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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
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
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, ItalyCorrespondence:
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.
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 (
). OS has been defined as an imbalance between the generation of reactive oxygen species (ROS) and antioxidant scavenging activities, in which the former prevails (
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 (
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 (
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 (
), 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 (
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 (
). 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) (
Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. Superoxide dismutase as major enzyme protectant against oxygen toxicity.
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, , and the subsequent Fenton reaction, Fe2+ + H2O2 → Fe3+ + HO− + HO) the formation of hydroxyl radical, which is a more dangerous oxidizing product (
). 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 (
). 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 (
). 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 (
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 (
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 (
Differential contribution of leucocytes and spermatozoa to the generation of reactive oxygen species in the ejaculates of oligozoospermic patients and fertile donors.
Journal of Reproduction and Fertility.1992; 94: 451-462
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 (
). 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 (
). 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 (
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 (
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.
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 (
). In this condition, ROS hyper-production damages sperm function, such as motility, capacitation, fertilization capability, acrosome reaction and DNA/chromatin integrity (
). 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 (
). 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 (
Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. Superoxide dismutase as major enzyme protectant against oxygen toxicity.
). 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 (
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 (
). 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 (
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 (
) 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 (
), 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 (
). 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 (
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.
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 (
). 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 (
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 (
). 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 (
). 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 (
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 (
). 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 (
). 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 (
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 (
). 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 (
) reported midpiece abnormalities, and some others showed that ROS-induced motility decrease is associated with a growth of lipid peroxidation measured as malondialdehyde (MDA) (
). 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) (
). 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 (
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 (
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 (
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 (
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.
); 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 (
). Several observations suggest that disorders in the DNA organization in the sperm nucleus are negatively related with the fertility competence of spermatozoa (
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 (
). 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 (
). 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 (
). 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 (
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 (
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 (
). 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 (
). 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 (
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 (
). 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 (
Seminal reactive oxygen species as predictors of fertilization, embryo quality and pregnancy rates after conventional in vitro fertilization and intracytoplasmic sperm injection.
International Journal of Andrology.2003; 26: 279-285
). 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 (
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 (
Seminal reactive oxygen species as predictors of fertilization, embryo quality and pregnancy rates after conventional in vitro fertilization and intracytoplasmic sperm injection.
International Journal of Andrology.2003; 26: 279-285
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 (
). 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 (
Seminal reactive oxygen species as predictors of fertilization, embryo quality and pregnancy rates after conventional in vitro fertilization and intracytoplasmic sperm injection.
International Journal of Andrology.2003; 26: 279-285
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 (
Transmission of de-novo mutations of the deleted in azoospermia genes from a severely oligozoospermic male to a son via intracytoplasmic sperm injection.
Y chromosome analysis of infertile men and their sons conceived through intracytoplasmic sperm injection: vertical transmission of deletions and rarity of de-novo deletions.
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) (
Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. Superoxide dismutase as major enzyme protectant against oxygen toxicity.
) 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 (
). 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 (
Protective role of superoxide dismutase in human sperm motility: superoxide dismutase activity and lipid peroxide in human seminal plasma and 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) (
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 (
) 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 (
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 (
). 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 (
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 (
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 (
Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. Superoxide dismutase as major enzyme protectant against oxygen toxicity.
). 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 (
). 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 (
). Generally, GSH is present in nanomolar concentrations in the cytosol, while its concentration is low in blood serum and in other biological fluids (
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 (
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 (
); 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 (
). 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 (
, 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 (
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 (
). 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 (
). 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 (
). 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 (
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 (
). 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 (
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 (
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 (
Some techniques, such as swim-up from semen or density-gradient protocols (Percoll or PureSperm), have been established to significantly improve motility and morphology (
Analysis of the relationship between reactive oxygen species production and leucocyte infiltrations in fractions of human semen separated on Percoll gradients.
International Journal of Andrology.1990; 13: 433-451
). 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 (
). 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.
) 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 (
) 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 (
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 (
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 (
). 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 (
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 (
). 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 (
). 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 (
). 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 (
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 (
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 (
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 (
). 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 (
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 (
). 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 (
Dietary antioxidant supplementation did not affect declining sperm function with age in the mouse but did increase head abnormalities and reduced sperm production.
), 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 (
). 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 (
Pharmacological action and therapeutic effects of glutathione on hypokinetic spermatozoa for enzymatic-dependent pathologies and correlated genetic aspects.
Clinical and Experimental Obstetrics and Gynecology.2003; 30: 130-136
). 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 (
). 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 (
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 (
). 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 (
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 (
). 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 (
). 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 (
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.
). 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 (
). 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 (
). 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 (
). 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 (
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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
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 (
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 (
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 (
). 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 (
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 (
Antioxidant treatment of patients with asthenozoospermia or moderate oligoasthenozoospermia with high-dose vitamin C and vitamin E: a randomized, placebo-controlled, double-blind study.
). 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 (
). 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 (
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 (
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 (
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 (
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