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Oxidative Stress and Male Infertility: From Research Bench to Clinical Practice

Center for Advanced Research in Human Reproduction, Infertility, and Sexual Function, Urological Institute, The Cleveland Clinic Foundation, Cleveland, Ohio.

Correspondence to: Ashok Agarwal PhD HCLD, Director, Center for Advanced Research in Human Reproduction, Infertility and Sexual Function Urological Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Desk A 19.1, Cleveland, OH 44195 (e-mail: agarwaa{at}ccf.org)

Received for publication February 28, 2002; accepted for publication June 10, 2002.

In recent years, the generation of reactive oxygen species (ROS) in the male reproductive tract has become a real concern because of their potential toxic effects at high levels on sperm quality and function. ROS are highly reactive oxidizing agents belonging to the class of free radicals (Aitken, 1994). A free radical is defined as "any atom or molecule that possesses one or more unpaired electrons" (Warren et al, 1987). Recent reports have indicated that high levels of ROS are detected in semen samples of 25% to 40% of infertile men (de Lamirande et al, 1995; Padron et al, 1997). However, a strong body of evidence suggests that small amounts of ROS are necessary for spermatozoa to acquire fertilizing capabilities (Aitken, 1999).

Spermatozoa, like all cells living in aerobic conditions, constantly face the oxygen (O₂) paradox: O₂ is required to support life, but its metabolites such as ROS can modify cell functions, endanger cell survival, or both (de Lamirande and Gagnon, 1995). Hence, ROS must be continuously inactivated to keep only a small amount necessary to maintain normal cell function. It is not surprising that a battery of different antioxidants is available to protect spermatozoa against oxidants (Sies, 1993).

Seminal oxidative stress (OS) develops as a result of an imbalance between ROS generating and scavenging activities (Sikka et al, 1995; Sharma and Agarwal, 1996; Sikka, 2001). Spermatozoa are particularly susceptible to OS-induced damage because their plasma membranes contain large quantities of polyunsaturated fatty acids (PUFAs; Alvarez and Storey, 1995) and their cytoplasm contains low concentrations of scavenging enzymes (Jones et al, 1979; Aitken and Fisher, 1994; de Lamirande and Gagnon, 1995; Sharma and Agarwal, 1996). In addition, the intracellular antioxidant enzymes cannot protect the plasma membrane that surrounds the acrosome and the tail, forcing spermatozoa to supplement their limited intrinsic antioxidant defenses by depending on the protection afforded by the seminal plasma (Iwasaki and Gagnon 1992; Zini et al, 1993). Oxidative stress attacks not only the fluidity of the sperm plasma membrane, but also the integrity of DNA in the sperm nucleus (Aitken, 1999).

Even though OS has been established as a major factor in the pathogenesis of male infertility, there is a lack of consensus as to the clinical utility of seminal OS testing in an infertility clinic. One important reason for the inability to use an OS test in clinical practice may be the lack of a standard protocol to assess seminal OS. The role of OS in male infertility has been the focus of our research center at the Cleveland Clinic Foundation for the past 10 years. The main objective of our research in this area was to transfer this important knowledge from the research bench to clinical practice. We designed studies with the following aims: 1) to understand the precise mechanism by which OS develops in semen, which we thought could help develop strategies to overcome the problem, 2) to establish assays for accurate and reliable assessment of seminal OS, and 3) to identify the clinical significance of testing seminal OS in the male infertility practice. In this review, we summarize the efforts of our program to explore the role of OS in male infertility. We also include detailed information on the protocols introduced recently by our center for the assessment of seminal OS in a clinical andrology laboratory.

Reactive Oxygen Species and Sperm Physiology

Until recently, ROS were exclusively considered toxic to human spermatozoa. The idea that limited amounts of ROS can intervene in a physiological manner in the regulation of some sperm functions was first evoked in a study by Aitken et al (1989). The authors found that low levels of ROS can enhance the ability of human spermatozoa to bind with zonae pellucida, an effect that was reversed by adding vitamin E. Other studies have found that incubating spermatozoa with low concentrations of hydrogen peroxide (H₂O₂) stimulates sperm capacitation, hyperactivation, and the ability of the spermatozoa to undergo the acrosome reaction and oocyte fusion (de Lamirande and Gagnon, 1993; de Lamirande et al, 1993; Griveau et al, 1994; Aitken et al, 1995,Aitken et al, 1995; Kodoma et al, 1996; Aitken, 1997). ROS other than H₂O₂, such as nitric oxide and superoxide anion (O₂^•-), have also been shown to promote sperm capacitation and the acrosome reaction (Griveau et al, 1995a; Zini et al, 1996).

Mechanism of Antioxidant Protection in Semen

It is interesting that seminal plasma is well endowed with an array of antioxidant defense mechanisms to protect spermatozoa against OS (Sikka, 1996; Smith et al, 1996; Armstrong et al, 1998). These mechanisms compensate for the deficiency in cytoplasmic enzymes in sperm (Donnelly et al, 1999). Seminal plasma contains a number of enzymatic antioxidants such as superoxide dismutase (SOD; Alvarez et al, 1987), the glutathione peroxidase/glutathione reductase (GPX/GRD) system (Chaudiere et al, 1984), and catalase (Jeulin et al, 1989). In addition, seminal plasma contains a variety of nonenzymatic antioxidants such as ascorbate (Fraga et al, 1991), urate (Thiele et al, 1995), -tocopherol (Aitken and Clarkson, 1988; Moilanen et al, 1993), pyruvate (de Lamirande and Gagnon, 1992), glutathione (Lenzi et al, 1994), taurine, and hypotaurine (Alvarez and Storey, 1983).

It has been reported that seminal plasma from fertile men has a higher total antioxidant capacity (TAC) than seminal plasma from infertile men (Lewis et al, 1995). However, pathological levels of ROS detected in semen from infertile men are more likely a result of increased ROS production rather than reduced antioxidant capacity of the seminal plasma (Zini et al, 1993). Antioxidant defense mechanisms include three levels of protection: 1) prevention, 2) interception, and 3) repair (Sies, 1993).

     Prevention— Prevention of ROS formation is the first line of defense against oxidative insult. An example is the binding of metal ions, iron, and copper ions in particular, which prevents them from initiating a chain reaction (Sies, 1993). Chelation of transition metals is a major means of controlling sperm lipid peroxidation (LPO) and DNA damage. When transition metals become loosely bound to oxygen reduction products, they can produce secondary and more reactive oxidants, particularly the hydroxyl radical (OH^•) (Halliwell, 1990).

     Interception— Free radicals have a tendency toward chain reaction (ie, a compound carrying an unpaired electron will react with another compound to generate an unpaired electron, "radical begets radical"). Hence, the basic problem is to break this chain reaction by the formation of nonradical end products (Sies, 1993). Alphatocopherol (vitamin E), a chain-breaking antioxidant, inhibits LPO in membranes by scavenging peroxyl (RO^•) and alkoxyl (ROO^•) radicals. The ability of -tocopherol to maintain a steady state rate of peroxyl radical reduction in the plasma membrane depends on the recycling of -tocopherol by external reducing agents such as ascorbate or thiols (Wefers and Sies, 1988). In this way, -tocopherol is able to function again as a free radical chain-breaking antioxidant, even though its concentration is low (Buettner, 1993). For efficient interception, the radical to be intercepted must have a relatively long half-life (Sies, 1993). The peroxyl radicals are major reaction partners because their half-life extends into the range of seconds (7 seconds). In contrast, the hydroxyl radical, with its high reactivity and extremely short half-life (10^-9 seconds), cannot be intercepted with reasonable efficiency (Sies et al, 1992).

     Repair— In some situations, the damage caused by oxidants may be repaired. Unfortunately, spermatozoa are unable to repair the damage induced by OS because they lack the cytoplasmic enzyme systems that are required to accomplish this repair. This is one of the features that make spermatozoa unique in their susceptibility to oxidative insult (Alvarez et al, 1987; Aitken et al, 1989).

Pathogenesis of Seminal Oxidative Stress

The term oxidative stress is applied when oxidants out-number antioxidants (Sies, 1993), peroxidation products develop (Spitteler, 1993), and when these phenomena cause pathological effects (Janssen et al, 1993). Oxidative stress has been implicated in numerous disease states such as cancer, connective tissue disorders, aging, infection, inflammation, acquired immunodeficiency syndrome, and male infertility (Clark et al, 1986; Aitken et al, 1992, 1995, 1995).

