Chapter 14 – Oxidative Stress Testing: Direct Tests


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).

Chapter 14 Oxidative Stress Testing: Direct Tests

Renata Finelli , Manesh Kumar Panner Selvam , Ashok Agarwal

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:

H2O2 + Fe2+ → Fe3+ + OH + OH•

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 [57]. 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).

Table 14.1 Direct and Indirect Tests for the Evaluation of Oxidative Stress

Direct tests Indirect tests
Chemiluminescence Myeloperoxidase or Endtz test
Nitroblue tetrazolium (NBT) Lipid peroxidation levels
Cytochrome c reduction test Chemokines and Interleukins
Electron spin resonance Antioxidants, micronutrients, vitamins (vitamin E, vitamin C)
Oxidation-Reduction Potential (ORP) Total antioxidant capacity – TAC
DNA damage

Table 14.2 Direct Tests Used to Evaluate Seminal Oxidative Stress: Principle, Advantages and Disadvantages.

Tests Principle Advantages Disadvantages
Chemiluminescence Use of fluorescent probes (luminol and lucigenin) • High sensitivity and specificity • Time consuming

• Large volume of sample required (800 µL)

• Probes are highly sensitive to light

• Frozen, azoospermic and hyperviscous samples cannot be analyzed
Nitroblue tetrazolium (NBT) NBT reaction by superoxide and NADPH oxidase to formazan • Whole ejaculates, washed sperm or leukocytes can be tested

• User friendly, fast and inexpensive

• Able to detect neutrophil concentration ≥ 0.5 x 106/mL
• Non-specific signal due to unspecific NBT reduction

• Interpretation of result is subjective
Cytochrome c reduction test Colorimetric assay based on the reduction of cyt c by NADPH-Cytochrome c reductase • Suitable test to quantify extracellular O2

• Good for high level of ROS production
• Cannot detect NADPH oxidase activity

• Cannot detect low concentrations or intracellular O2
Electron spin resonance Magnetic resonance spectroscopy • Good for high level of ROS production • Inference factors leading to possible neutralization of the spin-trap
Flow Cytometry detection of intracellular ROS Use of fluorescent probes (DCFDA and DHE) • Fast, accurate and the assay is reproducible

• Applicable to oligozoospermic samples
• Expensive instrument is required
Oxidation reduction potential (ORP) Measurement of redox potential by galvanostat-based instrument • User friendly device

• Fast and results are reliable

• Less quantity of sample (30 µL) required

• Assay can be performed on both fresh and frozen samples
Not suitable for azoospermic patients or high viscosity/poorly liquefied samples

14.5 Reactive Oxygen Species Measurement by Chemiluminescence Method

14.5.1 Principle

Chemiluminescence is the most widely used direct test for quantification of ROS in semen. The chemiluminescence is a phenomenon characterized by emission of light as a result of a chemical reaction [14, 15]. In this assay, fluorescent probes are used to investigate different ROS (including O2.-, H2O2, OH.) at the same time. Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) is a yellow-colored, membrane-permeable cyclic diacylhydrazide used to detect both, intra- and extracellular ROS. It cannot be used to differentiate between different types of oxidants and cannot be used in association with strong acids/bases or reductive agents. On the other hand, lucigenin (10,10’-dimethyl-9,9’-biacridiniumdinitrate) is membrane-impermeable, used to measure the extracellular O2. Both luminol and lucigenin form an unstable endoperoxide and dioxetane after being oxidized and reduced, respectively, followed by a rapid decomposition and emission of photons [14, 15].

14.5.2 Protocol

  • 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.

  • 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).

  • 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. 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].

14.5.4 Advantages/Disadvantages of the Test

Chemiluminescence methods for the ROS detection present many advantages. Some luminometers calculate the results in the integrated mode, with high sensitivity (76.4 percent) and specificity (53.3 percent). Modern luminometers are equipped with a user-friendly software, to help in the analysis and interpretation of results [16]. On the other hand, frozen, azoospermic, hyperviscous or poor liquefied semen samples cannot be analyzed with this technique and this represents a limitation of the technique. Moreover, the instrument is expensive, the assay is time-consuming and a minimum of 800 µL of semen is required to carry it out in duplicates. In addition, probes are light-sensitive, and many other factors such as variation in pH, centrifugation of the samples and the presence of other molecules (e.g. NADPH, cysteine, ascorbic acid or uric acid) can enhance or decrease chemiluminescent signals even in the absence of spermatozoa [1315].

14.5.5 Clinical Significance

High concentration of ROS is produced by morphologically abnormal sperm with residual cytoplasm. OS has been associated with lipid peroxidation and sperm DNA damage [17, 18]. Therefore, ROS analysis by chemiluminescence assay can provide information about the quality of spermatogenetic process and the sperm fertilizing potential [15].

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].

14.6.2 Protocol

  • Reagents – NBT is usually provided as a powder, dissolved in PBS at concentration of 0.01–0.1 percent.

  • NBT assay – There are several assay kits in commerce for performing NBT assay. The current protocol is based on the instructions provided by Oxisperm® kit (Halotech® DNA, Madrid, Spain). Semen samples are incubated in equal volume of NBT solution and the suspension allowed to gel at 37°C for 45 minutes. The color of solution is then compared with the color scheme provided by the kit.

  • NBT analysis – Alternatively, a spectrophotometer or a microplate reader can be used to quantify the resulting color reaction at wavelengths of 530–630 nm. Moreover, a light microscope (100× magnification) can be used to analyze the formazan staining in leukocytes and sperm on air-dried smears as reported in Esfandiari et al [21].

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Apr 6, 2021 | Posted by in GENERAL & FAMILY MEDICINE | Comments Off on Chapter 14 – Oxidative Stress Testing: Direct Tests

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