Oxidative Stress and Male Fertility: Promising Role of Nutraceuticals

Abstract

Oxidative stress is a key detrimental factor in male infertility under pathological or physiological conditions. A balanced oxidation-reduction process regulates the various functions linked to male fertility; however, oxidative stress leads to temporary infertility by affecting the hormonal pattern, sexual behavior, testicular milieu, functioning of accessory sex glands, and sperm quality. Currently, nutraceuticals are a common and popular way to mitigate the male fertility issues of pre-testicular, testicular, and post-testicular etiologies. Nutraceuticals possess multi-nutritional factors that improve metabolic activity, regulating hormonal profile, and sperm production. In addition, the antioxidant property of nutraceuticals agents combats oxidative stress, thus improving the hormonal release pattern, sexual behavior, testicular environment, and sperm quality in males.

1. Introduction

The incidences of male infertility have been rapidly increased during the last five decades perhaps due to the industrialization and mechanization. The rising surge in male infertility is multifactorial and could be linked to inevitable conditions such as congenital assaults (cryptorchidism), infections, age, varicocele, etc. or environmental contaminants, obesity, use of recreational drugs, occupational hazards, and irrational use of antimicrobial drugs [1]. The extent of infertility varies from region to region depending on the extensive industrialization, high mechanization, frequency of exposure to chemicals or radiation, and less physical activity [2]. Infertility is observed at pre-testicular, testicular, or post-testicular levels due to the mechanism of action of each influencing factor. These physiological or pathological abnormalities result in alteration in the activity of the hypothalamus, pituitary, or gonads is probably linked to the production of free radicals, which are manifested in the form of low libido, erectile dysfunction, debilitated accessory sex glands, impaired spermatogenesis, and sperm functions [3]. The onset of these conditions is chronic rather than acute states, which originated maximally due to oxidative stress. Oxidative stress is linked to physiological and pathological conditions, and its prevention or treatment is also long-term concerning its occurrence [4].

The nutrition is essential to body health since a healthy and balanced diet prevents the occurrence or cure different disease conditions. The use of nutraceuticals as potential ailments for male infertility has been recognized in the old world for centuries [5]. Every passing day, a new compound is being included in the medicinal list with its bioactive ingredients or antioxidant potential. With the understanding of oxidative damage to sexual behavior, reduced spermatogenesis, and low sperm quality, numerous nutraceuticals have been focused on their antioxidants potential for enhancing male fertility [6, 7]. However, utilizing a single agent with multiple properties could be better option for infertile males. On the other hand, patients are unaware about the potential effect of any therapeutic agent on the hypothalamus, pituitary, testes, or extra-testicular parts. This chapter proposed an overview of the underlying mechanism of pros and cons of oxidative stress on the male reproductive system and countering effects of nutraceuticals with antioxidant potential against pre-testicular, testicular, and post-testicular etiologies.

1.1 What is male fertility? Definitions

Male reproductive health is important for the maintenance of sexual activities including libido, optimum sperm production, copulation, and fertilizing ability during sexual life. All these processes are necessary for maintaining the possible genetic potential and progeny. Previously, the male reproductive health has been defined in different ways.

Generally, the male fertility in humans is defined as the ability of a person to impregnate a woman and produce offspring.

• The American Society for Reproductive Medicine defines male fertility as “the capacity of a man to produce normal sperm in sufficient quantities and with appropriate motility and morphology to result in pregnancy under normal conditions of sexual intercourse.”

• The World Health Organization defines male fertility as “the ability of a man to achieve a pregnancy in a fertile female partner.”

• The National Institute of Child Health and Human Development defines male fertility as “the ability of a man to cause pregnancy in a fertile female partner.”

In animals, the term fertility is being used to describe the ability of domestic animals to reproduce and produce viable offspring.

• In general, male fertility is defined as the ability of male animals to produce healthy and viable sperm that can fertilize female animals and result in the production of viable offspring.

All mentioned definitions emphasize the basic theme that male fertility is the ability of a male to contribute to a successful pregnancy in a fertile female of the same species.

1.2 What is male infertility? In light of the organizational perspective

Deviation from the normal male fertility process leads to the abnormal state known as “infertility” or “subfertility.” However, different tangible and intangible factors could influence the condition. Infertility could also be defined in many ways in humans and animals. Here are a few different definitions of male infertility:

• “The inability of a man to achieve a pregnancy in a fertile female after 12 months or more of regular unprotected sexual intercourse” (The World Health Organization).

• “The inability of a man to achieve a pregnancy in a fertile female after one year of regular unprotected intercourse, or the inability to produce a pregnancy after surgical, medical, or behavioral intervention” (The American Society for Reproductive Medicine).

• “The inability of a man to cause pregnancy in a fertile female partner” (The National Institute of Child Health and Human Development).

• “The inability of a man to achieve conception or to induce conception in a fertile female partner due to quantitative and/or qualitative deficiencies of his spermatozoa” (The European Society of Human Reproduction and Embryology).

In the case of animals, it is defined as:

• “The inability of a male animal to produce viable offspring due to abnormalities or dysfunction in its reproductive system.”

These definitions highlight a condition, where a male cannot contribute to a successful pregnancy with a fertile female partner. However, the variations in terms might be linked to the diagnosis process, the specific criteria for infertility, or other factors that result in male infertility.

2. Oxidation-reduction process in males

Oxidation-reduction reactions, also known as redox reactions, which are key determinants in male reproduction, alike many biological processes. Generally, redox reactions involve the transfer of electrons between molecules and result in the molecules’ oxidation state.

