**3. Oxidative stress in male infertility**

A decline in fertility rates is becoming an increasingly prevalent issue worldwide. Infertility affects up to 15% of the population globally [108], and furthermore, male infertility is responsible in about 20% of cases but may contribute to 40% of

infertile couples [109]. The leading cause of male infertility stems from a loss of spermatozoa function, ultimately resulting in a loss of fertilization potential [110]. This loss in function is causatively linked to oxidative stress within the spermatozoa driven by the presence and/or overproduction of intracellular ROS [111].

Several studies have shown conflicting results for the effect of the antioxidant therapy on male fertility, whilst a number of studies conveyed a favorable effect on basic semen parameters, advanced spermatozoa function tests and pregnancy rates. But, the ideal balance of the redox system necessary for optimal spermatozoa function is not known, and overconsumption of antioxidants may result in reductive stress that could cause detrimental effects on human health and well-being. Impairment of mitochondrial activity [112], reduction in blood–brain barrier permeability [113] and attenuation of endothelial cell proliferation [114] are consequences that have been reported to occur secondary to reductive stress. **Table 2** shows the mechanism of action of several commonly used antioxidants for the treatment of male infertility. The list of antioxidants used in treatment of male infertility is presented in **Table 3**.

Vitamin E is well accepted as the first line of defence against lipid peroxidation, protecting polyunsaturated fatty acids in cell membranes through its free radical quenching activity in biomembranes at an early state of free radical attack. MDA concentration was prevented by treatment with vitamin E; it may help in the prevention of against production of free radicals and quenches free hydroxyl radicals and superoxide anions, thereby reducing lipid peroxidation initiated by ROS at the level of plasma membranes [126]. Its antioxidant activity is similar to that of glutathione peroxidase. In infertility of male, the percentage of motile spermatozoa is significantly related to spermatozoa vitamin E content [127]. Lower levels of vitamin E were observed in the semen of infertile men [128]. Insufficient intake of vitamin E produced deleterious effects on the process of normal spermatozoa [129]. One of the earlier studies investigating vitamin E alone (300 mg daily) on infertile men reported significant improvement in spermatozoa motility [121]. Combined with clomiphene citrate treatments, vitamin E significantly improved spermatozoa concentration and motility of patients with idiopathic oligoasthenozoospermia (OAT) [130]. Another observational study investigated a daily regimen of vitamin E (400 mg) + selenium (200 lg), for a period of 100 days, on infertile men with idiopathic asthenoteratospermia. Results revealed that 52.6% of patients showed a significant improvement in spermatozoa motility, morphology or both [131]. On the other hand, a few other studies failed to reproduce any significant effect on semen


**81**

patients [118, 137].

other antioxidants [132].

*Proposed antioxidants in various clinical treatments.*

**Table 3.**

*Effects of Oxidative Stress on Spermatozoa and Male Infertility*

**Clinical applications Antioxidants daily References**

Asthenozoospermia Zinc (400 mg), vitamin E (20 mg) and vitamin C (10 mg) [120]

Teratozoospermia Selenium (200 mg) [117]

Improving DNA integrity Vitamin E (1 g) + vitamin C (1 g) [121]

E (10 mg), zinc (10 mg), folic acid (200 lg),

Improving ART vitamin E (200 mg) [33]

Improving live birth rate CoQ10 (300 mg) [116]

Vitamin C (400 mg), vitamin E (400 mg), b-carotene (18 mg), zinc (500 mmol) and selenium (1 mmol)

LC (1500 mg), vitamin C (60 mg), CoQ10 (20 mg), vitamin

LC (2 g) [115] CoQ10 (300 mg) [116] Selenium (200 mg) [117] Folic acid (5 mg) + zinc (66 mg) [118] Lycopene (2 mg) [119]

CoQ10 (300 mg) [116] Selenium (200 mg) [117] Lycopene (2 mg) [119]

Zinc (400 mg), vitamin E (20 mg) and vitamin C (10 mg) [120]

Vitamin E (600 mg) [122] Vitamin C (1 g) + vitamin E (1 g) [123]

Vitamin E (300 mg) [124] Zinc (5000 mg) [12] Vitamin E (1 g) + vitamin C (1 g) [121] Carnitines: LC (2 g) + LAC (1 g) [125]

