Advertisement

Oxidative stress and medical antioxidant treatment in male infertility

  • Francesco M Lanzafame
    Affiliations
    Territorial Center of Andrology, AUSL 8, via Brenta 1, 96100 Syracuse, Italy
    Search for articles by this author
  • Sandro La Vignera
    Affiliations
    Section of Endocrinology, Andrology and Internal Medicine and Master in Andrological and Human Reproduction Sciences, Department of Biomedical Sciences, University of Catania, Piazza S. Maria di Gesù, 95123 Catania, Italy
    Search for articles by this author
  • Enzo Vicari
    Affiliations
    Section of Endocrinology, Andrology and Internal Medicine and Master in Andrological and Human Reproduction Sciences, Department of Biomedical Sciences, University of Catania, Piazza S. Maria di Gesù, 95123 Catania, Italy
    Search for articles by this author
  • Aldo E Calogero
    Affiliations
    Section of Endocrinology, Andrology and Internal Medicine and Master in Andrological and Human Reproduction Sciences, Department of Biomedical Sciences, University of Catania, Piazza S. Maria di Gesù, 95123 Catania, Italy

    Correspondence:
    Search for articles by this author
Published:August 02, 2010DOI:https://doi.org/10.1016/j.rbmo.2009.09.014

      Abstract

      Oxidative stress (OS) has been recognized as one of the most important cause of male infertility. Despite the antioxidant activity of seminal plasma, epididymis and spermatozoa, OS damages sperm function and DNA integrity. Since antioxidants suppress the action of reactive oxygen species, these compounds have been used in the medical treatment of male infertility or have been added to the culture medium during sperm separation techniques. Nevertheless, the efficacy of such a treatment has been reported to be very limited. This may relate to: (i) patient selection bias; (ii) late diagnosis of male infertility; (iii) lack of double-blind, placebo-controlled clinical trial; and/or (iv) use of end-points that are not good markers of the presence of OS. This review considers the effects of the main antioxidant compounds used in clinical practice. Overall, the data published suggest that no single antioxidant is able to enhance fertilizing capability in infertile men, whereas a combination of them seems to provide a better approach. Taking into account the pros and the cons of antioxidant treatment of male infertility, the potential advantages that it offers cannot be ignored. Therefore, antioxidant therapy should remain in the forefront of preventive medicine, including human reproductive medicine.

      Keywords

      Introduction

      Widely accepted scientific evidence supports the role of oxidative stress (OS) as a causative factor in many human degenerative processes, diseases, syndromes and ageing processes (
      • Cutler R.G.
      Antioxidants and aging.
      ,
      • Davies K.J.
      Oxidative stress: the paradox of aerobic life.
      ,
      • Jacob R.A.
      • Burri B.J.
      Oxidative damage and defense.
      ,
      • Cutler R.G.
      • Plummer J.
      • Chowdhury K.
      • Heward C.
      Oxidative stress profiling: part II. Theory, technology, and practice.
      ). OS has been defined as an imbalance between the generation of reactive oxygen species (ROS) and antioxidant scavenging activities, in which the former prevails (
      • Sikka S.C.
      Relative impact of oxidative stress on male reproductive function.
      ).
      In recent years, OS and the role of ROS in the pathophysiology of human sperm function and male infertility have been explored intensively. Indeed, spermatozoa, from the moment that they are produced in the testes to being ejaculated into the female reproductive tract, are constantly exposed to oxidizing environments. They are extremely sensitive to ROS because of their high content of polyunsaturated fatty acids (PUFA) and their limited ability to repair DNA (
      • Griveau J.F.
      • Le Lannou D.
      Reactive oxygen species and human spermatozoa: physiology and pathology.
      ,
      • Shen H.
      • Ong C.
      Detection of oxidative DNA damage in human sperm and its association with sperm function and male infertility.
      ).
      Given the difficulty of reaching an accurate diagnosis, many antioxidant therapies have been used in the hope of improving sperm quality. Treatments have varied over the years involving the use of many different compounds, such as carnitines, phosphatidylcholine, kallikrein, pentoxifylline and vitamins A, E and C, without particular attention to counteracting the lipoperoxidative damage (
      • Mann T.
      • Lutwak-Mann C.
      Male Reproductive Function and Semen.
      ,
      • Lanzafame F.
      • Chapman M.G.
      • Guglielmino A.
      • et al.
      Pharmacological stimulation of sperm motility.
      ).
      The aim of this article is to review the pathways of sperm OS and antioxidant defences to better understand which conditions are at risk of disequilibrium, and which antioxidant therapies can lead to a real improvement of human sperm quality invitro and in vivo.

      Reactive oxygen species and spermatozoa

      Although necessary for survival, oxygen also leads to production of free radicals. These are atomic or molecular species with unpaired electrons on an otherwise open-shell configuration. Unpaired electrons are usually highly reactive, so radicals are likely to take part in chemical reactions that damage sperm plasma membrane lipids (
      • Jones R.
      • Mann T.
      Lipid peroxidation in spermatozoa.
      ,
      • Jones R.
      • Mann T.
      Toxicity of exogenous fatty acid peroxides towards spermatozoa.
      ), the so-called lipid peroxidation. This research area has received a great impulse and the importance of ROS generation and lipid peroxidation has been underlined as a mechanism that damages mammalian spermatozoa (
      • Jones R.
      • Mann T.
      Lipid peroxidation in spermatozoa.
      ,
      • Jones R.
      • Mann T.
      Toxicity of exogenous fatty acid peroxides towards spermatozoa.
      ,
      • Jones R.
      • Mann T.
      • Sherins R.
      Peroxidative breakdown of phospholipids in human spermatozoa, spermicidal properties of fatty acid peroxides, and protective action of seminal plasma.
      ,
      • Saleh R.
      • Agarwal A.
      Oxidative stress and male infertility: from research bench to clinical practice.
      ).
      ROS are highly reactive oxidizing agents. These include the superoxide anion radical, the hydroxyl radical, the peroxyl radical and a subclass of free radicals derived from nitrogen, which includes nitric oxide, peroxynitrite, nitroxyl anion and peroxynitrous acid (Table 1). Although hydrogen peroxide, singlet oxygen and hydrochlorous acid should not be classified as free radicals because they still contain a pair of electrons in the outer orbital, often these are also included as oxyradical species (
      • Forman H.J.
      • Boveris A.
      Superoxide radical and hydrogen peroxide in mitochondria.
      ,

      Pryor W 1984 The role of free radical reactions in biological systems. In: Pryor W, editor Free Radicals in Biology. New York: Academic Press; 1984.

      ,
      • Warren J.S.
      • Johnson K.J.
      • Ward P.A.
      Oxygen radicals in cell injury and cell death.
      ). The principal ROS produced by spermatozoa seems to be the superoxide anion radical, which generates hydrogen peroxide, spontaneously or following the activity of superoxide dismutase (SOD) (
      • Alvarez J.G.
      • Touchstone J.C.
      • Blasco L.
      • Storey B.T.
      Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. Superoxide dismutase as major enzyme protectant against oxygen toxicity.
      ). In the microenvironment of cell membranes, hydrogen peroxide is the most stable intermediate of oxygen reduction (
      • Aitken R.J.
      • Clarkson J.S.
      Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa.
      ,
      • Alvarez J.G.
      • Storey B.T.
      Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation.
      ).
      Table 1The most important classes of radical oxygen species.
      RadicalNotation
      Superoxide anionO2-
      HydroxylOH
      PeroxylROO
      Nitric oxideNO
      PeroxynitriteONOO
      Nitroxyl anionNO
      Peroxynitrous acidHOONO
      Hydrogen peroxideH2O2
      Singlet oxygen1O2
      Hydrochlorous acidHOCl
      In contrast to the superoxide anion radical, hydrogen peroxide can effortlessly go through the plasma membrane and, despite its weak oxidizing capacity, if the scavenger function is inadequate to eliminate completely hydrogen peroxide and Fe or Cu is present, it promotes (by the Haber–Weiss reaction, Fe3++O2-Fe2++O2, and the subsequent Fenton reaction, Fe2+ + H2O2 → Fe3+ + HO + HO) the formation of hydroxyl radical, which is a more dangerous oxidizing product (
      • Aitken R.J.
      • Harkiss D.
      • Buckingham D.
      Relationship between iron-catalysed lipid peroxidation potential and human sperm function.
      ). Hydroxyl radical is tremendously reactive and, hence, it can cause biological damage. Cellular homeostasis is normally regulated by the efficacy of the free-radical scavenger systems, by the concentrations of peroxidizable substances, such as PUFA, that are present in significant amounts and by an elevated concentration of docosahexaenoic acid (DHA) (C22:6 omega-3) fatty acids. In mature spermatozoa, the high concentration of unsaturated lipids is associated with a relative paucity of oxyradical scavenger enzymes. This relative deficiency is probably due to the virtual absence of cytoplasm in mature sperm cells (
      • Poulos A.
      • White I.G.
      The phospholipid composition of human spermatozoa and seminal plasma.
      ,
      • Jones R.
      • Mann T.
      • Sherins R.
      Peroxidative breakdown of phospholipids in human spermatozoa, spermicidal properties of fatty acid peroxides, and protective action of seminal plasma.
      ,
      • Bielski B.H.
      • Arudi R.L.
      • Sutherland M.W.
      A study of the reactivity of HO2/O2 with unsaturated fatty acids.
      ,
      • Ollero M.
      • Gil Guzman E.
      • Lopez M.C.
      • et al.
      Characterization of a subset of human spermatozoa at different stage of maturation: Implication and treatment of male infertility.
      ).
      In physiological amounts, ROS are involved in the control of normal sperm function (
      • De Lamirande E.
      • Gagnon C.
      A positive role for the superoxide anion in triggering hyperactivation and capacitation of human spermatozoa.
      ,
      • De Lamirande E.
      • Gagnon C.
      Human sperm hyperactivation in whole semen and its association with low superoxide scavenging capacity in seminal plasma.
      ,
      • De Lamirande E.
      • Gagnon C.
      Capacitation-associated production of superoxide ion by human spermatozoa.
      ,
      • Aitken R.J.
      • Fisher H.
      Reactive oxygen species generation and human spermatozoa: the balance of benefit and risk.
      ,
      • Griveau J.F.
      • Renard P.
      • Le Lannou D.
      An in vitro promoting role for hydrogen peroxide in human sperm capacitation.
      ,
      • Aitken R.J.
      Free radicals, lipid peroxidation and sperm function.
      ,
      • Griveau J.F.
      • Le Lannou D.
      Reactive oxygen species and human spermatozoa: physiology and pathology.
      ). Paradoxically, spermatozoa necessitate a slight intracellular production of superoxide anion radical to boost the capacitation process (
      • De Lamirande E.
      • Gagnon C.
      A positive role for the superoxide anion in triggering hyperactivation and capacitation of human spermatozoa.
      ,
      • De Lamirande E.
      • Gagnon C.
      Human sperm hyperactivation in whole semen and its association with low superoxide scavenging capacity in seminal plasma.
      ) and the acrosome reaction (
      • Griveau J.F.
      • Renard P.
      • Le Lannou D.
      Superoxide anion production by human spermatozoa as a part of the ionophore-induced acrosome reaction process.
      ). The short half-life and limited diffusion of these molecules is consistent with their physiological role in maintaining the stability between ROS production and the scavenger systems. The balance between the amounts of ROS produced and the amounts scavenged at any moment determines whether a given sperm function will be promoted or compromised (
      • Sharma R.K.
      • Agarwal A.
      Role of reactive oxygen species in male infertility.
      ). Recent data established that the upper cut-off value of normal semen samples that correlates with good semen quality is in the order of 0.075–0.1 × 106 counted photons/minute/10 million cells (
      • Das S.
      • Chattopadhyay R.
      • Jana S.K.
      • et al.
      Cut-off value of reactive oxygen species for predicting semen quality and fertilization outcome.
      ). In addition to the

      World Health Organization 1999 WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction (4th edition). Cambridge University Press, Cambridge, UK.

      semen analysis, a study showed that patients with asthenozoospermia, asthenoteratozoospermia or oligoasthenoteratozoospermia have a significantly lower seminal plasma level of total antioxidant capacity (TAC) compared with a group of 16 healthy males with normozoospermia (
      • Khosrowbeygi A.
      • Zarghami N.
      Levels of oxidative stress biomarkers in seminal plasma and their relationship with seminal parameters.
      ).
      Two different pathways contribute to ROS production and the ensuing male subfertility or infertility: (i) the reduced NADPH oxidase system at the level of the sperm plasma membrane (
      • Aitken R.J.
      • Buckingham D.
      • West K.
      • et al.
      Differential contribution of leucocytes and spermatozoa to the generation of reactive oxygen species in the ejaculates of oligozoospermic patients and fertile donors.
      ), which produces superoxide that is further converted to peroxide by the action of a SOD (
      • Griveau J.F.
      • Le Lannou D.
      Reactive oxygen species and human spermatozoa: physiology and pathology.
      ); and (ii) the reduced NAD-dependent oxido-reductase (diphorase) at the mitochondrial level (
      • Gavella M.
      • Lipovac V.
      NADH-dependent oxidoreductase (diaphorase) activity and isozyme pattern of sperm in infertile men.
      ).
      Very recently, the subcellular origin of sperm ROS has been further clarified. Disruption of mitochondrial electron transport flow in human spermatozoa results in the generation of ROS. The induction of ROS on the matrix side of the inner mitochondrial membrane at complex I causes a peroxidative damage of the midpiece and a loss of sperm movement. These findings suggest that sperm mitochondria contribute to the oxidative stress of defective human spermatozoa (
      • Koppers A.J.
      • De Iuliis G.N.
      • Finnie J.M.
      • et al.
      Significance of mitochondrial reactive oxygen species in the generation of oxidative stress in spermatozoa.
      ).