The redox reactions are involved in male reproduction through:

• Regulation of hormone levels

• Production of quality sperm

• Regulation of sperm maturation

• Promoting acrosome reaction for fertilization

• Prevent the sperm DNA

• Maintain the sperm mitochondrial potential

During sperm maturation, glucose-6-phosphate dehydrogenase protects the developing sperm from oxidative stress. This enzyme reduces the reactive oxygen species (ROS) by catalyzing the first step in the pentose phosphate pathway to produce a reducing agent, i.e., NADPH. In mature sperm, glutathione plays a vital role as an antioxidant and protects sperm cells from oxidative stress by forming the disulfide bonds in the tail region of sperm for motility.

2.1 Oxidative stress: story in male infertility

The oxidative stress is an imbalance between the production of ROS (by cellular respiration and catalytic responses of cytochrome steroidogenic enzymes P450) and the body’s antioxidant levels. It reacts and affects the lipids, proteins, and DNA that affect the biological process and lead to cellular affections. Oxidative stress is involved in the pathogenesis of diseases and the non-pathogenesis of clinical conditions. ROS production results from metabolic rate, polymorphisms or mutations in mitochondrial and nuclear DNA, lowered antioxidant production and repair rate, and metal ions and other toxins that affect the oxidation process [8].

Oxidative stress has been defined by different organizations as follows:

• “An imbalance between the production of free radicals (reactive oxygen and nitrogen species) and the ability of the body to counteract or detoxify their harmful effects through neutralization by antioxidants” (National Institute of Environmental Health Sciences).

• “A disturbance in the pro-oxidant-antioxidant balance in favor of the former, leading to potential damage” (The International Union of Biochemistry and Molecular Biology).

• “A condition that arises when the rate of production of reactive oxygen species (ROS) exceeds the rate of their elimination by antioxidant defenses, resulting in damage to cellular macromolecules and organelles” (The American Society for Biochemistry and Molecular Biology).

2.2 Oxidative stress and male behavior

2.2.1 Erectile dysfunction

The oxidative stress alters the morphology of blood vessels and associated nerves for erectile function, which further decreases the bioavailability of nitric oxide (NO) and induces mitochondrial dysfunction, inflammation, and endothelial NO synthase uncoupling. These alterations develop endothelial dysfunction by increasing the adhesion of monocytes to endothelial cells, impairing the angiogenic potential of endothelial cells, and inducing apoptosis. In addition, ROS-induced mitochondrial dysfunction contributes to the additional ROS production by altering mitochondrial metabolism, leading to the exacerbation of endothelial dysfunction [9, 10].

Oxidative stress has been linked with erectile dysfunction due to the excessive generation of free radicals in the cavernosal tissues. Superoxide combines with NO to form highly toxic peroxynitrite that induces lipid peroxidation and damages endothelial cells of penile vessels and cavernosal tissues. Recent trends in the management of erectile dysfunction involve increased NO levels using arginase inhibitors. Because in erectile dysfunction, there are elevated levels of arginase activity, which limits NO synthase activity, reduces NO biosynthesis, and increases arginine degradation [11]. The upregulation of the RhoA/ROCK pathway under oxidative stress damages corpus cavernosum smooth muscle function resulting in erectile dysfunction [12].

2.2.2 Reduced libido

During the process of steroidogenesis and spermatogenesis, normal level of ROS is produced by mitochondrial respiration and catalytic responses of cytochrome steroidogenic enzymes P450. The systemic hormones (FSH, LH, testosterone, E2, PRL) also regulate the total antioxidant capacity in animals. Testosterone and melatonin have a strong antioxidant capacity and protect sperm and testosterone-producing cells against damage by ROS. Damages to Leydig cells and the hypothalamic-pituitary-gonadal (HPG) axis due to excessive ROS decreases testosterone production. However, under oxidative stress condition, the hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-thyroid (HPT) axes are activated by the excessive production of ROS and stimulate the cortisol hormone and down-regulate the T3 and T4 synthesis. This hormone negatively affects the anterior pituitary gland through crosstalk between HPG and HPA axes and, finally, decreases the secretion of LH and FSH hormones. This lower level of LH, it is not enough to stimulate Leydig cells for testosterone production. This halted testosterone production altering the sialic acid levels in Sertoli and Leydig cells, decreasing the release of androgen-binding protein (ABP) to Sertoli cells, reducing T3 decreases StAR mRNA levels in Leydig cells, and increasing aromatase activity for testicular E2 hormones. Moreover, the ROS-induced alteration in Sertoli cells is associated with reduced hormonal secretions, blockage of gap junction communications, and damage to the blood-testis barrier. Increased oxidative stress also stimulates proinflammatory cytokines due to genital tract infection (tumor necrosis factor-α, interleukin-1β, and interleukin-6), which further harms the HPG axis and testosterone biosynthesis. ROS under obese conditions forcibly drives the adipocytes to secrete leptin along with insulin, negatively affecting the T3 and testosterone synthesis. Hence, ROS leads to male infertility by disrupting reproductive endocrine glands [8], and lower testosterone level “main regulator reproductive behavior” may suppress sexual activities in male.

