Human spermatozoa can produce ROS in one of two ways: (1) through the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system at the level of the sperm plasma membrane and (2) through the NADH-dependent oxidoreductase (diphorase) system at the level of mitochondria [13]. Damaged spermatozoa are an established source of excessive ROS production [14]. The increase in ROS generation is thought to result from excess residual cytoplasm (cytoplasmic droplets) typically present in abnormal spermatozoa. During spermatogenesis, defects in cytoplasmic extrusion result in the development of immature sperm containing a surplus of residual cytoplasm [15]. ROS generation has been positively correlated with the extent of residual cytoplasmic retention. This observation is thought to be mediated by the concurrent increase in the enzyme glucose-6-phosphate-dehydrogenase (G6PD) activity seen in abnormal spermatozoa. G6PD stimulates glucose influx through the hexose monophosphate shunt raising the intracellular availability of NADPH, a major source of electrons for spermatozoa used to fuel the generation of ROS [16].

Creatine kinase (CK), a biochemical marker of cytoplasmic space, has been correlated with the degree of oxidative damage in human sperm. Alani and El Yaseen [17] have found a significant positive correlation between CK activity and malondialdehyde (MDA) formation in sperm fractions from infertile men. Hallak et al. demonstrated an inverse relationship between CK levels and sperm morphological forms suggesting that CK levels can be used to reliably predict sperm quality and fertilizing potential in men with infertility [18].

Since spermatozoa are rich in mitochondria in order to supply the necessary energy needed for motility, the major source of ROS in spermatozoa in infertile men originates from the mitochondria. Mitochondrial dysfunction has been associated with increased ROS production [19], which results in more damage to the mitochondrial membrane and consequently fuels more ROS production.

Superoxide is the primary ROS generated in human spermatozoa [20]. This one-electron molecule is capable of reacting with itself in a dismutation reaction to generate H₂O₂ and O₂·. In the presence of transition metals such as iron and copper, it can interact to generate OH· through the Haber-Weiss reaction (·O₂ ⁻ +H₂O₂ ➔OH· +OH +O₂). Alternatively, OH· can be produced from H₂O₂ through the Fenton reaction, which requires ferrous ions as reducing agents (H₂O₂+Fe²⁺ ➔ Fe³⁺+OH· +OH). The OH· is thought to be the most hazardous ROS initiating the lipid peroxidation (LPO) cascade and causing loss of sperm functions [21].

Leukocytospermia has been largely considered as a major cause of male infertility [22]. Peroxidase-positive leukocytes include polymorphonuclear leukocytes and macrophages which represent 50 % and 30 % of all seminal leukocytes, respectively [23], while T lymphocytes constitute the remaining 10 % of seminal leukocytes [23]. They are mainly released with prostatic and seminal vesicle secretions and are capable of producing large amounts of ROS in response to infection or inflammation [22]. Once activated, the leukocytes’ myeloperoxidase system is stimulated leading to an oxidative burst that provides the early defense system against microbes in cases of infection.

Sperm damage from leukocyte-produced ROS can occur after removal of seminal plasma during preparations performed for assisted reproduction or when leukocytospermia exists. A recent study by Lobascio et al. [12] demonstrated the presence of a positive correlation between the number of seminal leukocytes and ROS levels (p < 0.001; n = 125). Moreover, they confirmed the negative consequence of such a relationship through finding significant negative correlations with sperm concentration (p = 0.01) and motility (p = 0.02) and a positive correlation with sperm DNA fragmentation (SDF) (p = 0.08) [12]. Mupfiga et al. investigated semen samples from 60 infertile patients and found higher ROS production and caspase activity, a marker of apoptosis, in samples containing a higher number of leukocytes [24]. Saleh et al. [25] compared semen samples from 48 infertile men with those from healthy volunteers. To objectively assess leukocyte-ROS production, samples from patients with no evidence of leukocytospermia (n = 32) were further incubated with blood neutrophils. In comparison with samples from healthy volunteers and non-leukoctospermic infertile men, significantly higher levels of ROS were detected in samples from leukocytospermic infertile men or after incubation with blood neutrophils. Furthermore, the authors demonstrated a significant decrease in sperm motility and increase in sperm DNA damage in leukocytospermic samples [25].