[22]

[29]

[32]

Oligozoospermia Vitamin E (180 mg), vitamin A (30 mg) and essential fatty

acids (600 mg)

parameters using vitamin E as a single treatment [123, 125] or in combination with

In the male reproductive system, vitamin C (ascorbic acid) is known to protect spermatogenesis and plays a key role in spermatozoa integrity and fertility both in men by increasing testosterone levels and preventing spermatozoa agglutination. It exists at a concentration 10 times higher in seminal plasma than in blood serum [133] and contributes up to 65% of the total antioxidant capacity of seminal plasma found intracellularly and extracellularly [134, 135]. Semen of infertile men with asthenozoospermia was found to contain lower vitamin C levels and higher ROS levels than those obtained from fertile controls [117]. Vitamin C as a single agent which is used to treat heavy smokers, with a daily dose of 200 or 1000 mg or placebo for 1 month, significantly improved spermatozoa quality [136]. Receiving 500 mg daily vitamin C with a combination of zinc, vitamin E and vitamin C for a total of 3 months after undergoing varicocelectomy significantly improved spermatozoa motility and morphology on varicocelectomy

*DOI: http://dx.doi.org/10.5772/intechopen.86585*

#### **Table 2.**

*Mechanism of action of commonly used antioxidants and clinical dosage.*



#### **Table 3.**

*Free Radical Medicine and Biology*

infertility is presented in **Table 3**.

infertile couples [109]. The leading cause of male infertility stems from a loss of spermatozoa function, ultimately resulting in a loss of fertilization potential [110]. This loss in function is causatively linked to oxidative stress within the spermatozoa

Several studies have shown conflicting results for the effect of the antioxidant therapy on male fertility, whilst a number of studies conveyed a favorable effect on basic semen parameters, advanced spermatozoa function tests and pregnancy rates. But, the ideal balance of the redox system necessary for optimal spermatozoa function is not known, and overconsumption of antioxidants may result in reductive stress that could cause detrimental effects on human health and well-being. Impairment of mitochondrial activity [112], reduction in blood–brain barrier permeability [113] and attenuation of endothelial cell proliferation [114] are consequences that have been reported to occur secondary to reductive stress. **Table 2** shows the mechanism of action of several commonly used antioxidants for the treatment of male infertility. The list of antioxidants used in treatment of male

Vitamin E is well accepted as the first line of defence against lipid peroxidation, protecting polyunsaturated fatty acids in cell membranes through its free radical quenching activity in biomembranes at an early state of free radical attack. MDA concentration was prevented by treatment with vitamin E; it may help in the prevention of against production of free radicals and quenches free hydroxyl radicals and superoxide anions, thereby reducing lipid peroxidation initiated by ROS at the level of plasma membranes [126]. Its antioxidant activity is similar to that of glutathione peroxidase. In infertility of male, the percentage of motile spermatozoa is significantly related to spermatozoa vitamin E content [127]. Lower levels of vitamin E were observed in the semen of infertile men [128]. Insufficient intake of vitamin E produced deleterious effects on the process of normal spermatozoa [129]. One of the earlier studies investigating vitamin E alone (300 mg daily) on infertile men reported significant improvement in spermatozoa motility [121]. Combined with clomiphene citrate treatments, vitamin E significantly improved spermatozoa concentration and motility of patients with idiopathic oligoasthenozoospermia (OAT) [130]. Another observational study investigated a daily regimen of vitamin E (400 mg) + selenium (200 lg), for a period of 100 days, on infertile men with idiopathic asthenoteratospermia. Results revealed that 52.6% of patients showed a significant improvement in spermatozoa motility, morphology or both [131]. On the other hand, a few other studies failed to reproduce any significant effect on semen

**Antioxidants Antioxidant mechanism Typical daily** 

Vitamin E Neutralizes free radicals 200–600 mg Vitamin C Neutralizes free radicals 200–1000 mg Selenium Enhancement of antioxidant enzyme activity 100–200 mg Zinc Inhibition of NADPH oxidase and scavenges hydroxyl radicals 15–40 mg Carnitines Neutralizes free radicals and acts as an energy source 1–3 g

system

Lycopene Scavenges free radicals 4–6 mg

*Vitamin E, tocopherol; vitamin C, ascorbic acid; NADPH, nicotinamide adenine dinucleotide phosphate.*

CoQ10 Scavenges free radicals of mitochondrial electron transport

*Mechanism of action of commonly used antioxidants and clinical dosage.*

**dose**

60–300 mg

driven by the presence and/or overproduction of intracellular ROS [111].

