Although reports have suggested the benefit of antioxidant treatment of infertile men, many studies also show no effect. Most studies in the literature are not randomized, placebo controlled, or double blinded in design, which makes it difficult to differentiate regression toward the mean from true positive treatment effects. The small patient sample sizes and varying male populations also add to the difficulty in comparing studies. Pregnancy, the most relevant outcome parameter, is rarely reported. Ideally, patients would be selected based on oxidative stress levels, and improvement in these levels would be correlated to improvement in pregnancy rates. Until those studies are performed, the use of antioxidants for the treatment of male infertility remains empiric.
Reactive oxygen species (ROS) are free radicals that are derived from the metabolism of oxygen. The production of ROS, such as superoxide (O 2 −), hydrogen peroxide (H 2 O 2 ), and the hydroxyl radical (OH − ), normally occurs in cells. ROS play an important role in multiple cellular physiologic processes, such as phagocytosis, and in many signaling processes. In the male reproductive tract, small amounts of ROS are involved in many sperm functions. Hydrogen peroxide stimulates sperm capacitation, the acrosome reaction, and hyperactivation. Free radicals are also involved in the fusion of spermatozoa with the oocyte .
Oxidative stress is a condition that occurs when high concentrations of ROS exist relative to antioxidant capacity. In 1943, John MacLeod first made the observation that oxidative stress could be a significant cause of male infertility . Neutrophils, macrophages, and immature spermatozoa are the major sources of ROS in the male reproductive tract. Leukocyte production of ROS in the setting of infection and inflammation may lead to the propagation of excessive ROS levels in surrounding tissues . In higher concentrations, ROS may cause varying degrees of sperm dysfunction, depending on the extent of oxidative stress. Damage from ROS occurs primarily through two routes: First, ROS may be responsible for the DNA fragmentation commonly seen in the spermatozoa of infertile men by causing single- and double-stranded DNA breaks . Aitken and colleagues described how a low concentration of hydrogen peroxide did not affect sperm motility but did suppress sperm-egg fusion. Second, higher levels of ROS also may cause damage through a chain of chemical reactions that result in lipid peroxidation of the sperm plasma membrane . Lipid peroxidation results in loss of the membrane fluidity, which is essential for sperm motility and sperm-oocyte fusion. Griveau and Le Lannou have shown that the acrosome reaction in human spermatozoa was susceptible to ROS. Several studies have shown that levels of ROS inversely correlate with sperm motility.
Antioxidants
Normally, antioxidant scavengers constantly inactivate ROS. Seminal plasma contains significant amounts of antioxidants. The human body uses three general systems of antioxidants for protection against free radicals: endogenous antioxidants, dietary antioxidants, and metal-binding proteins . Endogenous antioxidants can be categorized as low molecular weight molecules, such as bilirubin, thiols, uric acid, and coenzyme Q-10 (CoQ10), and larger molecular enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase. All three of the larger molecular enzymes are essential for adequate antioxidant defense. Dietary antioxidants include vitamin C, vitamin E, carotenoids, and flavonoids. The mechanism of action of the dietary antioxidants includes scavenging free radicals and interfering with the chain of chemical reactions that lead to lipid peroxidation. Antioxidants that act through the last mechanism are known as chain-breaking antioxidants. Metal-binding proteins, such as albumin, cerruloplasmin, metallothionein, transferrin, ferritin, and myoglobulin, inactivate the transition metal ions (eg, iron) that catalyze the production of free radicals . Twigg and colleagues demonstrated the protective effect of adding albumin to sperm in vitro by binding to lipid peroxides or binding to ferrous ion promoters .
Evaluation of oxidative stress
Oxidative stress occurs when there is an imbalance between ROS production and antioxidant capacity. High levels of seminal ROS have been found in 30% to 80% of infertile men . Elevated levels of ROS and decreased levels of total antioxidant capacity have been noted in patients with varicoceles . A positive correlation between ROS and varicocele grade also has been noted . With infection of the male reproductive tract, one can expect elevated levels of ROS caused by the presence of leukocytes. Patients with prostatovesiculoepididymitis have been shown to have higher ROS levels than patients without infection .