Sperm damage from leukocyte-derived ROS may happen even at leukocyte concentrations below the World Health Organization’s cutoff value for leukocytospermia, that is, greater than 1 × 10⁶ peroxidase-positive leukocytes/mL of semen [26]. In a comparative study, Agarwal et al. divided semen samples from 472 infertile men into three groups: group 1, no seminal leukocytes; group 2, low-level leukocytospermia (0.1–1.0 × 10⁶ WBC/mL); and group 3, frank leukocytospermia (>1.0 × 10⁶ WBC/mL). Results revealed significantly higher levels of ROS and sperm DNA fragmentation in group 2 samples (ROS, 1839.65 ± 2173.57RLU/s; DNA damage, 26.47 ± 19.64 %) compared with group 1 samples (ROS, 1101.09 ± 5557.54 RLU/s; DNA damage, 19.89 ± 17.31 %) (ROS, p = 0.002; DNA damage, p = 0.047), without significant differences detected between groups 2 and 3. Finally, seminal leukocytes may induce ROS production by human spermatozoa through a mechanism that is not clearly understood [25].

Apoptosis or programmed cell death is a noninflammatory response to tissue injury characterized by a series of biochemical changes leading to changes in cellular morphology and death. A stringently regulated apoptosis is essential for normal development of spermatozoa as well as the adjustment of the number of sperm cells that are produced [34]. On the other hand, a process termed “abortive apoptosis,” occurring during spermatogenesis, has been associated with male infertility [35–38].

While apoptosis may be triggered by several intrinsic and extrinsic factors, ROS levels appear to be directly correlated with the extent of sperm cell death. Mostafa et al. demonstrated the presence of higher levels of ROS which correlated with a higher percentage of apoptosis in the seminal plasma of infertile men compared to healthy volunteers [8]. After adding 100 μmole of H₂O₂ to 12 semen samples to induce OS, Mahfouz et al. reported a higher percentage of apoptosis after H₂O₂ exposure [39]. Wang et al. compared semen samples from 35 patients with idiopathic infertility with 8 samples from healthy volunteers. The authors detected significantly higher levels of ROS (4.15 × 10⁶ counted photons per minute [cpm] vs. 0.06 × 10⁶ cpm; P .01), cytochrome C (2.78 vs. 1.5; P .01), caspase 9 (2.52 vs. 6; P .006), and caspase 3 (0.56 vs. 1.69; P .01) in infertile men vs. healthy volunteers, respectively [40].

Lipid peroxidation (LPO) is the most extensively studied manifestation of ROS in biology. LPO is generally defined as oxidative induced damage of fatty acids that contain more than two carbon double bonds, also known as PUFA [41]. This is because most PUFA contain a double bond next to a methylene group weakening the methylene-carbon-hydrogen bond. As a result, free radicals are capable of capturing the hydrogen moiety from PUFA leaving an unpaired electron on the fatty acid that can be oxidized to form a peroxyl radical (Fig. 1.2). Lipid peroxides are unstable and decompose to form a complex series of compounds, which ultimately end up with MDA.

LPO measurement relies on the interaction of MDA with thiobarbituric acid (TBA). While this method is controversial in that it is quite sensitive, but not necessarily specific to MDA, it remains the most widely used means to determine LPO. The resulting membrane structure disturbance affects vital functions such as signal transduction and maintenance of ion and metabolite gradient necessary for optimal sperm function. Unlike other cells, spermatozoa are unable to overcome the resulting damage since they lack the necessary cytoplasmic enzymes involved in the repair process [42].

Normal sperm motility is an integral requirement for male fertility. The free radical’s ability to reduce sperm motility was first described by Jones et al. in 1979 [43], who linked ROS-induced LPO to reduction of sperm tail motion. ROS illicit a cascade of events that result in a decrease in axonemal protein phosphorylation and sperm immobilization [44]. H₂O₂ can inhibit the activity of G6PD decreasing the availability of NADPH thereby causing an accumulation of oxidized glutathione. The latter can reduce the antioxidant defenses of the spermatozoa which ultimately aggravate peroxidation of membrane phospholipids [44]. A direct cytotoxic effect for ROS on sperm mitochondria was also recognized as another cause for impaired sperm motility [27, 45, 46].

Clinical studies evaluating the link between ROS levels and defects in sperm motility have confirmed the presence of a negative association. du Plessis et al. [47] examined the influence of exogenous H₂O₂ addition to semen samples on sperm motility parameters and intracellular ROS and nitric oxide (NO) levels. After incubating human spermatozoa from ten donors with different exogenous H₂O₂ concentrations (0, 2.5, 7.5, and 15 μmole), they detected a significant inversely proportional relationship with sperm total motility (7.5 μmole 28.3 ± 4.01, P < 0.001; 15 μmole 16.67 ± 3.33, P < 0.001, versus control, 60.33 ± 6.86) and progressive motility (2.5 μmole 14 ± 1.65, P < 0.01; 7.5 μmole 10 ± 2.28, P < 0.001; 15 μmole 6.33 ± 1.68, P < 0.001, versus control, 29.33 ± 4.56). Higher concentrations of H₂O₂ significantly increased both NO (172.40 ± 22.341 % versus control; P < 0.05) and ROS levels (130.40 ± 7.108 % versus control; P < 0.05). The percentage of total motility was also inversely correlated with both endogenous NO (r ² = 0.99, P = 0.0041) and ROS (r ² = 0.965, P = 0.017) levels [47].

