Stesura Seveso 1Archivio Italiano di Urologia e Andrologia 2014; 86, 1 ORIGINAL PAPER Antioxidant cosupplementation therapy with vitamin C, vitamin E, and coenzyme Q10 in patients with oligoasthenozoospermia Yoshitomo Kobori, Shigeyuki Ota, Ryo Sato, Hiroshi Yagi, Shigehiro Soh, Gaku Arai, Hiroshi Okada Department of Urology, Dokkyo Medical University, Koshigaya Hospital, Japan. Objective: Overproduction of reactive oxygen species results in oxidative stress, a deleterious process that damages cell structure as well as lipids, proteins, and DNA. Oxidative stress plays a major role in various human diseases, such as oligoasthenozoospermia syndrome. Materials and methods: We evaluated the effectiveness of antioxidant co-supplementation therapy using vitamin C, vitamin E, and coenzyme Q10 in men with oligoastheno- zoospermia. Overall, 169 infertile men with oligoas- thenozoospermia received antioxidant therapy with 80 mg/day vitamin C, 40 mg/day vitamin E, and 120 mg/day coenzyme Q10. We evaluated spermiogram parameters at baseline and at 3 and 6 months of follow-up. Results: Significant improvements were evident in sperm concentration and motility following coenzyme Q10 therapy. Treatment resulted in 48 (28.4%) partner pregnancies, of which 16 (9.5%) were spontaneous. Significant improvements in sperm cell concentration and sperm motility were observed after 3 and 6 months of treatment. Conclusions: Vitamin C, vitamin E, and coenzyme Q10 supplementation resulted in a significant improvement in certain semen parameters. However, further studies are needed to empirically determine the effect of supplemen- tation on pregnancy rate. KEY WORDS: Vitamin C; Vitamin E; Coenzyme Q10; Male infertility. Submitted 27 August 2013; Accepted 5 October 2013 Summary INTRODUCTION There is much evidence to show that oxidant radicals and reactive oxygen species play a harmful role in human reproduction and male infertility (1). Testicular oxidative stress is important in a number of conditions known to be detrimental to male infertility. These include a broad spectrum of diseases and condi- tions due to lifestyle factors such as smoking, alcohol, and obesity (2), environmental hazards such as pesti- No conflict of interest declared. cides, plasticizers, and heavy metals (3), systemic infec- tions (4), chronic diseases and inflammation such as dia- betes, chronic renal failure, and varicocele (5), and a number of iatrogenic or idiopathic causes (6). A previous open, controlled pilot study of a cohort of infertile men with idiopathic asthenozoospermia showed that exogenous administration of coenzyme Q10 (CoQ10) increases the level of both CoQ10 and ubiquinol (QH2) in semen and is effective in improving sperm kinetics (7). In addition, administration of vitamins E and C significantly reduced hydroxyguanine levels in sperma- tozoa and led to an increased sperm count (8). We previously found that a relatively low intake of CoQ10 (30-60 mg/day) improved semen parameters (unpublished data). These data encouraged us to assess the possible effec- tiveness of this therapeutic approach by conducting a 6- month trial of cosupplementation antioxidant therapy (vitamin C, vitamin E, and CoQ10) in a cohort of infer- tile men with idiopathic oligoasthenozoospermia. Change in semen parameters and achievement of preg- nancy were evaluated after 6 months of treatment. MATERIALS AND METHODS Patients A total of 169 consecutive patients (mean age 36, range 25-58 years) with idiopathic oligoasthenoteratozoosper- mia were enrolled in the study. All presented with infer- tility after at least 2 years of unprotected intercourse. Male infertility was diagnosed if one or more standard semen parameters were below the cutoff levels according to the criteria of the World Health Organization, 1999 (sperm concentration < 20 ! 