2.2.3 Impaired sperm transport

Epididymis has an important role in sperm transportation, maturation, and storage. In addition, the epididymis provides essential proteins to spermatozoa via epididymosomes to maintain cellular functions and protect them from potential damages such as oxidative stress-dependent injuries. The peroxiredoxins (PRDXs) are a family of antioxidant enzymes highly expressed in yeast to humans. They are peroxidases that do not require cofactors such as heme group or selenium and contain one or two cysteines (Cys) residues in their active site, which are essential for their antioxidant function. PRDXs isoforms are divided into 2-Cys PRDXs (PRDX1-4), atypical PRDX (PRDX5), and 1-Cys PRDX (PRDX6). These enzymes are important antioxidants in spermatozoa that regulate the level of ROS, such as peroxides (H₂O₂ and organic hydroperoxides) and peroxynitrite (ONOO−), to avoid cellular toxicity [13].

The metabolic processes occurring within the epididymal tissue and the absorptive and secretory activities of epididymal epithelium, which provide an optimum internal milieu for sperm maturation, are regulated by androgens. Androgen deprivation leading to apoptosis in epididymal epithelium has been demonstrated [14]. Sialic acid, secreted by the principle cell, not only confers a negative charge to the sperm surface but also acts as a decapacitation factor protecting the spermatozoa from premature capacitation. Hence, sialic acid is essential for maturation and maintenance of plasma membrane structural integrity of spermatozoa in epididymis [15].

2.3 Oxidative stress and sperm production

Notwithstanding the antioxidant protection afforded to the testes to support its dual functions of steroidogenesis and sperm production, a wide variety of endogenous and exogenous factors are known to perturb these defenses and generate oxidative stress. Here are a few ways that oxidative stress can affect sperm production:

2.3.1 Reduced spermatogenesis

The diet, alcohol consumption, less physical activity, heat, radiation, chemical exposure, pollution, smoking, varicocele operation, and trauma influence sperm parameters and impair spermatogenesis [16]. Such changes are mostly linked to the high susceptibility of the unsaturated fatty acid content of sperm to oxidative stress. The lipid oxidation initiates the ROS that attacks the double bonds of an unsaturated fatty acid of the plasma membrane. Firstly, the ROS reacts with the adjacent lipid molecule triggering a chain reaction. By-products of lipid oxidation include mutagenic and genotoxic molecules malondialdehyde (MDA) and 4 hydroxy-nonenal (4-HNE) that cause the DNA damage. Secondly, DNA damage occurs due to free radical-initiated apoptosis, leading to caspase-mediated destruction of DNA [17, 18]. The molecular aspects in male germ cells, the epigenetic modification of the imprinting of paternal genes, DNA compaction, and silencing of the post-meiotic gene are necessary steps for spermatogenesis. However, altered methylation and acetylation of histone proteins H3 and H4 at specific lysine residues impair spermatogenesis [19].

2.3.2 Increased germ cell apoptosis

In general, high levels of ROS production induce apoptosis in the germ cells and clear the nonviable sperm from the testes. Under pathological conditions, a massive germ cell apoptosis affects the spermatocytes frequently, spermatogonia minutely, and spermatids rarely. The Sertoli cells are less prone to apoptosis as compared to the germ cells. The Fas-system, which is expressed in Sertoli cells, is responsible catering apoptosis in germ cells. In healthy males, the Sertoli cells express FasL, which triggers the apoptosis in Fas-positive germ cells in a paracrine manner among germ and Sertoli cells. The Bcl-2 (Bcl-2 and Bax) and caspase (caspase-3/-8/-9/-12) family members control the mitochondrial apoptosis process. An imbalance of the Bax and Bcl-2 ratio and mitochondrial potential depolarization activates the caspase-9 process and triggers apoptosis [20, 21].

2.3.3 Loss of Leydig cell function

The production of testosterone is major function of the Leydig cell, and is affected by several ways under increased ROS production. Firstly, the ROS affects the Leydig cell function by reacting with Leydig cell DNA and oxidize the DNA leads to DNA strands breakage and mutations. Secondly, Increased ROS is involved in oxidation of unsaturated fatty acids of Leydig cell plasma membrane, which further promote the ROS production. Thirdly, the depletion of antioxidants made the Leydig cells more prone to accumulated higher ROS levels. Lastly, provoking the release of cytokines by increased ROS levels influence the Leydig cell physiology. Under low testosterone conditions, the Leydig cells’ activity is hampered, and apoptosis occurs by stimulating caspase activity and DNA damage of Leydig cells. Altered testosterone and estradiol levels affect the availability of LH receptors on Leydig cells, changing Sertoli cells’ functionality for germ cell survival, and seminiferous tubular maturity [21].

2.3.4 Impaired testicular blood flow

Like other deleterious consequences of oxidative stress to the vascular system, impairment of testicular blood vessels minimizes the blood flow and oxygen delivery to the testes, and negatively impacting sperm production. Under pathological conditions of testicular torsion, testicular ischemia develops, and hence oxidative stress occurs in the testicles of the same side. Then the NO and H₂O₂ production increases leading to lipids peroxidation. The accumulation of isoprostane and decrease in antioxidant enzymes increase the apoptosis. Short periods of ischaemic conditions can also develop testicular oxidative stress, decreasing testicular antioxidants with reduced spermatogenesis [22]. During testicular varicose, endothelial NO synthase (eNOS) production increased, and blood flow toward testicular tissues also increased to compensate for the hypoxic condition due to venous stagnation. Increased NO concentrations react with superoxide free radicals and produce the reactive nitrogen species (peroxynitrite and peroxynitrous acid) that impair fertility. Moreover, increased leptin receptors (glial cell line-derived neurotrophic factor receptor-1 and voltage-dependent calcium channels) are also predisposing factors for oxidative stress under testicular varicose [23].