Because oxidative stress has been shown to play a key role in male infertility, it is important to be able to quantify the level of oxidative stress to determine if it is a significant contributor to sperm dysfunction in a given patient. There are numerous methods to determine the ROS levels in semen. Chemiluminescence, one of the most widely used methods to assess the level of ROS, discriminates between the production of superoxide and hydrogen peroxide by spermatozoa by the reagent used (luminol and lucigenin are the most common reagents) . Flow cytometry uses the principles of light scattering, light excitation, and emission of fluorochrome molecules to generate specific multiparameter data measures. By measuring the fluorescent intensity of a particular dye after oxidation by ROS, one can quantify the amount of ROS present in the sample . The total antioxidant capacity of the semen can be determined by either the enhanced chemiluminescence assay or the calorimetric assay. In both of these assays, ROS production is chemically induced, which results in the development of a chemiluminescence signal or a color change. The ability of the seminal plasma to inhibit the production of the signal or color change is compared with known amounts of a control antioxidant. The greater the ability of the seminal plasma to inhibit the production of the signal or color change, the greater the antioxidant capacity of the semen sample. By comparing the degree of inhibition of the reaction by seminal plasma to the ability of known amounts of the control antioxidant to inhibit the reaction, the amount of antioxidant capacity can be quantified .
Because the ultimate amount of oxidative stress and subsequent damage may be a function of the total amount of ROS and the total antioxidant capacity, an ROS-total antioxidant capacity score was developed. This measurement mathematically combines the ROS and total antioxidant capacity measurements of a patient’s semen and control semen. The controls consisted of healthy men with low ROS levels who had achieved pregnancy with their partner in the past 2 years . A low ROS-total antioxidant capacity score (< 30) indicates the presence of high oxidative stress and sperm dysfunction. Despite the different methods available to measure ROS, currently no standard exists for estimating oxidative stress. Allamaneni and colleagues defined the basal levels of ROS in normal donors in whole, unprocessed semen specimens and in mature and immature spermatozoa. It is hypothesized that the determination of what the basal level of ROS is in human semen may be used to identify pathologic ROS levels in infertile men and ultimately guide treatment.
Evaluation of oxidative stress
Oxidative stress occurs when there is an imbalance between ROS production and antioxidant capacity. High levels of seminal ROS have been found in 30% to 80% of infertile men . Elevated levels of ROS and decreased levels of total antioxidant capacity have been noted in patients with varicoceles . A positive correlation between ROS and varicocele grade also has been noted . With infection of the male reproductive tract, one can expect elevated levels of ROS caused by the presence of leukocytes. Patients with prostatovesiculoepididymitis have been shown to have higher ROS levels than patients without infection .
Because oxidative stress has been shown to play a key role in male infertility, it is important to be able to quantify the level of oxidative stress to determine if it is a significant contributor to sperm dysfunction in a given patient. There are numerous methods to determine the ROS levels in semen. Chemiluminescence, one of the most widely used methods to assess the level of ROS, discriminates between the production of superoxide and hydrogen peroxide by spermatozoa by the reagent used (luminol and lucigenin are the most common reagents) . Flow cytometry uses the principles of light scattering, light excitation, and emission of fluorochrome molecules to generate specific multiparameter data measures. By measuring the fluorescent intensity of a particular dye after oxidation by ROS, one can quantify the amount of ROS present in the sample . The total antioxidant capacity of the semen can be determined by either the enhanced chemiluminescence assay or the calorimetric assay. In both of these assays, ROS production is chemically induced, which results in the development of a chemiluminescence signal or a color change. The ability of the seminal plasma to inhibit the production of the signal or color change is compared with known amounts of a control antioxidant. The greater the ability of the seminal plasma to inhibit the production of the signal or color change, the greater the antioxidant capacity of the semen sample. By comparing the degree of inhibition of the reaction by seminal plasma to the ability of known amounts of the control antioxidant to inhibit the reaction, the amount of antioxidant capacity can be quantified .