**80**

**Table 2.**

*Proposed antioxidants in various clinical treatments.*

parameters using vitamin E as a single treatment [123, 125] or in combination with other antioxidants [132].

In the male reproductive system, vitamin C (ascorbic acid) is known to protect spermatogenesis and plays a key role in spermatozoa integrity and fertility both in men by increasing testosterone levels and preventing spermatozoa agglutination. It exists at a concentration 10 times higher in seminal plasma than in blood serum [133] and contributes up to 65% of the total antioxidant capacity of seminal plasma found intracellularly and extracellularly [134, 135]. Semen of infertile men with asthenozoospermia was found to contain lower vitamin C levels and higher ROS levels than those obtained from fertile controls [117]. Vitamin C as a single agent which is used to treat heavy smokers, with a daily dose of 200 or 1000 mg or placebo for 1 month, significantly improved spermatozoa quality [136]. Receiving 500 mg daily vitamin C with a combination of zinc, vitamin E and vitamin C for a total of 3 months after undergoing varicocelectomy significantly improved spermatozoa motility and morphology on varicocelectomy patients [118, 137].

Carnitines [L-carnitine (LC) and L-acetyl carnitine (LAC)] are water-soluble antioxidants involved in spermatozoa metabolism, fuelling important activities like spermatozoa motility [138]. The carnitine and acetylcarnitine can significantly improve spermatozoa motility or kinetics in patients with asthenozoospermia [120, 139]. In vitro studies of spermatozoa cultured in media containing carnitines had higher motility and viability. They exhibit their antioxidant activities through scavenging superoxide anions and hydrogen peroxide radicals, thereby inhibiting lipid peroxidation. A combined treatment of LC (2 g) and LAC (1 g) for 2 months' duration to placebo in men with OAT showed significant improvement in all semen parameters; however, the most significant increase was in spermatozoa motility. Low-grade varicocele and idiopathic infertility patients treated with LC and LAC in comparison with placebo had significant improvement in all semen parameters [140]. On the contrary, LC (1000 mg) and LAC (500 mg) daily treated asthenozoospermic men for 12 weeks and failed to show any significant improvement in spermatozoa motility [141].

CoQ10 is a vital antioxidant omnipresent in almost all body tissues. It is particularly present at high concentrations in spermatozoa mitochondria involved in cellular respiration and plays an integral role in energy production [142]. This contribution rationalizes its use as a promotility and antioxidant molecule. Furthermore, CoQ10 inhibits superoxide formation, delivering protection against OS-induced spermatozoa dysfunction. A significant negative correlation between CoQ10 levels and hydrogen peroxide has been reported, and a linear correlation between CoQ10 levels in seminal plasma spermatozoa count and motility was detected [115]. 300 mg CoQ10 for 26 weeks obtained a significant increase in sperm density and motility [143]. A systemic review of clinical trials on 332 infertile men revealed that treatment with CoQ10 (200–300 mg daily) resulted in a significant increase in spermatozoa concentration and motility [144].

Antioxidant properties of selenium are thought to stem from its ability to augment the function of glutathione. More than 25 selenoproteins exist, such as phospholipid hydroperoxide glutathione peroxidase (PHGPX) [145] and spermatozoa capsular selenoprotein glutathione peroxidase [146], to maintain spermatozoa structural integrity [147]. Selenium deficiency has been most commonly associated with morphological spermatozoa midpiece abnormalities and impairment of spermatozoa motility [148]. A significant increase (74%) in total normal spermatozoa concentration was noted amongst the subfertile group receiving combined therapy [116] with a combination of both folic acid and zinc for 26 weeks of treatment. Selenium has been less frequently investigated for the treatment of subfertile men. As previously noted, with selenium (200 mg) supplements for 26 weeks, results showed a significant improvement in all semen parameters. A strong correlation was seen between the sum of the selenium and mean spermatozoa concentration, motility and percentage normal morphology [149]. Furthermore, the combination of selenium with vitamin E resulted in an increase in spermatozoa motility [124, 150]. But in the contrary report, treatment with selenium (300 mg) daily for 48 weeks did not result in a significant influence on semen parameters of a group of normozoospermic men [122].