2.4 Oxidative stress and epididymis function

Compromised epididymal functions (removal of excess cytoplasm and acquiring motility and fertilization potential) due to oxidative stress is a leading cause of male infertility. The sperm maturation, storage, and transportation are down-expressed under oxidative stress conditions because epididymis is highly sensitive to oxidative stress due to the high levels of polyunsaturated fatty acids in its membranes and the presence of ROS-generating enzymes. The following lines show how oxidative stress can affect epididymis function:

2.4.1 Impairment in sperm maturation

In the epididymis, increased expression of antioxidant enzymes reduces oxidative damage to sperm through the secretion of epididymosomes. The high 4-HNE levels in caput and cauda epididymis result in lipid peroxidation and epididymal epithelium degeneration by oxidative stress and hamper sperm maturation. The increased production of PRDX6 and PRDX1 seems insufficient under oxidative stress to quench excessive ROS and maintain a conducive cellular environment for sperm maturation [13]. Increased production of immature sperm and increased ROS levels that severely affect epithelial cells of the epididymis. High ROS generation or failure of antioxidant systems provoke the eukaryotic cells to combat the deleterious by-products of redox that result in the oxidative injury of sperm.

2.4.2 Sperm DNA damage

The sperm at the testicular level are highly prone to ROS and induce DNA fragmentation in sperm. DNA fragmentation is relatively low during epididymal transit due to high ROS production. In the epididymis environment, sperm nuclear condensation and oxidation are interlinked processes. The DNA compaction is completed by creating inter- and intramolecular cross-links between nuclear protamines. For bridging sperm protamines, a balanced epididymis ROS and enzymes (protein disulfide isomerase and glutathione peroxidase) are required. Excessive ROS generation by immature sperm in the epididymis not only affects the sperm membranes but also multiplies ROS production that transiently increases sperm nuclear condensation and quickly reverses the DNA fragmentation due to nuclear decondensation and oxidative-induced DNA fragmentation [24].

This oxidative damage increases DNA fragmentation and lipid peroxidation in epididymal cells. In the epididymis, ROS oxidizes the guanosine to 8-deoxy-2′hydroxy-guanosine up-regulates the DNA mutation and damage to the paternal genome, leading to male infertility [25]. The disruption of molecular mechanisms driven by miRNAs or epigenetic changes can induce permanent sperm DNA damage in the epididymal climate [13].

2.4.3 Disrupted epididymal fluid composition

Oxidative stress alters the epididymal fluid composition and negatively impacts sperm function and viability. Induction of oxidative stress creates a disturbance of epididymal physiology, alterations in ion and fluid transporters within the epididymis, and abnormal water reabsorption result in significant changes in the luminal fluid composition [14, 26].

Obesity change in the epididymis’s microenvironment by increasing MDA expression in epididymal fluids with low glutathione levels that impair sperm quality. Epididymosomes secreted in the epididymal lumen communicates with the sperm by sharing the protein and noncoding RNA contents, lowering oxidative stress levels [27].

2.4.4 Inflammation in epididymis

The inflammation in the epididymis induces ROS that damages the tissue and impairs epididymal function. Although the presence of proinflammatory cytokines, tumor necrosis factor-alpha (TNF-α), interleukin-1 alpha (IL-1α), and interleukin-1 beta (IL-1β) cytokines in the epididymis are involved in certain physiological events, the increased TNF levels influence sperm motility, viability, and DNA fragmentation [28, 29].

2.5 Oxidative stress and accessory sex glands

Accessory sex glands (prostate gland, seminal vesicles, and bulbourethral gland glands) secrete the seminal plasma of the ejaculate that provides nutrients and protection to sperms. The seminal plasma contains enzymatic (superoxide dismutase; SOD, catalase; CAT, glutathione peroxidase; GPx) and nonenzymatic antioxidants (vitamins C and E, hypotaurine, taurine, uric acid, albumin, etc.). Among these enzymes, the SOD predominantly protects sperm against oxidative stress induced by NADPH treatment [30]. Oxidative stress affects the function of the prostate gland, seminal vesicles, and bulbourethral gland glands. The following lines indicate the deleterious effects of oxidative stress on accessory sex glands:

2.5.1 Altered seminal fluid production and composition

Increased ROS level damage the cells of accessory sex glands and affecting the production and composition of seminal fluid. Altered pH, ion concentration, and nutrient content in response to increased ROS negatively affect sperm function and viability. Seminal fluid hyperviscosity due to oxidative stress leads to low seminal plasma fructose, ascorbic acid, calcium, and zinc levels, which indicate low glandular activity. Changes in prostasomes (cholesterol, sphingomyelin, calcium, and different enzymes) due to high ROS generation reflect the low prostate gland activity [31]. The acidic pH of seminal fluid indicates high ROS, reduced sperm motility, and viability that is neutralized by bicarbonate produced by accessory sex glands. The bicarbonate levels in seminal plasma are regulated by the enzymatic activity of cytosolic carbonic anhydrase isoenzymes (CA-I, CA-II, and CA-III) in accessory sex glands [32].