Because the ultimate amount of oxidative stress and subsequent damage may be a function of the total amount of ROS and the total antioxidant capacity, an ROS-total antioxidant capacity score was developed. This measurement mathematically combines the ROS and total antioxidant capacity measurements of a patient’s semen and control semen. The controls consisted of healthy men with low ROS levels who had achieved pregnancy with their partner in the past 2 years . A low ROS-total antioxidant capacity score (< 30) indicates the presence of high oxidative stress and sperm dysfunction. Despite the different methods available to measure ROS, currently no standard exists for estimating oxidative stress. Allamaneni and colleagues defined the basal levels of ROS in normal donors in whole, unprocessed semen specimens and in mature and immature spermatozoa. It is hypothesized that the determination of what the basal level of ROS is in human semen may be used to identify pathologic ROS levels in infertile men and ultimately guide treatment.
Treatment of infertility caused by oxidative stress
Several approaches have been suggested to manage infertility caused by oxidative stress. Unfortunately, most clinical trials of antioxidants do not select patients based on measured levels of oxidative stress in semen, and many do not correlate treatment to changes in oxidative stress. Attempts may be made to decrease endogenous ROS production by changes in behaviors and lifestyle habits that have been associated with increased ROS production. Antioxidant supplements have been proposed and are commonly sold to increase the antioxidant capacity of semen, thereby scavenging excess ROS and resulting in less oxidative stress. Finally, specific laboratory techniques may be used to decrease ROS production, which is often stimulated when sperm are handled in the laboratory.
Behavior and lifestyle modification
Various behaviors and lifestyles have been associated with increased ROS production. Cigarette smoking has been shown to increase ROS in semen and decrease semen quality (density, total count, and motility) . Exposure to environmental pollution also has been linked to increased ROS production. A study of 85 middle-aged tollgate workers exposed to traffic pollutants showed significant decreases in total sperm viability, motility, and membrane function compared with age-matched controls living in the same area . Finally, several systemic diseases, such as diabetes mellitus, cancer, cardiovascular problems, and infection, are known to increase the production of ROS . These data suggest that behavior and lifestyle modification and treatment of a patient’s underlying pathology should be the first steps in reducing ROS. Unfortunately, few data link changes in these exposures to decreased oxidative stress and subsequent increases in human fertility. Although it is likely good medical practice to recommend modifications of unhealthy lifestyles or exposures, definitive evidence awaits further studies.
Dietary antioxidants
Dietary antioxidants are present in fruits and vegetables and daily dietary supplements. The National Academy of Sciences recommends 90 mg/d of vitamin C for an adult man and 15 mg/d of vitamin E . Carotenoids and selenium have recommended daily allowances of 900 and 55 μg/d, respectively. Despite the daily recommendations for dietary antioxidants, randomized trials have largely failed to show an effect of antioxidant vitamins on the risk of other disease processes, such as cardiovascular disease. The Women’s Antioxidant Cardiovascular Study was a randomized trial that tested the effects of vitamin C (500 mg/d), vitamin E (600 IU every other day), and beta-carotene (50 mg every other day) on the combined outcome of myocardial infarction, stroke, coronary revascularization, and cardiovascular disease–related death among 8171 female healthcare professionals. There were no overall effects of vitamin C, vitamin E, or beta-carotene on cardiovascular events among women at high risk for cardiovascular disease . This study suggested that at least for cardiovascular disease, antioxidants in pill form are not effective in modifying risk. The possibility of adverse events with therapy also should be considered.
A recent meta-analysis of trials of antioxidants to prevent gastrointestinal cancers found an increase in mortality with the use of most supplements . Menezo and colleagues reported that in an uncontrolled study, treatment with a combination of oral antioxidants led to decreased sperm DNA fragmentation and an increase in sperm DNA decondensation, which is likely not advantageous. The finding of a link between oxidative stress and sperm DNA fragmentation has led to studies looking for changes in semen DNA fragmentation as an outcome rather than just changes in semen parameters. Although the most desirable outcome measure is pregnancy rate, this is rarely reported. Although obtaining antioxidants through a diet rich in these compounds may be considered, a recent study reported no relationship between dietary antioxidant intake and the degree of sperm DNA fragmentation . A positive relationship between dietary antioxidant intake and better semen parameters has been reported, however . Unfortunately, comparison of individual studies is often difficult because many studies use combinations of different antioxidants and endpoints.
