM, El-Malik EM, Nasr MA. Lipid peroxidation and human sperm motility: Protective role of vitamin E. Journal of Andrology. 1996; 17:530-537
Ciftci H, Verit A, Savas M, Yeni E, Erel O. Effects of N-acetylcysteine on semen parameters and oxidative/antioxidant status. Urology. 2009; 74:73-76. DOI: 10.1016/j.urology.2009.02.034
Safarinejad MR, Safarinejad S. Efficacy of selenium and/or N-acetyl-cysteine for improving semen parameters in infertile men: A double-blind, placebo controlled, randomized study. Journal of Urology. 2009; 181:741-751. DOI: 10.1016/j.juro.2008.10.015
Ahmed SD, Karira KA, Jagdesh AS. Role of L-carnitine in male infertility. Journal of the Pakistan Medical Association. 2011; 61:732-736
Aliabadi E, Mehranjani MS, Borzoei Z, Talaei-Khozani T, Mirkhani H, Tabesh H. Effects of L-carnitine and L-acetyl-carnitine on testicular sperm motility and chromatin quality. Iranian Journal of Reproductive Medicine. 2012; 10:77-82
Holmes RP, Goodman HO, Shihabi ZK, Jarow JP. The taurine and hypotaurine content of human semen. Journal of Andrology. 1992; 13:289-292
Bidri M, Choay P. Taurine: A particular aminoacid with multiple functions. Annales Pharmaceutiques Francaises. 2003; 61:385-391
Zhao J, Dong X, Hu X, Long Z, Wang L, Liu Q, Sun B, Wang Q, Wu Q, Lia L. Zinc levels in seminal plasma and their correlation with male infertility: A systematic review and meta-analysis. Scientific Reports. 2016; 6:22386. DOI: 10.1038/srep22386
Santiago Y, Chan E, Liu PQ, Orlando S, Zhang L, Urnov FD, Holmes MC, Guschin D, Waite A, Miller JC, Rebar EJ, Gregory PD, Klug A, Collingwood TN. Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases. Proceedings of the National Academy of Sciences of USA. 2008; 105:5809-5814. DOI: 10.1073/pnas.0800940105
Powell SR. The antioxidant properties of zinc. Journal of Nutrition. 2000; 130:1447S-1454S
Boitani C, Puglisi R. Selenium, a key element in spermatogenesis and male fertility. Advances in Experimental Medicine and Biology. 2008; 636:65-73. DOI: 10.1007/978-0-387-09597-4_4
Bourdon E, Blache D. The importance of proteins in defense against oxidation. Antioxidants & Redox Signaling. 2001; 3:293-311
Elzanaty S, Erenpreiss J, Becker C. Seminal plasma albumin: Origin and relation to the male reproductive parameters. Andrologia. 2007; 39:60-65
Uysal O, Bucak MN. Effects of oxidized glutathione, bovine serum albumin, cysteine and lycopene on the quality of frozen-thawed ram semen. Acta Veterinaria Brunensis. 2007; 76:383-390. DOI: 10.2754/avb200776030383
Sedlak TW, Snyder SH. Bilirubin benefits: Cellular protection by a biliverdin reductase antioxidant cycle. Pediatrics. 2004; 113:1776-1782
Neuzil J, Stocker R. Free and albumin-bound bilirubin are efficient co-antioxidants for alpha-tocopherol, inhibiting plasma and low density lipoprotein lipid peroxidation. Journal of Biological Chemistry. 1994; 269:16712-16719
Sautin YY, Johnson RJ. Uric acid: The oxidant-antioxidant paradox. Nucleosides, Nucleotides & Nucleic Acids. 2008; 6:608-619. DOI: 10.1080/15257770802138558
Muraoka S, Miura T. Inhibition by uric acid of free radicals that damage biological molecules. Pharmacology & Toxicology. 2003; 93:284-289
de la Lastra CA, Villegas I. Resveratrol as an antioxidant and pro-oxidant agent: Mechanisms and clinical implications. Biochemical Society Transactions. 2007; 35:1156-1160
Baur JA, Sinclair DA. Therapeutic potential of resveratrol: The in vivo evidence. Nature Reviews Drug Discovery. 2010; 6:493-506
Shin S, Jeon JH, Park D, Jang MJ, Choi JH, Choi BH, Joo SS, Nahm SS, Kim JC, Kim YB. Trans-resveratrol relaxes the corpus cavernosum ex vivo and enhances testosterone levels and sperm quality in vivo. Archives of Pharmacal Research. 2008; 31:83-87
Tvrdá E, Kováčik A, Tušimová E, Massányi P, Lukáč N. Resveratrol offers protection to oxidative stress induced by ferrous ascorbate in bovine spermatozoa. Journal of Environmental Science and Health Part A. 2015; 50:1440-1451. DOI: 10.1080/10934529.2015.1071153
