Oxidative stress and male infertility: current knowledge of pathophysiology and role of antioxidant therapy in disease management

Abstract

Infertility is a global health problem involving about 15% of couples. Approximately half of the infertility cases are related to male factors. The oxidative stress, which refers to an imbalance in levels of reactive oxygen species (ROS) and antioxidants, is one of the main causes of infertility in men. A small amount of ROS is necessary for the physiological function of sperm including the capacitation, hyperactivation and acrosomal reaction. However, high levels of ROS can cause infertility through not only by lipid peroxidation or DNA damage but inactivation of enzymes and oxidation of proteins in spermatozoa. Oxidative stress (OS) is mainly caused by factors associated with lifestyle. Besides, immature spermatozoa, inflammatory factors, genetic mutations and altering levels of sex hormones are other main source of ROS. Since OS occurs due to the lack of antioxidants and its side effects in semen, lifestyle changes and antioxidant regimens can be helpful therapeutic approaches to overcome this problem. The present study aimed to describe physiological ROS production, roles of genetic and epigenetic factors on the OS and male infertility with various mechanisms such as lipid peroxidation, DNA damage, and disorder of male hormone profile, inflammation, and varicocele. Finally, the roles of oral antioxidants and herbs were explained in coping with OS in male infertility.

Introduction

Infertility is defined as an inability in pregnancy after 12 months of unprotected sexual intercourse. Almost 7% of couples worldwide are infertile, and half of the infertility is associated with male factors [1]. Various factors are involved in the pathophysiology of male infertility including OS. The male infertility is strongly associated with reactive oxygen species (ROS) [2]. ROS includes highly reactive free radicals including superoxide anions (O^·−₂), hydrogen peroxide (H₂O₂), proxyl (·ROO) and hydroxyl (·OH) [3]. Low levels of ROS are necessary for sperm capacitation, hyperactivation, acrosomal reaction, and sperm–ovule fertilization. However, high levels of ROS neutralize antioxidants in the seminal plasma and cause OS. Sperm damage by ROS is the main cause of infertility in 30–80% of infertile men; and these conditions ultimately damage DNA in the nucleus and mitochondria [4]. Sperm cells are highly sensitive to oxidative damages. On the other hand, these cells have cytoplasmic membranes rich in unsaturated fatty acids, and thus they are faced with the lipid peroxidation under the influence of ROS [2]. Moreover, these cells are incapable of repairing damage by oxidative stress because they suffer from lack of essential cytoplasmic enzymes. Finally, lower motility and death of sperm occurs due to the loss of adenosine triphosphate (ATP) caused by lipid peroxidation followed by axonemal injury [5]. OS indirectly affects hypothalamic axes and can disrupt the secretion of sex hormones. ROS reduces levels of male sex hormones and strikes their balance and can disrupt the reproductive system [6]. A wide range of factors including genetic, epigenetic, lifestyle-related factors such as over-consumption of alcohol, smoking, radiation, obesity and also infection and varicocele could increase the level of OS in the body and lead to infertility [7]. Therefore, the role of the antioxidant system is essential to cope with excessive-produced ROS. Antioxidant enzymes and molecules such as superoxide dismutase (SOD), glutathione (GSH) and catalase (CAT) are abundant in plasma or natural sperm cells [8]. Most of these enzymes are encoded by NRF2, SOD, CAT, glutathione S-transferase (GST), and glutathione peroxidase (GPx) genes. Genetic mutations in these genes, in turn, cause infertility caused by OS in men [9]. As a result of these injuries, rates of fertilization and implantation are reduced and fetal growth is disturbed, and the likelihood of abortion increases. Since the balance between antioxidant levels and ROS maintains the redox homeostasis, the use of oral antioxidants as well as herbal medicine can help treat men with infertility. These antioxidants remove extra ROS and provide the redox balance [10]. To overcome infertility, the mechanisms that cause sperm damage, as well as their therapeutic methods, should be considered more. Recently, some literature reviews describing the effect of OS on the male reproductive system were published. For instance, Bui et al. reported the role of excessive ROS levels in damage of genomic and mitochondrial DNA, shortening of telomere length, Y chromosomal microdeletions, and epigenetic changes [4]. Also, Agarwal et al. discussed about infection and inflammation-induced OS and male infertility [11]. In another report, Darbandi et al. described the mechanisms of ROS-mediated disturbance of male hormonal profiles [6]. But in the present review with a more comprehensive attitude, we discussed about different aspects of OS on male infertility including its pathophysiological and genetic aspects with an emphasis on the therapeutic use of antioxidants.

Physiological role of ROS in the male fertility

Reactive oxygen species can have both beneficial and destructive effects on sperm functions and these effects depend on the ROS level. Reactive species of oxygen and nitrogen should be regulated at appropriate levels to ensure physiological functions. A small amount of ROS is a product of normal metabolism in stem cells, sperm and ovum; and these reactive species regulate important reproductive processes such as capacitation, acrosomal reaction and hyperactivation. Sperm capacitation is a necessary process to obtain fertilizing facility. ROS such as superoxide, H₂O₂, nitrogen oxide (NO), and superoxide anion radical are contributed in this procedure [3]. Superoxide anion generation in neutrophils is correlated with some proteins tyrosine phosphorylation. Two chief phosphotyrosine-containing proteins, 81 and 105 kDa, are overexpressed in capacitating conditions. Catalase and superoxide dismutase eliminate both tyrosine phosphorylation of p81 and p105 and sperm capacitation, confirm the role of hydrogen peroxide and O^·−₂ in these two procedures. ADPH oxidase inhibitors could reduce both tyrosine phosphorylation of p81 and p105 and sperm capacitation whereas hydrogen peroxide induces these two procedures [12]. NADH and NADPH activate phosphorylation of p81 and p105 and sperm capacitation, but these procedures were not inhibited by catalase or SOD, nor were they correlated with an elevated production of O^·−₂. So, these cofactors seemed to apply their function by mechanisms altered from some other inducers [13]. Nitric oxide is another essential compound for sperm capacitation while inhibition of nitric oxide synthase (NOS) reduced the acrosome reaction percentage. In addition, nitric oxide could modulate tyrosine phosphorylation of proteins in sperm and there is a true association between nitric oxide-related tyrosine phosphorylation and sperm capacitation [14]. During human sperm capacitation, a double phosphorylation occurs in threonine–glutamine–tyrosine motif (P-Thr-Glu-Tyr-P) of 105 and 81 kDa proteins. This phosphorylation is specifically regulated by nitric oxide and the protein Tyr kinase, protein kinase C, and MEK or MEK-like kinase are involved in this process [15]. The phospho-MEK-like proteins are regulated by kinases and ROS and maybe display an intermediate phase among the initial events and the late tyrosine phosphorylation correlated with sperm capacitation [16]. However, The PI3K/Akt axis contributed in Tyr and Thr-Glu-Tyr phosphorylation and sperm capacitation but not MEK-like proteins [17]. Additionally, during capacitation, Ca2+ stimulates increased levels of cAMP. The cAMP, via an indeterminate serine/threonine phosphorylation, regulates the superoxide anion generation and this procedure could lead to elevation of p81 and p105 phosphorylation [18]. Cyclic adenosine monophosphate (cAMP) activates the protein kinase A (PKA) and could indirectly control the tyrosine phosphorylation of protein. Levels of cAMP are regulated by a balance of adenylyl cyclase (AC) and phosphodiesterases (PDEs) activities, which are responsible for its production and degradation, respectively. Lefièvre et al. [19] suggested that cAMP level is under the regulation of AC, since activity of PDE is steady throughout sperm capacitation. Moreover, they suggest that low cAMP concentrations are enough for PKA activation and sperm capacitation [19]. The above-mentioned contents strengthen the concept that differentially intricate pathways are required for sperm to develop capacitated. In addition, phosphorylation events may be involved in the acrosomal reaction which could be affected by OS. The acrosomal reaction is correlated with the generation of extracellular ROS by spermatozoa and both H₂O₂ and O^·−₂ might be involved in the control of this procedure. A possible mechanism of ROS effects on acrosomal reaction may be explained by tyrosine phosphorylation of sperm proteins such as 70–105 kDa and this effect could partially disappear by some ROS scavengers [13]. Acrosomal reaction and a partial pathway involved in sperm capacitation are depicted in Fig. 1.

