**7. Strategies to reduce oxidative stress in male reproduction**

Antioxidant supplementation has proven to be effective against male reproductive dysfunction *in vivo*. Recent reports have acclaimed significant attention due to the quality of their study design and demonstrated compelling evidence regarding the efficacy of antioxidants towards improving semen parameters. On the other hand, numerous clinical trials studying the effects of dietary antioxidants on semen parameters are still uncontrolled, focus on rather on healthy individuals or have indirect end-points of success. The dose and duration of antioxidant administration also need to be thoroughly examined and standardized. **Table 4** presents the most effective doses for the treatment of male subfertility based on currently available studies that explored the impact of antioxidant supplementation on sperm parameters.


ROS-induced damage may have significant clinical implications in the context of ARTs. Numerous reports have indicated that significantly increased ROS levels may occur in response to repeated cycles of centrifugation involved in conventional sperm preparation techniques used for ARTs [179]. Spermatozoa selected for ART often face OS and a high risk for DNA damage. When intrauterine insemination or *in vitro* fertilization (IVF) is used, such damage does not represent a cause of concern as damage to the sperm membrane lipids ensures that fertilization will not occur. However, in case of intracytoplasmic sperm injection is used, this natural selection barrier may be overlooked and sperm with DNA damage may

patients resulted in a higher sperm count and membrane integrity. ZnSO4

experimental group and TNF-α and antisperm antibodies decreased [176]

Coenzyme Q10 • Supplementation of 60 mg/day of coenzyme Q10 for 103 days led to an increase in the

with low fertilization rates due to male factor infertility [177]

**Table 4.** Most pronounced studies on the effects of oral antioxidant supplementation on male infertility.

an immunological role as T-helper cytokines and interleukin-4 levels increased in the

fertilization rate but had no effect on motility, morphology or concentration in 17 patients

• 60 patients with idiopathic asthenozoospermia who received 200 mg/day of coenzyme Q10 demonstrated significant improvement in motility after 6 months of treatment [178]

twice daily for 3 months to 50 asthenozoospermic

Physiological and Pathological Roles of Free Radicals in Male Reproduction

also played

139

http://dx.doi.org/10.5772/intechopen.70793

Selection of an effective sperm preparation technique is important to minimize ROS overgeneration and eventual oxidative insults to the male gamete. The density gradient technique is able to separate leukocytes and immature or damaged spermatozoa from normal spermato-

Assisted reproduction techniques may benefit from *in vitro* supplementation of antioxidants [180]. Various antioxidants such as vitamin E, vitamin C, cysteine, taurine and hypotaurine present in the culture medium have been shown to improve the developmental ability of the

In cases of IVF, incubation times of more than 16–20 hours have been correlated with increased oxidative damage. Shortening the insemination timeframes (up to 1–2 hours or less) may reduce ROS overgeneration in culture media and possibly improve fertilization, embryogen-

Because high levels of ROS have been associated with a decreased male infertility, measuring ROS levels in semen is an important part of the initial evaluation as well as follow-up of men with reproductive dysfunction [10–12]. Chemiluminescence and flow cytometry are currently

Chemiluminescence measures light emitted following administration of specific reagents to a semen sample. Two major probes currently used to assess ROS generation by spermatozoa

the most common techniques in clinical andrology to assess and study seminal OS.

be directly injected into the ovum [77].

Zinc sulfate (ZnSO4

esis and pregnancy rates [77, 179].

zoa, which may be subsequently used in ARTs [77, 179, 180].

) • Administration of 250 mg of ZnSO4

**8. Methods for detecting reactive oxygen species**

embryos by counteracting the effects of ROS [93, 180].


**Table 4.** Most pronounced studies on the effects of oral antioxidant supplementation on male infertility.

Vitamin C • 13 infertile patients received 1000 mg of vitamin C twice daily for a maximum of 2 months.

Vitamin E • 110 asthenozoospermic patients received 300 mg of vitamin E daily over a period of

Glutathione • 600 mg of GSH per day given to 11 men suffering from dyspermia associated with

improvement in sperm kinetics and higher sperm concentration [165]. Carnitine • 3 g/day of L-carnitine was administered to 100 asthenozoospermic men. After 4 months of

[160]

Vitamin C and vitamin E

138 Spermatozoa - Facts and Perspectives

Vitamins A, C, E, N-acetyl-cysteine and zinc

Selenium and Vitamins

the studied population [161]

morphology [162]

mL post-treatment [164]

morphology [166].

supplemented for 6 months [168]

1 g L-acetyl-carnitine [169]

200 μg/day of selenium [170]