2.5.2 Impaired glandular contraction

Increased ROS influences the contractility of the smooth muscle cells in the accessory sex glands that, in turn, reduce the seminal fluid volume at ejaculation time. However, anejaculation corresponds primarily to a lack of seminal fluid emission that may be caused by impaired seminal vesicle contraction and seminal fluid production. Alteration in the expression α1- adrenergic receptors in response to high ROS generation also affects the contraction of seminal vesicles and vas deferens [33]. Nerve dysfunction due to oxidative stress influences the sympathetic neuronal input, affecting the secretory pattern of the ductus ejaculatory closure resistance and contraction of the seminal vesicles smooth muscle cells [34]. In addition, cytotoxicity by oxidative stress reduces the contractility of the smooth muscle of accessory sex glands by expressing calcium channel blockers [35].

2.5.3 Increased inflammation

Oxidative stress can also induce inflammation in the accessory sex glands, damaging tissue, and impairing glandular function. Infiltration of leukocytes in infectious conditions provokes ROS overproduction. In response, the generation of proinflammatory cytokines (IL-1b, IL-6, IL-8, IL-10, and IL-12) modulates the activity of pro- and anti-oxidative systems, whereas the oxidative stress exerts its effects even at the end of infection that impair the sperm function [31, 36].

2.6 Oxidative stress and sperm function

Oxidative stress greatly affects sperm motility, morphology, and fertilization potential. The following lines reflect the effects of oxidative stress on motility, concentration, morphology, acrosome reaction, DNA, mitochondrial potential, etc.

2.6.1 Reduced sperm motility

Deviation in motility pattern coincides with the ROS-induced damages in the axonemal part of sperm, which are linked to the following mechanisms:

• Increased susceptibility of PUFAs in the plasma membrane of sperm leads to lipid peroxidation. This oxidative stress induces axonemal damage and reduced sperm viability with higher midpiece morphological defects, which affect sperm motility [37].

• The lower activity of G6PD along high cytokines levels is also one of the major causes of decreased sperm motility, which is indicative of oxidative stress in the seminal plasma due to increased MDA [38].

• An increase in S-glutathionylation and tyrosine nitration of sperm proteins by oxidative stress deleteriously affects sperm motility [39].

• Activation of mitochondrial oxidative phosphorylation in response to oxidative stress is involved in bioenergetic pathways for low sperm motility [40].

• The 4HNE influences the activity of metabolic enzymes in sperm, which provide the energy for sperm motility [41].

2.6.2 Altered sperm morphology

Increased ROS production leads to structural abnormalities in sperm, which halt the fertilization potential. Immature sperm or disrupted sperm morphology and cytoplasmic droplets are prime factors for increased ROS generation. The defected cytoplasm further stimulates abnormal sperms to produce endogenous ROS by activating the enzyme glucose-6-phosphate dehydrogenase [21]. In addition, oxidative stress in the male reproductive tract promotes apoptosis, elevating morphological defects in developing germ cells [42].

2.6.3 Reduced sperm count

Oxidative stress also affects sperm concentration in ejaculated semen, but the extent of the effect is not as great as seen in other sperm attributes. However, low sperm count is observed when males are exposed to prolonged exposure to oxidative during disease conditions that affect the seminiferous epithelium by elevated ROS production. Under such conditions, testicular atrophy is an indication due to ROS-induced damage in the seminiferous tubules [37].

2.6.4 Impaired acrosome reaction

Oxidative stress impairs the capacitation process and disrupts the acrosome integrity in sperm, which reduces the fertilization potential. Under normal circumstances, ROS facilitates the capacitation process by activating intracellular cAMP by downstream PKA that phosphorylates MEK (extracellular signal-regulated kinase)-like proteins, threonine-glutamate-tyrosine, and fibrous sheath proteins. This mechanism further promotes the capacitated sperm for the acrosome reaction [43]. To ensure fertilization, the hyperactive sperm cross the cumulus oophorous, bind to the oocyte, and create a pore in its extracellular matrix via the exocytotic release of proteolytic enzymes. These acrosome reactions are mediated through the phosphorylation of tyrosine proteins and Ca⁺² influx resulting in an intracellular rise in cAMP and PKA, enabling the spermatozoon to penetrate and fuse with the oocyte. ROS has been observed to facilitate actions on the zona pellucida of the spermatozoon by various means, including phosphorylation of three relevant plasma membrane proteins [44].

2.6.5 Sperm DNA fragmentation

ROS affects the sperm DNA integrity by base modification, telomere shortening, and epigenetic changes that interfere with DNA replication and transcription, chromosomal instability and genetic abnormalities in the sperm, and altered gene expression. ROS damage the sperm DNA by attacking the purine and pyrimidine bases, causing single and double-strand DNA breaks, cross-linkage, or chromosomal rearrangements. Moreover, deficiency in sperm protamination leads sperm DNA more prone to ROS effects. Secondly, ROS-initiated apoptosis damages sperm DNA by caspase-mediated DNA destruction. In ROS-induced sperm DNA damage, 8-hydroxy-2′-deoxyguanosine (8-OHdG) is the potential indicator of DNA damage. The ensuing oxidative stress drives the spermatozoa along the intrinsic apoptotic cascade, from loss of MMP to oxidative DNA adduct formation, DNA fragmentation, and ultimately, cell death [45].

2.6.6 Loss of sperm mitochondrial membrane potential (MMP)

Oxidative stress also significantly impacts the mitochondria in sperm, affecting sperm motility and function. Here are a few ways that oxidative stress can affect sperm mitochondria:

• Increased levels of polyunsaturated fatty acids (PUFAs) initiate sperm mitochondrial ROS production, which occurs due to the dual hydrophilic and hydrophobic properties of PUFA. Penetration of the inner mitochondrial membrane disrupts the ETC electron flow, which produces the superoxide anions; subsequently, oxidative stress develops [46].