In the context of human reproduction, a balance normally exists between ROS generation and scavenging in the male reproductive tract. As a result, only a minimal amount of ROS remains, which is needed to regulate normal sperm functions such as sperm capacitation, acrosome reaction, and sperm-oocyte fusion (Gagnon et al, 1991; de Lamirande and Gagnon, 1994; Griveau and Le Lannou, 1997). Excessive ROS production that exceeds critical levels can overwhelm all antioxidant defense strategies of spermatozoa and seminal plasma causing OS (Sikka et al, 1995; Sharma and Agarwal, 1996; de Lamirande et al, 1997; Sikka, 2001). All cellular components including lipids, proteins, nucleic acids, and sugars are potential targets for OS. The extent of OS-induced damage depends not only on the nature and amount of ROS involved but also on the moment and duration of ROS exposure and on extracellular factors such as temperature, oxygen tension, and the composition of the surrounding environment including ions, proteins, and ROS scavengers.

     Lipid Peroxidation of Sperm Plasma Membrane— Lipid peroxidation is broadly defined as "oxidative deterioration of PUFA" (ie, fatty acids that contain more than two carbon-carbon double bonds; Halliwell, 1984). The LPO cascade occurs in two fundamental stages: initiation and propagation. The hydroxyl radical (OH•) is a powerful initiator of LPO (Aitken and Fisher, 1994). Most membrane PUFAs have unconjugated double bonds that are separated by methylene groups. The presence of a double bond adjacent to a methylene group makes the methylene C-H bonds weaker and, therefore, hydrogen is more susceptible to abstraction. Once this abstraction has occurred, the radical produced is stabilized by the rearrangement of the double bonds, which forms a conjugated diene radical that can then be oxidized. This means that lipids, which contain many methylene-interrupted double bonds, are particularly susceptible to peroxidation (Blake et al, 1987). Conjugated dienes rapidly react with O₂ to form a lipid peroxyl radical (ROO•), which abstracts hydrogen atoms from other lipid molecules to form lipid hydroperoxides (ROOH). Lipid hydroperoxides are stable under physiological conditions until they contact transition metals such as iron or copper salts. These metals or their complexes cause lipid hydroperoxides to generate alkoxyl and peroxyl radicals, which then continue the chain reaction within the membrane and propagate the damage throughout the cell (Halliwell, 1984). Propagation of LPO depends on the antioxidant strategies employed by spermatozoa. One of the by-products of lipid peroxide decomposition is malondialdehyde. This by-product has been used in biochemical assays to monitor the degree of peroxidative damage in spermatozoa (Aitken et al, 1989; Aitken and Fisher, 1994). The results of such an assay exhibit an excellent correlation with the degree to which sperm function is impaired in terms of motility and the capacity for sperm-oocyte fusion (Aitken et al, 1993; Sidhu et al, 1998).

     Impairment of Sperm Motility— The increased formation of ROS has been correlated with a reduction of sperm motility Aitken et al, 1989; Iwasaki and Gagnon, 1992; Lenzi et al, 1993; Agarwal et al, 1994a; Armstrong et al, 1999). The link between ROS and reduced motility may be due to a cascade of events that result in a decrease in axonemal protein phosphorylation and sperm immobilization, both of which are associated with a reduction in membrane fluidity that is necessary for sperm-oocyte fusion (de Lamirande and Gagnon, 1995). Another hypothesis is that H₂O₂ can diffuse across the membranes into the cells and inhibit the activity of some enzymes such as glucose-6-phosphate-dehydrogenase (G₆PD). This enzyme controls the rate of glucose flux through the hexose monophosphate shunt, which in turn, controls the intracellular availability of nicotinamide adenine dinucleotide phosphate (NADPH). This in turn is used as a source of electrons by spermatozoa to fuel the generation of ROS by an enzyme system known as NADPH oxidase (Aitken et al, 1997). Inhibition of G₆PD leads to a decrease in the availability of NADPH and a concomitant accumulation of oxidized glutathione and reduced glutathione. This can reduce the antioxidant defenses of the spermatozoa and increase peroxidation of membrane phospholipids (Griveau et al, 1995a).

     Oxidative Stress-Induced DNA Damage— Two factors protect sperm DNA from oxidative insult: the characteristic tight packaging of the DNA and the antioxidants present in seminal plasma (Twigg et al, 1998a). Studies in which the sperm was exposed to artificially produced ROS resulted in a significant increase in DNA damage in the form of modification of all bases, production of base-free sites, deletions, frame shifts, DNA cross-links, and chromosomal rearrangements (Duru et al, 2000). Oxidative stress has also been correlated with high frequencies of single and double DNA strand breaks (Twigg et al, 1998a; Aitken and Krausz, 2001).

Strong evidence suggests that high levels of ROS mediate the DNA fragmentation commonly observed in spermatozoa of infertile men (Kodama et al, 1997; Sun et al, 1997). This information has important clinical implications, particularly in the context of assisted reproductive techniques (ART). Spermatozoa selected for ART most likely originate from an environment experiencing OS, and a high percentage of these sperm may have damaged DNA (Kodama et al, 1997; Lopes et al, 1998). There is a substantial risk that spermatozoa carrying damaged DNA are being used clinically in this form of therapy (Twigg et al, 1998b). When intrauterine insemination or in vitro fertilization is used, such damage may not be a cause of concern because the collateral peroxidative damage to the sperm plasma membrane ensures that fertilization cannot occur with a DNA-damaged sperm. However, when intracytoplasmic sperm injection (ICSI) is used, this natural selection barrier is bypassed and a spermatozoon with damaged DNA may be directly injected into the oocyte (Twigg et al, 1998b; Aitken, 1999).

Sources of Excessive Reactive Oxygen Species Production in Semen

Morphologically abnormal spermatozoa and seminal leukocytes have been established as the main sources of high ROS production in human ejaculates (Aitken and West, 1990; Kessopolou et al, 1992). Virtually every human ejaculate is contaminated with potential sources of ROS (Aitken, 1995). It follows that some sperm cells will incur oxidative damage and a concomitant loss of function in every ejaculate. Thus, the impact of OS on male fertility is a question of degree rather than the presence or absence of pathology.

Clear evidence suggests that human spermatozoa produce oxidants (Aitken et al, 1992; Hendin et al, 1999; Gil-Guzman et al, 2001). Spermatozoa may generate ROS in two ways: 1) the NADPH oxidase system at the level of the sperm plasma membrane (Aitken et al, 1992), and 2) the NADH-dependent oxido-reductase (diphorase) at the level of mitochondria (Gavella and Lipovac, 1992). The mitochondrial system is the major source of ROS in spermatozoa in infertile men (Plante et al, 1994).

     Effect of Sperm Morphology on ROS Production— Gomez et al (1998) have indicated that levels of ROS production by pure sperm populations were negatively correlated with the quality of sperm in the original semen. The link between poor semen quality and increased ROS generation lies in the presence of excess residual cytoplasm (cytoplasmic droplet). When spermatogenesis is impaired, the cytoplasmic extrusion mechanisms are defective, and spermatozoa are released from the germinal epithelium carrying surplus residual cytoplasm. Under these circumstances, the spermatozoa that are released during spermiation are believed to be immature and functionally defective (Huszar et al, 1997). Retention of residual cytoplasm by spermatozoa is positively correlated with ROS generation via mechanisms that may be mediated by the cytosolic enzyme G₆PD (Figure 1) (Aitken, 1999).

View larger version (23K): [in this window] [in a new window]   Figure 1. Mechanism of increased production of ROS by abnormal spermatozoa (spermatozoa with cytoplasmic retention).

Recent studies by Ollero et al (2001) and Gil-Guzman et al (2001) have shown that levels of ROS production in semen were negatively correlated with the percentage of normal sperm forms as determined by World Health Organization (WHO, 1999) classification and by Kruger's strict criteria (Kruger et al, 1987). The correlation of seminal ROS levels with morphologically abnormal sperm was also evident when morphology slides were scored using a sperm deformity index (SDI) (r = .31; P = .01; (unpublished data). The SDI was introduced by Aziz et al (1996) as a novel expression of sperm morphological abnormalities that were found to be highly correlated with fertilization in vitro. This new method for sperm morphology assessment uses a multiple entry scoring technique in which an abnormal sperm is classified more than once if more than one deformity is observed. SDI is calculated by dividing the total number of deformities observed by the number of sperm randomly selected and evaluated, irrespective of their morphological normality. In our study, SDI was significantly higher in a group of infertile men who had high levels of seminal ROS than in a group of infertile men with low ROS and in a group of fertile sperm donors (Table 1). The link between seminal ROS and high SDI may be causal and related to the greater capacity of morphologically abnormal spermatozoa to produce ROS.