      Potential aetiological factors for oxidative stress in semen

      There is much evidence that ROS are elevated in the male partners of infertile couples suffering from selected andrological diseases (
      • D’Agata R.
      • Vicari E.
      • Moncada M.L.
      • et al.
      Generation of reactive oxygen species in subgroups of infertile men.
      ,
      • Mazzilli F.
      • Rossi T.
      • Marchesini M.
      • et al.
      Superoxide anion in human semen related to seminal parameters and clinical aspects.
      ). It has been postulated that ROS hyperproduction is a major cause of idiopathic male infertility and a reduced antioxidant capacity can contribute to this disease (
      • Balercia G.
      • Armeni T.
      • Mantero F.
      • et al.
      Total oxyradical scavenging capacity toward different reactive oxygen species in seminal plasma and sperm cells.
      ). Indeed, about 40% of infertile patients have a high ROS production, whereas only a minority of fertile men have increased seminal ROS production (
      • Iwasaki A.
      • Gagnon C.
      Formation of reactive oxygen species in spermatozoa of infertile patients.
      ). The most relevant pathologies (proposed as aetiological factors) that can increase ROS concentrations are described below.

      Conditions in the scrotum

      Scrotal temperature is increased by fevers, modifications in microcirculation, venous stasis such as in the presence of varicoceles. Ischaemia and hypoxia also increase ROS concentrations (
      • Jung A.
      • Schuppe H.C.
      • Schill W.B.
      Fever as etiology of temporary infertility in the man.
      ,
      • Paul C.
      • Teng S.
      • Saunders P.T.K.
      A single, mild, transient scrotal heat stress causes hypoxia and oxidative stress in mouse testes, which induces germ cell death.
      ).

      Infection/inflammation of the male organs

      Infection/inflammation of the testis, epididymis, seminal vesicles and/or prostate may cause an increase in the number of seminal leukocytes (white blood cells; WBC) and/or WBC activation followed by an ROS burst, produced as a defence mechanism. This may be modulated via direct cell-to-cell contact or by soluble substances released by WBC (
      • Saleh R.A.
      • Agarwal A.
      • Kandirali E.
      • et al.
      Leukocytospermia is associated with increased reactive oxygen species production by human spermatozoa.
      ). Very recently, it has been shown that cytokines released during inflammation amplify the degree of OS initiated by WBC (
      • Fraczek M.
      • Sanocka D.
      • Kamieniczna M.
      • Kurpisz M.
      Proinflammatory cytokines as an intermediate factor enhancing lipid sperm membrane peroxidation in in vitro conditions.
      ). Alternatively, the antioxidant defence mechanisms can be overwhelmed resulting in OS (
      • Sikka S.C.
      Relative impact of oxidative stress on male reproductive function.
      ).

      Oestrogen disorders

      Oestrogens are either produced by an endogenous disequilibrium in androgen metabolism in the male reproductive tract or can access the reproductive tract via environmental exposure.
      • Bennetts L.E.
      • De Iuliis G.N.
      • Nixon B.
      • et al.
      Impact of estrogenic compounds on DNA integrity in human spermatozoa: evidence for cross-linking and redox cycling activities.
      reported that catechol oestrogens, quercetin, diethylstilbestrol and pyrocatechol were intensely active in stimulating redox activity, while genistein was only active at the highest doses tested and 17β-oestradiol, nonylphenol, bisphenol A and 2,3-dihydroxynaphthalene were inactive. It has been shown that ROS generation could be triggered by cis-unsaturated fatty acids including linoleic and DHA. This is of great importance because defective human spermatozoa contain abnormally high amounts of cis-unsaturated fatty acids, which may precipitate the OS encountered in male infertility (
      • Aitken R.J.
      • Wingate J.K.
      • De Iuliis G.
      • et al.
      Cis-unsaturated fatty acids stimulate reactive oxygen species generation and lipid peroxidation in human spermatozoa.
      ). In this condition, ROS hyper-production damages sperm function, such as motility, capacitation, fertilization capability, acrosome reaction and DNA/chromatin integrity (
      • Sikka S.C.
      Oxidative stress and role of antioxidants in normal and abnormal sperm function.
      ,
      • Aitken R.J.
      Molecular mechanisms regulating human sperm function.
      ) (Figure 1).
      Figure thumbnail gr1
      Figure 1Schematic representation of reactive oxygen species-induced cellular damage. CAT = catalase; GPx = glutathione peroxidase; GSH = reduced glutathione; SOD = superoxide dismutase.

      Chronic prostatitis

      Men with chronic prostatitis, with or without leukocytospermia, have OS (
      • Pasqualotto F.F.
      • Sharma R.K.
      • Potts J.M.
      • et al.
      Seminal oxidative stress in patients with chronic prostatitis.
      ). Production of ROS during the inflammatory processes of the testis and epididymitis are particularly harmful to spermatozoa. Indeed, a recent study, conducted in male Wistar rats, showed that repeated (1–2 weeks) experimentally induced exposure to the pro-oxidants tert-butyl hydroperoxide and cumene induced a marked dose-related enhancement of lipid peroxidation and increased ROS concentrations in both testis and epididymal spermatozoa (
      • Kumar T.R.
      • Muralidhara
      Induction of oxidative stress by organic hydroperoxides in testis and epididymal sperm of rats in vivo.
      ).

      Polymorphonuclear neutrophils

      Polymorphonuclear neutrophils appear to be the major source of ROS (
      • Tamura M.
      • Tamura T.
      • Tyagi S.R.
      • Lambeth J.D.
      The superoxide-generating respiratory burst oxidase of human neutrophil plasma membrane: phosphatidylserine as an effector of the activated enzyme.
      ,
      • Ochsendorf F.R.
      Infection in the male genital tract and reactive oxygen species.
      ,
      • Aitken R.J.
      • Baker M.A.
      Oxidative stress, sperm survival and fertility control.
      ). Although these data look very worrying, only very high numbers of ROS-producing WBC in the final ejaculate are detrimental to sperm function. An infective/inflammatory injury involving ROS in the prostate gland, seminal vesicles or epididymis could indirectly impair sperm function (
      • Ochsendorf F.R.
      Infection in the male genital tract and reactive oxygen species.
      ).

      Other factors

      ROS can also be produced by normal and especially abnormal spermatozoa (
      • Aitken R.J.
      • Clarkson J.S.
      Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa.
      ;
      • Alvarez J.G.
      • Touchstone J.C.
      • Blasco L.
      • Storey B.T.
      Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. Superoxide dismutase as major enzyme protectant against oxygen toxicity.
      ;
      • Fisher H.M.
      • Aitken R.J.
      Comparative analysis of the ability of precursor germ cells and epididymal spermatozoa to generate reactive oxygen metabolites.
      ,
      • Aitken R.J.
      • Baker M.A.
      Oxidative stress, sperm survival and fertility control.
      ). Immature spermatozoa with abnormal head morphology and cytoplasmic retention produce the highest amount of ROS, whereas mature spermatozoa and immature germ cells produce the lowest amount. The oxidative damage of mature spermatozoa by immature sperm-produced ROS during sperm migration from the seminiferous tubules to the epididymis may be another cause of male infertility (
      • Gomez E.
      • Buckingham D.W.
      • Brindle J.
      • et al.
      Development of an image analysis system to monitor the retention of residual cytoplasm by human spermatozoa: correlation with biochemical markers of the cytoplasmic space, oxidative stress, and sperm function.
      ). Recently, serum and seminal plasma Cu concentrations have been found higher in subfertile men than in fertile men. Moreover, subfertile men have significantly higher seminal plasma Fe concentrations. These findings suggest that Cu and Fe might be mediators of the effects of oxidative damage and play an essential role in spermatogenesis and male infertility (
      • Aydemir B.
      • Kiziler A.R.
      • Onaran I.
      • et al.
      Impact of Cu and Fe concentrations on oxidative damage in male infertility.
      ). In addition, it has recently been shown that men older than 40 years have significantly higher ROS concentrations compared with younger men and a positive correlation between seminal ROS concentrations and age (r = 0.20; P = 0.040) has been reported (
      • Cocuzza M.
      • Athayde K.S.
      • Agarwal A.
      • et al.
      Age-related increase of reactive oxygen species in neat semen in healthy fertile men.
      ).

      Genetic dispositions in sperm oxidative stress

      The discovery of specific genes and pathways affected by oxidants gives ROS a new function as second subcellular messengers in gene regulatory and signal transduction pathways (
      • Allen R.G.
      • Tresini M.
      Oxidative stress and gene regulation.
      ,
      • O’Flaherty C.
      • De Lamirande E.
      • Gagnon C.
      Positive role of reactive oxygen species in mammalian sperm capacitation: triggering and modulation of phosphorylation events.
      ) and specifically as physiological mediators that trigger phosphorylation events. The role of ROS as regulators of protein tyrosine phosphorylation has been known for a decade (
      • Leclerc E.
      • De Lamirande E.
      • Gagnon C.
      Regulation of protein-tyrosine phosphorylation and human sperm capacitation by reactive oxygen derivatives.
      ), but other novel, ROS-mediated phosphorylations have been recently reported. These include phosphorylation of protein kinase A substrates and subsequently the phosphorylation of mitogen-activated kinase-like proteins, proteins with the threonine–glutamine–tyrosine motif and, finally, fibrous sheath proteins (
      • O’Flaherty C.
      • De Lamirande E.
      • Gagnon C.
      Positive role of reactive oxygen species in mammalian sperm capacitation: triggering and modulation of phosphorylation events.
      ,
      • De Lamirande E.
      • O’Flaherty C.
      Sperm activation: role of reactive oxygen species and kinases.
      ). A recently published article has offered a different point of view about OS, suggesting that sperm susceptibility to OS is significantly greater in idiopathic infertile men with the glutathione S-transferase mull 1 (GSTM1) null genotype compared with those possessing the gene. Therefore, the GSTM1 polymorphism might be an important source of variation in susceptibility of spermatozoa to oxidative damage in patients with idiopathic infertility (
      • Aydemir B.
      • Onaran I.
      • Kiziler A.R.
      • et al.
      Increased oxidative damage of sperm and seminal plasma in men with idiopathic infertility is higher in patients with glutathione S-transferase Mu-1 null genotype.
      ).

      Sperm polyunsaturated fatty acid content

      Spermatozoa are very susceptible to OS by virtue of their high content of PUFA as major components of cellular and intracellular membranes, the low cytoplasmic concentrations of scavenging enzymes and the small cytoplasmic volume, which limits their scavenging capacities and the lack of DNA repair capacity (
      • Lenzi A.
      • Picardo M.
      • Gandini L.
      • Dondero F.
      Lipids of the sperm plasma membrane: from polyunsaturated fatty acids considered as markers of sperm function to possible scavenger therapy.
      ). The reactivity of ROS, particularly hydrogen peroxide and the superoxide anion radical, has been proposed as a major cause of PUFA peroxidation in the sperm plasma membrane, playing a key role in the aetiology of male infertility (
      • Sharma R.K.
      • Agarwal A.
      Role of reactive oxygen species in male infertility.
      ). The lipids of the spermatozoa have been suggested to be essential for their viability, maturity and function (
      • Davis B.K.
      Timing of fertilization in mammals: sperm cholesterol/phospholipid ratio as a determinant of the capacitation interval.
      ,
      • Sebastian S.M.
      • Selvaraj S.
      • Aruldhas M.M.
      • Govindarajulu P.
      Pattern of neutral and phospholipids in the semen of normospermic, oligospermic and azoospermic men.
      ). Phospholipids are the major structural components of membranes. Their fatty acid composition has been illustrated in a study by
      • Zalata A.A.
      • Christophe A.B.
      • Depuydt C.E.
      • et al.
      The fatty acid composition of phospholipids of spermatozoa from infertile patients.
      .
      In normozoospermic samples, PUFA content ranges between 25.6% and 34% of total fatty acids and phospholipids in the 47% and 90% Percoll fractions, respectively. DHA contributes to more to than 60% of total PUFA; palmitate (C16:0) and stearate (C18:0), predominate among the saturated fatty acids of spermatozoa phospholipids. The omega-6/omega-3 ratio increases significantly in both Percoll fractions of samples with oligozoospermia or with asthenozoospermia compared with normozoospermic samples (
      • Zalata A.A.
      • Christophe A.B.
      • Depuydt C.E.
      • et al.
      The fatty acid composition of phospholipids of spermatozoa from infertile patients.
      ). Plasma membrane fluidity, conferred by PUFA, is crucial to regulate some specific functions, such as the acrosome reaction and the spermatozoa–oocyte fusion. PUFA and cholesterol are the main targets for lipoperoxidation. Their degree of unsaturation is, therefore, an essential parameter for the ability of spermatozoa to preserve equilibrium in an oxidative environment (
      • Israelachvili J.N.
      • Marcelja S.
      • Horn R.G.
      Physical principles of membrane organization.
      ,
      • Meizel S.
      • Turner K.O.
      Stimulation of an exocytotic event, the hamster sperm acrosome reaction by cis-unsaturated fatty acids.
      ,
      • Alvarez J.G.
      • Storey B.T.
      Differential incorporation of fatty acids into and peroxidative loss of fatty acids from phospholipids of human spermatozoa.
      ,
      • Ollero M.
      • Gil Guzman E.
      • Lopez M.C.
      • et al.
      Characterization of a subset of human spermatozoa at different stage of maturation: Implication and treatment of male infertility.
      ). When ROS damage the double bonds associated with PUFA, a lipid peroxidation chain reaction begins. The most important outcome of this is a modification in membrane fluidity that can alter its function and consequently inhibits events during gamete fusion (
      • Lenzi A.
      • Picardo M.
      • Gandini L.
      • et al.
      Glutathione treatment of dyspermia: effect on the lipoperoxidation process.
      ).
      The lower proportion of DHA, total PUFA, total omega-3 fatty acids, and the double-bond index in spermatozoa from both Percoll fractions of oligozoospermic patients and in the 90% Percoll fraction of asthenozoospermic samples could be the consequence of excessive breakdown of PUFA due to the increased ROS production in these samples (
      • Aitken R.J.
      • Clarkson J.S.
      • Hargreave T.B.
      • et al.
      Analysis of the relationship between defective sperm function and the generation of reactive oxygen species in cases of oligozoospermia.
      ,
      • Zalata A.
      • Hafez T.
      • Comhaire F.
      Evaluation of the role of reactive oxygen species in male infertility.
      ,
      • Zalata A.A.
      • Christophe A.B.
      • Depuydt C.E.
      • et al.
      The fatty acid composition of phospholipids of spermatozoa from infertile patients.
      ). Most of the long-chain metabolites prejudice fertility in men with oligoasthenozoospermia, due to, at least in part, the reduced fluidity of the sperm membrane. Also, the significant increase of omega-6/omega-3 ratio in both oligozoospermic and asthenozoospermic, in comparison with normozoospermic samples, may suggest a physiological meaning of this ratio because of specific interactions of omega-6 and omega-3 fatty acids with certain membrane proteins and receptors (
      • Lee A.G.
      • East M.J.
      • Fround R.J.
      Are essential fatty acids essential for membrane function?.
      ). The higher the number of PUFA double bonds, the greater is the peroxidative damage induced by ROS; for this reason, the human sperm plasma membrane, which is very rich in PUFA and contains those with two or more double bonds, especially docosapentanoic acid (that contains six double bonds), is very vulnerable to peroxidation (
      • Kim J.G.
      • Parthasarathy S.
      Oxidation and the spermatozoa.
      ).
      In contrast to
      • Zalata A.A.
      • Christophe A.B.
      • Depuydt C.E.
      • et al.
      The fatty acid composition of phospholipids of spermatozoa from infertile patients.
      ,
      • Khosrowbeygi A.
      • Zarghami N.
      Levels of oxidative stress biomarkers in seminal plasma and their relationship with seminal parameters.
      reported a significant difference in oleic acid concentrations in spermatozoa from asthenozoospermic men compared with normozoospermic men. In spermatozoa from asthenoteratozoospermic and oligoasthenoteratozoospermic men, all the tested fatty acids are significantly higher than those found in normozoospermic men. Seminal plasma catalase (CAT) concentrations were significantly lower in all patients, while concentrations of free 15-F(2t)-isoprostane were significantly higher in all patients compared with normozoospermic men. These results let us postulate that spermatozoa from abnormal samples may have higher concentrations of PUFA, especially DHA, than spermatozoa from normozoospermic men. Therefore, lipid peroxidation would be higher in spermatozoa from abnormal samples than those from normozoospermic men.

