**1. Introduction**

In a landmark paper published in 1943, McLeod [1] demonstrated that the incubation of human spermatozoa under conditions of high oxygen tension precipitated a loss of motility that could be reversed by the presence of catalase. The clear implication of these findings, that human spermatozoa are vulnerable to hydrogen peroxide attack was confirmed many years later when human spermatozoa were exposed to the mixture of reactive oxygen species (ROS) generated by the xanthine oxidase system, a known mediator of sperm oxidative stress in vivo and in vitro [2,3]. Exposure in vitro was found to induce a loss of fertilizing potential and, ultimately, motility, via mechanisms that could be completely reversed by concomitant exposure to catalase, which specifically catalyzes the conversion of hydrogen peroxide to oxygen and water, but was exacerbated by superoxide dismutase, which catalyzes the dismutation of superoxide anion to hydrogen peroxide [3]. Excessively high levels of spontaneous ROS generation were subsequently shown to be associated with the defective sperm function encountered in cases of human infertility [4,5]. The mechanism by which such oxidative stress induced defective sperm function was further shown to be linked to the capacity of these reactive oxygen metabolites to stimulate lipid peroxidation in spermatozoa [6–8]. These cells are particularly vulnerable to this process because they are richly endowed with polyunsaturated fatty acids, the double bonds of which facilitate the hydrogen abstraction process that initiates the peroxidation cascade [8]. The causative links between oxidative stress, lipid peroxidation and sperm function have subsequently been confirmed many times by independent groups for human spermatozoa [9–13] and the spermatozoa of every other mammalian species examined including the bull [14], stallion [15,16], pig [17,18], dog [19], cat [20], etc. This fundamental concept, pioneered by such luminaries as Jones and Mann [21], Gagnon [22] and Storey [6] is fundamental to our understanding of the aetiology of defective sperm function. Of course, it is not the only factor responsible for compromising the fertilizing potential of mammalian spermatozoa and it is important not to overstate the case. Many other factors are potentially involved in suppressing a function such as sperm motility, including excess intracellular calcium [23], dephosphorylation of phosphatidyl-inositol-3-kinase [24,25], exposure to motility-inhibiting proteins in semen, including seminal amyloid [26] and seminogelin [27], and exposure to sperm immobilizing antibodies [28]. However, the relative significance of oxidative stress as a major factor in the aetiology of defective sperm function has been clearly demonstrated in the few antioxidant studies that have included measures of lipid peroxidation (malondialdehyde [MDA]) in their evaluation schedule as well as a placebo control group [29]. In one such study, patients exhibiting asthenozoospermia (motility ≤ 40%) were treated with antioxidants (100 mg vitamin E) or placebo, 3 times a day for either 6 months or until the patient's partner was diagnosed as pregnant, whichever came first. This study demonstrated that treatment with vitamin E significantly improved sperm motility while significantly reducing MDA levels in the spermatozoa in a manner that could not be replicated by administering a placebo formulation. A second, independent, placebo-controlled study drew the same conclusions; treatment with a combination of vitamin E and selenium for 3 months successfully decreasing MDA concentrations and concomitantly improving semen quality [30]. A further study which did not include a placebo control group but did measure MDA levels in seminal plasma, also confirmed the importance of lipid peroxidation in the pathophysiology of defective sperm function. Treatment for 90 days with an antioxidant preparation decreased seminal MDA and statistically improved all elements of the conventional semen profile including motility, concentration and morphology [31]. Another similar study, also concluded that antioxidant treatment (N-acetylcysteine in this case) for 3 months could simultaneously decrease seminal MDA levels while significantly improving sperm concentration and motility in the ejaculate [32]. Thus overall, there is a wide-ranging consensus based on thousands of patients that male infertility is associated with significantly elevated levels of MDA in semen which is, in turn, negatively correlated with levels of seminal antioxidant protection and key aspects of semen quality including sperm count, motility and morphology [33,34].

The apparent e ffectiveness of antioxidant therapy in suppressing oxidative stress while improving semen quality demonstrates the causative nature of these associations. It should be emphasized in passing that antioxidant trials that do not involve measurement of oxidative stress (sadly the majority) are essentially worthless for the very reason that not all defective sperm function is oxidatively induced. Clearly this is an area for further research involving properly controlled clinical trials with antioxidant formulations that are based upon a knowledge of sperm biochemistry and the bioavailability of the administered antioxidants within the male reproductive tract. This has been achieved in animal models where the cause of the infertility is unequivocally oxidative in nature. Thus, using the GPx5−/− knockout model, mice can be generated in which the reproductive pathology observed is entirely dependent on the creation of a localized stress within the epididymis. Treatment of these animals with a carefully formulated antioxidant preparation resulted in a return of oxidative DNA-damaged spermatozoa to control levels. In the same study, the loss of fertility observed as a consequence of scrotal heat stress could also be completely abrogated by pre-treating the animals with the same antioxidant formulation [35].

So, there is an overwhelming volume of evidence indicating a cascade of causal interactions between oxidative stress, peroxidative damage to sperm lipids and DNA and impaired sperm function. A number of questions arise in the wake of such findings including: what are the situations in which sperm oxidative stress occurs? Is the oxidative stress systemic or localized? Where does the oxidative stress come from in terms of cell type and biochemical pathway? What species of ROS are involved? How can we best measure them? In the remainder of this review, we shall attempt to address these key issues.

### **2. What Are the Situations in Which Sperm Oxidative Stress Occurs?**