     Effect of Sperm Preparation Techniques on ROS Production— Studies from our center have indicated that human spermatozoa significantly increase levels of ROS production in response to repeated cycles of centrifugation involved in the conventional sperm preparation techniques used for ART (Agarwal et al, 1994a). The duration of centrifugation was found to be more important than the force of centrifugation for inducing ROS formation by human spermatozoa (Shekarriz et al, 1995b). These data are significant because exposing spermatozoa to high levels of ROS may cause DNA fragmentation, which can have adverse consequences if they are used for ART, particularly ICSI (Lopes et al, 1998). This may be true particularly in light of a recent report by Zini and coworkers (2000) who found that improvement in sperm motility after Percoll processing was not associated with a similar improvement in sperm DNA integrity. The authors recommended that the current sperm preparation techniques be reevaluated with the goal of minimizing sperm DNA damage. In a recent study in our center, the recovery of sperm with intact nuclear DNA was found to be significantly higher after the swim-up technique than after the ISolate gradient technique (unpublished data).

Our group has also reported that ROS production by human sperm increases with sperm concentration and decreases with time (Shekarriz et al, 1995b, c). Results from our recent studies indicated that ROS production significantly varies in subsets of human spermatozoa at different stages of maturation (Ollero et al, 2001; Gil-Guzman et al, 2001). Following ISolate gradient (Irvine Scientific, Santa Ana, Calif) fractionation of ejaculated sperm, ROS production was found to be highest in immature sperm with abnormal head morphology and cytoplasmic retention and lowest in mature sperm and immature germ cells. The relative proportion of ROS-producing immature sperm was directly correlated with nuclear DNA damage values in mature sperm and inversely correlated with the recovery of motile, mature sperm. These interesting findings led to the hypothesis that oxidative damage of mature sperm by ROS-producing immature sperm during their comigration from seminiferous tubules to the epididymis may be an important cause of male infertility. If this is the case, perhaps interventions directed to 1) increase antioxidant levels in immature germ cell membranes during spermatogenesis, and 2) isolate spermatozoa with intact DNA by in vitro separation techniques should be of particular benefit to these patients in whom a defect in the normal regulation of spermiogenesis leads to an abnormal increase in the production of ROS-producing immature sperm.

     Effect of Temperature on ROS Production— Preliminary data from a recent unpublished study in our center indicate that levels of ROS production in semen may vary according to the temperature at which semen is incubated. For the purpose of this study, semen samples were collected from a nonselected group of infertility patients (n = 12) and from a group of healthy sperm donors (n = 12) after 2 to 3 days of sexual abstinence. We examined levels of ROS in washed sperm suspensions immediately after liquefaction (basal ROS) and compared the results with ROS levels in aliquots of the same samples after 1-hour incubation at 3 different temperatures (4°C, 25°C, and 37°C). Our results indicate that ROS levels are significantly lower in aliquots of semen incubated at 37°C for 1 hour compared to aliquots of the same samples incubated at 4°C and 25°C (Table 2). Sperm motion kinetics (curvilinear velocity, straight-line velocity, average path velocity, and amplitude of lateral head displacement) as determined by a computer-assisted semen analyzer (CASA) were significantly higher in samples incubated at 37°C than at 4°C (P = < .001, < .001, < .001, and < .001, respectively) although not significantly different from samples incubated at 25°C (P = .69, .51, .95, and .79, respectively). Therefore, it may be speculated that 37°C could be the optimum temperature at which semen can be collected and processed for different diagnostic and therapeutic purposes. Handling semen samples at 37°C may help avoid deleterious effects of ROS on human spermatozoa.

     Reactive Oxygen Species Production by Seminal Leukocytes— With respect to all nonsperm cells, the majority of the so-called "round cells" consist of immature germ cells with less than 5% leukocytes under normal conditions (Eggert-Kruse et al, 1992). Peroxidase-positive leukocytes were found to be the major source of high ROS production in semen (Tomlinson et al, 1993; Aitken et al, 1995,Aitken et al, 1995; Rajasekaran et al, 1995; Shekarriz et al, 1995a; Ochsendorf, 1999). Peroxidase-positive leukocytes include polymorphonuclear (PMN) leukocytes, which represent 50% to 60% of all seminal leukocytes and macrophages, which represent another 20% to 30% (Wolff and Anderson, 1988; Fedder et al, 1993; Thomas, et al, 1997). Peroxidase-positive leukocytes in semen are contributed largely by the prostate and the seminal vesicles (Wolff, 1995).

Activated leukocytes are capable of producing 100-fold higher amounts of ROS than nonactivated leukocytes (Plante et al, 1994). Leukocytes may be activated in response to a variety of stimuli including inflammation and infection (Pasqualotto et al, 2000a). Activated leukocytes increase NADPH production via the hexose monophosphate shunt. The myeloperoxidase system of both PMN leukocytes and macrophages is also activated, which leads to respiratory burst and production of high levels of ROS (Blake et al, 1987). Such an oxidative burst is an early and effective defense mechanism in cases of infection for killing the microbes (Saran et al, 1999).

Sperm damage from ROS that is produced by leukocytes occurs if seminal leukocyte concentrations are abnormally high (ie, leukocytospermia; Shekarriz et al, 1995a), if the patient has epididymitis, or if seminal plasma was removed during sperm preparation for assisted reproduction (Ochsendorf, 1999). However, Sharma et al (2001) observed that seminal leukocytes may cause OS even at concentrations below the WHO cutoff (ie, <1 x 10⁶ peroxidase positive leukocytes/mL semen) value for leukocytospermia. This may be due to the fact that seminal plasma contains large amounts of ROS scavengers but confers a variable (10% to 100%) protection against ROS generated by leukocytes (Kovalski et al, 1992).

Recent yet unpublished data from our center indicated that levels of ROS production by pure sperm suspensions from infertile men with a laboratory diagnosis of leukocytospermia were significantly higher than those in infertile men without leukocytospermia (Table 3). This observation led to the postulation that seminal leukocytes play a role in enhancing sperm capacity for excessive ROS production either by direct sperm-leukocyte contact or by soluble products released by the leukocytes. Studies are currently underway to investigate this hypothesis. However, this new finding may have significant implications for the fertility potential of sperm both in vivo and in vitro. Excessive production of ROS by sperm in patients with leukocytospermia implies that both the free-radical generating sperm themselves and any normal sperm in the immediate vicinity will be susceptible to oxidative damage. Furthermore, once the process of LPO is initiated, the self-propagating nature of this process ensures a progressive spread of the damage throughout the sperm population.

Assessment of Seminal Oxidative Stress in the Andrology Laboratory

Extensive research in the field of male infertility has been conducted to develop adequate indices of OS that would help determine, with accuracy, whether OS is a significant contributor to male infertility (Sharma et al, 1999). Levels of OS vary greatly in infertile men (Alvarez et al, 1987). Because OS is an imbalance between levels of ROS production and antioxidant protection in semen, it is conceivable that assessment of OS will rely on the measurement of ROS as well as TAC of semen.

     Protocol for Measurement of ROS by Chemiluminescence Assay— Levels of ROS can be measured in washed sperm suspensions using a chemiluminescence assay (Kobayashi et al, 2001; Figure 2). With this protocol, liquefied semen is centrifuged at 300 x g for 7 minutes, and the seminal plasma is separated and stored at -80°C for measurement of TAC. The pellet is washed with phosphate-buffered saline (PBS) and resuspended in the same washing media at a concentration of 20 x 10⁶ sperm/mL. Four-hundred-microliter aliquots of the resulting cell suspensions (containing sperm and leukocytes) are used to assess basal ROS levels. Eight microliters of horseradish peroxidase (HRP) (12.4 units of HRP Type VI, 310 U/mg; Sigma Chemical Company, St Louis, Mo) are added to the cell suspension. The HRP sensitizes the assay so that it measures the extracellular H₂O₂. Ten microliters of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione; Sigma), prepared as 5 mM stock in dimethyl sulfoxide (DMSO), are added to the mixture and serve as a probe.

View larger version (41K): [in this window] [in a new window]   Figure 2. Measurement of ROS in washed sperm suspensions (containing spermatozoa and seminal leukocytes) by chemiluminescence assay.

Luminol is an extremely sensitive oxidizable substrate that has the capacity to react with a variety of ROS at neutral pH. The reaction of luminol with ROS results in production of a light signal that is then converted to electrical signal (photon) by a luminometer. A negative control is prepared by adding 10 µL of 5 mM luminol to 400 µL of PBS. Levels of ROS are assessed by measuring the luminol-dependant chemiluminescence with the luminometer (model LKB 953; Wallac Inc, Gaithersburg, MD) in the integrated mode for 15 minutes. The results are expressed as x10⁶ counted photons per minute (cpm) per 20 x 10⁶ sperm. Normal ROS levels in washed sperm suspensions range from 0.10 to 1.0 x 10⁶ cpm per 20 x 10⁶ sperm.