Fig. 1.

Physiological and pathological roles of ROS in male reproductive system. Small amount of ROS plays an important role in acrosomal reaction and penetration of spermatozoa to the zona pellucida through a mild lipid peroxidation (a). ROS production is an early procedure in sperm capacitation. In the plasma membrane, an oxidase produces O^·−₂ in the extracellular region which helps sperm capacitation activators. Diphenyliodonium (DPI), an inhibitor of this oxidase could block production of O^.−₂ in oxidative stress condition. In addition, superoxide dismutate (SOD) could block the extra O^·−₂. Intration of H₂O₂ with a certain target in plasma membrane assists sperm capacitation and catalase could inhibit excessive H₂O₂. Nitric oxide (NO) produced by a NOS and induces capacitation. Adenylyl cyclase (AC) is a possible target for ROS which is responsible for producing cAMP (b). Sources of ROS in male infertility included obesity, immature sperm, radiation, leukocytosis, alcohol drinking, and smoking (c). The NADPH oxidase causes production of superoxide radical (O^·−₂) in sperm lipid membrane. The O^·−₂ turns into H₂O₂ in cytosol and this agent produces hydroxyl radical (OH·). H⁺ and OH.could initiate lipid peroxidation in the sperm membrane and excessive ROS in the sperm causes mitochondrial dysfunction and DNA damage (d)

ROS sources in seminal plasma

Various sources cause OS including genital tract infection, varicocele, metabolic syndromes, smoking, excessive consumption of alcohol and drugs, ionizing radiation, cell phone use, mental stress, severe exercise, spinal cord injury (SCI) and environmental pollution. In general, these sources are classified into endogenous and exogenous groups [20, 21].

Endogenous sources

Immature sperm

During the spermatogenesis, the sperm release may occur along with additional cytoplasm of the germinal epithelium; and spermatozoa are usually immature and non-functional. Sperms with excess cytoplasmic residues in the mid-piece are known as immature sperm. Additional cytoplasm in the mid-part of the sperm contains glucose-6-phosphate dehydrogenase (G6PD) enzyme. This cytoplasmic enzyme controls the production of intracellular β-nicotinamide adenine dinucleotide phosphate (NADPH) through a shunt hexose monophosphate. The NADPH is used to fuel the production of ROS via NADPH oxidase situated within the sperm membrane [22, 23]. In addition, there are abundant numbers of mitochondria in mid-piece that store energy for motility of sperm. Diaphorase, an enzyme at the mitochondrial respiratory chain, controls the ratio between reduced and oxidized forms of NADH; thus it contributes to the sperm energy balance. This oxidoreductase is integrated with the mitochondrial respiratory chain and generates superoxide anions, affecting the level of ROS [24]. Mitochondrial dysfunction could lead to ROS production and the ROS can influence mitochondrial integrity in sperm cells within a mutual process; ROS leads to damage of mitochondrial membrane and this causes an elevation in ROS generation production. Indeed, mitochondria and sperm membrane are two main regions for ROS production in immature sperms [25].

Leukocytes

There are few leukocytes in each natural ejaculation. According to the WHO criteria (World Health Organization), if the white blood cell count is greater than 1 million per ml of semen, it is defined as leukocytospermia [26]. The increased ROS in semen due to leukocytospermia leads to a decrease in concentration of antioxidants resulting in damage to DNA in sperms [27, 28]. The number of leukocytes increases in the seminal plasma due to the inflammation or infection of the reproductive tract. Positive peroxidase leukocytes can produce ROS 1000 times more than sperm by increasing the production of nicotinamide adenine dinucleotide phosphate (NADPH) through the transfer of hexose monophosphate [11]. Studies indicate that levels of ROS in fertile men are less than infertile men, and there is a direct relationship between OS and increased leukocyte count [29]. Seminal plasma leukocytes, except for macrophages, which are about 30%, are mainly polymorphonuclear (PMN). Leukocytes or white blood cells are created in response to stimuli during inflammation and infection and can create ROS up to 100 times more than inactive cells. Activation of the myeloperoxidase (MPO) system from PMN cells and macrophages leads to the respiratory burst and increases ROS products. Spermatic injury due to leukocyte occurs when the number of seminal plasma leukocytes such as leukocytospermia is significantly high [29]. The inflammation or infection should be taken into consideration in infertile men. Antibiotic treatment at low levels of leukocytospermia (0.2–1 × 10⁶) significantly increases spontaneous pregnancy [30].

Exogenous sources

Smoking

According to a study in Morocco in 2017, cigarette smoking is the most important factor affecting male infertility [31]. Nicotine and other chemicals in cigarettes disrupt levels of hormones and affect effective parameters in semen [32]. Smoking can damage the DNA of sperm in male reproductive cells through high accumulation of ROS in the sperm production site. Cigarette smoke contains a lot of toxic substances, carcinogens, and mutagenic substances in addition to stable and unstable free radicals and ROS in its particles. It also increases the production of superoxide anions, H₂O₂ causing oxidative damage to lipid membranes of cells, proteins, enzymes, and DNA and subsequently leads to infertility [33]. Cigarette smoking reduces creatine kinase (CK) in sperm as an energy reservoir for rapid buffering and rebuilding ATP and plays an important role in the sperm motility [34]. In addition, ROS-induced damage of mitochondrial DNA results in reduced ATP and energy accessibility, impending sperm motility [5]. Furthermore, cigarette smoking reduces the quality of semen, reduces acrosin activity, protein phosphorylation, disturbance of expression of micro-ribonucleic acids (miRNAs) and histone-to-protamine transition and ultimately leads to infertility [35]. Damage to DNA and methylation patterns due to the increased level of ROS in tissues of people, who have been indirectly exposed to cigarette smoke, is also another side effect of cigarettes [36].

Alcohol intake

Excessive alcohol consumption also has devastating effects on the quantity and quality of sperm parameters. Excessive alcohol consumption is associated with a decrease in sperm motility and concentration and also reduces the percentage of normal morphology of sperm [37]. Alcohol causes spermatic chromatin disorders through apoptosis, thereby affecting the sperm motility, nuclear maturation and DNA integrity [25]. According to an animal study in 2017, the sperm motility and count in rats have received alcohol decreased significantly, as well as the level of MDA and SDF increased, in addition to a decrease glutathione and superoxide dismutase levels have been observed in the testicles of animals that received alcohol [38]. Ethanol causes changes in the structure and function of mitochondria, reduces respiration and ATP levels, and increases the ROS production through its metabolism in the liver [39]. Alcohol consumption increases activities of cytochrome P450 enzymes (CYP2E), which in turn increase the activity of NADPH oxidase, and then changes levels of certain metals (especially copper- and iron-free ions) in the body, and ultimately increases the production of superoxide anions [39] in men who constantly consume alcohol; and nitric oxide, NO production increases via inducible nitric oxide synthase (iNOS) [40]. Nitrite oxide and its metabolite, peroxynitrite (ONOO-) act as mediators of mitochondrial dysfunction [40, 41].