Agarwal S, Rao AV. Tomato lycopene and its role in human health and chronic diseases. Canadian Medical Association Journal. 2000; 163:739-744
Filipcikova R, Oborna I, Brezinova J, Novotny J, Wojewodka G, De Sanctis JB, Radova L, Hajduch M, Radzioch D. Lycopene improves the distorted ratio between AA/DHA in the seminal plasma of infertile males and increases the likelihood of successful pregnancy. Biomedical Papers of the Medical Faculty of the University Palacký, Olomouc, Czech Republic. 2013; 157:1-6. DOI: 10.5507/bp.2013.007
Gupta NP, Kumar R. Lycopene therapy in idiopathic male infertility – A preliminary report. International Urology and Nephrology. 2002; 34:369-372
Zini A, San Gabriel M, Libman J. Lycopene supplementation in vitro can protect human sperm deoxyribonucleic acid from oxidative damage. Fertility and Sterility. 2010; 94:1033-1036. DOI: 10.1016/j.fertnstert.2009.04.004
Tvrdá E, Kováčik A, Tušimová E, Paál D, Mackovich A, Alimov J, Lukáč N. Antioxidant efficiency of lycopene on oxidative stress-induced damage in bovine spermatozoa. Journal of Animal Science and Biotechnology. 2016; 7:50. DOI: 10.1186/s40104-016-0113-9
Akmal M, Qadri JQ, Al-Waili NS, Thangal S, Haq A, Saloom KY. Improvement in human semen quality after oral supplementation of vitamin C. Journal of Medicinal Food. 2006; 9:440-442
Cyrus A, Kabir A, Goodarzi D, Moghimi M. The effect of adjuvant vitamin C after varicocele surgery on sperm quality and quantity in infertile men: A double blind placebo controlled clinical trial. International Brazilian Journal of Urology. 2015; 41:230-238. DOI: 10.1590/S1677-5538.IBJU.2015.02.07
Suleiman SA, Ali ME, Zaki ZM, El-Malik EM, Nasr MA. Lipid peroxidation and human sperm motility: Protective role of vitamin E. Journal of Andrology. 1996; 17:530-537
Rolf C, Cooper TG, Yeung CH, Nieschlag E. Antioxidant treatment of patients with asthenozoospermia or moderate oligoasthenozoospermia with high-dose vitamin C and vitamin E: A randomized, placebo-controlled, double-blind study. Human Reproduction. 1999; 14:1028-1033
Greco E, Iacobelli M, Rienzi L, Ubaldi F, Ferrero S, Tesarik J. Reduction of the incidence of sperm DNA fragmentation by oral antioxidant treatment. Journal of Andrology. 2005; 26:349-353
Paradiso Galatioto G, Gravina GL, Angelozzi G, Sacchetti A, Innominato PF, Pace G, Ranieri G, Vicentini C. May antioxidant therapy improve sperm parameters of men with persistent oligospermia after retrograde embolization for varicocele? World Journal of Urology. 2008; 26:97-102
Lenzi A, Culasso F, Gandini L, Lombardo F, Dondero F. Placebo-controlled, double-blind, cross-over trial of glutathione therapy in male infertility. Human Reproduction. 1993; 8:1657-1662
Costa M, Canale D, Filicori M, D'lddio S, Lenzi A. L-carnitine in idiopathic asthenozoospermia: A multicenter study. Italian study group on Carnitine and male infertility. Andrologia. 1994; 26:155-159
Lenzi A, Lombardo F, Sgrò P, Salacone P, Caponecchia L, Dondero F, Gandini L. Use of carnitine therapy in selected cases of male factor infertility: A double-blind crossover trial. Fertility and Sterility. 2003; 79:292-300
Lenzi A, Sgrò P, Salacone P, Paoli D, Gilio B, Lombardo F, Santulli M, Agarwal A, Gandini L. A placebo-controlled double-blind randomized trial of the use of combined l-carnitine and l-acetyl-carnitine treatment in men with asthenozoospermia. Fertility and Sterility. 2004; 81:1578-1584
Sigman M, Glass S, Campagnone J, Pryor JL. Carnitine for the treatment of idiopathic asthenospermia: A randomized, double-blind, placebo-controlled trial. Fertility and Sterility. 2006; 85:1409-1414
Iwanier K, Zachara BA. Selenium supplementation enhances the element concentration in blood and seminal fluid but does not change the spermatozoal quality characteristics in subfertile men. Journal of Andrology. 1995; 16:441-447