     Protocol for Measurement of Total Antioxidant Capacity in Semen by Enhanced Chemiluminescence Assay— Total antioxidant capacity in the seminal plasma can be measured using an enhanced chemiluminescence assay (Kolettis et al, 1999). Frozen samples of seminal plasma are thawed at room temperature and immediately assessed for TAC. Seminal plasma is diluted 1:20 with deionized water (dH₂O) and filtered through a 0.20-µm filter (Allegiance Healthcare Corporation, McGaw Park, Ill). Signal reagent is prepared by adding 30 µL of H₂O₂ (8.8 molar/L), 10 µL of para-iodophenol stock solution (41.72 µM), and 110 µL of luminol stock solution (3.1 mM) to 10 mL of Tris buffer (0.1 M pH 8.0). Horseradish peroxidase working solution is prepared from the HRP stock solution by making a dilution of 1:1 of dH₂O. Light emission occurs when the chemiluminescent substrate luminol is oxidized by H₂O₂ in a reaction catalyzed by HRP. HRP catalyzes the reaction between a hydrogen acceptor (oxidant) and a hydrogen donor. Under normal circumstances, this reaction produces low-intensity light emission that may decay rapidly. The characteristics of the reaction can be altered substantially by the addition of para-iodophenol as an enhancer that gives a more intense, prolonged, and stable light emission. The continuous light output depends on the constant production of free radical intermediates derived from para-iodophenol (enhancer), luminol (substrate), and H₂O₂ (oxidizer) in the presence of HRP.

Trolox (6-hydroxyl-2,5,7,8-tetramethylchroman-2-carboxylic acid), a water-soluble tocopherol analogue, is prepared as a standard solution (25, 50, and 75 µM) for TAC calibration. With the luminometer in the kinetic mode, 100 µL of signal reagent and 100 µL of HRP working solution are added to 700 µL of dH₂O and mixed. The mixture is equilibrated to the desired level of chemiluminescence output (between 2.8 and 3.2 x 10⁷ cpm) for 100 seconds. One hundred microliters of the standard Trolox solution is immediately added to the mixture, and the chemiluminescence is measured. Suppression of luminescence and the time from the addition of standard Trolox solution to 10% recovery of the initial chemiluminescence are recorded. Plotting the three concentrations of Trolox solution versus 10% recovery time results in a linear equation (Figure 3).

View larger version (15K): [in this window] [in a new window]   Figure 3. Linear relationship between the concentrations of standard Trolox solutions (25 µM, 50 µM, and 75 µM) and 10% recovery time (in seconds) of the initial chemiluminescence.

The same steps are repeated after the Trolox solutions are replaced with 100-µL aliquots of the prepared seminal plasma. The assay is conducted in a dark room because light affects the chemiluminescence. Seminal TAC levels are calculated using the following equation:

In this equation, M refers to the slope increase in the value of Trolox equivalent for 1-second increase of the recovery time, whereas C accounts for the daily background variability. The results are multiplied by the dilution (d) factor and expressed as molar Trolox equivalents (Sharma et al, 1999).

     Quality Control of Oxidative Stress Indices (ROS and TAC)— It was of utmost importance to standardize the measures that we used as indices for OS, including measurement of ROS in washed sperm suspensions and TAC in seminal plasma. We have demonstrated that the luminol-dependent chemiluminescence assay for ROS measurement in washed sperm suspensions is both accurate and reliable when the sperm concentration is greater than 1 x 10⁶/mL and when the samples are analyzed within 1 hour after collection (Kobayashi et al, 2000). Similarly, assessment of TAC in seminal plasma using the enhanced chemiluminescence assay was found to be both accurate and reliable, with very low intraassay, interassay, and interobserver variations (P .8) (unpublished data). The intraassay reliability was 91% [coefficient of variation (CV) = 5%], the interobserver reliability was 89% (CV = 13%), and interassay reliability was 92% (CV = 13%).

     ROS-TAC Score: An Accurate Method for Assessment of Seminal Oxidative Stress— The fact that neither ROS alone nor TAC alone can adequately quantify seminal OS led to the logical conclusion that combining these two variables may be a better index for the diagnosis of overall OS affecting spermatozoa. This conclusion led to the introduction of the ROS-TAC score as a new method for assessment of OS status in infertile men (Sharma et al, 1999). The new ROS-TAC score is a statistical formula derived from levels of ROS in washed sperm suspensions and TAC in seminal plasma using principal component analysis. The resulting score minimizes the variability in the individual parameters of OS (ROS alone or TAC alone). The ROS-TAC score is an accurate measure of seminal OS and low ROS-TAC scores indicate high seminal OS (Sharma et al, 1999). A cutoff value of 30 was determined as the lower limit of normal range for the ROS-TAC score, and individuals with scores below this cutoff value were found to be at particular risk for prolonged inability to initiate pregnancies.

     Assessment of ROS in Whole (Unwashed) Ejaculates: A Reliable Alternative for ROS-TAC Score— Our group has recently introduced an additional test of OS in which ROS levels are directly measured in the whole (unwashed) ejaculate following liquefaction (unpublished data). Semen samples in our study were collected from 34 infertile men and from 9 normal, healthy donors. Donors were selected on the basis of normal genital examination and normal standard semen parameters according to WHO (1999) guidelines. All semen specimens were produced by masturbation after the patients had abstained from sex for a period of 48 to 72 hours. Eight microliters of HRP were added to 400 µL-aliquots of liquefied semen. Levels of ROS were measured by the chemiluminescence assay using luminol (10 µL) as the probe with the luminometer set in the integrated mode for 15 minutes. The results were expressed as x10⁴ cpm per 20 x 10⁶ sperm. In addition, levels of ROS in the washed sperm suspensions and TAC in seminal plasma were measured in aliquots from the same samples in order to calculate the ROS-TAC scores.

Our results indicated that levels of ROS (median [25th and 75th percentiles]) in the whole ejaculate of the normal donors (x10⁴ cpm/20 million sperm/mL) were significantly lower than in infertile men (0.3 [0.2, 0.9] vs. 5.8 [1.0, 81.0] x 10⁴ cpm/20 million sperm/mL; P = .004). The maximum level of ROS in the whole ejaculate of normal donors was 1.5 x 10⁴ cpm/20 million sperm/mL. Sixty-eight percent (23 of 34) of infertile men had ROS levels >1.5 x 10⁴ cpm/20 million sperm/mL and were classified as OS-positive. The remaining 32% (11of 34) of infertile men had ROS levels 1.5 x 10⁴ cpm/20 million sperm/mL and were accordingly classified as OS-negative.

Leukocytospermia (concentrations greater than 1 x 10⁶ leukocytes/mL of semen) was found in 9% (1 of 11) of OS-negative samples and in 39% (9 of 23) of OS-positive samples. It is interesting that ROS-TAC scores were significantly higher (ie, lower OS, [49 {47, 52}] in the group classified as OS-negative based on ROS levels in whole [unwashed] ejaculates than the OS-positive group [36 {32, 44}], P = .001). The OS-negative group scores were not significantly different from the normal controls (53 [51, 55], P = 0.4). In addition, levels of ROS in whole (unwashed) ejaculate were correlated positively with levels of ROS in washed sperm suspensions and negatively with ROS-TAC scores (Figure 4).

View larger version (13K): [in this window] [in a new window]   Figure 4. Correlation of ROS levels in whole (unwashed) ejaculate with (A) levels of ROS in washed sperm suspensions (following cycles of simple wash and resuspension in PBS) (r = .87, P < .001), and (B) the statistical formula, reactive oxygen species—total antioxidant capacity score (r = -.75, P < .001) derived from levels of ROS in washed sperm suspensions and antioxidants in seminal plasma.

ROS levels in whole ejaculates were correlated negatively with sperm concentration (r = -.52, P = .0003), motility (r = -.41, P = .006) and morphology by the WHO method (r = -.34, P = .02), and positively with seminal leukocyte concentrations (r = .65, P < .0001). Furthermore, quality control studies of assessment of ROS in whole ejaculates indicated low variability (intraassay [CV = 17, with 99% reliability] and interassay [CV = 18, with 96% reliability]). Therefore, this test can be used as an alternative to ROS-TAC score for accurate and reliable assessment of seminal OS in the andrology laboratory. The strong positive correlation between levels of ROS in whole ejaculates and seminal leukocyte concentrations suggests that treatment of OS-positive patients should include plans for effective control of excessive leukocyte infiltration, antioxidant supplementation, or both.

