Studies have shown that physiological levels of ROS are required for many baseline bodily functions [44]. Moreover, appropriate concentrations of ROS allow for proper signal transduction, mediation of cytotoxic events, and facilitation of inflammation via prevention of platelet aggregation and neutrophil adherence to endothelial cells [44]. These free radical species also serve as signaling molecules or second messengers, as well as aid in the production of hormones, regulation of tight junctions, and mediation of apoptosis. In regard to the male reproductive system, low ROS levels are necessary for capacitation, hyperactivation, acrosome reaction, zona pellucida binding and fertilization capacity of spermatozoa and promote normal semen parameters, such as sperm motility, morphology, and viability [19, 28, 46].

Although ROS are necessary for normal physiological functions, excess levels overwhelm the body’s natural antioxidant capacity . Due to the unpaired electron in their outer orbit, ROS are highly reactive and interact with a variety of lipids, proteins, and nucleic acids in the body. Such reactions are extremely harmful for reproductive potential and possibly contribute to testicular dysfunction, decreased gonadotropin secretion, and abnormal semen parameters [47]. While ROS affect a variety of reproductive functions, their reactive nature leads to the generation of more free radicals. This, in turn, perpetuates a chain of reactions creating tissue damage in the form of oxidative stress [45] .

In addition to contributing to a variety of conditions , there is growing evidence that oxidative stress is involved in many aspects of UMI (Fig. 10.1). Studies have shown that when compared to fertile men, normozoospermic infertile males have elevated ROS levels measured by the malonaldehyde levels and protein carbonyl groups. [1, 50]. This is also evident by lower reactive oxygen species-total antioxidant capacity (ROS-TAC) scores in patients with UMI, which indicates elevated levels of seminal oxidative stress [51, 52]. Moreover, studies report high ROS dysfunction in UMI patients. This dysfunction can be in the form of reduced fertilization capacity, impairment of sperm metabolism, and lipid peroxidation of polyunsaturated fatty acids within the sperm plasma membrane [53] . Detrimental effects of high ROS on semen parameters such as motility, viability, morphology have been demonstrated by numerous studies [54, 55]. Not only does oxidative stress have negative consequences on sperm parameters but also on the DNA of sperm cells [50]. Studies have shown that patients with UMI have a significantly higher DNA fragmentation index ( > 30 %) induced by toxic levels of ROS when compared to those who are fertile [56]. Specifically, a fragmentation index > 30 % is associated with lower chances of achieving pregnancy by natural conception or by insemination [57]. This finding of increased ROS levels may indicate that seminal oxidative stress may be involved in the pathogenesis of sperm DNA damage in these patients [56]. It has also been suggested that specific genetic polymorphisms can promote the accumulation of oxidative stress within the seminal plasma, such as the glutathione S-transferase Mu-1 (GSTM1) gene polymorphism. Studies have reported that men with idiopathic and unexplained infertility who have the GSTM1 null genotype had significantly higher levels of seminal oxidative stress than those who possessed the GSTM1 gene. Therefore, in patients suffering from infertility, the GSTM1 polymorphism might be integral to determining the susceptibility of spermatozoa to oxidative damage [58]. Additionally, studies report that UMI patients with excess levels of ROS have numerous mutations on genetic analysis of sperm mitochondrial DNA. Examples of such alterations include nucleotide changes in the ATPase and nicotinamide adenine dinucleotide dehydrogenase genes. These DNA mutations may be the underlying etiology of a male’s unexplained infertility [59]. Furthermore, reactive oxygen species-induced DNA damage may accelerate the process of germ cell apoptosis, leading to the decline in sperm counts associated with male infertility [60]. Overall, it is clear that the oxidative stress may play a role in UMI via the mechanism of spermatozoa DNA damage .

The chemiluminescence assay is one of the most commonly used techniques to measure seminal ROS levels in clinical andrology laboratories . The two commonly used probes are luminol (5-amino-2,3-dihydro-1,4-phthalazinedione and 3-aminophthalic hydrazide) and lucigenin ( N, N’-dimethyl-9,9’-biacridinium dinitrate). Both H₂O₂ and O₂ ^●− are involved in luminol-dependent chemiluminescence because both catalase and SOD can disrupt the luminol signal very efficiently. A luminescent signal is produced with luminol through a one-electron oxidative event mediated by hydrogen peroxide (H₂O₂) and either endogenous peroxidase or by addition of horse radish peroxidase. Superoxide anion (O₂ ^●−)is an essential intermediate for the luminol-dependent chemiluminescence. Also, the redox cycling activity associated with this probe allows the significant amplification of the signal and allows easy measurement of H₂O_2. There are a variety of luminometers available and these may be single tube or multiple tube luminometers [61] .

Flow cytometry is a laboratory method used for analyzing the expression of cell surface markers and intracellular molecules. Oxidation of 2, 7 dichlorofluorescein diacetate (DCFH-DA) by ROS, which is generated within the cell, makes them highly fluorescent and can be used to measure formation of intracellular levels of hydrogen peroxide. Hydroethidine (HE) is another fluorescent probe that can be used for the measurement of intracellular levels of superoxide [61, 62].