14.1 Seminal Oxidative Stress

Seminal oxidative stress (OS) is a condition where the levels of oxidants overwhelm those of the antioxidants (reductants) present in the semen [1]. The most important oxidants are reactive oxygen species (ROS), a group of oxygen-based molecules including radicals (e.g. superoxide anion – O₂^-.; hydroxyl radical – OH^.; peroxyl radicals – ROO^.; alkoxyl radicals – RO^.; organic hydroperoxides – ROOH) and non-radical species (hydrogen peroxide – H₂O₂). Free radicals are molecules with one or more unpaired electrons in the outer orbit, which are highly reactive towards any kind of cellular components (lipids, proteins and DNA). H₂O₂ is not a “radical”, but it is classified as ROS because of its strong oxidizing characteristic and reactivity with ferrous ions in the Fenton reaction, leading to the production of hydroxyl radical:

Other important oxidants are derived from nitrogen (reactive nitrogen species – RNS), such as peroxynitrite (ONOO^–), hypochlorous acid (HOCl), nitric oxide (^.NO) [1].

Reactions between the oxidant and a recipient molecule results in the generation of a radical, triggering a chain reaction that perpetuates the cellular damage. ROS are considered primary mediators of OS, and their presence at physiological levels in semen are essential for important sperm functions including capacitation, hyperactivation, acrosome reaction and sperm-oocyte membrane fusion [1]. When the seminal ROS levels increase beyond a threshold for redox regulation, it disturbs the endogenous antioxidant system to counterbalance the redox potential. This redox imbalance results in OS that is harmful for the spermatozoa and negatively affects the reproductive male potential.

14.2 Sources of Oxidative Stress in Semen

The production of ROS can be classified as endogenous and exogenous (Figure 14.1) [2]. In the human semen, the majority of ROS are produced endogenously by leukocytes, and mitochondria of immature sperm. Mitochondria generate energy by means of oxidative phosphorylation. In this chemical pathway, four protein complexes are involved in the transfer of electrons from donors to acceptors, finally resulting in the reduction of molecular oxygen to H₂O. These redox reactions are coupled with the transfer of protons (H⁺) across the mitochondrial membrane in order to produce ATP [2]. Besides water as a final product of oxidative phosphorylation, around 1–2 percent of the oxygen is used by the enzymatic Complex I and III of the electron transport chain to synthetize O₂^-. following a single electron addition to the oxygen [3]. At the level of sperm plasma membrane, the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase catalyzes the synthesis of superoxide by transferring one electron to oxygen from NADPH [3].

Figure 14.1 Endogenous and exogenous sources of seminal reactive oxygen species [38].

Exogenous causes including social behaviors (high intake of alcohol/caffeine, smoking, increased BMI), exposure to pollutants, toxins or radiation, the misuse of drugs and/or medications as well as stress or aging also contribute to increase the levels of seminal ROS. Other sources of ROS include several pathologies such as varicocele, cancer or infections of male reproductive organs [2].

14.3 Role of Oxidative Stress in Male Infertility

The oxidant species are highly reactive molecules which can interact with every cellular component. Sperm membranes are rich in polyunsaturated fatty acids and are highly susceptible sites to ROS-mediated damage [1]. The “snatching” of an electron by ROS from the lipids affects the membrane integrity and leads to the production of lipid peroxidation products, such as malondialdehyde, a biochemical marker of OS. Malondialdehyde is a powerful electrophile that further oxidizes other cellular components, such as proteins, resulting in protein degradation, formation of protein-protein cross linkages and loss of function [2]. The damage to the sperm membrane can propagate into the cell from one molecule to the other in a self-perpetuating cycle. The inhibition of the enzyme glucose‑6‑phosphate dehydrogenase, for example, results in a reduced availability of NADPH and cellular capability to regenerate the antioxidant systems [4].