     Nitroblue Tetrazolium Test for Identification of ROS-Producing Cells in Semen— It is important to determine the source of ROS in a given semen sample because the clinical implications of infiltrating leukocytes are quite different from those of pathological conditions in which spermatozoa themselves are the source of ROS (Aitken, 1994). The luminol-dependent chemiluminescence assay that was described above helps assess levels of seminal OS by measuring the total amount of ROS in semen using a luminometer. This assay, however, does not provide information on the differential contribution of spermatozoa and leukocytes to ROS production in semen or on the state of activation of individual cells. Nitroblue tetrazolium (NBT) is a yellow, water-soluble dye that can be reduced by accepting electrons in the presence of free oxygen radicals to form a blue-black water-insoluble compound known as formazan (Baehner et al, 1967). The cytoplasmic NADH, which is produced by oxidation of glucose through the hexose monophosphate shunt, serves as an electron donor. The oxidase systems available in the cytoplasm help transfer electrons from NADH to NBT and reduce NBT into formazan (Baehner et al, 1976).

Thus, the NBT reaction indirectly reflects the ROS generating activity in the cytoplasm of cells and can help determine the cellular origin of ROS in a suspension containing a variety of cells such as semen. Earlier studies have shown that NBT reduction and formazan deposition in blood neutrophils are related to their phagocytic activity (Park et al, 1968; Segal and Levi, 1973). In the same studies, the state of activation of neutrophils was determined by scoring the blue-black formazan granules deposited in the cytoplasm. In a recent prospective study (unpublished data), we assessed ROS generating activity in semen from a group of infertile men using the NBT reduction test and also by the chemiluminescence assay. Nitroblue tetrazolium (0.1%) was prepared by adding 10 mg of NBT powder to 100 mL of PBS (pH 7.2) and stirring at room temperature for 1 hour. The NBT solution was filtered using a 0.2-µm filter. Equal volumes of NBT solution (0.1%) and unwashed semen were incubated for 30 minutes at 37°C. After another 30 minutes of incubation at room temperature, the mixture was centrifuged at 250 x g for 5 minutes. Smears were prepared from the pellet, air-dried, and stained with Wright stain. A total of 100 spermatozoa and 100 neutrophils were scored using 100x magnification. Leukocytes were scored as follows: cells filled with formazan (+++), intermediate density (++), scattered or few formazan granules (+), and no detectable formazan (-). Spermatozoa were scored as follows: formazan occupying 50% of cytoplasm (+) and >50% of cytoplasm (++).

The most important finding from our study was a significant increase in the percentage of NBT-positive leukocytes in semen from leukocytospermic samples (70%) as compared with nonleukocytospermic (14.5%) and donor samples (7%) (P = .02 and 0.03, respectively). In addition, a strong positive correlation was found between levels of ROS, as measured by chemiluminescence, and the percentage of NBT-positive neutrophils (r = .7; P < .0001) and sperm with cytoplasmic retention (r = .72; P < .0001). Furthermore, the results of the NBT test were inversely correlated with ROS-TAC scores. These results indicate that the NBT reduction test can help assess the differential contribution of leukocytes and defective spermatozoa to the amount of ROS generation in semen. The NBT test is easily performed and can be used as a routine andrology laboratory procedure to assess seminal OS without the need for expensive equipment such as the luminometer.

Clinical Significance of Assessment of Seminal Oxidative Stress

Currently, no single laboratory test can accurately and precisely assess a man's total fertility (Evenson et al, 1999). The entire purpose of performing a diagnostic test in the evaluation of the male partner of a suspected infertile couple is to learn whether or not his fertility is impaired (Matson, 1997). Therefore, any test must then have a threshold above and below which it will provide discrimination and predictive capabilities with little overlap between fertile and infertile men. Determining which tests are valid in the evaluation of male infertility is difficult for a number of reasons. First, the term "male infertility" does not constitute a defined clinical syndrome but rather a collection of disparate conditions with a variety of causes and prognoses (Aitken et al, 1995,Aitken et al, 1995). In addition, various clinical diagnoses are unable to determine the underlying cause of sperm dysfunction and pathophysiology of infertility. Relatively few men will have conditions causing absolute sterility such as azoospermia. Rather, the majority of infertile men have abnormal semen quality of varying severity and poorly understood etiology. Until the causes of male infertility are better understood, it is unlikely that any given descriptive test of sperm quality or sperm function will predict with absolute certainty that a man will be fertile or infertile in a given time period (Van Voorhis and Sparks, 1999).

The second reason that testing a man's fertility is difficult to do is that confounding factors are always present. Tests of semen are confounded by intrasubject variability in the quality of semen samples. Semen quality can vary a great deal in the same individual due to factors such as days of abstinence from ejaculation, febrile illness, stress, and even problems with collection of the sample. Another important factor is the female partner's relative fertility, which can vary tremendously (Van Voorhis and Sparks, 1999).

With these facts in mind, the ultimate goal of our extensive research in the area of OS was to reach a consensus as to the clinical significance of seminal OS testing in an infertility clinic. The first step toward reaching such a consensus was to standardize the protocols for assessing seminal OS indices (ROS and TAC). Next, we developed methods for accurately assessing seminal OS by calculating the ROS-TAC scores or measuring ROS levels in unwashed semen. These important initial steps helped our group to determine levels of seminal OS in different clinical settings (Table 4). We found that levels of ROS were significantly higher in men with spinal cord injury, which were associated with poor sperm motility and morphology (Padron et al, 1997). We also found elevated levels of ROS in infertile men with varicoceles (Hendin et al, 1999). In a recent study, varicocelectomy was associated with a significant increase in pregnancy and live birth rates for couples who underwent intrauterine insemination, although standard semen parameters were not improved in all patients (Daitch et al, 2001). This observation led to our speculation that an improvement in pregnancy rates following varicocelectomy may be due to a functional factor not tested during standard semen analysis such as seminal OS. Patients with varicocele also had low levels of TAC in their seminal plasma and, therefore, may benefit from antioxidant supplementation (Hendin et al, 1999). An earlier study on rats has indicated that free radical scavengers such as SOD can prevent free radical-mediated testicular damage (Agarwal et al, 1997).

Across all clinical diagnoses, the ROS-TAC score was a superior discriminator between fertile and infertile men than either ROS or TAC alone (Sharma et al, 1999). The average ROS-TAC scores for fertile men who underwent vasectomy reversal was nearly identical to those of normal fertile donors (Kolettis et al, 1999). Infertile men with an idiopathic or a male-factor diagnosis had significantly lower ROS-TAC scores compared to normal controls (Pasqualotto et al, 2000a; 2001). In addition, patients with a male-factor diagnosis who eventually initiated a successful pregnancy had significantly higher ROS-TAC scores than those who did not (Pasqualotto et al, 2001). Our results also indicated that infertile men with a diagnosis of chronic prostatitis or prostatodynia have significantly lower ROS-TAC scores than controls, irrespective of their leukocytospermia status (Pasqualotto et al, 2000a).

In a recent study (unpublished data), cigarette smoking in infertile men who had a normal genital examination was significantly correlated with higher levels of seminal OS, as evidenced by lower ROS-TAC scores (Table 5). Lower ROS-TAC scores in infertile smokers may be due to the significant increase in seminal ROS levels and decrease in TAC levels. The link between cigarette smoking and high seminal ROS may be at least in part related to the significant increase in leukocyte concentrations observed in semen of infertile smokers in the same study. An earlier study also reported an association between cigarette smoking in infertile men and greater leukocyte infiltration into semen (Close et al, 1990). The precise mechanism or mechanisms of greater seminal leukocyte infiltration into semen of infertile smokers is not clear. One potential explanation is that smoking metabolites may induce an inflammatory reaction in the male genital tract with a subsequent release of chemical mediators of inflammation. Inflammatory mediators such as interleukin (IL)-6 and IL-8 can recruit and activate leukocytes (Comhaire et al, 1999). In turn, activated leukocytes can generate high levels of ROS in semen, which may overwhelm the antioxidant strategies, resulting in OS (Aitken et al, 1995,Aitken et al, 1995).

Another theory is that toxic metabolites of cigarette smoke may impair spermatogenesis, and leukocytes infiltrate the male reproductive tract to eliminate defective spermatozoa by phagocytosis (Tomlinson et al, 1992). Blood and seminal plasma cotinine levels in fertile (n = 107) and infertile (n = 103) men who smoke were correlated with the number of cigarettes smoked per day (Wong et al, 2000). A statistically significant correlation was found between cotinine concentrations in seminal plasma and the percentage of abnormal sperm morphology. Chia et al (1994) examined the relationship between cigarette smoking, and blood and seminal plasma concentrations of cadmium, and sperm quality in 184 men who were undergoing initial screening for infertility. They found that levels of cadmium in blood and seminal plasma were negatively correlated with semen volume and sperm concentrations.