Radiation

Another exogenous factor is radiation that affecting male fertility and its biological consequences depend on the type of radiation, the amount of produced energy and duration of radiation [42]. Ionized and non-ionized radiation effectively affects spermatogenesis. Male fertility is heavily influenced by thermal, radioactive, radio frequency (RF) and other hazardous radiation [43]. In recent years, there is a significant increase in the use of mobile phones, laptops, wireless systems and microwave that have raised concerns about male infertility due to the electromagnetic radiation. Electromagnetic radiation leads to a lot of changes in reproductive parameters by an OS induction. Based on this hypothesis, the impact of 3G cell phone radiation was studied on the reproductive tract of male rats in 2018. After 3G cell phone radiation, decreased spermatogenic cells, sperm membrane changes, increased ROS rate and lipid peroxidation were observed along with a simultaneous reduction of sperm count and changes in morphology of sperm tail [44]. Cell phone radiation might affect cell plasma membrane and stimulates plasma membrane NADH oxidase which plays a key role in the various cellular harmful effects and leads to OS [45]. Another experimental study indicates that due to the electromagnetic radiation, the mitochondrial genome is damaged and leads to disturbances in the electron transport chain (ETC) resulting in OS [46]. Small changes in ROS may play an important role in the capacity of sperm, acrosome reaction, and fertilization, but the OS caused by radiofrequency radiation causes damage to spermatozoa [43]. Another study examined the impact of 2.45-GHz microwave on the histology and lipid peroxidation level in male rats. According to the present study, the exposure to microwave radiation for 2 hours per day for 35 days can cause oxidative changes in rats [47]. Other studies on the radio frequency radiation and its effects on male infertility reported the reduced glutathione level and disturbed sperm membrane integrity because of OS [48]. Therefore, researchers concluded that the ROS production could be a major pathway in men’s infertility and radiation pathophysiology; hence, the antioxidant therapy is a useful way to minimize its harmful effects (Fig. 1) [46].

Environmental sources

One of the most important environmental ROS source is genital heat stress. Exposure to heat radiant for a long time leads to scrotal hyperthermia and excessive ROS production [49]. Animal studies demonstrate that spermatogenesis, motility of spermatozoa, sperm concentration and sperm viability could be affected by heat stress [50, 51]. Also, studies demonstrate that heat stress and subsequently increased ROS may result in over-expression of caspase 3 that lead to apoptosis in various cell types including sertoli and leydig cells [52–54]. Another environmental source of ROS is pollution such as phthalates, air pollution, and heavy metals. Phthalates are the man-made chemical that exist in personal care products, plastics, and food packaging materials that influence the general population. Animal experiments demonstrated that a high level of ROS production, the lack of testicular antioxidant, and a decrease in hormone levels are the results of phthalates exposure [55, 56]. A human study shows that phthalates could increase the level of OS in the testis and subsequently cause mitochondrial dysfunction, and ultimately reduces the sperm function as a result of increased lipid peroxidation [57]. Air pollution can promote oxidative stress by affecting sperm lipid membrane and generate free radicals and decrease sperm parameters [58, 59]. In addition, heavy metals are considered to be another source of ROS. Cadmium and lead appear to cause testicular oxidative stress and also can be damaging to sperm DNA and the reduction of semen parameters [60–62].

Roles of genetic and epigenetic factors in OS affecting the male infertility

Environmental, genetic, and lifestyle factors affect male fertility. Both factors are associated with levels of sex hormones, chromatin stability, and sperm quality. About 20% of observed changes in sperm count, hormonal levels, sperm morphology, and sperm chromatin parameters are influenced by hereditary factors [63–65]. Since the ROS is affected by endogenous and environmental factors and can cause great damage to sperm quality and DNA, antioxidant enzymes play important roles in counteracting OS during spermatogenesis and fertilization. A number of antioxidant genes involved in spermatogenesis include NRF2, SOD, CAT, GPX, PRX, GRX, TRX, and NOS. Enzymes, which are encoded by these genes, are widely used in antioxidant responses, syntheses, and reduction of GSH and redox cycle of reducing thiol during spermatogenesis. Furthermore, most of these genes have ARE (antioxidant responsive element) motifs in promoter sites, thereby facilitating the NRF2 transcription pathway in the OS [66]. NRF2 is a key gene in the antioxidant defense because it is a nuclear transcription factor that can induce antioxidant enzymes through the ARE element [67]. In response to OS, NRF2 binds with AREs and regulate defensive mechanisms against OS. A study in mice lacking Nrf2 showed an important role for the NRF2 in prevention of oxidative damage in spermatogenesis [8]. However, Chan et al. reported that the function of NRF2 gene is not essential for mouse fertility [68]. SODs and CATs are genes that are regulated by NRF2-ARE signal pathway, and they protect sperm from oxidative damage by superoxide and H₂O₂. SOD cause superoxide radical destruction in molecular oxygen and hydrogen peroxide. Three families of SOD isozymes are found in humans: soluble SOD (SOD1), mitochondrial SOD (SOD2) and extracellular SOD (SOD3) [69] among which, SOD2 isozyme is more expressed in human sperms [70]. SOD gene has several varieties, and studies indicate that rs4880 polymorphism is effectively associated with an increased risk of infertility in men [71]. CAT is a heme enzyme that is encoded by CAT genes. This enzyme converts H₂O₂ to oxygen and water, and eventually maintains normal levels of ROS and protects sperm against ROS [72, 73]. CAT gene also has a single-nucleotide C-262T mutation in the promoter site and is effectively associated with increased infertility in men [72]. However, an animal study revealed that the fertility rate was normal at 3 months in wild-type, Sod^−/− or Cat^−/− knockout mice. But, the fertility rate was significantly decreased in all mice at 12 months [74]. NOSs constitute a family of enzymes that catalyze the production of nitric oxide (NO) from l-arginine and confront with ROS at low levels. NO plays role in the sperm motility, survival and acrosome reaction [75]. Three NOS isozymes have been found in mammals including neuronal NOS (nNOS; NOS1), inducible NOS (iNOS; NOS2) and endothelial NOS (eNOS; NOS3) [76]. These three isoforms can be found in various testicular cells including sertoli cells, germ cells in seminiferous epididymis, leydig cells, myofibroblasts, endothelial cells, and spermatozoa. The effective presence of the above isoforms in testes indicates the importance of NOS in the spermatogenesis [77]. A study evaluated relationships of different nitrite oxide polymorphisms with male infertility. The results indicated significant relationships between T-786C and G894T polymorphisms, reduced sperm parameters, and increased OS [78]. Glutathione S-transferases (GSTs) are other proteins that catalyze the GSH binding to xenobiotic electrophilic substrates as causes of ROS [79]. GST family includes three large subgroups namely cytosolic, mitochondrial, and microsomal GSTs [80]. In humans, GSTs include mitochondrial GSTK1, microsomal MGST1-MGST3, and cytosolic GSTA1-GSTA5, GSTZ1, GSTM1-GSTM5, GSTO1-GSTO2, GSTP1, and GSTT1-GSTT4 [81]. Studies on different GST genotypes indicate that the removal of GSTT1 and GSTM1 genes, as well as point mutations in GSTP1 gene, is associated with an increase in the male infertility risk [82]. TPX, PRX, and GRX enzymes are involved in reducing cellular thiols. GRXs and TRXs jointly catalyze the reduction of protein-mixed disulfides [83]. TRX1 isozyme exists in the cytosol and nucleus, while TRX2 is specifically expressed in mitochondria [84]. PRX enzymes constitute a group of peroxidases that destroy organic hydroperoxides and H₂O₂. GPX family catalyzes the reduction of thiol with glutathione [85]. Among its isozymes, GPX4 is dominant in testes and is vital to spermatogenesis [86]. GPX5 is expressed in the epididymis caput and probably maintains sperm DNA integrity [87]. As mentioned above, the genetic polymorphisms found in the genes involved in the OS system can affect male infertility. Genetic polymorphisms, depending on their position on the gene map, can alter the expression and structure of a protein [88, 89]. Genetic variations can occur on the coding or non-coding regions of the genes. Non-coding region mutations, as well as mutations in the coding regions, can affect the gene expression and RNA splicing. Mutations in promoter regions can be directly related to gene expression [90–92]. Studying the molecular effects of polymorphisms in vitro and in vivo conditions are time-consuming and costly. Some previous studies have shown that computational analyses with bioinformatics tools can be a quick and cost-effective way in molecular analyzes, especially in investigating the molecular effects of genetic mutations [93, 94]. The important genes associated with the OS system and the role of their genetic variation in male infertility are summarized in Table 1.