Vézina D, Mauffette F, Roberts KD, Bleau G, Selenium-vitamin E. Supplementation in infertile men. Effects on semen parameters and micronutrient levels and distribution. Biological Trace Element Research. 1996; 53:65-83
Scott R, MacPherson A, Yates RW, Hussain B, Dixon J. The effect of oral selenium supplementation on human sperm motility. British Journal of Urology. 1998; 82:76-80
Keskes-Ammar L, Feki-Chakroun N, Rebai T, Sahnoun Z, Ghozzi H, Hammami S, Zghal K, Fki H, Damak J, Bahloul A. Sperm oxidative stress and the effect of an oral vitamin E and selenium supplement on semen quality in infertile men. Archives of Andrology. 2003; 49:83-94
Comhaire FH, Christophe AB, Zalata AA, Dhooge WS, Mahmoud AM, Depuydt CE. The effects of combined conventional treatment, oral antioxidants and essential fatty acids on sperm biology in subfertile men. Prostaglandins, Leukotrienes & Essential Fatty Acids. 2000; 63:159-165
Ciftci H, Verit A, Savas M, Yeni E, Erel O. Effects of N-acetylcysteine on semen parameters and oxidative/antioxidant status. Urology. 2009; 74:73-76. DOI: 10.1016/j.urology.2009.02.034
Omu AE, Al-Azemi MK, Kehinde EO, Anim JT, Oriowo MA, Mathew TC. Indications of the mechanisms involved in improved sperm parameters by zinc therapy. Medical Principles and Practice. 2008; 17:108-116. DOI: 10.1159/000112963
Lewin A, Lavon H. The effect of coenzyme Q10 on sperm motility and function. Molecular Aspects of Medicine. 1997; 18:S213-S219
Balercia G, Buldreghini E, Vignini A, Tiano L, Paggi F, Amoroso S, Ricciardo-Lamonica G, Boscaro M, Lenzi A, Littarru G. Coenzyme Q10 treatment in infertile men with idiopathic asthenozoospermia: A placebo-controlled, double-blind randomized trial. Fertility and Sterility. 2009; 91:1785-1792. DOI: 10.1016/j.fertnstert.2008.02.119
Agarwal A, Said TM, Bedaiwy MA, Banerjee J, Alvarez JG. Oxidative stress in an assisted reproductive techniques setting. Fertility and Sterility. 2006; 86:503-512
Sikka SC. Role of oxidative stress and antioxidants in andrology and assisted reproductive technology. Journal of Andrology. 2004; 25:5-18
Kobayashi H, Gil-Guzman E, Mahran AM. Quality control of reactive oxygen species measurement by luminol-dependent chemiluminescence assay. Journal of Andrology. 2001; 22:568-574
Aitken RJ, De Iuliis GN, Baker MA. Direct methods for the detection of reactive oxygen species in human semen samples. In: Agarwal A, Aitken RJ, Alvarez JG, editors. Studies on Men’s Health and Fertility, Oxidative Stress in Applied Basic Research and Clinical Practice. 1st ed. New York: Springer Science+Business Media, LLC; 2012. p. 275-299. DOI: 10.1007/978-1-61779-776-7_14
Rothe G, Valet G. Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2′, 7′-dichlorofluorescein. Journal of Leukocyte Biology. 1990; 47:440-448
De Iuliis GN, Wingate JK, Koppers AJ, McLaughlin EA, Aitken RJ. Definitive evidence for the nonmitochondrial production of superoxide anion by human spermatozoa. Journal of Clinical Endocrinology and Metabolism. 2006; 91:1968-1975
Myhre O, Andersen JM, Aarnes H, Fonnum F. Evaluation of the probes 2′,7′-dichlorofluorescein diacetate, luminol, and lucigenin as indicators of reactive species formation. Biochemical Pharmacology. 2003; 65:1575-1582
Mancini A, Milardi D, Bianchi A. Increased total antioxidant capacity in seminal plasma of varicocele patients: A multivariate analysis. Archives of Andrology. 2007; 53:37-42
Stadtman ER, Oliver CN. Metal-catalyzed oxidation of proteins. Physiological consequences. Journal of Biological Chemistry. 1991; 266:2005-2008
Jiang ZY, Hunt JV, Wolf SP. Detection of lipid hydroperoxides using fox method. Analytical Biochemistry. 1992; 202:384-389
Takagi Y, Nikaido T, Toki T, Kita N, Kanai M, Ashida T, Ohira S, Konishi I. Levels of oxidative stress and redox-related molecules in the placenta in preeclampsia and fetal growth restriction. Virchows Archiv. 2004; 444:49-55