Vitamin C supplementation improved sperm count, motility and morphology [159] • 115 men with clinical varicocele and abnormal semen analyses were recruited. After surgery, the subjects received 250 mg vitamin C for 2 months. Vitamin C supplementation following surgery resulted in a better motility and morphology. Prior to surgery, vitamin C was not effective on the sperm count, but it improved sperm motility and morphology

26 weeks. At the end of the experiment, sperm motility increased, while LPO decreased in

• 1000 mg vitamin C and 800 mg vitamin E were administered to 31 subjects diagnosed with asthenozoospermia and normal or only moderately reduced sperm concentration for a period of 56 days. The treatment did not affect sperm concentration, motility and

• 64 men with unexplained infertility and an elevated percentage of DNA-fragmented spermatozoa received 1 g vitamin C and 1 g vitamin E daily for 2 months. No differences in basic sperm parameters were found following antioxidant treatment; however, the

• 20 post-varicocelectomy oligospermic patients were subjected to a daily administration of 0.06 IU/kg of vitamin A, 3 mg/kg of vitamin C, 0.2 mg/kg of vitamin E, 10 mg/kg of NAC and 0.01 mg/kg of zinc over a period of 13 weeks. Sperm count increased by 20-fold, and of the 20 subjects, 6 of the originally infertile men had sperm counts greater than 20million/

unilateral varicocele or germ-free genital tract inflammation over 2 months lead to an

treatment, a significant improvement was observed in sperm concentration, motility and

asthenozoospermia who underwent 6 months of daily treatment with 2 g L-carnitine and

• 9 oligoasthenoteratozoospermic men were supplemented for a period of 6 months with selenium and vitamin E, leading to improvements in sperm motility, morphology and

• 46 oligoasthenoteratozoospermic and 16 subfertile patients received selenium alone or in combination with vitamins A, C and E at daily doses of 100 μg, 1 mg, 10 mg and 15 mg, respectively. No improvement was observed in sperm concentration after 3 months,

• 28 infertile men were supplemented daily by vitamin E (400 mg) and selenium (225 μg) during 3 months. Following treatment, a significant decrease of LPO was observed

• Supplementation of 600 mg/day of NAC to 60 patients diagnosed with idiopathic infertility seemed to improve sperm motility, volume, viscosity and seminal oxidative status [175].

• 2 g/day of carnitine administered for 6 months led to a significant improvement in sperm concentration and motility in 100 patients with oligoasthenoteratozoospermia [167], while the following year an increased sperm count and motility were found in 56 infertile men after a combined daily treatment with 2 g carnitine and 1 g acetyl-L-carnitine

• No improvements in semen quality was detected in 26 men diagnosed with

Selenium • No positive effects were found in 33 subfertile men following 3 months of treatment with

although the motility was increased in the treated subjects [172]

together with an improvement of sperm motility [173] N-acetyl-cysteine • No improvements in sperm parameters were observed after 3 months of 600 mg/day

administration of NAC to 27 infertile men [174].

viability, although the concentration did not change significantly [171]

percentage of DNA-fragmented spermatozoa was markedly reduced [163].

ROS-induced damage may have significant clinical implications in the context of ARTs. Numerous reports have indicated that significantly increased ROS levels may occur in response to repeated cycles of centrifugation involved in conventional sperm preparation techniques used for ARTs [179]. Spermatozoa selected for ART often face OS and a high risk for DNA damage. When intrauterine insemination or *in vitro* fertilization (IVF) is used, such damage does not represent a cause of concern as damage to the sperm membrane lipids ensures that fertilization will not occur. However, in case of intracytoplasmic sperm injection is used, this natural selection barrier may be overlooked and sperm with DNA damage may be directly injected into the ovum [77].

Selection of an effective sperm preparation technique is important to minimize ROS overgeneration and eventual oxidative insults to the male gamete. The density gradient technique is able to separate leukocytes and immature or damaged spermatozoa from normal spermatozoa, which may be subsequently used in ARTs [77, 179, 180].

Assisted reproduction techniques may benefit from *in vitro* supplementation of antioxidants [180]. Various antioxidants such as vitamin E, vitamin C, cysteine, taurine and hypotaurine present in the culture medium have been shown to improve the developmental ability of the embryos by counteracting the effects of ROS [93, 180].

In cases of IVF, incubation times of more than 16–20 hours have been correlated with increased oxidative damage. Shortening the insemination timeframes (up to 1–2 hours or less) may reduce ROS overgeneration in culture media and possibly improve fertilization, embryogenesis and pregnancy rates [77, 179].