Urata et al. [48] incubated sperm from 37 healthy volunteers with lipopolysaccharide (LPS), an OS inducer, with and without antioxidant scavengers. Sperm motility was inhibited by 15 % in the presence of 0.1 μg/mL, 21 % in the presence of 1 μg/mL, and 50 % in the presence of 10 μg/mL dose of LPS after 60 minutes of incubation, compared with the control groups (P < 0.05). LPS-treated groups had a significantly higher ROS production in comparison to the control groups (P < 0.05). The addition of ROS scavengers such as superoxide dismutase and glutathione restored the motility index and suppressed ROS production in the LPS-treated semen samples [48]. Kourouma et al. [49] examined the in vitro administration of nonylphenol, a chemical known to induce OS by generating H₂O₂ and ^.O₂ ⁻, on epididymal sperm from 24 Sprague Dawley rats. Results showed a significant decline in the percentage of motile spermatozoa (P < 0.001) in a dose-related manner [49].

Sperm DNA is uniquely structured to keep its nuclear chromatin highly stable, compact, and protected against assaults. It undergoes timely de-condensation to ensure the proper transfer of the packaged genetic material to the ovum during the fertilization process. Sperm DNA damage can occur as a consequence of OS and is thought to occur secondary to dysregulated apoptosis factors (Fig. 1.3). ROS can alter DNA integrity through modification of nucleic bases resulting in deletions, cross-links, frameshifts, and chromosomal rearrangements [50–52]. Such changes destabilize the DNA backbone, causing single- and double-strand DNA breaks [53]. The significance of sperm DNA fragmentation (SDF) has been acknowledged in male infertility. High SDF has been found to decrease the chances of natural pregnancy [54], increase the likelihood of miscarriages [55], and decrease the outcomes of assisted reproductive techniques, specifically intrauterine insemination [56] and conventional in vitro fertilization [57].

The relationship between OS and DNA damage has been proven in numerous reports. Lommiello et al. investigated semen samples from 56 infertile men and revealed a significant positive correlation between ROS levels and the degree of SDF (p = 0.037) [58]. In another study, semen collected from 63 patients attending an IVF unit was tested for physical characteristics along with ROS levels and SDF. The authors reported a strong positive correlation between intrinsic ROS levels and SDF measurements [59].

Varicocele is a common medical condition that has long been implicated as a major cause of male infertility [60, 61]. It is prevalent in about 15 % of the general male population, 35 % of men with primary infertility, and up to 80 % in men with secondary infertility [62, 63]. OS is now widely believed to be the common underlying pathophysiology causing infertility in varicocele patients [64, 65]. Studies involving OS markers in the semen of men with varicocele detected a significant increase of such markers in varicocele patients compared to controls [66].

Seminal ROS levels measured by chemiluminescence were significantly higher in infertile men with varicocele than fertile controls. Moreover, Allamaneni et al. [67] reported a positive correlation between seminal ROS levels and varicocele grade meaning that men with larger varicoceles had significantly higher seminal ROS levels than men with small varicoceles. Among healthy fertile men, the presence of varicocele was associated with a significant increase in ROS levels in comparison to men without varicocele [68]. Higher seminal levels of specific free radicals, namely, NO and NO synthase, have been detected in infertile men with varicocele compared with fertile men without varicocele [69–72]. Seminal levels of H₂O₂ and extracellular seminal O₂ were also found to be significantly higher in infertile men with varicocele in comparison to fertile healthy controls [73, 74].

Interestingly, surgical treatment of varicocele has been shown to reduce seminal OS in varicocele patients [75]. In one study, Sakamoto et al. [69] found that a time lag of approximately 6 months is required to achieve a marked improvement in seminal ROS markers after varicocele repair. Mostafa et al. [76] demonstrated a significant reduction of markers of seminal OS and an elevation of antioxidant levels 3 and 6 months after varicocele ligation. Finally, Hurtado de Catalfo et al. [77] reported normalization of seminal levels of antioxidant enzymes compared with age-matched fertile controls after varicocele ligation.