      Oxidative stress damages sperm function

      Several reports suggest that an increased production of ROS and/or modification in the levels of antioxidant defences are implicated in the occurrence of many sperm defects. These include reduction of sperm motility (
      • De Lamirande E.
      • Gagnon C.
      Reactive oxygen species and human spermatozoa.
      ,
      • De Lamirande E.
      • Gagnon C.
      Reactive oxygen species and human spermatozoa.
      ,
      • Aitken R.J.
      • Buckingham D.
      • Harkiss D.
      Use of a xanthine oxidase free radical generating system to investigate the cytotoxic effects of reactive oxygen species on human spermatozoa.
      ,
      • Sikka S.C.
      Oxidative stress and role of antioxidants in normal and abnormal sperm function.
      ,
      • Aitken R.J.
      Molecular mechanisms regulating human sperm function.
      ), spermatozoa–oocyte fusion (
      • Aitken R.J.
      • Irvine D.S.
      • Wu F.C.
      Prospective analysis of sperm oocyte fusion and reactive oxygen species generation as criteria for the diagnosis of infertility.
      ,
      • Griveau J.F.
      • Le Lannou D.
      Reactive oxygen species and human spermatozoa: physiology and pathology.
      ) and acrosine activity (
      • Zalata A.A.
      • Ahmed A.H.
      • Allamaneni S.S.R.
      • et al.
      Relationship between acrosin activity of human spermatozoa and oxidative stress.
      ). It has been reported that more than half (55%) of the oligozoospermic patients who display a spermatozoa–oocyte penetration rate lower than 25% have an elevated ROS production (
      • Aitken R.J.
      • Clarkson J.S.
      • Hargreave T.B.
      • et al.
      Analysis of the relationship between defective sperm function and the generation of reactive oxygen species in cases of oligozoospermia.
      ). Furthermore, spermatozoa of oligozoospermic patients have been confirmed as a very important source of ROS (
      • Aitken R.J.
      • Clarkson J.S.
      • Hargreave T.B.
      • et al.
      Analysis of the relationship between defective sperm function and the generation of reactive oxygen species in cases of oligozoospermia.
      ,
      • Zalata A.
      • Hafez T.
      • Comhaire F.
      Evaluation of the role of reactive oxygen species in male infertility.
      ). In addition, a strong correlation between sperm function, including motility, and the percentage of ROS-producing spermatozoa has been reported (
      • Gil-Guzman E.
      • Ollero M.
      • Lopez M.C.
      • et al.
      Differential production of reactive oxygen species by subset of human spermatozoa at different stages of maturation.
      ).
      Some studies (
      • Rao B.
      • Soufir J.C.
      • Martin M.
      • David G.
      Lipid peroxidation in human spermatozoa as related to midpiece abnormalities and motility.
      ,
      • Kim J.G.
      • Parthasarathy S.
      Oxidation and the spermatozoa.
      ) reported midpiece abnormalities, and some others showed that ROS-induced motility decrease is associated with a growth of lipid peroxidation measured as malondialdehyde (MDA) (
      • Suleiman S.A.
      • Ali M.E.
      • Zaki Z.M.
      • et al.
      Lipid peroxidation and human sperm motility: protective role of vitamin E.
      ,
      • Chen C.S.
      • Chao H.T.
      • Pan R.L.
      • Wei Y.H.
      Hydroxyl radical-induced decline in motility and increase in lipid peroxidation and DNA modification in human sperm.
      ,
      • Hsieh Y.Y.
      • Chang C.C.
      • Lin C.S.
      Seminal malondialdehyde concentration but not glutathione peroxidase activity is negatively correlated with seminal concentration and motility.
      ) and DNA modifications (
      • Chen C.S.
      • Chao H.T.
      • Pan R.L.
      • Wei Y.H.
      Hydroxyl radical-induced decline in motility and increase in lipid peroxidation and DNA modification in human sperm.
      ). Recently, it has been reported that the percentage of immotile spermatozoa correlate positively with MDA seminal plasma concentrations (r = 0.50, P < 0.01), while sperm concentration displays a significant negative correlation (r = −0.63, P < 0.001) (
      • Saraniya A.
      • Koner B.C.
      • Doureradjou P.
      • et al.
      Altered malondialdehyde, protein carbonyl and sialic acid levels in seminal plasma of microscopically abnormal semen.
      ). On the contrary, a decrement of MDA corresponds to an increase of the pregnancy rate (
      • Suleiman S.A.
      • Ali M.E.
      • Zaki Z.M.
      • et al.
      Lipid peroxidation and human sperm motility: protective role of vitamin E.
      ) and an augmentation of ROS to fertility reduction in vivo (
      • Aitken R.J.
      • Irvine D.S.
      • Wu F.C.
      Prospective analysis of sperm oocyte fusion and reactive oxygen species generation as criteria for the diagnosis of infertility.
      ). Very recently it has been shown that age affects the epidydimal antioxidant defence with an increased ROS production and consequent lipid peroxidation in Brown Norway rats (
      • Weir C.P.
      • Robaire B.
      Spermatozoa have decreased antioxidant and increased reactive oxygen species in the Brown Norway rat.
      ).
      It has also been postulated that OS could be a cause for hyperviscosity of seminal plasma in infertile males (
      • Aydemir B.
      • Onaran I.
      • Kiziler A.R.
      • et al.
      The influence of oxidative damage on viscosity of seminal fluid in infertile men.
      ).

      Oxidative stress and sperm chromatin and DNA integrity

      Usually, sperm chromatin is condensed and insoluble; these features protect the genetic integrity and facilitate the transfer of the paternal genome through the male and female reproductive tracts. Furthermore, a special kind of protection against OS induced by metals is conferred to protamine by its capacity to trap some of them (
      • Manicardi G.C.
      • Tombacco A.
      • Bizzaro D.
      • et al.
      DNA strand breaks in ejaculated human spermatozoa: comparison of susceptibility to the nick translation and terminal transferase assays.
      ,
      • Liang R.
      • Senturker S.
      • Shi X.
      • et al.
      Effects of Ni(II) and Cu(II) on DNA interaction with the N-terminal sequence of human protamine P2: enhancement of binding and mediation of oxidative DNA strand scission and base damage.
      ). Despite this tight DNA packaging and the seminal plasma protection from oxidative damage, many correlations have also been observed between ROS generation and DNA alteration (
      • Lee A.G.
      • East M.J.
      • Fround R.J.
      Are essential fatty acids essential for membrane function?.
      ,
      • Manicardi G.C.
      • Tombacco A.
      • Bizzaro D.
      • et al.
      DNA strand breaks in ejaculated human spermatozoa: comparison of susceptibility to the nick translation and terminal transferase assays.
      ,
      • Twigg J.
      • Irvine D.S.
      • Houston P.
      • et al.
      Iatrogenic DNA damage induced in human spermatozoa during sperm preparation: protective significance of seminal plasma.
      ,
      • Zalata A.A.
      • Christophe A.B.
      • Depuydt C.E.
      • et al.
      The fatty acid composition of phospholipids of spermatozoa from infertile patients.
      ,
      • Liang R.
      • Senturker S.
      • Shi X.
      • et al.
      Effects of Ni(II) and Cu(II) on DNA interaction with the N-terminal sequence of human protamine P2: enhancement of binding and mediation of oxidative DNA strand scission and base damage.
      ). The exposure of spermatozoa to iatrogenically induced ROS significantly increases DNA fragmentation, modification of all bases, production of base-free sites, deletions, frame shifts, DNA cross-links and chromosomal rearrangements above that of the normal population (
      • Aitken R.J.
      • Gordon E.
      • Harkiss D.
      • et al.
      Negative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa.
      ,
      • Barroso G.
      • Morshedi M.
      • Oehringer S.
      Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa.
      ). Indeed, ROS have been shown to induce: (i) DNA protein cross-linking in chromatin (
      • Nackerdien Z.
      • Rao G.
      • Cacciuttolo M.A.
      • et al.
      Chemical nature of DNA-protein cross-links produced in mammalian chromatin by hydrogen peroxide in the presence of iron or copper ions.
      ,
      • Oliski R.
      • Nackerdien Z.
      • Dizdaroglu M.
      DNA protein cross linking between thymine and tyrosine in chromatin of gamma irradiated and hydrogen peroxide treated cultured human cells.
      ,
      • Twigg J.P.
      • Irvine D.S.
      • Aitken R.J.
      Oxidative damage to DNA in human spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm injection.
      ); (ii) significant positive correlation with DNA fragmentation; (iii) high frequency of DNA single- and double-strand breaks (
      • Liang R.
      • Senturker S.
      • Shi X.
      • et al.
      Effects of Ni(II) and Cu(II) on DNA interaction with the N-terminal sequence of human protamine P2: enhancement of binding and mediation of oxidative DNA strand scission and base damage.
      ,
      • Barroso G.
      • Morshedi M.
      • Oehringer S.
      Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa.
      ,
      • Dizdaroglu M.
      Oxidative damage to DNA in mammalian chromatin.
      ,
      • Chiu S.M.
      • Xue L.Y.
      • Friedman L.R.
      • Oleinick N.L.
      Differential dependence on chromatin structure for copper and iron ion induction of DNA doublestrand breaks.
      ); and (iv) oxidative DNA base changes, in a wide variety of mammalian cell types, especially in asthenozoospermic infertile and normozoospermic infertile subjects compared with fertile men (
      • Hughes C.M.
      • Lewis S.E.
      • McKelvey-Martin V.J.
      • Thompson V.
      A comparison of baseline and induced DNA damage in human spermatozoa from fertile and infertile men, using a modified comet assay.
      ,
      • Kodama H.
      • Yamaguchi R.
      • Fukuda J.
      • et al.
      Increased deoxyribonucleic acid damage in the spermatozoa of infertile male patients.
      ). DNA fragmentation seems to be inversely correlated with sperm count, morphology, motility and fertilization rate (
      • Sun J.G.
      • Jurisicova A.
      • Casper R.F.
      Deletion of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro.
      ,
      • Twigg J.P.
      • Irvine D.S.
      • Aitken R.J.
      Oxidative damage to DNA in human spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm injection.
      ,
      • Shen H.
      • Ong C.
      Detection of oxidative DNA damage in human sperm and its association with sperm function and male infertility.
      ,
      • Aitken R.J.
      • Krausz C.
      Oxidative stress, DNA damage and the Y chromosome.
      ). Several observations suggest that disorders in the DNA organization in the sperm nucleus are negatively related with the fertility competence of spermatozoa (
      • Evenson D.P.
      • Jost L.K.
      • Marshall D.
      • et al.
      Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic.
      ,
      • Host E.
      • Lindenberg S.
      • Ernst E.
      • Christensen F.
      DNA strand breaks in human spermatozoa: a possible factor, to be considered in couples suffering from unexplained infertility.
      ,
      • Host E.
      • Lindenberg S.
      • Smidt-Jensen S.
      DNA strand breaks in human spermatozoa: correlation with fertilization in vitro in oligozoospermic men and in men with unexplained infertility.
      ,
      • Host E.
      • Lindenberg S.
      • Smidt-Jensen S.
      The role of DNA strand breaks in human spermatozoa used for IVF and ICSI.
      ,
      • Shen H.M.
      • Chia S.E.
      • Ong C.N.
      Evaluation of oxidative DNA damage in human sperm and its association with male infertility.
      ,
      • Spanò M.
      • Bonde J.P.
      • Hjollund H.I.
      • et al.
      Sperm chromatin damage impairs human fertility.
      ).
      A higher percentage of DNA-damaged cells has been reported in the raw semen samples of patients with male accessory gland infection (
      • Comhaire F.H.
      • Mahmoud A.M.
      • Depuydt C.E.
      • et al.
      Mechanism and effects of male genital tract infection on sperm quality and fertilizing potential: the andrologist’s view point.
      ). It was also described that chromatin alterations were higher in immature spermatozoa (
      • Alvarez J.G.
      • Sharma R.K.
      • Ollero M.
      • et al.
      Increased DNA damage in sperm from leukocytospermic semen samples as determined by the sperm chromatin structure assay.
      ). It is suspected that DNA damage may lead to an amplified risk of miscarriage and chromosomal abnormalities (
      • Griveau J.F.
      • Le Lannou D.
      Reactive oxygen species and human spermatozoa: physiology and pathology.
      ).
      Within the fertilized oocyte, sperm DNA damage can be repaired during the period between sperm entry into the cytoplasm and the beginning of the next S phase, by virtue of pre- and post-replication mechanisms (
      • Matsuda Y.
      • Tobari I.
      Repair capacity of fertilized mouse eggs for X-ray damage induced in sperm and mature oocytes.
      ,
      • Genesca A.
      • Caballin M.R.
      • Miro R.
      • et al.
      Repair of human sperm chromosome aberrations in the hamster egg.
      ,
      • Evans M.D.
      • Dizdaroglu M.
      • Cooke M.S.
      Oxidative DNA damage and disease: induction, repair and significance.
      ). Consequently, the biological impact of abnormal sperm chromatin structure depends on the combined effects of chromatin damage in the spermatozoa and the capability of the oocyte to repair that pre-existing damage. However, if spermatozoa are selected from samples with extensively damaged DNA for use in assisted reproduction treatment such as intracytoplasmic sperm injection (ICSI) or IVF, the oocyte’s repair capacities might be inadequate, leading to fragmentation and a low rate of embryonic development that results in a high rate of early pregnancy loss (
      • Ahmadi A.
      • Ng S.C.
      Developmental capacity of damaged spermatozoa.
      ,
      • Ahmadi A.
      • Ng S.C.
      Fertilizing ability of DNA-damaged spermatozoa.
      ) or a poor blastocyst development (
      • Seli E.
      • Gardner D.K.
      • Schoolcraft W.B.
      • et al.
      Extent of nuclear DNA damage in ejaculated spermatozoa impacts on blastocyst development after in vitro fertilization.
      ). Sperm DNA fragmentation does not correlate with the fertilization rate, but there is a significantly reduced pregnancy rate in IVF patients inseminated with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling-positive spermatozoa. The same study showed a similar tendency in ICSI patients (
      • Henkel R.
      • Kierspel E.
      • Hajimohammad M.
      • et al.
      DNA fragmentation of spermatozoa and assisted reproduction technology.
      ). This implies that spermatozoa with damaged DNA are able to fertilize oocytes, but at the time when the paternal genome is switched on, further development stops (
      • Evenson D.P.
      • Larson K.L.
      • Jost L.K.
      Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with the other techniques.
      ). Some reports have indicated that when >30% of spermatozoa have damaged DNA, natural pregnancy is not possible (
      • Evenson D.P.
      • Larson K.L.
      • Jost L.K.
      Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with the other techniques.
      ,
      • Angelopoulou R.
      • Plastira K.
      • Msaouel P.
      Spermatozoal sensitive biomarkers to defective protaminosis and fragmented DNA.
      ,
      • Evenson D.P.
      • Wixon R.
      Data analysis of two in vivo fertility studies using sperm chromatin structure assay-derived DNA fragmentation index vs. pregnancy outcome.
      ). Other studies have, however, failed to confirm that this cut-off point affects treatment outcome (
      • Payne J.F.
      • Raburn D.J.
      • Couchman G.M.
      • et al.
      Redefining the relationship between sperm deoxyribonucleic acid fragmentation as measured by the sperm chromatin structure assay and outcomes of assisted reproductive techniques.
      ). It is noteworthy that the American Society for Reproductive Medicine recently reported that none of the current methods available to evaluate sperm DNA integrity predicts treatment outcome (Practice Committee of American Society for Reproductive