• Oxidative stress in the sperm also induces the generation of mitochondrial ROS. Adducts of aldehydes, by-products of lipid peroxidation (e.g., 4-hydroxynonenal), and mitochondrial proteins within the electron transport chain (ETC) further stimulate the mitochondrial ROS production [39, 47].

• Increased mitochondrial ROS levels induce oxidative stress by promoting apoptotic activity through a truncated apoptotic pathway [46].

• Increased mitochondrial permeability increases the intracellular Ca²⁺ that loss of MMP by lowering the ATP content and increased ROS, which deteriorates the plasma membrane integrity [48].

Spontaneous mitochondrial ROS production also results in loss of MMP, increased lipid peroxidation, and impaired sperm motility [49], which is linked to disruption of the mitochondrial electron transport, formation of adducts with mitochondrial proteins, reduced mitochondrial expression of prohibitin, the opening of the mitochondrial permeability transition pore, and induction of apoptosis in exposed sperm (reviewed in Aitken [46]).

In addition, nitric oxide radicals (by diffusion or generated within the mitochondria) react with superoxide anions (derived from the inner mitochondrial membrane) to form the peroxynitrite, which further produces adduct, that is. 4-hydroxynonenal protein adducts. Formation of such adducts leads to reduced motility via mitochondrial dysfunction [50, 51, 52, 53, 54].

2.6.7 Damaged sperm plasma membrane

Oxidative stress affects the membrane of sperm through protein or lipid peroxidation, reducing membrane fluidity. The sperm plasma membrane is crucial in sperm motility, capacitation and fertilization. Here are a few ways that oxidative stress can affect the sperm membrane:

• High contents of PUFA in the sperm plasma membrane make its susceptibility to membrane lipid peroxidation. However, PUFAs are necessary for membrane fluidity and fusogenicity of sperm membranes because the lowest carbon-hydrogen dissociation energies at the bisallylic methylene position in PUFA are easy targets of ROS [52].

• PUFA-rich sperm plasma membrane regulates membrane fluidity and is a target site of ROS to promote lipid peroxidation cascade, which decreases membrane fluidity [51].

• ROS produced by the NADPH oxidase system also induces damage in the sperm plasma membrane [55].

3. Nutraceuticals

Nutraceuticals are categorized as food or components used for health benefits beyond basic nutrition. Nutraceuticals could be in dietary supplement forms, functional foods (fortified or enhanced with additional nutrients), or bioactive compounds. Nutraceuticals are present in a single or in combination with vitamins, minerals, herbal supplements, probiotics, prebiotics, and omega-3 fatty acids. Mostly, nutraceutical remedies reduce inflammation, boost immune function, improve cardiovascular health, and promote healthy aging.

The popularity of nutraceuticals has been boosted in recent years on the eve of global antimicrobial resistance, and scientists are moving toward alternative approaches to assure health and well-being. However, it is noteworthy that many nutraceuticals possess promising health benefits, but scientific evidence about their safety and mechanism is limited. Similarly, the use of nutraceuticals to improve sexual health is also widespread under physiological or pathological infertility. Nutraceuticals to improve male infertility against oxidative stress have been extensively used and partially followed the plausible involved mechanisms.

3.1 Use of nutraceuticals against male infertility: underlying mechanisms

While there is limited research specifically examining the effects of nutraceuticals on copulation, there is some evidence to suggest that certain nutraceuticals may positively impact sexual function and libido in both men and women. Some examples of nutraceuticals that have been studied in this context include:

3.1.1 Nutraceuticals and erectile dysfunction

• L-arginine is an amino acid responsible for nitric oxide in the biological cycle that involves dilating penile vessels for blood flow to enhance penile erection. Arginase enzyme regulates NO synthase generation in corpus cavernosal tissues, leading to penile erection. Under erectile dysfunction, high arginase activity diminishes the arginine level. It is not available for synthesis NO, the substrate of endothelial NO synthase and neuronal NO synthase [56] for penile erection by provoking the relaxation of vascular and nonvascular cavernosal tissues via cGMP activation [57]. L-arginine promotes the NO and cyclic guanosine monophosphate production for erectile function. Under oxidative stress, arginine is converted to NO, which also scavenges the excessive ROS from penile tissues and improves erectile function [58, 59].

• Horny goat weed is a nutraceutical, which is being widely used for erectile dysfunction and possesses the active bioactive ingredient icariin. Icariin has the potency of a PDE5 inhibitor and boosting properties of testosterone. Moreover, icariin increases smooth muscle proliferation and has neurotrophic effects, which are useful for erectile dysfunction due to oxidative stress-induced endothelial cell damage [60].

• In addition, maca root is used as an aphrodisiac. Although the exact mechanism of action how it mitigate oxidative stress and improves penile erection is not clear, the antioxidant properties could be speculated about it [61].

• Muira Puama and Saw Palmetto are potential nutraceuticals that improve erectile dysfunction by inhibiting PDE5 activity and quenching excessive ROS in penile tissues. Administration of maca root is a choice supplement in case of erectile dysfunction by active ingredients of maca roots, i.e. macamides and macaenes, etc., which potentially boost the antioxidant level in male reproduction. Moreover, the erectile-enhancing effect of maca roots is linked to glucosinolate extracts that activate the androgen binding receptors in penile tissues [62]. However, the particular mechanism of maca root for erectile dysfunction is still unexplored.