Table 1.

Common genetic polymorphisms in genes involved in OS and their role in male infertility

Gene	Polymorphisms	Effects	Population	Sample size (case/control)	References
Paraoxonase 1	rs662	The variation is associated with decreased risk of male infertility	Iran	450 (220/230)	[95]
Superoxide dismutase 2	lle58Thr	The rs1001179 is associated with increased risk of male infertility	Spain	393 (313/80)	[96]
	rs4880
Catalase	rs1001179
Glutathione peroxidase 1	rs1050450
Glutathione S-transferase	GSTT1 (null or non-null)
	GSTM1 (null or non-null)
Nitric oxide synthase 3	rs2070744	Intron variant 4a/4b is associated with increased risk of male infertility	Serbia	262 (131/131)	[97]
	rs1799983
	Intron variant 4a/4b
Aryl hydrocarbon receptor	rs2066853	The variation is associated with decreased sperm parameters as well as increased seminal OS	Egypt	170 (50/120)	[97]
Glutathione peroxidase 1	rs1050450	There is no association between the variation and idiopathic male infertility	Iran	250 (100/150)	[98]
Nitric oxide synthase 3	rs1799983	There is a significant association between rs1799983 and rs2070744 and decreased sperm parameters and increased seminal OS	Egypt	300 (80/220)	[78]
	rs2070744
Nitric oxide synthase 3	rs2682826	The rs1799983 polymorphism could cause oxidative sperm DNA damage, and contributing to male infertility	China	1160 (580/580)	[99]
	rs1047735
	rs2297518
	rs10459953
	rs1799983
Human catalase	rs1001179	This variation is associated with male infertility	Iran	385 (90/95)	[72]
Superoxide dismutase 2	rs4880	The rs4880 and rs1799983 variations were significantly associated with male infertility	France	179 (69/110)	[71]
Myeloperoxidase	rs2333227
Glutathione peroxidase 1	rs1050450
Catalase	rs1001179
Nitric oxide synthase	rs2070744
	rs1799983
8-Oxoguanine DNA glycosylase 1	rs1052133	Interaction between rs1052133 polymorphism and smoking is related with male infertility	China	1100 (480/620)	[100]
Glutathione S-transferase	GSTM1 (null or non-null)	The rs1695 is associated with idiopathic male infertility. Also, individuals carrying genotype GSTT1-null and GSTP1 (Ile/Val + Val/Val) were at a high risk for male infertility	China	713 (234/479)	[82]
	GSTT1 (null or non-null)
	GSTP1 rs1695
Glutathione S-transferase	GSTM1	Men with GSTM1 (-/-)/GSTT1 (-/-) genotype are at a high risk for increased risk of reproductive problems	Russia	264 (104/160)	[101]
	GSTT1
Luteinising hormone β-subunit	Gly102Ser	This polymorphism is relatively common among infertile Egyptian men	Egypt	89 (50/39)	[102]
Paraoxonase 1	rs662	The rs662 variation is not associated with male infertility	China	732 (403/329)	[103]
N-Acetyltransferase-2	rs1799929	Male with homozygous and heterozygous variations of GSTP1 (rs1138272, rs1695) and NAT2 (rs1799929, rs1799930) genotypes have an increased risk of infertility	Vietnam	300 (150/150)	[104]
	rs1799930
Glutathione S-transferase	rs1138272
	rs1695

Epigenetic regulations are precise mechanisms that ensure that gene expression is regulated in normal growth [105]. Epigenetic changes regulate the gene expression and are not associated with the direct change in the DNA sequence. Epigenetic mechanisms including DNA methylation, histone changes, and performance of non-coding RNAs are essential for a number of biological processes including genomic imprinting and chromosome X inactivation [106]. Epigenetic markers, which play major roles in the gamete viability in sperms, are obtained during the cell growth and differentiation. Sperm DNA epigenetic planning happens at numerous important steps of spermatogenesis. In male rodents, expression of DNA methyltransferase 1 (DMT1) is upregulated in primary meiotic germ cells [107]. Testicular tissues from fertile male display restricted expression of DMT1 gene in pachytene spermatocytes, spermatogonia, and round spermatids [108]. It is reported that genes involved in spermatogenesis could experience epigenetic re-programming during maturation in epididymis. So, testicular and epididymal pathology could play a possible role in disruption of normal DNA sperm methylation program [109]. ROS leads to hypomethylation which is associated with sertoli cell-only syndrome, testis cancer, and hypospermatogenesis in humans [110]. However, some possible mechanisms for disrupted methylation of sperm DNA may exist including failures in the folate metabolizing pathway and OS [111]. OS results in breaks of the DNA strands and the generation of DNA base adducts such as O⁶-methylguanine and 8-OH-dG, both described to affect the interaction of the DNA and DNA methyltransferases [112]. The O⁶-methylguanine [113] or 8-OHdG presence in CpG sequences [114], mainly prevents the adjacent cytosine methylation and eventually results in total DNA hypomethylation [111]. The folate/homocysteine metabolic pathway is necessary for the production of methyl donors and consequently is crucial for the DNA methylation process in all cell types [115]. Failings in the folate pathway could lead to reducing the levels of S-adenosylmethionine (SAM) and have been associated with hypomethylation of DNA [116]. Methylenetetrahydrofolate reductase (MTHFR) is an important enzyme in folate pathway which converts the 5,10-methylene tetrahydrofolate to 5-methyl tetrahydrofolate. The latest compound provides methyl groups for conversion of homocysteine to methionine [117]. OS and hyper-homocysteinaemia have been associated with hypomethylation of somatic cell DNA. However, aberrations in metabolism of homocysteine could still possibly be correlated with changes in methylation of sperm DNA [111]. Further studies are necessary to access precise mechanisms of intermediary damage by ROS on epigenetic processes.

Effect of OS in cellular components

The role of OS in DNA damage

A high level of DNA damage usually occurs in sperms simultaneous with infertility [46, 118]. Undoubtedly, factors that enhance the level of ROS can affect the integrity of sperm chromatin. Guanine base (G) is the most common DNA’s organic base that is exposed to oxidative attack by free radicals and converts to 8-hydroxyguanine (8-OHG). According to conducted surveys on DNA of infertile men’s sperms and their comparison with fertile individuals, it is found that 8-OHG in infertile individuals is about 100 times more than fertile ones. OS leads to the production of oxidized DNA base compounds such as the 8-hydroxy-2-deoxyguanosine (8-OHdG) especially in genome locations that are less protamine [119].

Spermatozoon has 8-oxoguanine DNA glycosylase enzymes in the base excision repair (BER). This glycosylase is connected to the nucleus and mitochondria of sperm and can actively separate 8OHdG and release additional base compounds into the extracellular space. Spermatozoa do not have downstream compounds of BER pathway including apurinic endonuclease 1 (APE1) and XRCC1. Due to this incomplete repair capacity of DNA, abasic sites arise in locations that are under the influence of 8OHdG. These sites destabilize the ribose-phosphate and lead to the b-elimination or opening ribose cycle response and DNA strand fragment. This biochemical aspect of DNA is known as the main cause of the onset of cancer in other types of cells. Therefore, the base oxidative DNA damage not only is a mutagen but also indirectly fragments DNA [120]. The deficiency in the protection by antioxidants due to exogenous and endogenous factors leads to the oxidative damage in sperm and eventually DNA damage [121]. DNA damage causes a number of changes such as a single or double-strand fragment, DNA fragmentation, the creation of free-base sites, changes in purine, pyrimidine, deoxyribose, and DNA crosslinking [46]. These changes can lead to stopping or starting gene transcription, induction of final transduction pathways, increased degradation of telomeric DNA, replication errors, genomic instability, and transversion from CG to AT [122]. Sperms of patients with severe DNA oxidative damage are degenerated and absorbed through the spermatogenesis and sperm maturation in the epididymis resulting in reduced sperm count in these patients. DNA fragmentation is the final stage of ROS damage and is often seen in spermatozoa of infertile men [123]. There is a close relationship between ROS and sperm DNA fragmentation (SDF). SDF can occur for several reasons including varicocele, infection, aging, heat, lifestyle-related factors, incomplete protamination, and occasionally for no reason. A large number of them are mediated by ROS, and thus ROS is the main cause of SDF creation. ROS also can damage mitochondrial DNA of sperm. Since mtDNA have a circular structure with few DNA base pairs, lack of histones and absence of nucleotide excision repair (NER) pathway is more susceptible to ROS attack. In addition, ROS can damage the inner mitochondrial membrane and thus directly damage mtDNA and simultaneously cause electron leakage from the electron transport chain, which in turn increases the production of OS [4].