      Practice Committee of American Society for Reproductive Medicine 2008 The clinical utility of sperm DNA integrity testing. Fertility and Sterility 90, S178–S180.

      ). Therefore, in the presence of low OS, spermatozoa are still able to fertilize the oocytes, but at higher levels, DNA damage occurs. Repair of this kind of damage in the zygote can be anomalous and may lead to mutations linked with pre-term pregnancy loss and many pathologies in the offspring, including childhood cancer (
      • Aitken R.J.
      • Baker M.A.
      Oxidative stress, sperm survival and fertility control.
      ).

      Smoking, ROS production and sperm damage

      Other causes may increase ROS production. Many studies have been carried out on the effect of cigarette smoking and show a decline in sperm count, motility, citric acid concentration and a rise in the number of abnormal cells (
      • Sofikitis N.
      • Miyagawa I.
      • Dimitriadis D.
      • et al.
      Effects of smoking on testicular function, semen quality and sperm fertilizing capacity.
      ,
      • Saleh R.
      • Agarwal A.
      • Sharma R.
      • et al.
      Effect of cigarette smoking on levels of seminal oxidative stress in infertile men: a prospective study.
      ,
      • Kunzle R.
      • Mueller M.D.
      • Hanggi W.
      • et al.
      Semen quality of male smokers and nonsmokers in infertile couples.
      ). Men who smoke cigarettes present a 48% increase in WBC number, 107% higher ROS concentrations and a 10-point decrease in TAC scores with respect to infertile non-smokers. Using the sperm chromatin structure assay, it has been reported that the DNA fragmentation index is significantly higher in infertile men who smoke (
      • Potts R.J.
      • Newbury C.J.
      • Smith G.
      • et al.
      Sperm chromatin damage associated with male smoking.
      ). It has also been seen that cigarette smoke causes oxidative DNA damage in spermatozoa due to its high content of oxidants and its depletion of antioxidants.
      Concentrations of Cd, Pb, MDA, protein carbonyls and ROS concentrations in infertile men who smoke have been reported to be significantly higher than those in fertile and non-smoking infertile men. Reduced glutathione (GSH) concentrations and glutathione S-transferase activity are lower in infertile smoker men than in fertile or non-smoking infertile men. Positive correlations have been found between seminal plasma Cd and seminal plasma protein carbonyls and between seminal plasma Pb and spermatozoon ROS concentrations in subfertile smokers, while there was a significant positive correlation between blood Cd and ROS concentrations in fertile smokers. A significant negative correlation between blood Cd concentrations and sperm and seminal plasma GSH concentrations have been reported (
      • Kiziler A.R.
      • Aydemir B.
      • Onaran I.
      • et al.
      High levels of cadmium and lead in seminal fluid and blood of smoking men are associated with high oxidative stress and damage in infertile subjects.
      ). Recently, cigarette smoking has been negatively correlated with a decrease of antioxidant activity, measured against SOD in the seminal plasma (
      • Pasqualotto F.F.
      • Umezu F.M.
      • Salvador M.
      • et al.
      Effect of cigarette smoking on antioxidant levels and presence of leukocytospermia in infertile men: a prospective study.
      ). It has been shown that smokers have decreased seminal plasma vitamin E and vitamin C concentrations (
      • Fraga C.G.
      • Motchnik P.A.
      • Wyrobek A.J.
      • et al.
      Smoking and low antioxidant levels increase oxidative damage to sperm DNA.
      ,
      • Mostafa T.
      • Tawadrous G.
      • Roaia M.M.
      • et al.
      Effect of smoking on seminal plasma ascorbic acid in infertile and fertile males.
      ).
      In view of this, spermatozoa from smoking men exhibit augmented DNA damage. This may result in sperm DNA mutations that predispose offspring to greater hazard of malformations, cancer and genetic diseases (
      • Ji B.T.
      • Shu X.O.
      • Linet M.S.
      • et al.
      Paternal cigarette smoking and the risk of childhood cancer among offspring of nonsmoking mothers.
      ,
      • Sépaniak S.
      • Forges T.
      • Monnier-Barbarino P.
      Cigarette smoking and fertility in women and men.
      ). Accordingly, epidemiological studies of childhood cancer have established that a paternal smoking habit is the most important identifiable risk factor linked with the beginning of the disease (
      • Sorahan T.
      • Lancashire R.J.
      • Hulten M.A.
      • et al.
      Childhood cancer and parental use of tobacco: Deaths from 1953 to 1955.
      ,
      • Sorahan T.
      • Prior P.
      • Lancashire R.J.
      • et al.
      Childhood cancer and parental use of tobacco: deaths from 1971 to 1976.
      ).

      The sperm protective system against oxidative stress

      Seminal plasma plays a crucial, protective role against ROS; its removal during sperm preparation may be hazardous to sperm DNA integrity (
      • Jeulin C.
      • Soufir J.C.
      • Weber P.
      • et al.
      Catalase activity in human spermatozoa and seminal plasma.
      ,
      • Villegas J.
      • Kehr K.
      • Soto L.
      • et al.
      Reactive oxygen species induce reversible capacitation in human spermatozoa.
      ). The use of spermatozoa for ICSI will carry the same hazard by excluding the protective role of the seminal plasma (
      • Liu J.
      • Nagy Z.
      • Joris H.
      • et al.
      Intracytoplasmic sperm injection does not require special treatment of the spermatozoa.
      ,
      • Zorn B.
      • Vidmar G.
      • Meden-Vrtovec H.
      Seminal reactive oxygen species as predictors of fertilization, embryo quality and pregnancy rates after conventional in vitro fertilization and intracytoplasmic sperm injection.
      ). These studies indicate that spermatozoa from patients with abnormal sperm count, motility and morphology have increased degrees of DNA damage. These data are in keeping with the similarly low value (∼20%) reported by another study conducted using a group of unselected semen donors (
      • Evenson D.P.
      • Baer R.K.
      • Turner T.
      • Schrader S.
      Individuality of DNA denaturation patterns in human sperm as measured by the sperm chromatin structure assay.
      ). Moreover, human seminal plasma appears to contain sufficient free Fe and Cu to catalyse the ROS-generating process (
      • Kwenang A.
      • Kroos M.J.
      • Koster J.F.
      • Van Eijk H.G.
      Iron, ferritin and copper in seminal plasma.
      ).
      The fact that generation of ROS is liable to be elevated in severely oligozoospermic patients treated by ICSI only exacerbates the amount of oxidative damage which spermatozoa undergo with this form of treatment (
      • Aitken R.J.
      • Clarkson J.S.
      • Hargreave T.B.
      • et al.
      Analysis of the relationship between defective sperm function and the generation of reactive oxygen species in cases of oligozoospermia.
      ,
      • Zalata A.
      • Hafez T.
      • Comhaire F.
      Evaluation of the role of reactive oxygen species in male infertility.
      ). However, functionally competent spermatozoa are not a prerequisite for ICSI (
      • Zorn B.
      • Vidmar G.
      • Meden-Vrtovec H.
      Seminal reactive oxygen species as predictors of fertilization, embryo quality and pregnancy rates after conventional in vitro fertilization and intracytoplasmic sperm injection.
      ) and OS does not appear to interfere with fertilization rates achieved with this therapeutic technique (
      • Twigg J.P.
      • Irvine D.S.
      • Aitken R.J.
      Oxidative damage to DNA in human spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm injection.
      ).
      However, the ability of genetically damaged spermatozoa to achieve normal fertilization following ICSI could have adverse consequences. These may appear during the post-implantation development of the offspring, rather than before (
      • Twigg J.P.
      • Irvine D.S.
      • Aitken R.J.
      Oxidative damage to DNA in human spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm injection.
      ). High seminal ROS concentrations are associated with impaired fertilizing ability and lower pregnancy rates after IVF. In ICSI, a negative association of ROS with embryo development to the blastocyst stage has been observed (
      • Zorn B.
      • Vidmar G.
      • Meden-Vrtovec H.
      Seminal reactive oxygen species as predictors of fertilization, embryo quality and pregnancy rates after conventional in vitro fertilization and intracytoplasmic sperm injection.
      ).
      The vulnerability of ICSI arises from the fact that, with conventional IVF, the kind of OS that damages the genome also leads to collateral peroxidative damage to the sperm plasma membrane that may prevent spermatozoa–oocyte fusion from taking place. However, this fertilization block is removed when ICSI is performed. Thus, spermatozoa exhibiting severe DNA oxidative damage are able to produce normal rates of nuclear decondensation and pronucleus formation following ICSI (
      • Twigg J.P.
      • Irvine D.S.
      • Aitken R.J.
      Oxidative damage to DNA in human spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm injection.
      ). It is likely that the oocyte and cleavage-stage embryo is competent to repair a certain degree of DNA damage (
      • Genesca A.
      • Caballin M.R.
      • Miro R.
      • et al.
      Repair of human sperm chromosome aberrations in the hamster egg.
      ). Evidence suggests the possibilities of the male infertility transmission to offspring (
      • Bofinger M.K.
      • Needham D.F.
      • Saldana L.R.
      • et al.
      45,X/46,X,r(Y) karyotype transmitted by father to son after intracytoplasmic sperm injection for oligospermia. A case report.
      ,
      • Jiang M.C.
      • Lien Y.R.
      • Chen S.U.
      • et al.
      Transmission of de-novo mutations of the deleted in azoospermia genes from a severely oligozoospermic male to a son via intracytoplasmic sperm injection.
      ,
      • Cram D.S.
      • Ma K.
      • Bhasin S.
      • et al.
      Y chromosome analysis of infertile men and their sons conceived through intracytoplasmic sperm injection: vertical transmission of deletions and rarity of de-novo deletions.
      ,
      • Aboulghar H.
      • Aboulghar M.
      • Mansour R.
      • et al.
      A prospective controlled study of karyotyping for 430 consecutive babies conceived through intracytoplasmic sperm injection.
      ).
      Under physiological conditions, protection against OS is supplied to spermatozoa by numerous antioxidants, which are present both in the seminal plasma and spermatozoa. GSH, glutathione peroxidase (GPX), glutathione reductase (GRD) (
      • Li T.K.
      The glutathione and thiol content of mammalian spermatozoa and seminal plasma.
      ,
      • Alvarez J.G.
      • Storey B.T.
      Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation.
      ), SOD (
      • Alvarez J.G.
      • Touchstone J.C.
      • Blasco L.
      • Storey B.T.
      Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. Superoxide dismutase as major enzyme protectant against oxygen toxicity.
      ), glucose-6-phosphate dehydrogenase (
      • Griveau J.F.
      • Dumont E.
      • Renard P.
      • et al.
      Reactive oxygen species, lipid peroxidation and enzymatic defence systems in human spermatozoa.
      ), ascorbate (
      • Lewis S.E.
      • Sterling E.S.
      • Young I.S.
      • Thompson W.
      Comparison of individuals antioxidants of sperm and seminal plasma in fertile and infertile men.
      ), α-tocopherol (
      • Therond P.
      • Auger J.
      • Legrand A.
      • Jouannet P.
      Alpha-tocopherol in human spermatozoa and seminal plasma: relationships with motility, antioxidant enzymes and leukocytes.
      ), taurine and hypotaurine (
      • Holmes R.P.
      • Goodman H.O.
      • Shihabi Z.K.
      • Jarow J.P.
      The taurine and hypotaurine content of human semen.
      ) and coenzyme Q10 (CoQ10) (
      • Lewin A.
      • Lavon H.
      The effect of coenzyme Q10 on sperm motility and function.
      ) are the constituents of the antioxidant activity of the spermatozoon. An additional contribution is performed by lactoferrin that coats sperm heads, but it is secreted by seminal vesicles (
      • Buckett W.M.
      • Luckas M.J.M.
      • Gazvani M.R.
      • et al.
      Seminal plasma lactoferrin concentrations in normal and abnormal semen samples.
      ).