This assay measures the oxidative end products of the interaction between ROS and certain reagents, which results in an emission of light that can be measured with a luminometer (Fig. 1.4) [80]. Two reagents are available, luminol and lucigenin. In contrast to lucigenin, which detects only the superoxide anion [81, 82], luminol has few advantages such as: (1) it has the ability to react with different ROS, including superoxide anion, hydroxyl radical, and hydrogen peroxide; (2) it measures both intra- and extracellular free radicals; and (3) it conducts a fast reaction allowing rapid measurement [83]. To ensure accurate readings, semen samples should contain sperm concentration 1 × 10⁶/mL or greater and be analyzed within the first hour of collection. Despite its clinical applicability, the widespread use of the chemiluminescence assay has been hampered by equipment expenses and the presence of assay confounders such as incubation time, leukocyte, and seminal plasma contamination [84].

Flow cytometry is an alternative method that can also be used to measure intracellular sperm ROS [85]. It quantifies the amount of fluorescence per cell. Excited by a light source, cells emit light that is passed through optical filters before reaching optical detectors. Optical filters allow light of specific wavelengths to pass, thereby producing waves of specific colors. Flow cytometry is also an expensive tool that is not practical for widespread clinical use.

NBT is a cost-effective user-friendly assay. Using a light microscope, it can accurately predict ROS levels and provide insight on the potential source of OS, i.e., spermatozoa or leukocytes. It is based on the ability of NBT to interact with the superoxide present within spermatozoa or leukocytes converting into a blue pigment called diformazan, which can be measured and correlated with intracellular ROS concentration [78, 86].

The most commonly used method of assessing sperm membrane peroxidation is MDA level measurement via the TBA assay. Sensitive high-pressure liquid chromatography (HPLC) equipment [87, 88] or spectrofluorometric measurement of iron-based promoters [89] may be utilized for detection of the low sperm MDA concentrations. On the other hand, seminal plasma MDA levels are five to tenfold higher than sperm, making measurement with standard spectrophotometers possible [90]. The clinical relevance of MDA measurement emerges from its significant positive correlation with seminal ROS levels in men with infertility, compared with fertile controls or normozoospermic individuals [90–92]. Furthermore, in vitro studies have linked ROS-induced abnormalities in motility, sperm DNA integrity, and sperm-oocyte fusion with an increase in MDA concentration [33, 89].

Oxidation-reduction potential (ORP), also known as the redox potential, is a measure of the potential for electrons to move from one chemical species to another [93]. ORP is a measure of this relationship between oxidants and antioxidants, providing a comprehensive measure of OS. Recently, a novel technology based on a galvanostatic measure of electrons has been developed (MiOXSYS) and utilized to evaluate changes in OS in trauma patients and as a function of extreme exercise [94–96]. The MiOXSYS is a simple, rapid, and inexpensive system composed of the analyzer and disposable test sensor (Fig. 1.5). It measures the electron transfer from reductants (antioxidants) to oxidants under a steady low-voltage reducing current. Thus, it provides an aggregate measure of all current oxidant activity and antioxidant activity in a sample. Higher ORP values (millivolts, mV) indicate a higher oxidant activity relative to the antioxidant activity and therefore greater state of OS. Recent studies have confirmed the reliability of the MiOXSYS System in measuring ORP levels in semen and seminal plasma demonstrating the presence of significant negative correlations between ORP results and abnormalities of semen parameters [97].

A number of routine laboratory tests have been suggested as possible predictors for the presence of OS in the semen [98]. While any abnormality in routine semen parameters (count, motility, morphology) may be associated with OS, asthenozoospermia is probably the best surrogate marker for OS in a routine semen analysis [99, 100]. The presence of an exaggerated number of round cells in the semen may represent leukocytes and hence possible OS [26]. These cells, however, may represent immature germ cells and hence need to be tested with ancillary tests such as peroxidase test, CD45 staining, or measurement of seminal elastase activity [101, 102]. Poor sperm viability detected by the hypoosmotic swelling test or dye exclusion assays has been linked with the presence of sperm OS [103]. Additionally, macroscopic semen parameters have also been considered. Hyperviscosity of the seminal plasma, an observation that is commonly seen with infection, has been linked to increased levels of seminal plasma MDA [104] and reduced seminal plasma antioxidant status [105].

The accompanying stresses of the modern world have caused an increase in negative behaviors such as smoking, substance abuse, and obesity, all of which have been shown to contribute to OS, and, therefore, minimizing such unfavorable behavior is likely to aid in alleviating OS [106].

Environmental exposures to heat, pollution, toxins, and heavy metals should be minimized as these can result in development of OS. Moreover, activities capable of increasing scrotal temperature such as hot baths, saunas, extended periods of driving, and long and sedentary office hours should be avoided. Finally, adequate protective equipment and aeration should be ensured at workplaces to limit exposure to any chemical or vapor that may cause OS.