The role of OS in spermatic membrane lipid peroxidation

Sperms are particularly susceptible to injuries by ROS because, on the one hand, their plasma membranes are full of polyunsaturated fatty acids (PUFA) with multiple double bonds, and on the other hand, the cytoplasm rates of antioxidant enzymes in sperms are extremely low. Induction of lipid peroxidation cascades is a result of the exceeded level of ROS in stressed sperms leading to reduced sperm function [124]. Extensive lipid peroxidation changes the accumulation, structure, and dynamics of lipid membranes, and since these lipids are highly reactive compounds, it can produce a large amount of ROS [125]. Hydroxyl radical (·OH) is a powerful lipid peroxidation initiator (Fig. 1). Most of the unsaturated fatty acids of the sperm membrane are non-conjugated double bonds separated by methylene groups. The presence of double bonds near the methylene group weakens H–C bonds of methylene, thereby increasing the likelihood of hydrogen separation. When hydrogen is separated, free radicals are created and the location of double bonds changes in such a way that the created radical is stabilized. This radical contains two double bonds that are separated by a single bond. Therefore, lipids containing a large number of double bonds in the vicinity of methylene groups are highly susceptible to O2 peroxidation for radical creation. Conjugated radicals rapidly react with lipid peroxyl radicals (·ROO) that in turn separate hydrogen atoms from other lipid molecules to create lipid hydroperoxide (ROOH) [126]. Increased levels of ROS during a series of responses including the oxidation of sulfhydryl groups (SH-) decrease the phosphorylation of the axonal protein, thereby decreasing the sperm motility. Hydrogen peroxide (a kind of ROS) can pass through the membrane of sperm and enter the cytoplasm. Hydrogen peroxide in the sperm cytoplasm inhibits activities of certain enzymes including G6PD. This enzyme regulates the entry of glucose into the hexose monophosphate path (pentose phosphate pathway). Under normal conditions, this pathway produces reduced NADPH for cellular reduction responses. Inhibition of hexose monophosphate shunt reduces the production of NADPH as a reduction equivalent in sperm. Glutathione peroxidase (GPx) is a major antioxidant enzyme in the sperm and uses reduced glutathione (SH-G) for reduction of ROS. As a result of the action of glutathione peroxidase, the reduced glutathione is converted into its oxidized form (G-S-S-G). Cells need the NADPH for re-reduction of oxidized glutathione; hence, the reduced NADPH level due to the inhibition of G6PD enzyme reduces the activity of glutathione peroxidase enzyme as an antioxidant defense. Therefore, the level of phospholipids peroxidation increases in the sperm membrane, thereby reducing the fluidity of the membrane and reducing the sperm motility. Malondialdehyde (MDA) is a sub-product of lipid peroxidation that is considered as a measure of peroxidation damage to spermatozoa in various biochemical tests [124]. Also, removing electrons from lipids of plasmid membrane by ROS can cause lipid peroxidation. This leads to the creation of a series of reduction–oxidation reactions that ultimately result in highly mutagenic and genotoxic electrophilic aldehydes such as malondialdehyde (MDA), 4-hydroxynonenal (4-HNE) and acrolein. 4-HNE is derived from lipid hydroperoxides (LOOHs) of fatty acids ω-6 such as linoleic acid and arachidonic acid [4]. 4-HNE is a very genotoxic by-product, while MDA is very mutagenic. 4-HNE and acrolein significantly increase lipid peroxidation, ROS production in the mitochondria, DNA fragmentation and apoptosis [127, 128]. High levels of ROS could interrupt mitochondrial membranes, resulting in caspases’ activation and eventually apoptosis. Cytochrome c release throughout the apoptosis further increases ROS levels, raising damage and fragmentation of DNA, and potentially enhances the apoptotic cycle [3, 103]. Therefore, the sperm plasma membrane is a key target for ROS, which by stimulating cascade signals can damage the genetic composition of these cells. A recent study investigated the comparison of sperm parameters with MDA level as a factor for measuring the lipid peroxidation, NO, ascorbic acid and GSH in both infertile and unknown reproductive potential groups. Comparison of variables indicates that the sperm quality decreases in the infertile group, while NO, GSH and ascorbic acid levels increase. The results indicate that GSH and ascorbic acid levels probably increased to cope with the harmful effects of ROS and prevention of lipid peroxidation [129]. Given the above cases, determination of MDA level and total antioxidant capacity (TAC) may be useful in the diagnosis and treatment of male infertility.

The role of OS on male reproductive hormones

Testicles’ OS, which may be due to the damage to Leydig cells and other endocrine structures such as the anterior pituitary, decreases testosterone production [130]. In normal steroidogenesis, ROS is mainly produced by mitochondrial respiration and catalytic responses of cytochrome steroidogenic enzymes P450 [131]. OS increases the number of immature sperms through indirect effects on the production of male hormones during spermatogenesis [132]. Systemic hormones (FSH, LH, testosterone, E₂, PRL) adjust the total antioxidant capacity [133]. Some hormones like testosterone and melatonin act as antioxidants and protect sperm and other testosterone cells against damage by ROS [134]. Hypothalamic–pituitary–adrenal (HPA) axis is activated by the production of ROS and cortisol hormone is released in response to stress. This hormone negatively affects the anterior pituitary gland through cross-talk between hypothalamic–pituitary–gonadal (HPG) and HPA axes, and finally, decreases secretes of LH and FSH hormones. As a result of low LH, it cannot stimulate Leydig cells to secrete testosterone.

Furthermore, the reduced FSH decreases the release of androgen-binding protein (ABP) to sertoli cells; hence, a general decrease in testosterone level occurs during the OS. ROS also affects the hypothalamo–pituitary–thyroid (HPT) axis and reduces secretes of T3 and T4. Reduced T3 decreases StAR mRNA levels in Leydig cells and decreases testosterone production. Increased OS also decreases insulin secretion from the pancreas and negatively affects the secretion of T₃, thereby affecting the biosynthesis of testosterone. Conditions like obesity not only involve HPA and HPT axes but also cause the secretion or non-secretion of several metabolic hormones manifesting the sequence of ROS induction in male reproduction. ROS due to obesity can force adipocytes to secrete more leptin. This hormone together with insulin negatively adjusts the secretion of T₃, and thus inhibits the performance of testes. Secreted leptin by adipocytes inhibits the emission of gonadotropin-releasing hormone (GnRH) from the hypothalamus. After exposure to ROS, the aromatase activity increases resulting in excessive production of testicular E₂ hormones. OS also increases the secretion of inhibitory hormones; hence, E₂ hormones and the inhibitor prevent the secretion of testosterone. The exposure to ROS increases the PRL secretion from the anterior pituitary, and thus decreases GnRH secretion. The genital tract infection can produce some proinflammatory cytokines such as tumor necrosis factor alpha (TNF)-α, interleukin (IL)-1b and IL-6, thereby re-inhibiting the secretion of GnRH and testosterone hormones. Therefore, ROS can reduce the production of testosterone by affecting the hormonal axis alone or end up in cross-talk between different endocrine systems. Reducing testosterone disrupts spermatogenesis and produces immature sperm. Lack of testosterone as the main regulator of male reproductive behavior may lead to the suppression of sexual behavior among men. Therefore, ROS leads to male infertility by disrupting reproductive endocrine glands (Fig. 2) [6].