Vitamin C (ascorbic acid), a major antioxidant present in extracellular fluid, is present at a high concentration in seminal compared with blood plasma (364 versus 40 μM) and is present in detectable amounts in the sperm themselves. Vitamin C is known to be an effective scavenger of hydroxyl, superoxide, and hydrogen peroxide radicals. It also has been shown to recycle tocopherol (vitamin E) by repairing its tocopheroxyl radical, thereby allowing vitamin E to function as a free radical chain-breaking antioxidant . Vitamin C has been found in reduced quantity in the seminal plasma of infertile men . In a randomized controlled trial of 75 fertile, heavy smokers divided into placebo, 200-mg, and 1000-mg vitamin C supplementation group, both supplementation arms of the study showed a significant improvement in sperm concentration, morphology, and viability ( P < .01 and P < .001 for the 200- and 1000-mg groups, respectively) . Although an uncontrolled study reported an improvement in semen parameters of infertile men after treatment with vitamins C and E, a randomized controlled trial demonstrated no effect ( Table 1 ) .
| Study | Antioxidant | Study design | N | Study population | Dose and duration | Outcome |
|---|---|---|---|---|---|---|
| Greco et al | vitamin C, vitamin E | R/C/B | 64 | infertile men with elevated percentage of DNA-fragmented spermatozoa | 1 g vitamin C, 1 g vitamin E daily, 2 mo | no difference in semen parameters; significant reduction in percentage of DNA-fragmented spermatozoa |
| Greco et al | vitamin C, vitamin E | NR/NC/NB | 38 | men with an elevated percentage of DNA-fragmented spermatozoa with one failed ICSI attempt | 1 g vitamin C, 1 g vitamin E daily, 2 mo | no difference in cleavage or fertilization rates or embryo morphology; significant improvement in pregnancy and implantation rates |
| Keskes-Ammar et al | vitamin E and selenium | R/C/NB | 53 | infertile male volunteers | 400 mg vitamin E, 225 μg selenium daily, 3 mo | improved motility, decreased MDA concentration |
| Comhaire et al | vitamin E, vitamin A, acetyl-cysteine | NR/C/NB | 27 | infertile men | 600 mg acetylcysteine or 30 mg β-carotene, 180 mg vitamin E daily, 6 mo | decreased ROS, decreased 8-hydroxy-deoxyguanosine; no improvement in concentration, morphology, or motility |
| Rolf et al | vitamin C, vitamin E | R/C/B | 33 | asthenozoospermia | 1000 mg vitamin C and 80 mg vitamin E daily, 56 d | no improvement in concentration, motility, morphology, viability |
| Kodama et al | vitamin E, vitamin C, glutathione | NR/NC/NB | 36 | infertile men | 200 mg vitamin C, 200 mg vitamin E, 400 mg glutathione daily, 2 mo | improved concentration, decreased MDA concentration, decreased DNA damage; no improvement in morphology or motility |
| Geva et al | vitamin E | NR/NC/NB | 15 | ART patients | 200 mg/d, 3 mo | decreased MDA concentration; no improvement in morphology |
| Suleiman et al | vitamin E | R/C/B | 110 | asthenozoospermia | 300 mg/d, 6 mo | motility, decreased MDA concentration |
| Kessopoulou et al | vitamin E | R/C/B | 30 | infertile men with high ROS | 600 mg/d, 3 mo | improved zona-binding assay; no improvement in motility, concentration, morphology or ROS |
In a randomized, placebo-controlled trial, Greco and colleagues found that treatment of men with unexplained infertility associated with elevated sperm DNA fragmentation (≥ 15%) with oral vitamin C and E, led to decreased DNA fragmentation without a change in semen parameters. It is important to note that these studies did not choose patients based on abnormal ROS levels. An uncontrolled study of 38 men with an elevated percentage of fragmented spermatozoa (≥ 15%) and one prior failed intracytoplasmic sperm injection (ICSI) attempt who underwent oral supplementation with vitamins E and C demonstrated a significant improvement in pregnancy (48.2% versus 6.9%) and implantation (19.6% versus 2.2%) when compared with their prior ICSI attempt . No current randomized controlled trials show an improvement in the semen parameters or pregnancy rates of healthy infertile men who take oral supplementation of vitamin C.
