*6.2. Vitamins*

The interest of vitamin E and its use as antioxidant is due to its protective activity against ROS which subsequently decreases LPO, and therefore exerts positive e ffects on sperm functions, such as sperm concentration and motility [58]. However, its e ffects in fertility are less clear. For example, in a small clinical trial (*n* = 30), oral administration of vitamin E (300 mg twice daily) for three months raised the levels of vitamin E in blood serum, although human seminal plasma levels were not modified, questioning its possible e ffects on reproductive parameters [50]. Nevertheless, in this clinical trial, vitamin E treatment achieved an improvement of the zona pellucida binding test without any other improvement described, including ROS level [50]. Similarly, 15 normospermic infertile men after one month of daily consumption of 200 mg of vitamin E improved their fertilization rate (19.3 ± 23.3 pretreatment versus 29.1 ± 22.2 post-treatment) after IVF. Those results were associated with lower sperm LPO levels in comparison with preintervention values [52]. In another work, oral administration of vitamin E (100 mg thrice daily) to patients with asthenospermia (*n* = 52) established three di fferent groups of men according to the results: (i) men without improvement of their sperm motility (40%); (ii) men with improved sperm motility but did not achieve pregnancy (39%); (iii) men with improved motility and achieved pregnancy (21%), of which 81.8% of pregnancies finished in live birth. The placebo control group did not achieve any pregnancies [47]. Later, daily intake of a combination of vitamin E and C (1 mg of each component) for two months in patients where intracytoplasmic sperm injection (ICSI) had previously failed was studied (*n* = 38). The results showed two di fferent populations: (i) those where the antioxidant treatment decreased the percentage of sperm

DNA damage (*n* = 29) and (ii) those where the treatment did not a ffect this parameter (*n* = 9) [49]. The most interesting result was observed in the responsive group that after ICSI, the pregnancy rate (6.9 vs. 49.3%) and implantation rate (2.2 vs. 19.2%) were improved compared with the pretreatment group, although no di fferences were found in embryo quality [49]. In a nonplacebo-controlled and nondouble-blind design trial, daily intake of a combination of selenium (200 μg) and vitamin E (400 UI) followed for 3.5 months by infertile men (*n* = 690) achieved 10.8% spontaneous pregnancy [50].

Several studies have been performed looking for beneficial e ffects from a combination compounds with antioxidant activity. For example, a formulation using a mix of several compounds with antioxidant activity (vitamin C, vitamin E, carnitine, folic acid, lycopene, selenium, and zinc) was evaluated using a mouse Gpx5 knock-out (KO) subjected to a second stress: scrotal heat (KO + SH) (42 ◦C for 30 min) [58]. Although the exact ingestion quantity of this antioxidant combination could not be determined, their e ffects include the reversion of sperm DNA oxidation induced in KO + SH animals and protection of seminiferous tubules. The results showed that animals supplemented with KO + SH versus the nonsupplemented animals had double the fertilization rate (73.7 vs. 35.2%) and fetus reabsorption was halved (8.9 vs. 17.8%) [58]. In another trial, infertile human patients with oligoand/ or astheno- and/or teratozoospermia with or without varicocele (*n* = 104) using a combination of antioxidants (vitamin C 90 mg, vitamin B12 1.5 μg, LC 1mg, fumarate 725 mg, LAC 500 mg, fructose 1000 mg, CoQ10 20 mg, zinc 10 mg, and folic acid 200 μg) were studied for six months. The results showed that the individuals from the treated group, regardless of whether they su ffered from varicocele or not, presented improved sperm concentration total sperm motility [43]. Moreover, after treatment, 22.2% (10/45) of supplemented patients achieved pregnancy, while in the control group, only 4.1% (2/49) of the couples were pregnan<sup>t</sup> [43]. A close analysis of the men from the supplemented group revealed that only 4.8% (1/21) of patients su ffering varicocele improved after treatment, while the nonvaricocele group achieved 37.5% (9/24) pregnancy [43]. A di fferent group studied the e ffect of a commercial multiantioxidant supplement (vitamin E 400 IU, vitamin C 100 mg, lycopene 6 mg, zinc 25 mg, selenium 26 μg, folate 0.5 mg, garlic 1000 mg) for three months on 60 men with high levels of DNA fragmentation and poor sperm motility and membrane integrity [51]. The treatment achieved doubled pregnancy rate (63.9 vs. 37.5%), implantation rate (46.2 vs. 24%), and viable pregnancy rate (38.5 vs. 16%) versus the placebo group without any modification of any sperm parameters, fertilization, or embryo quality rates [51]. However, this work was later criticized because of the experimental design, particularly the low number of individuals in the trial, unequal distribution of individuals between the placebo (*n* = 16) and treatment groups (*n* = 36) and the suitability of the statistical analysis used [59].

Contradictory results were found when men were supplemented with di fferent oral antioxidants after varicocelectomy. Oral intake of vitamin E (300 mg twice/day) for 12 months (*n* = 40) improved the sperm parameters of sperm concentration and the percentage of motile spermatozoa, although these data were not significant compared with control [60]. Recently, a multiple antioxidant combo was tested (l-carnitine fumarate 1 g, acetyl-l-carnitine HCl 0.5 g, fructose 1 g, citric acid 50 mg, vitamin C 90 mg, zinc 10 mg, folic acid 200 μg, selenium 50 μg, coenzyme Q-10 20 mg, and vitamin B12 1.5 μg) after varicocelectomy (*n* = 90) for six months [45]. Surgery improved the following sperm parameters: sperm concentration, percentage of motile spermatozoa or progressive motility, and spermatozoa with normal morphology. Moreover, treated men achieved 29% pregnancy versus 17.9% in the placebo group [45].