      Antioxidant activity of the seminal plasma

      Seminal plasma affords spermatozoa with a key defence against OS by several forms of ROS (
      • Lenzi A.
      • Picardo M.
      • Gandini L.
      • Dondero F.
      Lipids of the sperm plasma membrane: from polyunsaturated fatty acids considered as markers of sperm function to possible scavenger therapy.
      ,
      • Balercia G.
      • Armeni T.
      • Mantero F.
      • et al.
      Total oxyradical scavenging capacity toward different reactive oxygen species in seminal plasma and sperm cells.
      ), surrounding spermatozoa with a highly specialized scavenger system that preserves the cell membrane (
      • Kim J.G.
      • Parthasarathy S.
      Oxidation and the spermatozoa.
      ). Seminal plasma represents the most important defence against free-radical toxicity. It contains high and low molecular weight factors. They include enzymatic ROS scavengers, such as SOD (
      • Nissen H.P.
      • Kreysel H.W.
      Superoxide dismutase in human semen.
      ,
      • Kobayashi T.
      • Miyazaki T.
      • Natori M.
      • Nozawa S.
      Protective role of superoxide dismutase in human sperm motility: superoxide dismutase activity and lipid peroxide in human seminal plasma and spermatozoa.
      ), which have been correlated with the liquefaction process of the seminal plasma and with the redox cycle of vitamin C, CAT (
      • Jeulin C.
      • Soufir J.C.
      • Weber P.
      • et al.
      Catalase activity in human spermatozoa and seminal plasma.
      ,
      • Siciliano L.
      • Tarantino P.
      • Longobardi F.
      • et al.
      Impaired seminal antioxidant capacity in human semen with hyperviscosity or oligoasthenozoospermia.
      ) and non-enzymatic chain-breaking antioxidants such as vitamin C (
      • Thiele J.J.
      • Friesleben H.J.
      • Fuchs J.
      • Ochsendorf F.R.
      Ascorbic acid and urate in human seminal plasma: determination and interrelationships with chemiluminescence in washed semen.
      ), vitamin E (
      • Therond P.
      • Auger J.
      • Legrand A.
      • Jouannet P.
      Alpha-tocopherol in human spermatozoa and seminal plasma: relationships with motility, antioxidant enzymes and leukocytes.
      ), uric acid (
      • Ronquist G.
      • Niklasson F.
      Uridine, xanthine, and urate contents in human seminal plasma.
      ,
      • Thiele J.J.
      • Friesleben H.J.
      • Fuchs J.
      • Ochsendorf F.R.
      Ascorbic acid and urate in human seminal plasma: determination and interrelationships with chemiluminescence in washed semen.
      ), albumin (
      • Elzanaty S.
      • Erenpreiss J.
      • Becker C.
      Seminal plasma albumin: origin and relation to the male reproductive parameters.
      ), carnitine, carotenoids and flavonoids (
      • Tremellen K.
      • Miari G.
      • Froiland D.
      • Thompson J.
      A randomised control trial examining the effect of an antioxidant (Menevit) on pregnancy outcome during IVF-ICSI treatment.
      ) and the amino acids taurine and hypothaurine (
      • Holmes R.P.
      • Goodman H.O.
      • Shihabi Z.K.
      • Jarow J.P.
      The taurine and hypotaurine content of human semen.
      ), Zn (
      • Gavella M.
      • Lipovac V.
      In vitro effect of zinc on oxidative changes in human semen.
      ) and Cu (
      • Nissen H.P.
      • Kreysel H.W.
      Superoxide dismutase in human semen.
      ). A recent additional study has confirmed the protective role of SOD against lipid peroxidation (
      • Tavilani H.
      • Goodarzi M.T.
      • Doosti M.
      • et al.
      Relationship between seminal antioxidant enzymes and the phospholipid and fatty acid composition of spermatozoa.
      ).
      A special antioxidant attribute of the seminal plasma is the relatively high concentration of another non-enzymatic antioxidant: GSH. This is a tripeptide thiol constituting a cofactor of the selenium containing GPX, the main enzyme involved in converting H2O2 to alcohol and a substrate in reactions catalysed by glutathione transferase (an enzyme which catalyses covalent reactions of GSH with electrophilic substances such as quinones) (
      • Aydemir B.
      • Onaran I.
      • Kiziler A.R.
      • et al.
      Increased oxidative damage of sperm and seminal plasma in men with idiopathic infertility is higher in patients with glutathione S-transferase Mu-1 null genotype.
      ). In different biological systems, the glutathione redox cycle, involving the enzymes GPX and GRD, has an important role in protecting cells against oxidative damage (
      • Reglinski J.
      • Hoey S.
      • Smith W.E.
      • Sturrock R.D.
      Cellular response to oxidative stress at sulfhydryl group receptor sites on the erythrocyte membrane.
      ,
      • Williams A.C.
      • Ford W.C.L.
      Functional significance of the pentose phosphate pathway and glutathione reductase in the antioxidant defenses of human sperm.
      ,
      • Luberda Z.
      The role of glutathione in mammalian gametes.
      ,
      • Tavilani H.
      • Goodarzi M.T.
      • Doosti M.
      • et al.
      Relationship between seminal antioxidant enzymes and the phospholipid and fatty acid composition of spermatozoa.
      ) through its thiolic group, which can react directly with hydrogen peroxide and the superoxide anion and hydroxyl radicals, and through its sulphydryl group, which can react with alkoxyl radicals and hydroperoxides, producing alcohols. Together, the enzyme scavengers and low-molecular weight antioxidants make up the TAC of the seminal plasma (
      • Smith R.
      • Vantman D.
      • Ponce J.
      • et al.
      Total antioxidant capacity of human seminal plasma.
      ).
      One more defence is conferred by the prostasomes, secreted by the prostate into the seminal plasma. These organelles have the ability to interact with neutrophils and to reduce their capacity to produce superoxide anion radicals (
      • Saez F.
      • Motta C.
      • Boucher D.
      • Grizard G.
      Antioxidant capacity of prostasomes in human semen.
      ). Prostasomes, in fact, can rigidify the plasma membrane of neutrophils and this results in the inhibition of the NADPH oxidase activity of neutrophils by lipid transfer from the prostasome to the plasma membrane of neutrophils (
      • Saez F.
      • Motta C.
      • Boucher D.
      • Grizard G.
      Prostasomes inhibit the NADPH oxidase activity of human neutriphils.
      ).
      • Rhemrev J.P.
      • Van Overveld F.W.
      • Haenen G.R.
      • et al.
      Quantification of the nonenzymatic fast and slow TRAP in a post-addition assay in human seminal plasma and the antioxidant contribution of various seminal compounds.
      showed that the high antioxidant capacity of seminal plasma protects spermatozoa from OS, indicating also a different role of antioxidants contributing, respectively, to slow and fast total radical-trapping potential capacity. Seminal plasma has also been shown to be able to scavenge all of the considered oxyradicals with a similar efficiency for peroxyl and hydroxyl radicals, but with a slightly lower efficiency for peroxynitrite (
      • Balercia G.
      • Armeni T.
      • Mantero F.
      • et al.
      Total oxyradical scavenging capacity toward different reactive oxygen species in seminal plasma and sperm cells.
      ).

      Antioxidant activity of spermatozoa

      The existence of one alternative defence mechanism that safeguards spermatozoa from the action of ROS has been proven. The sperm heads have another kind of defence: lactoferrin. This is an iron-binding protein that coats the head avoiding the peroxidative action of the transition metal (
      • Buckett W.M.
      • Luckas M.J.M.
      • Gazvani M.R.
      • et al.
      Seminal plasma lactoferrin concentrations in normal and abnormal semen samples.
      ).
      SOD and GPX/GRD pair play an important role against the deleterious effects of superoxide anion radical and hydrogen peroxide (
      • Li T.K.
      The glutathione and thiol content of mammalian spermatozoa and seminal plasma.
      ,
      • Nissen H.P.
      • Kreysel H.W.
      Superoxide dismutase in human semen.
      ,
      • Alvarez J.G.
      • Touchstone J.C.
      • Blasco L.
      • Storey B.T.
      Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. Superoxide dismutase as major enzyme protectant against oxygen toxicity.
      ,
      • Alvarez J.G.
      • Storey B.T.
      Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation.
      ) but also SOD, GPX and glucose-6-phosphate dehydrogenase (
      • Griveau J.F.
      • Dumont E.
      • Renard P.
      • et al.
      Reactive oxygen species, lipid peroxidation and enzymatic defence systems in human spermatozoa.
      ) act together against the heart of hydrogen peroxide (
      • Irvine D.S.
      Glutathione as a treatment for male infertility.
      ). These enzymes seem to be present only in the cytoplasm of the sperm midpiece. Because of this localization, it seems unlikely that they can protect the sperm head plasma membrane and the tail.
      The oxidation of ascorbic acid to dehydroascorbic acid produces the generation of both ascorbyl radicals and hydrogen peroxide. Since the concentration of CAT in spermatozoa and seminal plasma is low, GSH and GPX are the main agents that can eradicate the hydrogen peroxide generated (
      • Luberda Z.
      The role of glutathione in mammalian gametes.
      ). The antioxidant system acts in an integrated fashion. SOD dismutates the superoxide anion radical into hydrogen peroxide. Produced hydrogen peroxide during the reactions has to be removed by the action of both CAT and GPX (
      • Alvarez J.G.
      • Storey B.T.
      Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation.
      ), GRD (
      • Williams A.C.
      • Ford W.C.L.
      Functional significance of the pentose phosphate pathway and glutathione reductase in the antioxidant defenses of human sperm.
      ), α-tocopherol, vitamin C (
      • Lewis S.E.
      • Sterling E.S.
      • Young I.S.
      • Thompson W.
      Comparison of individuals antioxidants of sperm and seminal plasma in fertile and infertile men.
      ), vitamin E (
      • Therond P.
      • Auger J.
      • Legrand A.
      • Jouannet P.
      Alpha-tocopherol in human spermatozoa and seminal plasma: relationships with motility, antioxidant enzymes and leukocytes.
      ), albumin (
      • Elzanaty S.
      • Erenpreiss J.
      • Becker C.
      Seminal plasma albumin: origin and relation to the male reproductive parameters.
      ) and taurine and hypotaurine (
      • Holmes R.P.
      • Goodman H.O.
      • Shihabi Z.K.
      • Jarow J.P.
      The taurine and hypotaurine content of human semen.
      ). Generally, GSH is present in nanomolar concentrations in the cytosol, while its concentration is low in blood serum and in other biological fluids (
      • Li T.K.
      The glutathione and thiol content of mammalian spermatozoa and seminal plasma.
      ).
      Spermatozoa contain also the CoQ10, an energy-promoting agent with antioxidant properties, concentrated in the mitochondria of the midpiece. Its reduced form, ubiquinol, also acts as an antioxidant (
      • Lewin A.
      • Lavon H.
      The effect of coenzyme Q10 on sperm motility and function.
      ).