Fig. 2.

Effect of oxidative stress on production of testosterone. The hypothalamic–pituitary–gonadal axis (HPG; green arrow) regulates production of testosterone pathway in mammalian. The hypothalamus produces gonadotropin-releasing hormone (GnRH) and this hormone stimulates anterior lobe of the pituitary gland to secrete luteinizing hormone (LH) and follicle-stimulating hormone (FSH). FSH affects sustentacular cells (SC) to release androgen-binding protein (ABP) which this protein concentrates testosterone near spermatogenic cells. In addition, LH affects interstitial cells (IC) to release testosterone. Excessive production of ROS activates hypothalamic–pituitary–adrenal axis (HPA; blue arrow) and affects hypothalamus to release corticotropin-releasing hormone (CRH) which stimulates the anterior lobe of pituitary gland to release adrenocorticotropic hormone (ACTH) and this hormone affects adrenal gland. The adrenal releases cortisol hormone in response to OS. This hormone negatively affects the anterior pituitary gland and finally decreases secretion of LH and FSH hormones. In another pathway, obesity increases production of ROS. Excessive ROS stimulates adipocyte cells to produce leptin hormone and this hormone inhibits the HPG axis. On the other hand, excess ROS negatively affect pancreas to decrease insulin production, which results in decrease of testosterone production

Effect of OS in pathophysiological process and male infertility

Inflammation and OS

Inflammation is the natural response of the host to microbial attack or tissue damage and ultimately leads to arterial regeneration and improvement of its function [135]. Following the inflammation, neutrophils are the first immune cells presenting at the infection area; though macrophages also play an important role in the inflammatory response. Macrophages and other stimulated inflammatory cells secrete a large amount of prostaglandin E2 (PGE2), cytokine, and NO. However, inflammation affects steroidogenesis and spermatogenesis. In addition to inflammation, there is a significant reduction in testosterone levels and luteinizing hormone in the blood [136]. Inflammation also stops spermatogenesis and inhibits the sperm maturation process. Spermatocytes and spermatids are the main targets of inflammation. Epididymitis may be also affected by inflammatory attacks on testes. The inflammatory response is caused by leukocytes that penetrate semen and secrete antisperm antibodies. Decreasing lipid components of sperm flagella membrane, the inflammation increases its hardiness and decreases sperm motility resulting in sperm agglutination and asthenozoospermia. As a result of these events, maturation processes such as acrosome reaction, capacitation, and oocyte penetration encounter problems [137]. However, evidence suggests that the relationship between inflammation and OS is very high in sperm. Semen of infertile men has ROS in addition to high levels of proinflammatory factors and cytokines. Despite the fact that some external pathogenic factors such as bacteria may produce ROS, leukocytes are also the most important sources of semen ROS [138]. There are direct and indirect ways to increase ROS level by leukocytes. Leukocytes indirectly increase ROS levels by producing proinflammatory cytokines. In general, cytokines are proteins that act as signal molecules to regulate cellular responses such as inflammation. These proteins activate the xanthine oxidase system, raise ROS levels, and create OS.

On the other hand, by stimulating phagocytes and initiating the phagocytosis process, a high level of ROS is produced that reacts with the spermatozoa membrane and increases the ratio of oxidants-to-antioxidants. The OS caused by these conditions continues even after the elimination of pathogens [136]. Studies indicate that an increase in TNF-α of semen is associated with decreased number and mobility of sperm and its morphological defect. Increased cytokine levels in semen due to the proliferation and differentiation of beta cells, the proliferation of T cells and natural lethal cells cause apoptosis. Interleukin 1 alpha and interleukin 1 beta also induce apoptosis through the proliferation and differentiation of beta cells, the chemical absorption of leukocytes into inflammation site, induction of neutrophils and production of monocytes. These cytokines also negatively affect the quality of semen. A study suggests that increased levels of interleukin 1 beta are associated with reduced sperm motility [139, 140]. These results are accompanied by a simultaneous increase in ROS and MDA levels. Concentrations of similar cytokines, which act as immune-regulating elements for gonads, increase with infection and damage to semen tissue. Participation of these cytokines in the inflammation has a direct relationship with leukocytospermia. In inflammatory conditions, white blood cells in semen penetrate the inflammation site. In normal conditions, leukocytes are prevented from going to the center of the seminal vesicle. In inflammatory sites, arteries which feed testes are dilated and allow leukocytes to go to the vascular center and exit the vascular endothelium. In addition to these changes, the increased permeability leads to the fluid accumulation followed by swelling, pain and also the accumulation of immunoglobulins and other semen proteins in the reproductive system. The excretion of leukocytes from the seminal vesicle depends on the crosslinking responses that are activated by the release of inflammatory mediators. Therefore, the inflammation or physical damage to the reproductive system causes phagocytic cells to be absorbed into the injury site. Activated neutrophils by TNF-α produce oxygen and oxide nitride radicals and release their stored granular content resulting in the host defense and local tissue degradation. Since the male infertility is often caused by infection or damage to testes; and inflammatory response in the male reproductive system has a negative effect on the sperm quality and fertility, and the inflammation process produces ROS, all possible measures to eliminate or reduce inflammation reduce OS and improve pregnancy (Fig. 3) [141].

Fig. 3.

Schematic illustration of spermatogenesis in the seminiferous epithelium in an inflammatory condition. In the inflammatory reaction, sertoli cells, mast cells, and resident macrophages release chemokine and cytokines such as IL1β, IL6, IL8, TNFα, and TGFβ. These proinflammatory molecules cause the accumulation of leukocytes in the seminiferous tubules. Leukocytes also increase the level of ROS and OS up to 1000 times compared to normal conditions, and result in failed spermatogenesis

Varicocele and oxidative stress

Varicocele is a main cause of infertility in men; and its incidence is 15% in the whole men and 40% in infertile men [142]. OS is a major contributor to the pathophysiology of this disease. Increased level of ROS and reduced concentrations of antioxidants simultaneously intensify varicocele [143]. In men with varicocele, ROS is released from three types of reproductive system cells under heat and hypoxic stress conditions. These cells include main epididymal cells, endothelial cells in the dilated pampiniform plexus and testicular cells such as developing germ cells, Leydig cells, macrophages, and peritubular cells. In mitochondria, heat and hypoxic stress can directly activate complex III of the electron transport chain and cause the release of ROS. On the other hand, NO is released from testicular and endothelial cells in testis with varicocele and can nitrosylate complexes I and IV, and simulates complex III to further release ROS [144]. OS plays an important role in causing damage to testicular tissue. A great number of studies have measured OS markers in semen of infertile men with varicocele and compared their values to men with idiopathic infertility. The results indicate that infertile men with varicoceles have higher levels of ROS, free radicals such as NO, nitric oxide synthase and H₂O₂, high levels of MDA and hexanoyl-lysine (another product of lipid peroxidation) [145]. Spermatic veins are the main components of the pathogenesis of varicocele. Endothelial cells can produce a lot of ROS by certain stimuli. Plasma levels of NO and iNOS are higher in spermatic veins of infertile men with varicocele than their other veins [146]. Enzymatic and non-enzymatic antioxidants have been also measured to investigate OS in infertile men with varicoceles. Results indicate lower levels of seminal TAC in infertile men with varicocele than healthy participants [144]. However, some studies have reported that an early increase in concentrations of antioxidants such as SOD and CAT is due to the application of a defense mechanism to prevent the disease [147]. ROS level is high both in infertile men with varicocele and infertile men without varicocele, but it is much higher in the first group indicating that varicocele increases the production of ROS and subsequently the oxidative damage. Varicocele is also associated with the severity of OS; and the highest degree of varicocele leads to the highest OS [145].