Vitamin E (α-tocopherol), which is present within the cell membrane, is one of the major membrane protectants against ROS. It neutralizes hydrogen peroxide and protects the plasma membrane from lipid peroxidation. Suleiman and colleagues showed that oral administration of 300 mg/d of vitamin E in 52 asthenospermic patients significantly decreased malondialdehyde concentration (a measure of lipid peroxidation) in spermatozoa and improved sperm motility versus placebo. Furthermore, 11 of the 52 spouses in the treatment group became pregnant in the 6-month treatment period, but there was none in the placebo group. In a double-blind, randomized, placebo cross-over controlled trial, Kessopoulou and colleagues showed that oral administration of vitamin E (600 mg/d) in a population of 30 men with high levels of ROS in their semen (measured using the chemiluminescent method) improved sperm function as assessed by the zona pellucida binding assay. It should be noted that a randomized, placebo-controlled, double-blind study by Rolf and colleagues did not show an improvement in conventional semen parameters or 24-hour sperm survival rate in 32 patients with asthenozoospermia or moderate oligoasthenozoospermia treated with high-dose oral vitamins C and E for an 8-week period. Studies on oral vitamin C and vitamin E supplementation for treatment of male infertility are summarized in Table 1 . Although more well-designed studies (randomized controlled trials) examine the effects of oral vitamin E supplementation than any other antioxidant, the studies showed conflicting results when the endpoint was improvement in semen parameters ( Table 1 ) .
Selenium, a trace element, is necessary for the synthesis of glutathione peroxidase. Vitamin E has been closely linked to selenium metabolism, and it has been shown that vitamin E and selenium work synergistically as antiperoxidants. Keskes-Ammar and colleagues found that 225 μg/d of oral selenium in combination with 400 mg/d of oral vitamin E over a 3-month period significantly decreased malondialdehyde concentrations (a lipid peroxidation marker) in seminal plasma and improved sperm motility. These findings were not confirmed in another study, however . Few studies looking at the effects of selenium supplementation on male infertility, and the results of these studies are conflicting.
Carotenoids, such as beta-carotene and lycopene, are an important component of antioxidant defense . Beta-carotenes protect the plasma membrane against lipid peroxidation . Lycopene, which is found in tomatoes, has a suggested daily intake of 5 to 10 mg/d. Lycopene has been shown to be twice as potent as beta-carotene and ten times more potent than vitamin E in scavenging singlet oxygen and inhibiting lipid peroxidation in serum plasma . A recent study by Goyal and colleagues showed a significant increase in seminal plasma levels of lycopene after oral supplementation with a processed form of tomatoes (22.8 mg of lycopene) daily for a 2-week period. No net increase in the radical scavenging capacity of the oral lycopene-enriched seminal plasma was noted, however. Astaxanthin, a carotenoid extracted from the algae Hemaococcus pluvialis, is an antoxidant that is a much higher singlet molecular oxygen quencher than vitamin E. A small, double-blinded, randomized controlled trial of men with infertility who received 16 mg/d of Astaxanthin for 3 months showed significantly higher sperm linear velocity and total and per cycle pregnancy rates when compared with placebo . Of note, this study included patients who achieved pregnancies by intercourse whereas others followed IUI. Other treatments, including varicocele repair, anti-estrogens, and antibiotics were used in some patients. The number of studies investigating oral supplementation of carotenoids or lycopene in the setting of male infertility is sparse. More studies are needed to determine if carotenoid or lycopene supplementation significantly affects semen parameters in men who have infertility.