An additional factor that may explain why semen from smokers had higher levels of ROS may be that cigarette smoke itself contains high levels of ROS such as superoxide anion, H₂O₂, and hydroxyl radicals (Stone and Bermudes, 1986; Church and Pryor, 1990). The finding of higher levels of seminal OS in association with cigarette smoking is of significance and may have important implications in the fertilizing potential of infertile men.

Strategies to Reduce Seminal Oxidative Stress

Reducing levels of seminal OS is of great importance for natural and assisted fertility. Long-term strategies must determine the cause of the enhanced ROS generation by spermatozoa in infertile men. An insight into the molecular basis of defective spermatogenesis is vital in order to identify the underlying etiology of sperm pathology. If the error in spermatogenesis that leads to such atypical activity of excessive ROS production could be defined, it would provide an important lead in determining the etiology of male infertility and helping researchers develop appropriate therapeutic strategies.

The seminal leukocyte population should also be considered potentially detrimental and carefully monitored. It is important to minimize the interaction between ROS-producing cells in semen (eg, PMN leukocytes) and spermatozoa that have a potential to fertilize. This may help protect spermatozoa from the detrimental effects of high levels of ROS and enhance their capacity to fertilize. It is also important for clinicians to know that sperm preparation techniques used for ART, which include repeated cycles of centrifugation, may induce ROS generation by spermatozoa. This may expose spermatozoa to OS-induced damage, particularly after the antioxidant-rich seminal plasma is removed.

Summary

Production of very low amounts of ROS in semen appears to play a physiological role in regulating normal sperm functions, whereas high levels of ROS endanger sperm function and viability. Oxidative stress due to excessive production of ROS, impaired antioxidant defense mechanisms, or both precipitates a range of pathologies that are currently believed to negatively affect the male reproductive function. Oxidative stress-induced damage to sperm may be mediated by lipid peroxidation of the sperm plasma membrane, reduction of sperm motility, and damage to the DNA in the sperm nucleus. Despite the established role of OS in the pathogenesis of male infertility, there is a lack of consensus as to the clinical utility of seminal OS testing in an infertility clinic. One important reason for the inability to utilize the OS test in clinical practice is related to the lack of a standard protocol for assessment of seminal OS.

Introduction of ROS-TAC score as an accurate measure of seminal OS by our program has helped in our effort to translate the important information gained on OS from the research bench to the clinical practice of andrology. In addition, results of our recent studies indicate the availability of alternate protocols for assessment of seminal OS by measuring ROS directly in the whole (unwashed) ejaculate or assessing the differential contribution of spermatozoa and leukocytes to ROS production in semen using the NBT reduction test.

Our results indicate that the diagnostic and prognostic capabilities of the seminal OS test are beyond those of conventional tests of sperm quality and function. The OS test can accurately discriminate between fertile and infertile men and identify patients with a clinical diagnosis of male-factor infertility that are likely to initiate a pregnancy when followed over a period of time. In addition, the test can help select subgroups of infertile men in whom OS is a significant factor and who might benefit from antioxidant supplementation. We strongly believe that incorporating such a test into the routine andrology workup is an important step for the future of the male infertility practice.

Acknowledgments

We gratefully thank the following members of our research team for contributing to the studies described in this review: Rakesh Sharma, Navid Esfandiari, David Nelson, and Anthony J. Thomas, Jr. We also thank members of the clinical andrology laboratory, Karen Seifarth, Cheryl Wellstead, and Lora Cordek, who provided technical support for these studies. We also thank Robin Verdi for providing secretarial support.

References

Agarwal A, Ikemoto I, Loughlin KR. Relationship of sperm parameters to levels of reactive oxygen species in semen specimens. J Uro. 1994a;152:107 -110.

Agarwal A, Ikemoto I, Loughlin KR. Levels of reactive oxygen species before and after sperm preparation: comparison of swim-up and L4 filtration methods. Arch Andro.1994b; 32:169 -174.

Agarwal A, Ikemoto I, Loughlin KR. Prevention of testicular damage by free-radical scavengers. Urolog.1997; 50:759 -763.

Aitken RJ. Free radicals, lipid peroxidation, sperm function. Reprod Fertil De.1995; 7:659 -668.

Aitken RJ. Molecular mechanisms regulating human sperm function. Mol Hum Repro.1997; 3:169 -173.[Free Full Text]

Aitken RJ. The Amoroso lecture. The human spermatozoon—a cell in crisis? J Reprod Ferti.1999; 115:1 -7.

Aitken RJ, Buckingham D, Brindle J, Gomez E, Baker G, Irvine S. Analysis of sperm movement in relation to the oxidative stress created by leukocytes in washed sperm preparations and seminal plasma. Hum Repro. 1995;10:2061 -2071.[Abstract/Free Full Text]

Aitken RJ, Buckingham DW, West KM. Reactive oxygen species and human spermatozoa: analysis of the cellular mechanisms involved in luminol- and lucigenin-dependent chemiluminescence. J Cell Physio. 1992;151:466 -477.[Medline]

Aitken RJ, Clarkson JS. Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques. J Andro. 1988;9:367 -376.

Aitken RJ, Clarkson JS, Fishel S. Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biol Repro. 1989;40:183 -197.

Aitken RJ, Fisher H. Reactive oxygen species generation and human spermatozoa: the balance of benefit and risk. Bioassay. 1994;16:259 -267.[Medline]

Aitken RJ, Fisher H, Fulton N, Gomez E, Knox W, Lewis B, Irvine DS. Reactive oxygen species generation by human spermatozoa is induced by exogenous NADPH and inhibited by flavoprotein inhibitors diphenylene iodinium and quinacrine. Mol Reprod De.1997; 47:468 -482.

Aitken RJ, Harkiss D, Buckingham D. Relationship between iron-catalyzed lipid peroxidation potential and human sperm function. J Reprod Ferti.1993; 98:257 -265.

Aitken RJ, Krausz C. Oxidative stress, DNA damage and the Y chromosome. Reproductio.2001; 122:497 -506.

Aitken RJ, Krausz C, Buckingham D. Relationship between biochemical markers for residual sperm cytoplasm, reactive oxygen species generation and the presence of leukocytes and precursor germ cells in human sperm suspension. Mol Reprod De.1994; 39:268 -279.

Aitken RJ, Paterson M, Fisher H, Buckingham DW, Van Dubin M. Redox regulation of tyrosine phosphorylation in human spermatozoa is involved in the control of human sperm function. J Cell Sc.1995; 108:2017 -2025.[Abstract]

Aitken RJ, West KM. Analysis of the relationship between reactive oxygen species production and leukocyte infiltration in fractions of human semen separated on Percoll gradients. Int J Andro.1990; 3:433 -451.

Alvarez JG, Storey BT. Taurine, hypotaurine, epinephrine and albumin inhibit lipid peroxidation in rabbit spermatozoa and protect against loss of motility. Biol Repro.1983; 29:548 -555.[Abstract]

Alvarez JG, Storey BT. Differential incorporation of fatty acids into and peroxidative loss of fatty acids from phospholipids of human spermatozoa. Mol Reprod De.1995; 42:334 -346.

Alvarez JG, Touchstone JC, Blasco L, Storey BT. Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa: superoxide dismutaseas major enzyme protectant against oxygen toxicity. J Andro.1987; 8:336 -348.

Armstrong JS, Rajasekaran M, Chamulitrat W, Gatti P, Hellstrom WJ, Sikka SC. Characterization of reactive oxygen species induced effects on human spermatozoa movement and energy metabolism. Free Radic Biol Me. 1999;26:869 -880.[Medline]

Armstrong JS, Rajasekaran M, Hellstrom WJ, Sikka SC. Antioxidant potential of human serum albumin: role in the recovery of high quality human spermatozoa for assisted reproductive technology. J Andro. 1998;19:412 -419.

Aziz N, Buchan I, Taylor C, Kingsland CR, Lewis-Jones I. The sperm deformity index: a reliable predictor of the outcome of oocyte fertilization in vitro. Fertil Steri.1996; 66:1000 -1008.

Baehner RL, Boxer LA, Davis J. The biochemical basis of nitroblue tetrazolium reduction in normal human and chronic granulomatous disease polymorphonuclear leukocytes. Bloo.1976; 48:309 -313.[Abstract/Free Full Text]

Blake DR, Allen RE, Lunec J. Free radical in biological systems-a review oriented to inflammatory processes. Br Med Bul.1987; 43:371 -385.[Abstract/Free Full Text]

Buettner GR. The pecking order of free radicals and antioxidants, lipid peroxidation, alpha-tocopherol and ascorbate. Arch Biochem Biophy. 1993;300:535 -543.[Medline]

Chaudiere J, Wilhelmsen EC, Tappel AL. Mechanism of selenium-glutathione peroxidase and its inhibition by mercaptocarboxylic acids and other mercaptans. J Biol Che.1984; 259:1043 -1050.[Abstract/Free Full Text]

Chia SE, Xu B, Ong CN, Tsakok FM, Lee ST. Effect of cadmium and cigarette smoking on human semen quality. Int J Fertil Menopausal Stu. 1994;39:292 -298.

