Oxidative Stress and Male Subfertility: Recent Detection Methodologies (2025)

1.1 Free radicals and oxidative stress

Oxygen is often referred to as “Janus gas,” the two-faced ancient Greco-Roman god, as it exhibits both positive benefits and potentially harmful side effects on biological systems. Within the body, oxygen is involved, among others, in the structure of various molecules, known as “reactive oxygen species” (ROS). ROS are oxygen-negative ions (Ο₂⁻², Ο₂⁻) or more complex ions with other chemical elements (OH⁻, RO⁻, ROO⁻). All above components include also electrically neutral organic compounds (i.e., ROOH, HOCl). ROS are a key component of oxidative stress and are related to a group of free radicals and neutral molecules that oxidize macromolecules in the organism [1]. Oxidative radical species also include nitrogen molecules (NO, NO₂, HNO₄) and nitrogen derivatives (ozone), which are called reactive nitrogen species (RNS). Recently, sulfur ions (RSR⁻, GSSG⁻) as well as sulfur neutral compounds (SO₂, SO₃) have also been found to belong in the above group, called reactive sulfur species (RSS). Inside an organism, free radicals are converted, under appropriate conditions, into the corresponding neutral compounds. Thus, the term reactive species (RS) includes all ROS, RNS, and RSS, while the term free radicals (FR) includes all the negative ions of ROS, RNS, and RSS (Table 1).

Depending on their concentration, the RS act either beneficially or harmfully within the organism. RS are produced in all body cells and take part in key pathways of cellular metabolism. In general, FR (Table 1, part 1) act via their charge bind to counter ions of various macromolecules of the organism, thus substantially influencing their function. Their conversion to the electrically neutral compounds (Table 1, part 2) inhibits their function, but they can be converted back to free radicals if specific chemical conditions promote the conversion. The importance of RSS in the overall oxidative stress activity was first reported in 2001 and documented relatively recently. RSS are formed by the sulfur effect on ROS and depending on conditions can exhibit oxidative or reductive activity on proteins [4].

The general public knows RS mainly for their pathological activity, since they attack transmembrane cellular proteins, lipids, and DNA molecules. The harmful oxidative “attack” of ROS/RSS/RNS molecules on the organism macromolecules is characterized as “oxidative distress.” In contrast, the beneficial effect of the active compounds is called “oxidative eustress.” Their high concentration and chronic action leads to oxidative stress, which plays a central and promoting role in the pathophysiology of many different disorders/diseases, including complications of pregnancy, as well as, a decrease in male fertility (Table 2) [7]. For instance, mitochondrial ROS are involved in high-energy electron transfers and support the production of large amounts of adenosine-5-triphosphate (ATP) via oxidative phosphorylation. In addition to the production of ATP molecules, which provide the necessary energy to the body, ROS have a beneficial role in the body’s defense against microorganisms and in cell signaling [8]. ROS/RSS/RNS produced by metabolic processes and the human environment (diet, pollutants, etc.) are also an important source of ROS (Table 3).

1.2 RS action in the body

FR were discovered in 1900 [10], while their oxidizing activity was discovered many years later [11]. FR’s strong activity is considered incompatible with life, but in the 1950s, it was shown that FRs are also present in biological systems [12]. In 1985, Sies formulated the first definition of oxidative stress [13]. Numerous scientific papers followed concerning its impact on various biological systems, including male and female reproductive organs.

As already stated, the normal concentration of RS has a beneficial role in cell signaling, apoptosis, gene expression, and ion transport in the cells of the organism. On the contrary, high RS have a negative binding effect on very crucial macromolecules. The most important action of RS is redox signaling through post-translational modifications. The main redox signaling agents are hydrogen peroxide (H₂O₂) and superoxide anion radical (O₂⁻⁻), which are under the control of growth factors, cytokines, and enzymes (mainly NADPH oxidases and mitochondrial electron transport chain enzymes) [14]. Among all RS, the most active is H₂O_2, acting in concentrations of nmole. Nitric oxide, hydrogen sulfide, and so on are also involved in redox signaling. Recent methodological developments have allowed the evaluation of molecular interactions of specific RS molecules with specific targets in redox signaling pathways, as shown in Figure 1.

Picture from “Commons Wikipedia” with license Creative Commons, created by Dan Cojocari. https://commons.wikimedia.org/w/index.php?curid=46529393

RS and organism macromolecule bonding are controlled by the body’s and external natural antioxidants, such as vitamins C, E, polyphenols, uric acid, various enzymes, and so on. Natural and artificial antioxidants can be got by common foods or food supplements (Table 4). There are two ways to distinguish the antioxidants [15]. The first is according to their function:

• Enzymatic antioxidants (superoxide dismutase, catalase [CAT], glutathione peroxidase (GPX)),

• Nonenzymatic antioxidants (glutathione (GSH), vitamins C, E, ubiquinones, carotenoids, β-carotene, lycopene, polyphenols, uric acid, and mineral antioxidants: zinc, copper, selenium).

The second is according to its line of defense:

• First line: the enzymes GSH, SOD, CAT,

• Second line: the vitamins A, C, D, E,

• Third line: carotenoids, the bioflavonoid, and coenzyme Q10.