      Epididymal antioxidant system

      The epididymis also contains an enzymatic antioxidant system corresponding to GPX, SOD (
      • Perry A.C.
      • Jones R.
      • Niang L.S.
      • et al.
      Genetic evidence for an androgen-regulated epididymal secretory glutathione peroxidase whose transcript does not contain a selenocysteine codon.
      ,
      • Perry A.C.
      • Jones R.
      • Hall L.
      Isolation and characterization of a rat cDNA clone encoding a secreted superoxide dismutase reveals the epididymis to be a major site of its expression.
      ) and CAT. In the mouse, a more specific type, such as indoleamine dioxygenase among these many kinds of GPX, plays an important role during the epididymal sperm transit (
      • Drevet J.R.
      The antioxidant glutathione peroxidase family and spermatozoa: a complex story.
      ); SOD, GPX, glutathione transferase and the hexose monophospate shunt are present in the rat testis. These are variously expressed during the different stages of spermatogenesis (
      • Yoganathan T.
      • Eskild W.
      • Hansson V.
      Investigation of detoxification capacity of rat testicular germ cells and Sertoli cells.
      ,
      • Peltola V.
      • Huhtaniemi I.
      • Ahotupa M.
      Antioxidant enzyme activity in the maturing rat testis.
      ). CAT is localized in peroxisomes, while GPX has been identified in the same subcellular organelles as SOD.
      The role of vitamin E is to end the free-radical cascade in cellular membranes. Tocopheryl radicals are produced during the oxidation of vitamin E, which can then be reduced by ubiquinone or by ascorbic acid. The oxidation of vitamin C gives rise to ascorbyl radicals, which can be reduced by GSH and produce thiyl radicals and oxidized glutathione. This last step can then be reversed by GRD. Thus, the whole system has to work simultaneously, and an alteration of one of the components can lead to a potentially damaging accumulation of free radicals.
      In spermatozoa of patients with oligozoospermia, GSH concentrations are significantly lower than in controls. Sperm GSH content in normozoospermic men shows a large variation. A significant association between the intracellular GSH content and the aptitude to penetrate bovine cervical mucus has been reported. The intracellular GSH concentrations correlate significantly with the GSH concentrations in seminal plasma. The GSH concentration in seminal plasma does not differ between the various groups, however, it correlates significantly with FSH serum concentrations (
      • Ochsendorf F.R.
      • Buhl R.
      • Bastlein A.
      • Beschmann H.
      Glutathione in spermatozoa and seminal plasma of infertile men.
      ). Analogous findings have been reported by
      • Lewis S.E.
      • Sterling E.S.
      • Young I.S.
      • Thompson W.
      Comparison of individuals antioxidants of sperm and seminal plasma in fertile and infertile men.
      , who described decreased concentrations of ascorbate in the seminal plasma of asthenozoospermic men and improved ROS activity. Higher ROS production was observed in 16 of the 18 patients (88.8%, P < 0.0001 versus controls). Seminal plasma SOD, CAT, GPX and total sulphydryl-group concentrations in infertile patients were significantly lower than in controls (
      • Alkan I.
      • Simsek F.
      • Haklar G.
      • et al.
      Reactive oxygen species production by the spermatozoa of patients with idiopathic infertility: relationship to seminal plasma antioxidants.
      ,
      • Pasqualotto F.F.
      • Umezu F.M.
      • Salvador M.
      • et al.
      Effect of cigarette smoking on antioxidant levels and presence of leukocytospermia in infertile men: a prospective study.
      ). It has been shown, that seminal plasma, TAC is generally lower in men with varicocele than in healthy subjects (
      • Barbieri E.R.
      • Hidalgo M.E.
      • Venegas A.
      • et al.
      Varicocele-associated decrease in antioxidant defenses.
      ,
      • Hendin B.N.
      • Kolettis P.N.
      • Sharma R.K.
      • et al.
      Varicocele is associated with elevated spermatozoal reactive oxygen species production and diminished seminal plasma antioxidant capacity.
      ).
      While SOD may play a physiological role in maintaining a balance between superoxide anion radical and hydrogen peroxide, high concentrations of this enzyme are linked with impaired sperm function because: (i) there is excessive generation of hydrogen peroxide, which causes peroxidative damage; (ii) it impairs the fertilizing potential of the spermatozoa by removing superoxide anion radical; and (iii) high SOD activities reflect errors during the spermatogenesis associated with germ cell exfoliation and the retention of excess residual cytoplasm by the spermatozoa (
      • Aitken R.J.
      • Buckingham D.W.
      • Carreras A.
      • Irvine D.S.
      Superoxide dismutase in human sperm suspensions: relationship with cellular composition, oxidative stress, and sperm function.
      ). When the scavenging capacity of the seminal plasma was related to sperm motility parameters, a significant relationship was found with total oxyradical scavenging capacity values towards hydroxyl radicals, demonstrating a lower protection against toxicity of these specific ROS in seminal plasma of individuals with reduced motility of sperm cells (
      • Balercia G.
      • Armeni T.
      • Mantero F.
      • et al.
      Total oxyradical scavenging capacity toward different reactive oxygen species in seminal plasma and sperm cells.
      ). However, the literature reports contradictory evidence of the occurrence of oxidative damage to human spermatozoa and modification of single antioxidant defence. The latter have been reported to increase, decrease or even remain constant in seminal plasma and spermatozoa of individuals influenced by a range of infertility problems (
      • Alleva R.
      • Scararmucci A.
      • Mantero F.
      • et al.
      Protective role of ubiquinol content against formation of lipid hydroperoxide in human seminal fluid.
      ,
      • Lewis S.E.
      • Sterling E.S.
      • Young I.S.
      • Thompson W.
      Comparison of individuals antioxidants of sperm and seminal plasma in fertile and infertile men.
      ,
      • Ochsendorf F.R.
      • Buhl R.
      • Bastlein A.
      • Beschmann H.
      Glutathione in spermatozoa and seminal plasma of infertile men.
      ,
      • Zini A.
      • Garrels K.
      • Phang D.
      Antioxidant activity in the semen of fertile and infertile men.
      ). In this respect, it should be considered that OS embraces a complex set of phenomena; thus, it is highly doubtful that the analysis of a single antioxidant can elucidate specific relationships between a mixture of stressors and cellular damage (
      • Wayner D.D.
      • Burton G.W.
      • Ingold K.U.
      • et al.
      The relative contributions of vitamin E, urate, ascorbate and proteins to the total peroxyl radical-trapping antioxidant activity of human blood plasma.
      ). An enhancement of oxidative damage to sperm membranes, proteins and DNA is linked with modification in signal transduction mechanisms that involve fertility (
      • Sikka S.C.
      • Rajasekaran M.
      • Hellstrom W.J.
      Role of oxidative stress and antioxidants in male infertility.
      ). Despite the above-reported contrasting results, an effort to measure the antioxidant defences has been made. The concentrations of SOD and MDA both in the seminal plasma and spermatozoa were similar. With regard to GPX, it is about 13 times higher in spermatozoa than in the seminal plasma. Nitric oxide is also slightly higher in spermatozoa when compared with the seminal plasma (
      • Gallardo J.M.
      Evaluation of antioxidant system in normal semen.
      ).

      In-vitro antioxidant supplements in human sperm preparation techniques

      It has been confirmed that the techniques utilized for preparing spermatozoa have an effect on ROS production in human sperm suspensions and this inversely correlates with the fertilizing potential of spermatozoa in vitro (
      • Aitken R.J.
      • Clarkson J.S.
      Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques.
      ). Therefore, it has been shown that IVF success rate is significantly improved when ROS production declines (
      • Sukcharoen N.
      • Keith J.
      • Irvine D.S.
      • Aitken R.J.
      Prediction of the in vitro fertilization (IVF) potential of human spermatozoa using sperm function tests - the effect of the delay between testing and IVF.
      ). Adding medium before liquefaction may prevent the binding of bacteria and detritus to the sperm surface and subsequently decrease the DNA damage triggered by ROS (
      • Zollner U.
      • Zollner K.P.
      • Dietl J.
      • Steck T.
      Semen sample collection enhances the implantation rate following ICSI in patients with severe oligoasthenoteratozoospermia.
      ).
      Some techniques, such as swim-up from semen or density-gradient protocols (Percoll or PureSperm), have been established to significantly improve motility and morphology (
      • Aitken R.J.
      • West K.
      Analysis of the relationship between reactive oxygen species production and leucocyte infiltrations in fractions of human semen separated on Percoll gradients.
      ) and to reduce the proportion of spermatozoa with DNA fragmentation (
      • Colleu D.
      • Lescoat D.
      • Gouranton J.
      Nuclear maturity of human spermatozoa selected by swim-up or Percoll gradient centrifugation procedures.
      ,
      • Sakkas D.
      • Manicardi G.C.
      • Tomlinson M.
      • et al.
      The use of two density gradient centrifugation techniques and the swim-up method to separate spermatozoa with chromatin and nuclear DNA anomalies.
      ) and, consequently, to be a valid aid for semen preparation (
      • Benchaib M.
      • Lornage J.
      • Mazoyer C.
      • et al.
      Sperm deoxyribonucleic acid fragmentation as a prognostic indicator of assisted reproductive technology outcome.
      ). When density-gradient centrifugation and swim-up were compared, the results showed the latter approach to result in a better rate of curvilinear and straight-line velocity, hyperactivation, acrosome reaction (
      • Poulos A.
      • White I.G.
      The phospholipid composition of human spermatozoa and seminal plasma.
      ) and DNA integrity (
      • Zini A.
      • Finelli A.
      • Phang D.
      • Jarvi K.
      Influence of semen processing technique on human sperm DNA integrity.
      ). In contrast, repeated centrifugation of washed sperm preparations and the isolation of spermatozoa from seminal plasma have been shown to increase ROS production and to damage sperm DNA, possibly due to the mechanical activation of cell membrane oxidative systems in addition to contact with damaged spermatozoa and WBC.
      Although some studies on bovine oocytes (
      • Blondin P.
      • Coenen K.
      • Sirard M.A.
      The impact of reactive oxygen species on bovine sperm fertilizing ability and oocyte maturation.
      ) and embryos (
      • Dalvit G.C.
      • Cetica P.D.
      • Beconi M.T.
      Effect of alpha-tocopherol and ascorbic acid on bovine in vitro fertilization.
      ) reported contradictory results, many data showed that supplementation of culture media with antioxidants can improve sperm quality and reduce OS in some animal species. Studies have been conducted on ram spermatozoa with different compounds at various concentrations showing improved sperm functions and pregnancy rates (
      • Maxwell W.M.
      • Stojanov T.
      Liquid storage of ram semen in the absence or presence of some antioxidants.
      ,
      • Mara L.
      • Accardo C.
      • Pilichi S.
      • et al.
      Benefits of TEMPOL on ram semen motility and in vitro fertility: a preliminary study.
      ). In bovines, disulphide-reducing agents or divalent cation chelators prolong the motility of spermatozoa after freezing–thawing (
      • Lindemann C.B.
      • O’Brien J.A.
      • Giblin F.J.
      An investigation of the effectiveness of certain antioxidants in preserving the motility of reactivated bull sperm models.
      ), vitamins E and C alone or in combination play a relevant role in improving oocyte fertilization (
      • Blesbois E.
      • Grasseau I.
      • Blum J.C.
      Effects of vitamin E on fowl semen storage at 4 °C.
      ) and CAT reverses the reduction of the oocyte penetration rate induced by ROS (
      • Blondin P.
      • Coenen K.
      • Sirard M.A.
      The impact of reactive oxygen species on bovine sperm fertilizing ability and oocyte maturation.
      ). In rat spermatids, GSH can avoid the damage resulting from exposure to peroxidizing agents (
      • Den Boer P.J.
      • Poot M.
      • Verkerk A.
      • et al.
      Glutathione-dependent defence mechanisms in isolated round spermatids from the rat.
      ).
      The presence of antioxidants can suppress the generation of ROS (
      • Aitken R.J.
      • Clarkson J.S.
      Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques.
      ,
      • Donnelly E.T.
      • McClure N.
      • Lewis S.E.
      The effects of ascorbate and alpha-tocopherol supplementation in vitro on DNA integrity and hydrogen peroxide-induced DNA damage in human spermatozoa.
      ) and antioxidants may protect sperm DNA. When added in vitro, vitamin C (600 mmol/l), α-tocopherol (30 and 60 mmol/l) and urate (400 mmol/l) each have been described to give significant protection (P < 0.001) from subsequent DNA damage by X-irradiation. Thus, the supplementation of the culture medium with antioxidant compounds separately can beneficially affect sperm DNA integrity (
      • Hughes C.M.
      • Lewis S.E.
      • McKelvey-Martin V.J.
      • Thompson W.
      The effects of antioxidant supplementation during Percoll preparation on human sperm DNA integrity.
      ). Some antioxidants such as ascorbate and α-tocopherol are able to provide significant protection against DNA damage (
      • Donnelly E.T.
      • McClure N.
      • Lewis S.E.
      The effects of ascorbate and alpha-tocopherol supplementation in vitro on DNA integrity and hydrogen peroxide-induced DNA damage in human spermatozoa.
      ) and exhibit anti-apoptotic effects in a variety of cell culture systems, including granulosa cells and antral follicles (
      • Kolodecik T.R.
      • Aten R.F.
      • Behrman H.R.
      Ascorbic acid-dependent cytoprotection of ovarian cells by leukocyte and nonleukocyte peroxidases.
      ,
      • Tarín J.J.
      • Vendrell F.J.
      • Ten J.
      • Cano A.
      Antioxidant therapy counteracts the disturbing effects of diamide and maternal ageing on meiotic division and chromosomal segregation in mouse oocytes.
      ).
      Vitamin C is a water-soluble ROS scavenger with high potency. It is capable of downgrading peroxidation outside the cell but has little effect in the membrane or inside the cell. Vitamin C has two different actions: at concentrations below 1000 μmol/l it protects spermatozoa from free-radical damage as shown by improvement in their motility and viability. Concomitantly, there is also depletion of malondialdehyde generation (an end product of lipoperoxidase). At a concentration higher than 1000 μmol/l vitamin C is, however, a pro-oxidant, as shown by an abrupt fall in sperm motility and viability and concomitant increase in lipid peroxidation (
      • Verma A.
      • Kanwar K.C.
      Human sperm motility and lipid peroxidation in different ascorbic acid concentrations: an in vitro analysis.
      ).
      Effectiveness of antioxidants is often linked to the cause of the ROS production.
      • Parinaud J.
      • Le Lannou D.
      • Vieitez G.
      • et al.
      Enhancement of motility by treating spermatozoa with an antioxidant solution (Sperm-Fit) following ejaculation.
      have shown that the supplementation of antioxidants (Sperm-Fit) during sperm centrifugation significantly reduces WBC-mediated motility loss. Moreover, the supplementation of albumin in the culture medium has been shown to protect spermatozoa from the detrimental action of ROS, mainly when ROS come from spermatozoa (
      • Storey B.
      Biochemistry of the induction and prevention of lipoperoxidative damage in human spermatozoa.
      ) and to increase the recovery of higher quality spermatozoa compared with Percoll (
      • Armstrong J.S.
      • Rajasekaran M.
      • Hellstrom W.J.
      • Sikka S.C.
      Antioxidant potential of human serum albumin: role in the recovery of high quality human spermatozoa for assisted reproductive technology.
      ). In vitro studies on spermatozoa have established that supplementation of culture media with antioxidants counteracts asthenozoospermia (
      • Parinaud J.
      • Le Lannou D.
      • Vieitez G.
      • et al.
      Enhancement of motility by treating spermatozoa with an antioxidant solution (Sperm-Fit) following ejaculation.
      ). Addition of GSH and hypotaurine, either singly or in combination, to sperm preparation medium had no significant effect on sperm progressive motility or baseline DNA integrity (
      • Donnelly E.T.
      • McClure N.
      • Lewis S.E.
      Glutathione and hypotaurine in vitro: effects on human sperm motility, DNA integrity and production of reactive oxygen species.
      ).
      Spermatozoa of patients with asthenozoospermia incubated with 50 μmol/l of CoQ10 show a significant increase in motility, while no effect is reported in spermatozoa with normal motility (
      • Lewin A.
      • Lavon H.
      The effect of coenzyme Q10 on sperm motility and function.
      ). Data on ferulic acid suggest that it is beneficial to sperm viability and motility in both fertile and infertile individuals, leading to a decline of lipid peroxidative damage to sperm membranes and increase of intracellular cAMP and cGMP (
      • Zheng R.L.
      • Zhang H.
      Effects of ferulic acid on fertile and asthenozoospermic infertile human sperm motility, viability, lipid peroxidation, and cyclic nucleotides.
      ). Ferulic acid, a trans-cinnamic acid derivative, is an organic compound of plant cell walls. As a component of lignin, ferulic acid is a precursor in the manufacture of other aromatic compounds. With dihydroferulic acid, it is a lignocellulose component, serving to cross-link the lignin and polysaccharides, thereby conferring rigidity to the cell walls. Ferulic acid can also be found in plants seeds such as rice, wheat and oats, as well as in coffee, apple, artichoke, peanut, orange and pineapple.
      The use of GSH and SOD in vitro has also been proposed for improving sperm function. SOD increases hyperactivation and acrosome reaction rates, while GSH has been effective in improving acrosome reaction. CAT did not show any significant effect on these parameters (
      • Griveau J.F.
      • Le Lannou D.
      Effects of antioxidants on human sperm preparation techniques.
      ). These results denote that GSH safeguards sperm motility in vitro during pelleting, when they come into contact with seminal ROS, produced by WBC or damaged spermatozoa (
      • Lenzi A.
      • Gandini L.
      • Picardo M.
      Debate: Is antioxidant therapy a promising strategy to improve human reproduction? A rationale for glutathione therapy.
      ).
      Sperm SOD activity confirms a significant correlation with the number of motile spermatozoa, whereas seminal plasma SOD activity does not relate to sperm concentration or motility. MDA sperm concentration is significantly associated with the number of immotile spermatozoa. A decline in the motility of spermatozoa incubated in medium devoid of seminal plasma is observed after 120 min while the MDA concentration of the spermatozoa increased. Supplementation of exogenous SOD (400 U/ml) to the sperm suspension significantly reduced this loss of motility and the augmentation of the MDA concentration. These findings propose a significant role for SOD in sperm motility. It seems that lipid peroxidation of human spermatozoa may cause loss of motility and that SOD may avoid this lipid peroxidation. These results suggest that SOD may have a possible clinical application in the use of spermatozoa prepared for assisted reproduction treatment (
      • Kobayashi T.
      • Miyazaki T.
      • Natori M.
      • Nozawa S.
      Protective role of superoxide dismutase in human sperm motility: superoxide dismutase activity and lipid peroxide in human seminal plasma and spermatozoa.
      ).
      Isoflavones (genistein and equol) are plant compounds. Their physiological effects include antioxidant activity. Compared with vitamin C and α-tocopherol, genistein was the most potent antioxidant, followed by equol. Genistein and equol, when added in combination, were more protective than when added singularly. Based on these preliminary data, these compounds may play a role in antioxidant protection against sperm DNA damage (
      • Sierens J.
      • Hartley J.A.
      • Campbell M.J.
      • et al.
      In vitro isoflavone supplementation reduces hydrogen peroxide induced DNA damage in sperm.
      ).
      Pentoxifylline has a stimulating effect on Fe-induced lipid peroxidation, which usually acts positively on membrane fluidity and physiological destabilization. However, it can also stimulate a damaging peroxidation chain reaction when the spermatozoa are weaker than usual or when incubation is too long (
      • Gavella M.
      • Lipovac V.
      Effect of pentoxifylline on experimentally induced lipid peroxidation in human spermatozoa.
      ). Spermatozoa from 15 asthenozoospermic patients whose spermatozoa formed high concentrations of ROS at steady state were treated in vitro with pentoxifylline to verify its effect on ROS production and sperm motion parameters. Pentoxifylline diminished ROS generation by spermatozoa in these patients and preserved the decrease of curvilinear velocity and beat cross-frequency for 6 h in vitro (
      • Okada H.
      • Tatsumi N.
      • Kanzaki M.
      • et al.
      Formation of reactive oxygen species by spermatozoa from asthenospermic patients: response to treatment with pentoxifylline.
      ).
      In a recent study, spermatozoa washed with Ham’s F-10 media, incubated with EDTA and various CAT concentrations generated a significantly lower amount of ROS compared with spermatozoa incubated without these compounds. CAT significantly increased sperm acrosome reaction rate. Both the antioxidants significantly reduced the DNA fragmentation rate of the spermatozoa, whereas no effect on lipid peroxidation was observed (
      • Chi H.J.
      • Kim J.H.
      • Ryu C.S.
      • et al.
      Protective effect of antioxidant supplementation in sperm-preparation medium against oxidative stress in human spermatozoa.
      ). Another recent study on Boer bucks spermatozoa has shown that motility is improved and DNA damage is reduced after incubation with α-lipoic acid at a concentration of 0.02 mmol/ml (
      • Ibrahim S.F.
      • Osman K.
      • Das S.
      • et al.
      A study of the antioxidant effect of alpha lipoic acids on sperm quality.
      ).