Church DF, Pryor WA. Free radical chemistry of cigarette smoke and its toxicological implications. Environ Health Perspec. 1990;87:9741 -9745.

Clark IA, Hunt NH, Cowden WB. Oxygen-derived free radicals in the pathogenesis of parasitic disease. Adv Parasito.1986; 25:1 -44.

Close CE, Roberts PL, Berger RE. Cigarettes, alcohol and marijuana are related to pyospermia in infertile men. J Uro.1990; 144:900 -903.

Comhaire FH, Mahmoud AM, Depuydt CE, Zalata AA, Christophe AB. Mechanisms and effects of male genital tract infection on sperm quality and fertilizing potential: the andrologist's viewpoint. Hum Reprod Updat. 1999;5:393 -398.[Abstract/Free Full Text]

de Lamirande E, Gagnon C. Reactive oxygen species and human spermatozoa. II. Depletion of adenosine triphosphate (ATP) plays an important role in the inhibition of sperm motility. J Andro.1992; 13:379 -386.

de Lamirande E, Gagnon C. Human sperm hyperactivation and capacitation as parts of an oxidative process. Free Radic Biol Me. 1993;14:255 -265.

de Lamirande E, Gagnon C. Reactive oxygen species (ROS) and reproduction. Adv Exp Med Bio.1994; 366:185 -197.[Medline]

de Lamirande E, Gagnon C. Impact of reactive oxygen species on spermatozoa: a balancing act between beneficial and detrimental effects. Hum Repro.1995; 10:15 -21.

de Lamirande E, Eiley D, Gagnon C. Inverse relationship between the induction of human sperm capacitation and spontaneous acrosome reaction by various biological fluids and the superoxide scavenging capacity of these fluids. Int J Andro.1993; 16:258 -266.

de Lamirande E, Jiang H, Zini A, Kodoma H, Gagnon C. Reactive oxygen species (ROS) and sperm physiology. Rev Repro.1997; 2:48 -54.

de Lamirande E, Leduc BE, Iwasaki A, Hassouna M, Gagnon C. Increased reactive oxygen species formation in semen of patients with spinal cord injury. Fertil Steri.1995; 64:637 -642.

Daitch JA, Bedaiway MA, Pasqualotto FF, et al. Varicocelectomy improves intrauterine insemination success rates among men with varicocele. J Uro. 2001;165:1510 -1513.

Donnelly ET, McClure N, Lewis S. Antioxidant supplementation in vitro does not improve human sperm motility. Fertil Steri. 1999;72:484 -495.

Duru NK, Morshedi M, Oehninger S. Effects of hydrogen peroxide on DNA and plasma membrane integrity of human spermatozoa. Fertil Steri. 2000;74:1200 -1207.

Eggert-Kruse W, Bellman A, Rohr G, Tilgen W, Rumiebaum B. Differentiation of round cells in semen by means of monoclonal antibodies and relationship with male infertility. Fertil Steri.1992; 58:1046 -1055.

Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K, Angelis PD, Claussen OP. Utility of sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Hum Repro. 1999;14:1039 -1049.[Abstract/Free Full Text]

Fedder J, Askjaer SA, Hjort T. Nonspermatozoal cells in semen: relationship to other semen parameters and fertility status of the couple. Arch Andro.1993; 31:95 -103.

Fraga GG, Motchnik PA, Shigenaga MK, Helbrock JH, Jacob RA, Ames B. Ascorbic acid protects against endogenous oxidative DNA damage in human sperm. Proc Natl Acad Sci US.1991; 88:11003 -11006.[Abstract/Free Full Text]

Gagnon C, Iwasaki A, de Lamirande E, Kovalski N. Reactive oxygen species and human spermatozoa. Ann NY Acad Sc.1991; 637:436 -444.[Medline]

Gavella M, Lipovac V. NADH-dependent oxido-reductase (diaphorase) activity and isozyme pattern of sperm in infertile men. Arch Andro. 1992;28:135 -141.

Gil-Guzman E, Ollero M, Lopez MC, Sharma RK, Alvarez JG, Thomas AJ Jr, Agarwal A. Differential production of reactive oxygen species by subsets of human spermatozoa at different stages of maturation. Hum Repro. 2001;16:1922 -1930.[Abstract/Free Full Text]

Gomez E, Irvine DS, Aitken RJ. Evaluation of a spectrophotometric assay for the measurement of malondialdehyde and 4-hydroxyalkenals in human spermatozoa: relationships with semen quality and sperm function. Int J Andro.1998; 21:81 -94.

Griveau JF, Renard P, Le Lannou D. An in vitro promoting role for hydrogen peroxide in human sperm capacitation. Int J Andro. 1994;17:300 -307.

Griveau JF, Dumont E, Renard B, Callegari JP, Lannou DL. Reactive oxygen species, lipid peroxidation and enzymatic defense systems in human spermatozoa. J Reprod Ferti.1995a; 103:17 -26.

Griveau JF, Renard P, Le Lannou D. Superoxide anion production by human spermatozoa as a part of the ionophore-induced acrosome reaction in vitro. Int J Andro.1995b; 18:67 -74.

Griveau JF, Le Lannou D. Reactive oxygen species and human spermatozoa. Int J Androl1997; 20:61 -69.[Medline]

Halliwell B. Tell me about free radicals, doctor: a review. J Roy Soc Me.1984; 82:747 -752.

Halliwell B. How to characterize a biological antioxidant. Free Radic Res Commu.1990; 9:1 -32.

Hendin B, Kolettis P, Sharma RK, Thomas AJ Jr, Agarwal A. Varicocele is associated with elevated spermatozoal reactive oxygen species production and diminished seminal plasma antioxidant capacity. J Uro. 1999;161:1831 -1834.

Huszar G, Sbracia M, Vigue L, Miller DJ, Shur BD. Sperm plasma membrane remodeling during spermiogenic maturation in men: relationship among plasma membrane beta 1,4-galactosyltransferase, cytoplasmic creatine phosphokinase and creatine phosphokinase isoform ratios. Biol Repro. 1997;56:1020 -1024.[Abstract]

Iwasaki A, Gagnon C. Formation of reactive oxygen species in spermatozoa of infertile patients. Fertil Steri.1992; 57:409 -416.

Janssen YM, Van-Houton B, Borm PJ, Mossuran BT. Cell and tissue responses to oxidative damage. Lab Inves.1993; 69:261 -274.[Medline]

Jeulin C, Soufir JC, Weber P, Laval-Martin D, Calvayrac R. Catalase activity in human spermatozoa and seminal plasma. Gamete Re. 1989;24:185 -196.

Jones R, Mann T, Sherins RJ. Peroxidative breakdown of phospholipids in human spermatozoa: spermicidal effects of fatty acids peroxidatives and protective action of seminal plasma. Fertil Steri. 1979;31:531 -537.

Kessopoulou E, Tomlinson MJ, Banat CLR, Bolton AE, Cooke ID. Origin of reactive oxygen species in human semen-spermatozoa or leukocytes. J Reprod Ferti.1992; 94:463 -470.

Kobayashi H, Gil-Guzman E, Mahran AM, Sharma RK, Nelson DR, Thomas AJ Jr, Agarwal A. Quality control of reactive oxygen species measurement by luminol-dependent chemiluminescence assay. J Andro.2001; 22:568 -574.

Kodama H, Kuribayashi Y, Gagnon C. Effect of sperm lipid peroxidation on fertilization. J Andro.1996; 16:151 -157.

Kodama H, Yamaguchi R, Fukuda J, Kasai H, Tanaka T. Increased oxidative deoxyribonucleic acid damage in the spermatozoa of infertile male patients. Fertil Steri.1997; 65:519 -524.

Kolettis P, Sharma RK, Pasqualotto F, Nelson D, Thomas AJ Jr, Agarwal A. The effects of seminal oxidative stress on fertility after vasectomy reversal. Fertil Steri.1999; 71:249 -255.

Kovalski NN, de Lamirande E, Gagnon C. Reactive oxygen species generated by human neutrophils inhibit sperm motility: protective effects of seminal plasma and scavengers. Fertil Steri.1992; 58:809 -816.

Kruger TF, Acosta AA, Simmons KF, Swanson RJ, Matta JF, Veeck LL, Morshedi M, Brugo S. New method of evaluating sperm morphology with predictive value for human in vitro fertilization. Urolog.1987; 30:248 -251.