• Ginseng contains potential aphrodisiacs because of active agents (ginsenosides and ginseng saponins). These compounds are observed to increase nitric oxide (NO) synthase activity, increasing the blood flow to the penile corpora cavernosa. The Antioxidants property of ginseng is also the mechanism to combat erectile dysfunction [63].

3.1.2 Nutraceuticals and male libido

The effect of each nutraceutical is linked to various potential mechanisms to improve the libido.

• The use of Korean red ginseng is established in boosting sex hormones by providing an environment of antioxidants in the testes because of the enzymatic and nonenzymatic characteristics of ginsenosides [64].

• The Ashwagandha (Withania somnifera) improves libido through strong cellular antioxidant effects. It reduces the ROS level by improving the metal cofactors of SOD, and catalase has a key role in steroidogenesis. Moreover, it restores the sex hormone level by promoting antioxidant vitamins and minimizing cortisol under stressful conditions [65]. Another involved mechanism of W. Somniferous for improving sexual hormones via activating the Nrf2/HO-1 pathway and inhibiting the NF-κB levels is explained [66].

• Tribulus terrestris improves the libido level by improving the testosterone level. It directly increases the testosterone, dihydrotestosterone, and dehydroepiandrostenedione, which improve libido. It is also linked to the proliferation of testicular cells through the increased conversion of testosterone into dihydrotestosterone by the action of 5-α reductase [67].

• Tongkat Ali’s also improves the libido, but the mechanism of its action for increased testosterone is unclear.

• Supplemental zinc improves hypogonadism by boosting testosterone levels due to its potential action of antioxidant and cofactor of the enzyme involved in steroidogenesis [68].

• Fenugreek seed extract, composed of numerous enzymes, amino acids (including arginine), vitamins, and lipids, improve the libido by maintaining the glucose and cholesterol level for steroidogenesis [69].

3.1.3 Nutraceuticals and spermatogenesis

• Vitamin D regulates the testicular functions by enabling the Sertoli, germ cells, Leyding cells, and epithelial cells lining the male reproductive tract to utilize the calcium for metabolism [70].

• Vitamin E is an antioxidant that may help to protect testicular cells from oxidative damage. It consists of biologically active compounds of tocopherols and tocotrienols that scavenge the ROS and prevent spermatogonium degeneration, testicular dysfunction, and seminiferous tubule shrinkage. The antioxidant activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) in testicular cell membranes and mitochondria are promoted by vitamin E integration. Along with boosting testosterone levels, Zinc is involved in anatomical configuration and normal functioning of male reproductive organs, sperm maturation during spermatogenesis and preventing the germinative epithelium from ROS affections [6].

• Coenzyme Q10 (CoQ10) is an antioxidant involved in energy production in the body, testicular function, and testosterone production. CoQ10 supplementation affects spermatogenesis by lowering FSH levels and increasing inhibin B levels [71, 72, 73, 74].

• Ashwagandha contains numerous bioactive compounds, which improve spermatogenesis due to its antioxidative properties. In addition, its supplementation ensures the availability of different metabolites, that is. iron, alanine, aspartate, fructose, lactate, glutamine, etc., for testicular functions [75, 76].

• Beyond the antioxidant or aphrodisiac effects, Tongkat Ali is involved in minimizing the inhibitory effects of estrogen on spermatogenic cells during spermatogenesis [77].

• Korean red ginseng improves spermatogenesis through testicular antioxidants homeostasis (glutathione, vitamin C and E, and expressions of phosphatidylinositol transfer protein, fatty acid-binding protein-9, and triosephosphate isomerase-1 proteins) and ginsenosides (dammarane-type triterpene saponin) effects on re-establishing of sperm maturation process [78].

3.1.4 Nutraceuticals and sperm motility

• Carnitine is an amino acid regulating intracellular metabolism through b-oxidation and buffers the acetyl-coenzyme A (CoA) to CoA ratio by transporting long-chain fatty acids into the mitochondria. This process is involved in energy production in sperm for sperm motility and maturation. Moreover, the antioxidant property of carnitine protects the sperm against excessive ROS and maintains motility [79].

• Vitamin C is an antioxidant that improves sperm motility by reducing oxidative stress. It is a key cofactor for hydroxylation and amidation reactions and is involved in the synthesis of collagen, components of the intercellular matrix, and proteoglycans for cellular metabolism. Its antioxidant property prevents ROS attack on sperm plasma and mitochondrial membrane and maintains motility [80].

• CoQ10 has strong antioxidant potential and plays a role in cell energy production. Improvement in sperm motility by CoQ10 is maintained by providing energy. Mitochondrial bioenergetics is known for sperm motility and requires a high energy expenditure, which is fulfilled by CoQ10 when oxidative stress exists [74, 81].

• Omega-3 fatty acids are important for reproductive health. Repair of plasma and mitochondrial membrane and lipid metabolism by Omega-3 fatty acids supplementation under oxidative stress provides sperm membrane integrity that, in turn, is helpful for sperm motility [82]. Promotion of the sperm lactate dehydrogenase isoenzymatic form by omega-3 PUFA supplements maintains the catalytic conversion of pyruvate to lactate in the energy metabolism of sperm [83].

• Zinc is an essential mineral that is important for male reproductive health. It helps to increase sperm motility. Zinc is an enzyme cofactor for DNA transcription and protein synthesis that might improve sperm metabolism and maintain motility [84, 85].