Zinc plays a vital role in the metabolism of RNA and DNA, signal transduction, gene expression and regulation of apoptosis. Its antioxidant properties are thought to result from its ability to decrease production of hydrogen peroxide and hydroxyl radicals through antagonizing redox-active transition metals, such as iron and copper [151]. Zinc concentrations of seminal plasma were found to be significantly lower in subfertile men [152]. Spermatozoa flagellar abnormalities, such as hypertrophy and hyperplasia of the fibrous sheath, axonemal disruption, defects of the inner microtubular dynein arms and abnormal or absent midpiece, are all associated with zinc deficiency [153]. Zinc given for 3 months in men with asthenozoospermia obtained a significant improvement in spermatozoa concentration, progressive motility and fertilizing capacity and a reduction in the incidence of anti-spermatozoa antibodies [153]. Oral zinc supplementation

**83**

China

*Effects of Oxidative Stress on Spermatozoa and Male Infertility*

sperm/mL [119].

successfully restored seminal catalase-like activity and improved spermatozoa concen-

Lycopene is a naturally synthesized carotenoid presented in fruits and vegetables. Its powerful ROS quenching abilities make it a major contributor to the human redox defense system [155]. Lycopene is detected at high concentrations in human testes and seminal plasma with levels that tend to be lower in infertile men [156]. The treatment with 2 mg lycopene twice daily for 3 months significantly improves spermatozoa concentration and motility in 66% of patients, respectively. However, the effects were only significant in patients who had baseline spermatozoa concen-

Spermatozoa possess an inherent but limited capacity to generate ROS which may help the fertilization process. Antioxidants improve the motility and fertilizing ability of spermatozoa. A balance between the benefits and risks from ROS and antioxidants appears to be necessary for the survival and normal functioning of spermatozoa. Antioxidants in extenders may minimize the detrimental effect of ROS and improve the quality of frozen–thawed spermatozoa in animals and human. From the other point of view, the divergent effect of each antioxidant supplementation, improving different parameters of frozen–thawed sperm quality, is attributed to animal species, extender medium and type of molecule and concentration used for each species. Although a beneficial influence was generally observed for antioxidants in reversing ROS-induced spermatozoa dysfunction and in improving pregnancy rates, evaluation of ROS and the use of antioxidants are not routine in clinical practice. The dose and duration of these antioxidants should also be determined and standardized. There should be an effort to develop optimum combinations of antioxidants to supplement spermatozoa media. Finally, this study suggests that further research should be done to determine the appropriate antioxidant compounds as well as certain dose of antioxidants whether used clinical practices or cryopreservation. Moreover the future studies should concern

the spermatozoa fertilization and pregnancy rate as a research emphasis.

1 Grassland Agri-Husbandry Research Center, College of Grassland Science,

2 Jilin Provincial Key Laboratory of Grassland Farming, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun, Jilin,

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

tration and progressive motility in a group of asthenozoospermic men [154].

*DOI: http://dx.doi.org/10.5772/intechopen.86585*

trations of >5 × 106

**4. Conclusion**

**Author details**

Yi Fang1,2\* and Rongzhen Zhong1,2

Qingdao Agricultural University, Qingdao, China

\*Address all correspondence to: fangyi@iga.ac.cn

provided the original work is properly cited.

#### *Effects of Oxidative Stress on Spermatozoa and Male Infertility DOI: http://dx.doi.org/10.5772/intechopen.86585*

successfully restored seminal catalase-like activity and improved spermatozoa concentration and progressive motility in a group of asthenozoospermic men [154].

Lycopene is a naturally synthesized carotenoid presented in fruits and vegetables. Its powerful ROS quenching abilities make it a major contributor to the human redox defense system [155]. Lycopene is detected at high concentrations in human testes and seminal plasma with levels that tend to be lower in infertile men [156]. The treatment with 2 mg lycopene twice daily for 3 months significantly improves spermatozoa concentration and motility in 66% of patients, respectively. However, the effects were only significant in patients who had baseline spermatozoa concentrations of >5 × 106 sperm/mL [119].