Due to a very high amount of polyunsaturated fatty acids in the plasma membrane, spermatozoa are highly susceptible to OS, which is a well-established cause of male infertility. Excess levels of ROS impairs sperm production and motility [1]. Genomic and mitochondrial sperm DNA integrity are also affected by OS. Oxidation of nitrogen bases, such as guanosine, leads to the synthesis of 8-hydroxy-2’-deoxyguanosine (8OHdG), and cause single- or double-strand breaks in sperm DNA [2]. Strong correlation exists between the extent of sperm DNA fragmentation (SDF) and different OS markers, such as intracellular ROS, malondialdehyde or 8-OHdG [5–7]. High rates of sperm DNA damage not only lead to a higher mutation rate but can also have negative impact on the fertility potential and embryo development in spontaneous pregnancies and in cycles of artificial reproductive techniques [8, 9]. Idiopathic infertile men show altered semen quality without any apparent cause of infertility, and recently, it has been reported that in 80 percent of cases, the cause could be referred to increasing levels of OS [11]. On the other hand, the percentage of men affected by unexplained male infertility is more uncertain and it could be between 10–30 percent [12]. Varicocele patients show higher ROS and decreased antioxidants levels, as well as increased SDF [10].

14.4 Tests Used to Measure Oxidative Stress

Seminal OS can be measured by both direct and indirect tests (Table 14.1) [13]. Direct tests measure the concentration of oxidant molecules, while indirect tests measure the concentration of antioxidants or analyzes the ROS-induced damage on cellular components, such as DNA, proteins and lipids. Currently, there is no “gold standard” test for the evaluation of seminal OS. Each test has its own advantages and disadvantages (Table 14.2). In this chapter, we focus on direct tests used for the measurement of OS. Direct laboratory tests include measurement of ROS by chemiluminescence method, nitro blue tetrazolium (NBT) assay, cytochrome C reduction test, electron spin resonance technique and oxidation-reduction potential (ORP).

14.5 Reactive Oxygen Species Measurement by Chemiluminescence Method

14.5.2 Protocol

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  Reagent – Luminol: 100 mM stock solution in dimethyl sulfoxide (DMSO). A 5 mM working solution should be prepared fresh and stored at room temperature. Since luminol is light-sensitive, tubes need to be covered with aluminum foil.

  
  
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  Sample preparation – Blank (400 µL PBS), a negative (400 µL PBS + 10 µL Luminol) and a positive (400 µL PBS + 10 µL Luminol + 50 µL H₂O₂) controls are included. The liquefied semen samples (400 µL) are mixed with luminol (10 µL) (Figure 14.2A).

  
  
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  ROS determination – The analysis is conducted using a luminometer (Figure 14.2B). The instrument measures sample light output and it consists of: a) a sample chamber, which holds a test tube, microplate, or other type of sample container; b) detection device, which can be photodiodes and photomultiplier tubes; c) signal processing method; d) signal output display. The test is run in triplicate.

Figure 14.2 A) Processing of sample for ROS analysis by chemiluminescence assay; B) external view of the luminometer connected to the monitor for real-time display of ROS production on the monitor screen [13].

14.5.3 Interpretation of Results

The chemiluminescent signals measured by a luminometer are expressed in relative light units (RLU). The average RLU is calculated for both the analyzed tests and controls tubes. The results for ROS samples are obtained by subtracting the average RLU of negative control, and after normalization for sperm concentration/mL (Figure 14.3).

Figure 14.3 ROS calculation. RLU, relative light units; ROS, reactive oxygen species.

14.5.3.1 Reference Value

Reference values are obtained from the analysis of a large cohort of patient samples. Agarwal et al. analyzed 258 infertile men and 92 controls and suggested a cut-off of <102.2 RLU/s/10⁶ sperm/mL to discriminate between fertile and infertile men [16].

14.6 Nitroblue Tetrazolium

14.6.1 Principle

Nitroblue Tetrazolium (NBT) (2,2’-bis (4-nitrophenyl)-5,5’-diphenyl-3,3’-(3,3’-dimethoxy-4,4’-diphenylene) ditetrazolium chloride) is a yellow water-soluble molecule which is reduced by superoxide and NADPH oxidase to water-insoluble formazan crystals [19] and allows the determination of cytoplasmic ROS. In the sperm cytoplasm, the hexose monophosphate pathway is responsible for the synthesis of NADPH by means of glucose-6-phosphate dehydrogenase. The NADPH contributes to the synthesis of superoxide anions by NADPH oxidase. The same enzyme in turn catalyzes the reduction of NBT into formazan and indirectly provides the measure of ROS generation in cytoplasm. The reduced formazan is bright purple-blue colored, and it is easily detected microscopically or spectrophotometrically (Figure 14.4) [20].

Figure 14.4 Microscopic evaluation of formazan precipitates in sperm [20].

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