Obesity and oxidative stress

Obesity is a metabolic disorder characterized by environmental, genetic, lifestyle and physical activity factors. Approximately 1.9 billion of the world’s population are overweight (BMI > 25 kg/m²) or obese (BMI > 30 kg/m²). Laboratory and clinical data indicate the negative effect of obesity on the male reproductive function. Determinants of sperm quality including the sperm count and its motility are lower and the DNA damage is higher in obese or overweight people compared with those with normal BMIs [148]. Consequently, obesity increases the risk of infertility by more than 20% [36]. Obesity along with the chronic inflammation increases metabolic rate and ROS in the tissue of testes and sperm. OS in sperm and testis has a positive correlation with increasing BMI and sperm DNA damage, but a negative correlation with reduced sperm motility and acrosome reaction [149]. In addition, gonad temperature in obese men may also help to change sperm parameters. The optimum temperature of spermatogenesis is between 34 and 35 °C in humans, and the spermatogenesis process is put at risk at higher temperatures. In obese men, the increased scrotal fat directly increases the internal temperature of the gonad [150]. Typically, increasing testicular heat can significantly exacerbate OS, thereby reducing the mobility and concentration of sperm and increase DNA damage [151]. It has been recently found that the OS of testicular tissue in obese rats is used to the increased MDA level and reduced SOD level. Obesity is associated with an increase in serum-free fatty acids, and unsaturated fatty acids are sensitive to ROS attacks, and thus the SOD rate decreases and MDA increases by peroxidation. Therefore, the long-term effects of obesity can reduce fertility in men due to increased OS [152]. OS is a common complication in many diseases such as infertility in men, and reactive oxygen species play important roles in spermatogenesis and reproduction. The normal functioning of sperm requires a balance between ROS levels and antioxidants; hence, the use of natural antioxidants, healthy lifestyles, regular exercise, and proper diets lead to the weight loss and gradual BMI reduction that helps reduce OS and provide sperm health [153].

Effect of antioxidants and herbal therapy on improvement of sperm quality

Antioxidant system including enzymatic factors (superoxide dismutase, catalase, and glutathione peroxidase), non-enzymatic factors, and low-molecular weight compounds (glutathione, n-acetylcysteine, vitamin E, A and C, coenzyme Q10, carnitines, myo-inositol, lycopene and …), and nutrients (selenium, zinc, and copper) can protect the body against OS. Lack of one of these leads to a reduction in the antioxidant capacity of the plasma [154]. Many urologists prescribe oral antioxidants for infertility [155], probably as the OS in semen is due to the lack of antioxidants and its side effects. Ideally, an oral antioxidant should reach a high concentration in the reproductive tract and restore vital elements for spermatogenesis. In addition, antioxidant supplementation should increase the seminal plasma clearance capacity and decrease ROS level in semen [156]. Nowadays, the use of herbal therapy has also been considered to prevent infertility because these antioxidants can reduce the destructive effects of oxidative stress [157].

Enzymatic factors

SOD metalloenzymes are effective antioxidant enzymes in the body. These enzymes are the first body defense against the ROS. SODs convert the superoxide anion into H₂O₂, and then the produced H₂O₂ is eliminated by activities of glutathione peroxidase or catalase. SOD protects spermatozoa against O₂ toxicity, lipid peroxidation and ultimately OS [158]. There is no consensus on SOD consumption dosage; however, the most commonly proposed treatment for oral consumption is 150 IU per day for at least 3 months [159]. Catalase exists in peroxisomes and converts H₂O₂ into the water and molecular oxygen in two steps. This enzyme plays a major role in the capacitation of nitric oxide-induced sperm [154]. Glutathione peroxidase converts H₂O₂ to water or alcohol, provides the main protection against low levels of oxidative damage, and plays an important role in the defense of sperm [160]. Glutathione is a tripeptide containing sulfur and can be seen in GSH and GSSG. The decreased glutathione in the human seminal plasma leads to instability in the middle of spermatozoa and ultimately causes motion impairment [161]. Glutathione supplement plays a pathological role in infertile men in situations where reactive oxygen species or other toxic substances such as unilateral varicoceles or genital inflammation result in significant improvement in sperm parameters (number, mobility, and morphology). GSH also plays an important protective role against cell membrane lipid damage. Its effective dose is intramuscular 600 mg per day for 2–3 months [162]. N-Acetylcysteine (NAC) is a glutathione precursor and stimulates the sperm motility. It effectively improves the volume of seminal fluid and its viscosity and increases the sperm motility in humans [163]. The most commonly used dose of NAC is 600 mg per day as a combination with other antioxidants for at least 3 months [164].

Animal experiments

NAC protects sperm DNA against oxidative damage and can improve sperm parameters in mice [165]. The catalase present in the sperm of ram has a potential role in controlling oxidative stress by catalyzing H₂O₂ [166].

Non-enzymatic factors

Carotenoids are natural antioxidants that protect the integrity of the cell membrane, regulate the differentiation of epithelial cells and contribute to the regulation of spermatogenesis. Lack of carotenoids in the diet can reduce sperm motility [154]. In humans, low serum levels of retinol reduce sperm quality [167]; hence the use of vitamin A can be used to treat male infertility [168]. The amount of vitamin C in seminal plasma is ten times more than serum [169]. In humans, ascorbic acid levels are positively correlated with percentages of normal sperms [170] and negatively with the DNA fragmentation [171]. These findings support the consumption of vitamin C for treating male infertility. Proper vitamin C dose for increasing sperm quality is oral 1 g per day [172]. Vitamin E protects the sperm cell membrane against the OS, prevents lipid peroxidation and traps free radicals of hydroxyl and superoxide [154]. A clinical trial indicates that sperm function improves after consuming 600 mg of vitamin E per day for 3 months. Other studies used lower doses (200 mg/300 mg) [173]. A study reported that the sperm motility increased in infertile men after oral consumption of vitamin E. Improvement of sperm motility is associated with the reduction of MDA production as the final product of lipid peroxidation. In addition, during 6 months of treatment, 21% of patients achieved pregnancy [174]. In general, these findings indicate that vitamin E can be a good choice for treating male infertility [175]. Coenzyme Q10 is a fat-soluble antioxidant that endogenously synthesizes and prevents the oxidation and lipid peroxidation. It also regulates the mitochondrial electron transfer chain [176]. A number of clinical studies found a positive effect of coenzyme Q10 on male fertility. This antioxidant improves all sperm parameters [177] and has a protective effect against OS and DNA damage [178]. Moreover, the treatment with CoQ10 improves sperm parameters in people with varicocele [176]. CoQ10 can regenerate other antioxidants such as vitamins E and C [179]. Selenium is a necessary nutrient for normal growth of testicles, spermatogenesis, and sperm motility and function. The selenium deficiency leads to the epithelium atrophy of seminiferous, spermatogenesis and sperm maturation impairment, reduced volume of testes, decreased sperm motility and changed sperm shape (especially in the head and middle part) [180]. There is no fully precise mechanism by which selenium reduces OS and improves sperm parameters, but is likely to be achieved by selenoenzyme such as GPX. An appropriate dose of selenium for oral use is 80–300 micrograms per day alone or with other antioxidants for 3 months [181]. Low amounts of ROS are essential for capacitation and acrosome reaction. Therefore, the excess reduction of ROS concentration due to the use of antioxidants may have reversible effects on fertility [182]. However, antioxidants neutralize the OS; and most studies indicate that improvement of sperm parameters occurs when several antioxidants are used in combination. However, there is no definitive consensus on dose and duration of use of these substances; and there is a need for further studies [10].

Animal experiments

A study on cows indicated that retinol stabilizes sperm acrosome membrane in the OS condition due to the high temperature [167]. Vitamin C in mice in a dose equivalent to human dose (10 m/kg) can reduce MDA level and increase the number of sperms [183]. Another study shows that treatment with ascorbic acid reduces peroxidation levels and histological damage on seminiferous tubules in diabetic rats [184].