3.1.5 Nutraceuticals and sperm concentration

• CoQ10 supplements regarding sperm count, lower FSH, and higher inhibin B enhance the Sertoli cell function, improving sperm concentration [86].

• Vitamin B12 is involved in sperm metabolic activity. It is a coenzyme that reduces ribonucleotides to deoxyribonucleotides. It also stimulates growth and maintains synthesis and sperm maturation. It prevents the methylmalonic acid that increases ROS-induced incidences in sperm [87].

• Zinc plays a role in testicular development and spermatogenesis. It has a role in DNA function regulation in sperm cells and indirectly improves spermatogenesis by promoting testosterone production. Moreover, the antioxidant effect of Zn protects the Leydig cells from ROS-induced damage and maintains normal sperm concentration [88, 89].

• L-acetyl carnitine protects sperm mitochondria from free radicals. It stabilizes spermatogonial production through antiapoptotic action and maintains sperm count under oxidative stress [90, 91].

3.1.6 Nutraceuticals and sperm morphology

• Omega-3 fatty acids possess the α-linolenic acid (ALA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA). The supplementation of these under oxidative stress stabilizes the cell membrane composition by incorporation into the sperm cell membrane and maintains the sperm morphology [83, 89].

• L-carnitine is an amino acid that is important for energy production in cells. It may help to improve sperm morphology by providing energy to the sperm cells. Irrational incidence of apoptosis during spermatogenesis due to excessive ROS leads to sperm morphological abnormalities, which is ameliorated by L-carnitine supplement. It seems that the antioxidant mechanism of L-carnitine is involved in reducing sperm morphological defects [90, 92].

• Vitamin D, a membrane-bound antioxidant, has a potential scavenger capacity. Oxidative stress marker (4-hydroxynonenal) is the main indicator of vitamin D deficiency which increases sperm morphological defects; hence, vitamin D supplement and expression of vitamin D receptors in the male reproductive tract could minimize sperm morphological defects [93].

• Zinc uptake can minimize oxidative stress and maintain Leydig cell integrity and steroid hormone synthesis, which improves sperm morphology [88].

• CoQ10 supplementation improves sperm morphology by providing energy to the sperm cells. Moreover, it transports electrons in the mitochondrial respiratory chain to promote energy in the mid-piece region of sperm. It also acts as a lipid-soluble antioxidant for the lipoprotein-rich cell membrane and stabilizes sperm morphology against oxidative stress [74].

3.1.7 Nutraceuticals and sperm mitochondrial integrity

• CoQ10 supplement mitigates the excessive ROS production during the mitochondrial oxidative phosphorylation process due to its antioxidant potential. Promoting the mitochondrial electron transport chain by CoQ10 preserves ATP production and mitochondrial transcription, maintaining sperm motility [94].

• L-carnitine is involved in energy metabolism within sperm mitochondria. L-carnitine supplements improve motility by protecting sperm mitochondria against ROS [95].

• Omega-3 fatty acids, docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) possess antioxidant effects, which reduce oxidative stress in sperm mitochondria and improve sperm motility. PUFA supplementation reduces the excessive ROS by maintaining the βeta-oxidation in sperm mitochondria for sustainable sperm functions [96].

• Myoinositol is a component of the vitamin B complex and maintains sperm mitochondrial potential for motility. Myoinositol might increase cytosolic Ca⁺² in sperm, which also increases mitochondrial Ca⁺² necessary for mitochondrial potential and sperm motility [94].

3.1.8 Nutraceuticals and sperm DNA

• Vitamin C mitigates the excessive ROS in sperm and reduces the disulfide bridges of cysteine residues and improves the testicular antioxidants, which protect the sperm from chromatin damage [97].

• Zinc is an integral element in DNA synthesis. Zn is also required for correct sperm DNA condensation/decondensation. Chromatin stability of the ejaculated sperm is Zn-regulated and controls the disulfide bridge formation [89, 98].

• CoQ10 is a strong antioxidant, and its reduced form, ubiquinol, and prevents oxidative stress in sperm DNA [73, 74, 99].

• L-Carnitine is a powerful antioxidant and reduces sperm DNA decondensation by scavenging O₂⁻ and H₂O₂ and inhibiting iron-mediated ROS production [97, 100].

• Folic acid is intrinsically involved in purine and pyrimidine production that plays an important role in DNA synthesis and proper sperm function. It is also a potent free radical scavenger by providing methyl donors methionine and S-adenosylmethionine, decreasing the frequency of sperm DNA abnormalities [87].

4. Conclusions

The current evidence shows how oxidative stress influences male fertility by impairing the male reproductive tract environment, reducing the hormonal levels, and damaging the testicular or sperm functions. At the same time, numerous biological agents such as minerals, vitamins, and herbs possess single or multiple active compounds that directly improve the enzymatic antioxidant activity or indirectly enhance antioxidant levels through cellular metabolism and scavenge the excessive ROS from the male reproductive system. It is evident how these antioxidants clear the excessive ROS for better libido and penile erection. The involved antioxidative mechanism linked to famous nutraceuticals compounds for spermatogenesis, motility, concentration, morphology, mitochondrial function, and DNA integrity of sperm is separately depicted in this chapter.

In future, use of nutraceuticals in male infertility conditions needs validation by exploring molecular mechanics, long-term effects with high safety profiles, specific pathways, and target sites of nutraceuticals.