      Antioxidant therapy in human male infertility

      Despite contrasting results (
      • Ten J.
      • Vendrell F.J.
      • Cano A.
      • Tarìn J.J.
      Dietary antioxidant supplementation did not affect declining sperm function with age in the mouse but did increase head abnormalities and reduced sperm production.
      ,
      • Ménézo Y.J.
      • Hazout A.
      • Panteix G.
      • et al.
      Antioxidants to reduce sperm DNA fragmentation: an unexpected adverse effect.
      ), antioxidant therapy appears to be efficient not only in vitro but also in vivo as an efficient strategy to improve the reproductive function. Experimental data in laboratory and farm animals support this contention (
      • Chew B.P.
      Effects of supplemental beta-carotene and vitamin A on reproduction in swine.
      ,
      • Luck M.R.
      • Jeyaseelan I.
      • Scholes R.A.
      Ascorbic acid and fertility.
      ). After exposure to ROS, the sperm membrane becomes more fragile and antioxidant treatment may prevent lipid peroxidation of sperm membranes (
      • Lenzi A.
      • Gandini L.
      • Picardo M.
      Debate: Is antioxidant therapy a promising strategy to improve human reproduction? A rationale for glutathione therapy.
      ). GSH therapy has a crucial role in increasing sperm motility of spermatozoa and consequently in improving fertilization in bulls with asthenozoospermia due to varicocele and in rabbits with dyspermy caused by cryptorchidism (
      • Tripodi L.
      • Tripodi A.
      • Mammi C.
      • et al.
      Pharmacological action and therapeutic effects of glutathione on hypokinetic spermatozoa for enzymatic-dependent pathologies and correlated genetic aspects.
      ). In lead-injected mice, the administration of vitamin C, at a concentration equivalent to the human therapeutic dose (10 mg/kg body weight), is able to significantly reduce the testicular MDA content with a simultaneous rise in sperm count and a significant reduction in the proportion of abnormal sperm population. Vitamin E (100 mg/kg body weight) treatment has a similar but lower efficacy than vitamin C. The co-administration of both vitamins at the above-mentioned doses leads to the most significant drop in MDA content along with elevation of sperm count and a decrease in the percentage of abnormal spermatozoa (
      • Mishra M.
      • Acharya U.R.
      Protective action of vitamins on the spermatogenesis in lead-treated Swiss mice.
      ). Vitamin E treatment has a similar effect against mercury-induced alteration of sperm number and functions (
      • Rao M.V.
      • Sharma P.S.
      Protective effect of vitamin E against mercuric chloride reproductive toxicity in male mice.
      ). A high amount of dietary α-tocopheryl acetate significantly increases vitamin E semen concentrations and its oxidative stability after cryopreservation. When the seminal plasma ascorbate concentration decreases to 7.3 μg/ml, the fertilization rate and the hatching rate of embryos decreases significantly. When associated with higher vitamin E concentrations, ascorbate increased seminal plasma α-tocopherol concentrations and the oxidative stability of semen, while both parameters decrease with lower vitamin E concentrations. Their combination significantly improves the viability and the kinetics of spermatozoa with an increase in fertility rate (
      • Castellini C.
      • Lattaioli P.
      • Bernardini M.
      • Dal Bosco A.
      Effect of dietary alpha-tocopheryl acetate and ascorbic acid on rabbit semen stored at 5 °C.
      ).
      The trials conducted on animal models indicate that antioxidant therapies can be successful in humans. Some of these studies suggest that a first-line therapy is prevention and this must be assured by an adequate dietary intake. Controversial data often result from the many uncontrolled studies carried out to support such a treatment and its efficacy is not yet proven (
      • Agarwal A.
      • Said T.M.
      Carnitines and male infertility.
      ). Unfortunately, andrologists often see patients with fertility problems many years after the beginning of their pathology mainly because andrological diseases have subclinical effects and few or no symptoms. In this manner the sperm damage becomes often irreversible.
      In vivo trials in humans have shown that administration of antioxidants improves sperm quality in heavy smokers (
      • Dawson E.B.
      • Harris W.A.
      • Teter M.C.
      • Powell L.C.
      Effect of ascorbic acid supplementation on the sperm quality of smokers.
      ) and in patients with male factor infertility (
      • Lenzi A.
      • Culasso F.
      • Gandini L.
      • et al.
      Placebo-controlled, double blind, crossover trial of glutathione therapy in male infertility.
      ) as well as increasing the fertilizing potential of healthy men with high seminal ROS concentrations (
      • Kessopoulou E.
      • Powers H.J.
      • Sharma K.K.
      • et al.
      A double-blind randomized placebo crossover controlled trial using the antioxidant vitamin E to treat reactive species associated male infertility.
      ) and fertile normozoospermic men with low fertilization rates in previous IVF cycles (
      • Geva E.
      • Bartoov B.
      • Zabludovsky N.
      • et al.
      The effect of antioxidant treatment on human spermatozoa and fertilization rate in an in vitro fertilization program.
      ).
      Whether antioxidant therapy in men can be improved is an unsolved question, as high doses of certain antioxidants, including vitamin A, may have embryotoxic and teratogenic effects (
      • Geelen J.A.
      Hypervitaminosis A induced teratogenesis.
      ,
      • Tzimas G.
      • Nau H.
      The role of metabolism and toxicokinetics in retinoid teratogenesis.
      ). This review cannot exclude the possibility that selected patients with elevated ROS generation or with reduced protective scavenging capacity in the seminal plasma may benefit from antioxidant treatment as suggested by Kessopoulou and Lenzi and their colleagues (
      • Lenzi A.
      • Culasso F.
      • Gandini L.
      • et al.
      Placebo-controlled, double blind, crossover trial of glutathione therapy in male infertility.
      ,
      • Kessopoulou E.
      • Powers H.J.
      • Sharma K.K.
      • et al.
      A double-blind randomized placebo crossover controlled trial using the antioxidant vitamin E to treat reactive species associated male infertility.
      ). But as yet, it is unknown whether ROS production can be used as a criterion to select men for antioxidative therapy, since intracellular sperm antioxidant status, sperm count, abstinence time and other confounding factors must also be considered. Also, no reliable, predictive and low-priced tests are available to evaluate the ROS exposure or to measure the TAC of the patient. Therefore, a valid approach would be to remove all causes that can amplify ROS production and/or to decrease seminal plasma scavenging activity.
      To identify which markers can be useful to measure OS before starting any antioxidant treatment or, alternatively, which markers can better measure ROS-induced damage in the plasma membrane would be very useful. A method to quantify the OS-induced damage will allow us to better evaluate the post-treatment efficacy and to understand how the injury will benefit from the antioxidant treatment. The main antioxidant compounds used in humans and their effect on the reproductive function are reviewed below.