Glutathione is one of the most common antioxidants found in the body. It plays an important role in protecting lipids, proteins, and nucleic acids against oxidative damage. It combines with vitamin E and selenium to form glutathione peroxidase (the main enzyme involved in removing hydrogen peroxides in the epididymis). It is found at physiologically significant concentrations in seminal plasma and, although it cannot cross cell membranes, its concentration can be increased in biologic fluids by parenteral administration . In a placebo-controlled, double-blinded crossover trial, 600 mg glutathione was administered for 2 months by intramuscular injection in 20 infertile men. Glutathione therapy significantly increased sperm motility, particularly forward progression .
CoQ 10 , an energy-promoting agent, is concentrated in the mitochondria in the sperm midpiece. CoQ 10 recycles vitamin E and prevents its pro-oxidant activity . The reduced form of CoQ 10 , ubiquinol, also acts as an antioxidant preventing lipid peroxidation. CoQ 10 has been shown to inhibit hydrogen peroxide formation in the seminal fluid and seminal plasma of infertile men . In an in vitro study on semen samples of men with asthenozoospermia, incubation with 50 μM of CoQ 10 significantly increased sperm motility. In the same uncontrolled study, in vivo supplementation of 60 mg/d of oral CoQ 10 for a mean of 103 days in 17 infertile men improved their fertilization rate via ICSI without changing their semen parameters . An open, uncontrolled study of 22 men with idiopathic asthenozoospermia who were given 400 mg/d of oral CoQ 10 for 6 months also showed a significant improvement in forward motility compared with pretreatment results . The administration of oral CoQ 10 may be beneficial in the treatment of asthenozoospermia because of its role in mitochondrial respiratory chain and as an antioxidant; however, randomized controlled trials are needed to further substantiate the positive effect of oral CoQ 10 supplementation on sperm motility.
Zinc and copper are trace elements that constitute a part of the antioxidant enzyme superoxide dismutase. Adequate intake of these elements is important to maintain the function of these enzymes. The estimated average daily intake in the United States is 12.3 mg zinc and 900 μg of copper. Although both of these metals are important constituents of an antioxidant enzyme, high levels of these metals may catalyze reactions that lead to an increase in ROS. In an in vitro study on salmon sperm DNA, Lloyd and colleagues showed that at concentrations of 20 to 50 μM and more, these metal ions caused maximum DNA strand breaks. Currently, no studies area available in humans, and the in vivo dosage required for these concentrations in seminal plasma is still unknown.
Carnitine is a dietary antioxidant that decreases ROS by removing extracellular toxic acetyl-CoA that is responsible for mitochondrial ROS . Seventy-five percent of carnitine that is present in humans is derived from diet . The highest concentration of carnitine occurs in the epididymis, with epididymal concentrations approximately 2000-fold higher than in plasma . A multicenter, uncontrolled clinical trial by Costa and colleagues showed that oral administration of l -carnitine (3 g/d over a 4-month period) in 134 patients with asthenozoospermia led to a significant improvement in sperm motility, linearity index, rapid linear progression, and mean velocity. A placebo-controlled, double-blinded, randomized trial of the use of combined l -carnitine (2 g/d) and l -acetyl-carnitine (1 g/d) treatment in a 2-month period in 56 men with asthenozoospermia found no statistically significant improvement in sperm concentration or motility . A placebo-controlled, double-blinded cross-over trial of 86 infertile patients showed that l -carnitine supplementation (2 g/d over a 2-month period) led to a significant increase in motility (11% increase) versus placebo (8.8% increase), but only after the exclusion of 5 patients deemed outliers. With the inclusion of these 5 patients, no significant increase in motility was observed .
A smaller randomized, double-blinded, placebo-controlled trial by Sigman and colleagues reported that oral carnitine supplementation (2 g l -carnitine and 1 g l -acetyl-carnitine daily) in men with idiopathic asthenospermia demonstrated no clinically or statistically significant effect on sperm motility or total motile count. It should be noted that oral carnitine therapy has not been shown to increase seminal plasma or sperm carnitine or acetyl carnitine levels . Although earlier uncontrolled studies have shown an increase in sperm motility with carnitine supplementation, randomized controlled studies have not shown any consistent significant increase in sperm motility or count ( Table 2 ) .
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