The function of antioxidants can:

• Neutralize free radicals by exchanging electrons with them,

• Convert free radicals into other, less active RS (Table 2, Part 2),

• Act in combination, until they are completely inactivated [2].

At present, the role of all oxidants in human physiology and in diseases of various systems such as cardiovascular, immune system, skeletal muscles, metabolic regulation, aging, cancer, and reproduction is well established. Furthermore, oxidative stress is not the only cause of DNA damage. Long time before the role of RS in DNA damage was highlighted, the harmful effects of radiation and exogenous chemicals were already known. Once DNA damage is done by any cause, metabolic pathways are similar [16].

2.1 Trace elements which influence oxidative stress

Trace elements are defined as specific inorganic elements involved in the body’s metabolic processes in very small quantities. They are classified as essential and non-essential. Essential trace elements are absolutely necessary for the body’s functions. Some of them (Ca, Na, K, Mg, Mn, Zn, Cu, Se) play an important role in human reproduction (Figure 3). Their deficiency reduces male fertility since it impairs the normal function of spermatogenesis (Table 8). The spermatogenesis decrease can be direct (deterioration of parameters of basic semen analysis) (Table 7) or indirect (decrease in steroid/testosterone production). Three of these trace elements affect male fertility by increasing or decreasing oxidative stress. These are selenium (Se), zinc (Zn), and copper (Cu) whose deficiency increases oxidative stress [25]. In Figure 3, Tables 6 and 8offer details regarding chemical element contribution in male reproduction and sources of trace elements.

3.1 Measurement of oxidative stress in seminal fluid with commercial kits

One of the commercial methods to determine oxidative stress in semen is the Oxisperm method. The Oxisperm kit is produced by Halotech DNA and has IVD certification according to EC Directive 746/2017.

The method is based on the ability of water-soluble nitro blue tetrazolium (NBT) to be converted under the influence of peroxide anions (O₂⁻²) of oxidative stress into water-soluble blue crystals, known as formazan. Formazan crystals are visible in the cell membranes of spermatozoa. They can be observed by microscopy at 400X magnification under a bright field of view [45]. In addition, formazan crystals produce a characteristic color effect when reacted with agarose gel (Reactive Gel), which varies from yellow to purple-blue. The intensity of the color is proportional to the level of oxidative stress (Figure 4). The assessment of the color intensity can be done either visually or by measuring the absorbance of the color in a photometer at a wavelength of 400–600nm.

A different approach to assessing oxidative stress has been proposed by BRED Life Science Technology, which assesses the effect of ROS on sperm through the effect of superoxide anions on sperm mitochondria. According to this method, the mitochondria of living spermatozoa that have been exposed to the effect of superoxide anions are labeled differently from the rest of the spermatozoa (Figure 5). The result can be seen by flow cytometry. The advantage of this method is that the determination of oxidative stress is carried out in living spermatozoa. However, special analytical equipment (flow cytometry analyzer) is required [46].

3.2 Determination of DNA fragmentation index

DNA Fragmentation Index (DFI) expresses the percentage of spermatozoa that retain their DNA intact. In healthy men, there is a percentage of spermatozoa (<15%) where their DNA is damaged even though the spermatozoa retain good morphology and/or other microscopic characteristics. Oxidative stress increases DFI % by more than 15%. The high values of DFI decrease male fertility.

The reasons of high values of RS are the main indications for the measurement of DFI in infertile men, who have/are:

• history of multiple miscarriages with their partner,

• unexplained infertility for more than 6months from both male and female factors,

• over 40years old,

• smoking habits,

• obesity,

• history of cancer,

• medications for various reasons,

• exposed to toxic agents or long-term exposure to high temperatures,

• urogenital infection,

• failed to develop healthy embryos in IVF cycles.

A common method for the determination of DFI is Sperm Chromatin Dispersion (SCD) [51, 52]. The principle of the method includes the fixing of spermatozoa in an agarose gel and then on a slide. The preparation is then treated with a denaturing agent to decompose the fragmented DNA. Lysis solution is then added to remove the cytoplasm of the spermatozoa. The remaining nuclear material is then stained. The level of DNA fragmentation is determined by the halo observed at the head of the spermatozoa. Spermatozoa with high DNA fragmentation have no halo or minimal halo, whereas normal spermatozoa release DNA chains and create a moderate to large diameter halo (Figure 6) [53].

3.3 The determination of sperm apoptosis

Apoptosis (cell death) is another consequence of oxidative stress. A common method to determine apoptosis is TUNEL [38]. TUNEL stands for “terminal deoxynucleotidyl transferase dUTP nick end labeling.” In the TUNEL method, fragments of sperm DNA are detected after being initially labeled with deoxyuridine triphosphate (dUTP). This is followed by binding to the final tracer, which can be either fluorochrome (Figure 7) or biotin-streptavidin-peroxidase (HRP) complexes that react with a chromogenic substrate of HRP. TUNEL is capable of directly assessing both single-strand and double-strand breaks, so the more DNA strand fragments present, the greater the cell labeling [54].