Other nutrients

In addition to antioxidants, there are a variety of therapies around the world for male infertility management. The recent therapies as assisted reproductive techniques (ARTs) are not only costly but also have a success rate of only 10–30%. Accordingly, the use of herbal medicines as an alternative therapy or complementary technique has been taken into account more for coping with male infertility [157]. The active substances of plants, which improve fertility, include various chemical groups such as saponins, phytosterols, carotenoids, oxygenated volatile compounds, phenols, lignans and alkaloids [185]. Withania somnifera (ashwagandha) is the root of evergreen shrub in India. Studies indicate that the oral intake of this plant increases the quality of semen, prevents the lipid peroxidation, regulates levels of reproductive hormones, and also decreases the risk of infertility in men by reducing the level of OS [157]. Curcumin is a Chinese herb monomer that is extracted from yellow turmeric and has antioxidant and anti-inflammatory properties. According to a study on the effect of curcumin on sperm quality in patients with leukocytospermia, results indicate that curcumin significantly improves the sperm motility in patients and decreases H₂O₂. Therefore, it overcomes male infertility by reducing OS [186]. Cinnamon has antioxidant, anti-inflammatory and anti-diabetic properties. Therefore, the use of cinnamon has potential effects on the oxidative and antioxidant balance of testes and sperm quality; and its use is beneficial for men with asthenozoospermia [187]. Crocus sativus is a plant of iridaceae family and is traditionally used for diseases of the digestive system, reproductive tract, and depression. Saffron plays a protective role against DNA damage, chromatin anomalies and provides the membrane integrity [188, 189]. Nigella sativa (black caraway) is a herbal medicine that is applicable for a wide range of diseases such as pulmonary problems, headache, rheumatism, gastrointestinal problems, and male sexual disorders [185]. In one clinical trial, the use of seed oil for infertile men with different factors of infertility resulted in the enhancement of all semen parameters [190]. Pomegranate seeds contain fatty acids and phenolic compounds with antioxidant properties. Green tea can improve gametes in men and women by the ability of catechin polyphenol through eliminating ROS. Epigallocatechin-3-gallate (EGCG) is considered as an active antioxidant that can potentially improve reproductive health by reducing the OS [191]. There is little information about exact mechanisms of these plants and infertility, but most findings have pointed out the antioxidant properties of plants.

Animal experiments

A study examined the long-term effects of cinnamon crust oil on antioxidant levels of testes and sperm quality of mature adult rats. The results indicated a significant reduction in the level of MDA and a significant increase in the activity of glutathione peroxidase and catalase [187]. A study on rats demonstrated that dietary Zn deficiency decreases serum testosterone levels, increases OS and results in apoptosis of germ cells [192]. Catechins are the main components of green tea, which have higher antioxidant activity than vitamin C. Catechins in rats were observed to regulate gene expression and reduce ROS levels [193]. Animal studies indicate that the use of Crocus sativus (saffron) improves sperm parameters due to its antioxidant properties [194]. In addition, it promotes semen quality by increasing testosterone levels [195]. Animal studies show that the alcoholic extract of black caraway seed improves the sperm survival and also increases the sperm motility. The precise mechanism of black caraway function is still unknown, but it seems that it increases the activity of beta-hydroxy steroid dehydrogenase (a key enzyme in the synthesis of testosterone) due to the presence of unsaturated fatty acids, and thus it is useful for the fertility [196] Sesamum indicum (sesame) is an annual plant and old crop which could improve the quality of sperm parameters in rat. Furthermore, sesame helps to make better spermatogenesis process by affecting the hypothalamus–pituitary–testis axis and increasing the blood testosterone [197]. Pomegranate seeds can improve the integrity of the sperm membrane and protect it against oxidative damage, and prevent the DNA damage by antioxidant properties by eliminating free radicals [198]. Ionidium suffruticosum (L.) ging (Violaceae) is an important herbal medicine that is widely used as traditional medicine. According to a study on infertile rats treated with this herbal medicine, results indicated that spermatogenesis, sperm count, and oxidative biomarkers of SOD and CAT improved after a treatment period. Therefore, the use of this herbal medicine for the treatment of male infertility was recommended in traditional medicine [199]. Grape seed extract acts as an anti-inflammatory, antioxidant and antibacterial agent and also has protective effects on the heart, liver, and nerve. Grape seed contains a special flavonoid called anthocyanin oligomers. This compound increases intracellular vitamin C level and thus destructs ROS and free radicals, and it has more antioxidant properties than vitamins C and E. In a study, the impairment of testicular activity was induced by aluminum chloride, and the sperm number and motility increased after intervention with grape seed extract. It also protects sperm against DNA damage and protects testicles by reducing the NO activity [200].

Conclusion

OS is now considered as the main cause of male infertility. Despite the need for low levels of ROS for the physiological function of sperm, its increased level disturbs sperm functions, thereby leading to male infertility by mechanisms such as lipid peroxidation and DNA damage. Therefore, the early diagnosis of infertility is necessary to prevent its irreversible damage due to the OS in the long term. There are several tests for early diagnosis of high levels of OS factors. Results of measuring levels of ROS in semen as well as the determination of MDA and TAC levels can affect the male infertility diagnosis and treatment. Besides, the lifestyle change is the primary action to balance levels of ROS and antioxidants in the body and prevent oxidative stress. Reduced smoking and alcohol, non-exposure to radiation, and selection of a proper diet along with planned physical activity have significant effects on reducing levels of OS and thus male infertility. Moreover, the use of oral antioxidants can also help to reduce OS, but more studies are necessary about their doses and duration.

Abbreviations

·OH

Hydroxyl

·ROO

Proxyl

4-HNE

4-Hydroxynonenal

8-OHdG

8-Hydroxy-2-deoxyguanosine

ABP

Androgen-binding protein

ARE

Antioxidant responsive element

ARTs

Assisted reproductive techniques

ATP

Adenosine triphosphate

BER

Base excision repair

cAMP

Cyclic adenosine monophosphate

CAT

Catalase

CK

Creatine kinase

CYP2E

Cytochrome P450 enzymes

ETC

Electron transport chain

G6PD

Glucose-6-phosphate dehydrogenase

GnRH

Gonadotropin releasing hormone

GPx

Glutathione peroxidase

GRX

Glutaredoxin

GSH

Glutathione

GST

Glutathione s-transferase

H₂O₂

Hydrogen peroxide

HPA

Hypothalamic–pituitary–adrenal

HPG

Hypothalamic–pituitary–gonadal

HPT

Hypothalamo–pituitary–thyroid

IL

Interleukin

LOOHs

Lipid hydroperoxides

MDA

Malondialdehyde

miRNAs

Micro-ribonucleic acids

MPO

Myeloperoxidase

NAC

N-Acetylcysteine

NADPH

Adenine dinucleotide phosphate

NO

Nitrogen oxide

NOS

Nitric oxide synthase

NRF2

Nuclear factor erythroid 2-related factor 2

O^·−₂

Superoxide anion

ONOO-

Peroxynitrite

OS

Oxidative stress

P-Tyr

Phosphotyrosine

PGE2

Prostaglandin E2

PKA

Protein kinase A

NER

Nucleotide excision repair

PMN

Polymorphonuclear

P-PKA

Phospho-PKA

PRX

Peroxiredoxin

PUFA

Polyunsaturated fatty acids

RF

Radio frequency

ROOH

Hydroperoxide

ROS

Reactive oxygen species

SCI

Spinal cord injury

SDF

Sperm DNA fragmentation

SH-

Sulfhydryl groups

SOD

Superoxide dismutase

SOD3

Extracellular SOD

TAC

Total antioxidant capacity

TNF-α

Tumor necrosis factor alpha

TRX

Thioredoxin

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.