Lenzi A, Cualosso F, Gandini L, Lombardo F, Dondero F. Placebo-controlled, double-blind, cross-over trial of glutathione therapy, in male infertility. Hum Reprod.1993; 9:2044 -2050.

Lenzi A, Picardo M, Gandini L, Lombardo F, Terminali O, Passi S, Dondero F. Glutathione treatment of dyspermia: effect on the lipoperoxidation process. Hum Reprod.1994; 9:2044 -2050.[Abstract/Free Full Text]

Lewis SEM, Boyle PM, McKinney KA, Young IS, Thompson W. Total antioxidant capacity of seminal plasma is different in fertile and infertile men. Fertile Steril.1995; 64:868 -870.

Lopes S, Jurisicova A, Sun J, Casper RF. Reactive oxygen species: a potential cause for DNA fragmentation in human spermatozoa. Hum Reprod. 1998;13:896 -900.[Abstract/Free Full Text]

Matson PL. Clinical value of tests for assessing male infertility. Clin Obstet Gynecol.1997; 11:641 -654.

Moilanen J, Hovatta O, Lindroth L. Vitamin E levels in seminal plasma can be elevated by oral administration of vitamin E in infertile men. Int J Androl.1993; 16:165 -166.[Medline]

Ochsendorf FR. Infections in the male genital tract and reactive oxygen species. Hum Reprod.1999; 5:399 -420.

Ollero M, Gil-Guzman E, Lopez MC, et al. Characterization of subsets of human spermatozoa at different stages of maturation: implications in the diagnosis and treatment of male infertility. Hum Reprod. 2001;16:1912 -1921.[Abstract/Free Full Text]

Padron OF, Brackett NL, Sharma RK, Kohn S, Lynne CM, Thomas AJ Jr, Agarwal A. Seminal reactive oxygen species, sperm motility and morphology in men with spinal cord injury. Fertil Steril.1997; 67:1115 -1120.[Medline]

Park BH, Fikrig SM, Smithwick EM. Infection and nitroblue-tetrazolium reduction by neutrophils. A diagnostic acid. Lancet. 1968;2:532 -534.[Medline]

Pasqualotto FF, Sharma RK, Agarwal A, Nelson DR, Thomas AJ Jr, Potts JM. Seminal oxidative stress in chronic prostatitis patients. Urology. 2000a;55:881 -885.[Medline]

Pasqualotto FF, Sharma RK, Nelson DR, Thomas AJ Jr, Agarwal A. Relationship between oxidative stress, semen characteristics, and clinical diagnosis in men undergoing fertility investigation. Fertil Steril. 2000b;73:459 -464.[Medline]

Pasqualotto FF, Sharma RK, Kobayashi H, Nelson DR, Thomas AJ Jr, Agarwal A. Oxidative stress in normospermic men undergoing infertility evaluation. J Androl.2001; 73:459 -464.

Plante M, de Lamirande E, Gagnon C. Reactive oxygen species released by activated neutrophils, but not by deficient spermatozoa, are sufficient to affect normal sperm motility. Fertil Steril. 1994;62:387 -393.[Medline]

Rajasekaran M, Hellstrom WJ, Naz RK, Sikka SC. Oxidative stress and interleukins in seminal plasma during leukocytospermia. Fertil Steril. 1995;64:166 -171.[Medline]

Saran M, Beck-Speier I, Fellerhoff B, Bauer G. Phagocytic killing of microorganisms by radical processes: consequences of the reaction of hydroxyl radicals with chloride yielding chlorine atoms. Free Radic Biol Med. 1999;26:482 -490.[Medline]

Segal AW, Levi AJ. The mechanism of the entry of dye into neutrophils in the nitroblue tetrazolium (NBT) test. Clin Sci Mol Med. 1973;45:817 -826.[Medline]

Sharma RK, Agarwal A. Role of reactive oxygen species in male infertility. (Review). Urology.1996; 48:835 -850.[Medline]

Sharma RK, Pasqualotto FF, Nelson DR, Thomas AJ Jr, Agarwal A. The reactive oxygen species—total antioxidant capacity score is a new measure of oxidative stress to predict male infertility. Hum Reprod. 1999;14:2801 -2807.[Abstract/Free Full Text]

Sharma R, Pasqualotto FF, Nelson DR, Thomas AJ Jr, Agarwal A. Relationship between seminal white blood cell counts and oxidative stress in men treated at an infertility clinic. J Androl.2001; 22:575 -583.[Abstract]

Shekarriz M, Sharma RK, Thomas AJ Jr, Agarwal A. Positive myeloperoxidase staining (Endtz test) as an indicator of excessive reactive oxygen species formation in semen. J Assist Reprod Genet. 1995a;12:70 -74.[Medline]

Shekarriz M, Thomas AJ Jr, Agarwal A. A method of human semen centrifugation to minimize the iatrogenic sperm injuries caused by reactive oxygen species. Eur Urol.1995b; 28:31 -35.[Medline]

Shekarriz M, Thomas AJ Jr, Agarwal A. Incidence and level of seminal reactive oxygen species in normal men. Urology. 1995c;45:103 -107.[Medline]

Sidhu RS, Sharma RK, Thomas AJ Jr, Agarwal A. Relationship between creatine kinase activity and semen characteristics in sub-fertile men. Int J Fertil Womens Med.1998; 43:192 -197.[Medline]

Sies H. Strategies of antioxidant defence. Eur J Biochem. 1993;215:213 -219.[Medline]

Sies H, Stahl W, Sundquist RA. Antioxidant function of vitamins. Vitamins E and C, beta-carotenoids, and other carotenoids. Ann NY Acad Sci. 1992;669:7 -20.[Medline]

Sikka SC. Oxidative stress and role of antioxidants in normal and abnormal sperm function Front Biosci.1996; 1:78 -86.

Sikka SC. Relative impact of oxidative stress on male reproductive function. Curr Med Chem.2001; 8:851 -862.[Medline]

Sikka SC, Rajasekaran M, Hellstrom WJG. Role of oxidative stress and antioxidants in male infertility. J Androl.1995; 16:464 -468.[Abstract/Free Full Text]

Smith R, Vantman D, Escobar J, Lissi E. Total antioxidant capacity of human seminal plasma. Hum Reprod.1996; 11:1655 -1660.[Abstract/Free Full Text]

Spitteler G. Review: on the chemistry of oxidative stress. J Lipid Mediators.1993; 7:77 -82.

Stone KK, Bermudes E. Aqueous extracts of cigarette tar containing free radicals cause DNA nicks in mammalian cells. Environ Health Perspect. 1986;102:173 -177.

Sun JG, Jurisicova A, Casper RF. Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol Reprod.1997; 56:602 -607.[Abstract]

Thiele JJ, Freisleben HJ, Fuchs J, Oschendorf FR. Ascorbic acid and urate in human seminal plasma: determination and interrelationship with chemiluminescence in washed semen. Hum Reprod.1995; 10:110 -115.[Abstract/Free Full Text]

Thomas J, Fishel SB, Hall JA, Green S, Newton TA, Thornton SJ. Increased polymorphonuclear granulocytes in seminal plasma in relation to sperm morphology. Hum Reprod.1997; 12:2418 -2421.[Abstract/Free Full Text]

Tomlinson MJ, Barrat GLR, Cooke ID. Prospective study of leukocytes and leukocyte subpopulations in semen suggests that they are not a cause of male infertility. Fertil Steril.1993; 60:1069 -1075.[Medline]

Tomlinson MJ, White A, Barratt CL, Bolton AE, Cooke ID. The removal of morphologically abnormal sperm forms by phagocytes: a positive role for seminal leukocytes. Hum Reprod.1992; 7:517 -522.[Abstract/Free Full Text]

Twigg J, Irvine DS, Aitken RJ. Oxidative damage to DNA in human spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm injection. Hum Reprod.1998a; 13:1864 -1871.[Abstract/Free Full Text]

Twigg J, Irvine DS, Houston P, Fulton N, Michael L, Aitken RJ. Iatrogenic DNA damage induced in human spermatozoa during sperm preparation: protective significance of seminal plasma. Mol Hum Reprod 1998b;4:439 -445.[Abstract/Free Full Text]

Van Voorhis BJ, Sparks AET. Semen analysis: what tests are clinically useful? Clin Obstet Gynecol.1999; 42:957 -971.[Medline]

Warren JS, Johnson KJ, Ward PA. Oxygen radicals in cell injury and cell death. Pathol Immunopathol Res.1987; 6:301 -315.[Medline]

Wefers H, Sies H. The protection by ascorbate and glutathione against microsomal lipid peroxidation is dependent on vitamin E Eur J Biochem. 1988;174:353 -357.

Wolff H. The biologic significance of white blood cells in semen. Fertil Steril.1995; 63:1143 -1157.