      Ascorbic acid (vitamin C)

      In seminal plasma, vitamin C concentrations are 10-fold higher than in serum (
      • Jacob R.A.
      • Pianalto F.S.
      • Agee R.E.
      Cellular ascorbate depletion in healthy men.
      ). Vitamin C is a powerful antioxidant when peroxyl radicals are present in the aqueous phase (
      • Frei B.
      • England L.
      • Ames B.N.
      Ascorbate is an outstanding antioxidant in human blood plasma.
      ), but the vitamin is a weak scavenger for ROS within the lipid membrane (
      • Doba T.
      • Burton G.W.
      • Ingold K.U.
      Antioxidant and co-antioxidant activity of vitamin C. The effect of vitamin C, either alone or in the presence of vitamin E or a water-soluble vitamin E analogue, upon the peroxidation of aqueous multilamellar phospholipid liposomes.
      ). In semen samples with ROS hyper-production, ascorbate concentrations in seminal plasma are significantly reduced (
      • Lewis S.E.
      • Sterling E.S.
      • Young I.S.
      • Thompson W.
      Comparison of individuals antioxidants of sperm and seminal plasma in fertile and infertile men.
      ). Seminal ascorbic acid concentration is also significantly lower in leukospermic samples. A significantly greater percentage of samples with abnormal DNA fragmentation index has been detected in samples with low seminal ascorbic acid concentrations compared with those with normal or high concentration of ascorbic acid (
      • Song G.J.
      • Norkus E.P.
      • Lewis V.
      Relationship between seminal ascorbic acid and sperm DNA integrity in infertile men.
      ). Interestingly, at low concentrations, vitamin C is an antioxidant, but at high concentrations it can start an auto-oxidation process (
      • Wayner D.D.
      • Burton G.W.
      • Ingold K.U.
      The antioxidant efficiency of vitamin C is concentration-dependent.
      ). In addition, plasma saturation of vitamin C takes place in humans at a daily amount of 1 g and higher doses may stimulate the formation of kidney stones because of the increased excretion as oxalate (
      • Levine M.
      • Conry-Cantilena C.
      • Wang Y.
      • et al.
      Vitamin C pharmacokinetics in healthy volunteers: evidence for a recommended dietary allowance.
      ).
      A 2.2-fold increase in plasma ascorbic acid concentration is achieved with a supplementation dose of vitamin C (1 g/day) (
      • Wen Y.
      • Cooke T.
      • Feely J.
      The effect of pharmacological supplementation with vitamin C on low-density lipoprotein oxidation.
      ). Furthermore, its seminal plasma concentrations correlated positively with the percentages of morphologically normal spermatozoa (
      • Thiele J.J.
      • Friesleben H.J.
      • Fuchs J.
      • Ochsendorf F.R.
      Ascorbic acid and urate in human seminal plasma: determination and interrelationships with chemiluminescence in washed semen.
      ) and this evidence can also indicate that vitamin C is a protective vitamin in the epididymis.
      In previous studies, attempts have been made to improve semen parameters of infertile men by vitamin C supplementation (1 g/day) (
      • Dawson E.B.
      • Harris W.A.
      • Rankin W.E.
      • et al.
      Effect of ascorbic acid on male fertility.
      ,
      • Dawson E.B.
      • Harris W.A.
      • Teter M.C.
      • Powell L.C.
      Effect of ascorbic acid supplementation on the sperm quality of smokers.
      ). An elevated intake of vitamin C was related with improved semen quality, as indicated in the higher mean sperm count, sperm concentration and total progressive motile sperm count (
      • Eskenazi B.
      • Kidd S.A.
      • Marks A.R.
      • et al.
      Antioxidant intake is associated with semen quality in healthy men.
      ). In a placebo-controlled study in smokers, the groups receiving vitamin C at a dose of 200 or 1000 mg/day, had sperm parameter improvement, and the most relevant improvement was observed in the group receiving the highest dose for 4 weeks (
      • Dawson E.B.
      • Harris W.A.
      • Teter M.C.
      • Powell L.C.
      Effect of ascorbic acid supplementation on the sperm quality of smokers.
      ). Vitamin C protects human spermatozoa from endogenous oxidative DNA damage (
      • Fraga C.G.
      • Motchnik P.A.
      • Shigenaga M.K.
      • et al.
      Ascorbic acid protects against endogenous oxidative DNA damage in human sperm.
      ).

      α-Tocopherol (vitamin E)

      A single-blind study has been carried out with vitamin E. Eight patients receiving vitamin E at the dose of 100 mg three times a day for 120 days failed to show any improvement (
      • Giovenco P.
      • Amodei M.
      • Barbieri C.
      • et al.
      Effects of kallikrein on the male reproductive system and its use in the treatment of idiopathic oligozoospermia with impaired motility.
      ). Administration of 300 mg/day of vitamin E determines a small rise in seminal plasma vitamin E concentration (
      • Moilanen J.
      • Hovatta O.
      • Lindroth L.
      Vitamin E levels in seminal plasma can be elevated by oral administration of vitamin E in infertile men.
      ). Its seminal plasma concentrations become faintly more elevated in infertile men when vitamin E is given at doses of 300 and 1200 mg/day for 3 weeks (
      • Moilanen J.
      • Hovatta O.
      Excretion of alpha-tocopherol into human seminal plasma after oral administration.
      ). The concentration of α-tocopherol in spermatozoa is independent from the concentration and the total α-tocopherol amount in the seminal plasma; the percentage of motile spermatozoa is significantly related to sperm α-tocopherol content (
      • Therond P.
      • Auger J.
      • Legrand A.
      • Jouannet P.
      Alpha-tocopherol in human spermatozoa and seminal plasma: relationships with motility, antioxidant enzymes and leukocytes.
      ).
      Efforts have been made to improve semen parameters of infertile men by vitamin E (600 mg/day) administration (
      • Kessopoulou E.
      • Powers H.J.
      • Sharma K.K.
      • et al.
      A double-blind randomized placebo crossover controlled trial using the antioxidant vitamin E to treat reactive species associated male infertility.
      ). In a double-blind, randomized, placebo crossover controlled trial, 30 healthy men with high semen ROS concentrations and a normal female partner received vitamin E (600 mg/day) or placebo tablets for 3 months. Vitamin E increased significantly blood serum vitamin E concentrations and improved the in vitro sperm function as assessed by the zona-binding test (
      • Kessopoulou E.
      • Powers H.J.
      • Sharma K.K.
      • et al.
      A double-blind randomized placebo crossover controlled trial using the antioxidant vitamin E to treat reactive species associated male infertility.
      ). Other studies used lower doses of vitamin E, such as 300 mg/day (
      • Giovenco P.
      • Amodei M.
      • Barbieri C.
      • et al.
      Effects of kallikrein on the male reproductive system and its use in the treatment of idiopathic oligozoospermia with impaired motility.
      ,
      • Moilanen J.
      • Hovatta O.
      • Lindroth L.
      Vitamin E levels in seminal plasma can be elevated by oral administration of vitamin E in infertile men.
      ) or 200 mg/day (
      • Geva E.
      • Bartoov B.
      • Zabludovsky N.
      • et al.
      The effect of antioxidant treatment on human spermatozoa and fertilization rate in an in vitro fertilization program.
      ). A placebo controlled double-blind study showed that sperm MDA concentration was higher in asthenozoospermic and oligoasthenozoospermic patients and that vitamin E administration significantly reduced MDA concentration and enhanced sperm motility in asthenozoospermic men (
      • Suleiman S.A.
      • Ali M.E.
      • Zaki Z.M.
      • et al.
      Lipid peroxidation and human sperm motility: protective role of vitamin E.
      ). Furthermore, 11 (21%) of 52 spouses of the treatment group became pregnant in the course of the 6-month treatment period; resulting in nine normal-term deliveries, whereas the other two aborted in the first trimester. No pregnancies were reported in the placebo group (
      • Suleiman S.A.
      • Ali M.E.
      • Zaki Z.M.
      • et al.
      Lipid peroxidation and human sperm motility: protective role of vitamin E.
      ).
      In a prospective study, 15 fertile normozoospermic men, who had low fertilization rates in their earlier IVF cycles, were treated with 200 mg/day of vitamin E for 3 months. The high MDA concentrations significantly declined to normal and the fertilization rate per cycle improved significantly after 1 month of treatment (
      • Geva E.
      • Bartoov B.
      • Zabludovsky N.
      • et al.
      The effect of antioxidant treatment on human spermatozoa and fertilization rate in an in vitro fertilization program.
      ).
      In another study, 97 healthy, non-smoking men were interviewed on dietary habits and their semen was analysed. A high intake of daily nutrients and supplements with antioxidant quality was associated with a better semen quality; for example, vitamin E intake and progressive motility and total progressive motile sperm count; and between β-carotene intake and sperm concentration and progressive motility (
      • Eskenazi B.
      • Kidd S.A.
      • Marks A.R.
      • et al.
      Antioxidant intake is associated with semen quality in healthy men.
      ).

      Ascorbic acid (vitamin C) and α-tocopherol (vitamin E)

      Vitamin C and vitamin E may operate synergistically in vivo to reduce the peroxidative damage on spermatozoa, by joining their hydrophilicity and lipophilicity. In addition, if these agents act directly on spermatozoa to avoid damage by ROS, such improvement may be fast, provided that the vitamins gain access to spermatozoa either at ejaculation or within the epididymis. In patients with asthenozoospermia, a prominent production of seminal plasma ROS and a higher ROS-mediated injury of sperm membranes has been discovered, but the source of these effects is unidentified (
      • De Lamirande E.
      • Gagnon C.
      Reactive oxygen species and human spermatozoa.
      ,
      • De Lamirande E.
      • Gagnon C.
      Reactive oxygen species and human spermatozoa.
      ,
      • Agarwal A.
      • Ikemoto I.
      • Loughlin K.R.
      Relationship of sperm parameters with levels of reactive oxygen species in semen specimens.
      ). Neither is it known at which point the peroxidative damage to spermatozoa takes place, whether within semen (during the time required for liquefaction), during the epididymal transit or within the testis. By altering membrane integrity, ROS may prejudice sperm motility as well as sperm viability (
      • Davis B.K.
      Timing of fertilization in mammals: sperm cholesterol/phospholipid ratio as a determinant of the capacitation interval.
      ,
      • Sebastian S.M.
      • Selvaraj S.
      • Aruldhas M.M.
      • Govindarajulu P.
      Pattern of neutral and phospholipids in the semen of normospermic, oligospermic and azoospermic men.
      ).
      In a single-centre, double-blind, placebo-controlled randomized study, simultaneous daily administration of high vitamin C (1 g) and vitamin E (800 mg) doses for 8 weeks did not improve semen parameters or 24-h sperm survival rate in patients with asthenozoospermia or moderate oligoasthenozoospermia (
      • Rolf C.
      • Cooper T.G.
      • Yeung C.H.
      • 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.
      ). These disappointing results agree with those reported by some (
      • Giovenco P.
      • Amodei M.
      • Barbieri C.
      • et al.
      Effects of kallikrein on the male reproductive system and its use in the treatment of idiopathic oligozoospermia with impaired motility.
      ,
      • Moilanen J.
      • Hovatta O.
      • Lindroth L.
      Vitamin E levels in seminal plasma can be elevated by oral administration of vitamin E in infertile men.
      ) but are at variance with those reported elsewhere in the literature (
      • De Lamirande E.
      • Gagnon C.
      Reactive oxygen species and human spermatozoa.
      ,
      • Geva E.
      • Bartoov B.
      • Zabludovsky N.
      • et al.
      The effect of antioxidant treatment on human spermatozoa and fertilization rate in an in vitro fertilization program.
      ). It is possible that the relatively short treatment time utilized in this study explains why no improvement was found, especially if the effect takes place within the testis.
      In another study, 64 men with unexplained infertility and an elevated percentage (15%) of DNA-fragmented spermatozoa in the ejaculate were randomly divided into two groups. One group received vitamin C (1 g) and vitamin E (1 g) daily and the other placebo. After 2 months of treatment, the percentage of DNA-fragmented spermatozoa was significantly reduced in the antioxidant-treated group, whereas no difference was observed in the placebo group (
      • Greco E.
      • Iacobelli M.
      • Rienzi L.
      • et al.
      Reduction of the incidence of sperm DNA fragmentation by oral antioxidant treatment.
      ). Another study was conducted on 38 men with an elevated proportion (15%) of DNA-fragmented spermatozoa in the ejaculate. They were treated with vitamin C (1 g) and vitamin E (1 g) daily for 2 months after one ICSI cycle failure. In 29 of these cases (76%), the antioxidant treatment led to a reduction in the percentage of DNA-fragmented spermatozoa and a second ICSI effort produced a large improvement in the clinical pregnancy (48.2% versus 6.9%) and implantation (19.6% versus 2.2%) rates (
      • Greco E.
      • Romano S.
      • Iacobelli M.
      • et al.
      ).

      α-Tocopherol (vitamin E) and selenium

      There is only one study that attempted to treat, in an open randomized trial, 28 men with a daily administration of vitamin E (400 mg) and selenium (225 μg) for 3 months. In this study, another 26 patients received vitamin B (4.5 g/day) for the same duration. In these patients, vitamin E and selenium supplementation produced a significant decrease in MDA concentrations and an improvement of sperm motility (
      • Keskes-Ammar L.
      • Feki-Chakroun N.
      • Rebai T.
      • et al.
      Sperm oxidative stress and the effect of an oral vitamin E and selenium supplement on semen quality in infertile men.
      ).

      Glutathione

      GSH seems to be the most frequently used compound, owing to its demonstrated antitoxic and antioxidant action in other degenerative pathologies. Although it cannot cross cell membranes, the concentration this antioxidant in biological fluids can increase after systemic administration. GSH is able to reach the seminal plasma and concentrate there. In this fluid, it protects spermatozoa from oxidative stress, suggesting that its supplementation may play a therapeutic role in some andrological disease, particularly during inflammation (
      • Lenzi A.
      • Culasso F.
      • Gandini L.
      • et al.
      Placebo-controlled, double blind, crossover trial of glutathione therapy in male infertility.
      ).
      In a 2-month pilot study, GSH (600 mg/day i.m.) was administrated to a group of patients with dyspermia associated with various selected andrological pathologies. A significant discrepancy was seen in the proportion of spermatozoa with forward motility and in the parameters of the sperm motility evaluated by computer analysis. Sperm motility increased, particularly in patients with chronic inflammation of the genital tract and in patients with varicocele (
      • Lenzi A.
      • Lombardo F.
      • Gandini L.
      • et al.
      Glutathione therapy for male infertility.
      ), two conditions i