106/ml, sperm motility < 50%, normal morphology < 30%, and/or semen vol- ume < 2 ml) based on at least two semen analyses per- formed 3 months apart to eliminate accidental and pos- sible adverse effects of exogenous factors on spermatoge- nesis. After providing a complete medical and reproduc- tive history exploring all aspects that might be related to fertility, patients underwent physical examination and DOI: 10.4081/aiua.2014.1.1 Kobori_Stesura Seveso 26/03/14 10:10 Pagina 1 Archivio Italiano di Urologia e Andrologia 2014; 86, 1 Y. Kobori, S. Ota, R. Sato, H. Yagi, S. Soh, G. Arai, H. Okada 2 serum chemical and hematological laboratory tests. Testicular volume was measured using an orchidometer. Serum follicle stimulating hormone, luteinizing hor- mone, and testosterone levels were measured in all patients. Patients with infection, liver dysfunction, renal dysfunction, or a metabolic disease (e.g., diabetes melli- tus) were excluded, as were patients with malignant neo- plasm and those with spouses with diseases or condi- tions that may affect conception. Study design All patients underwent antioxidant therapy with 120 mg CoQ10, 80 mg vitamin C, and 40 mg vitamin E daily (two tablets of SO support; Partners, Yokohama, Japan). Semen parameters were evaluated before and at 3 and 6 months of treatment with co-supplementation. Pregnancy out- come and use of assisted reproduction technology was evaluated after 3 and 6 months of treatment. Ethics All patients provided informed consent. The study design was approved by the institutional review board. Statistical analysis Statistical analysis was performed using SPSS 17.0 (SPSS Inc., Chicago, IL). Data are expressed as mean ± SD val- ues. Differences between groups were estimated using the paired t-test. P < 0.05 was considered statistically sig- nificant for hypothesis testing. RESULTS Baseline patient characteristics are shown in Table 1. The mean duration of infertility was 2.3 years. Table 2 shows mean (± SD) semen parameters before and at 3 and 6 months of treatment. The t-test performed on single variables for the homogeneity at baseline showed that there were no significant differences regard- ing atypical sperm cells and semen volume. On the con- trary, significant improvements in sperm cell concentra- tion and sperm motility were observed after 3 and 6 months of treatment. A total of 48 (28.4%) pregnancies were achieved, includ- ing 16 (9.5%) spontaneous pregnancies as follows: seven after 3 months, eight after 6 months, and one after 9 months of treatment. Overall, 32 pregnancies were achieved using assisted reproductive technology. Six couples used artificial insemination by husband (AIH), eight couples used conventional in vitro fertilization (IVF) and 18 couples used intracytoplasmic sperm injection (ICSI). Oral administration of CoQ10, vitamin C, and vitamin E was generally well tolerated, and no adverse effects or laboratory abnormalities were observed. DISCUSSION Several approaches have been proposed for the manage- ment of infertility caused by oxidative stress. Once an individual has been identified as having oxidative stress- related infertility, treatment should be aimed at identifi- cation and amelioration of the underlying cause before considering antioxidant treatment. Lifestyle behaviors such as smoking, poor diet, alcohol abuse, pollution and environmental toxins, obesity, and psychological stress have all been linked to oxidative stress. While the effec- tiveness of eliminating these lifestyle triggers on oxida- tive stress has not been formally tested, it is likely that Characteristic Range Mean ± SD Age (yrs) 25-58 36 ± 9 Age of wife (yrs) 22-44 34 ± 8 Serum hormones Testosterone (ng/dl) 169-988 406 ± 88 LH (IU/l) 1.1-11.5 3.5 ± 2.4 FSH (IU/l) 1.6–26.8 5.0 ± 4.1 Testicular volume (ml) Right 8-26 18 ± 5 Left 4-26 16 ± 5 FSH: follicle stimulating hormone; LH: luteinizing hormone Table 1. Baseline patient demographics, serum hormone levels, and semen parameters. Baseline 3 months 6 months Sperm variable Mean ± SD Mean SD Mean SD Sperm concentration (n x 106/ml) 26.3 ± 36.0 37.5 54.0 49.0 ± 59.0 P value 0.03 < 0.001 Sperm motility (%) 25.2 ± 18.1 39.1 ± 20.3 41.3 ± 22.1 P value < 0.001 < 0.001 Atypical sperm cells (%) 25.4 ± 10.0 22.6 ± 10.3 23.4 ± 12.0 P value 0.43 0.44 Semen volume (ml) 3.1 ± 1.9 3.1 ± 2.2 4.3 ± 2.9 P value 0.78 0.08 Table 2. Descriptive statistics of sperm variables throughout the study. Kobori_Stesura Seveso 26/03/14 10:10 Pagina 2 making positive lifestyle changes such as changing to a diet high in fruit and vegetables, maintaining normal weight, and reducing smoking or alcohol intake would have at least some beneficial effects on sperm health. Several studies have reported that levels of reactive oxy- gen species within semen can be reduced by augmenting the scavenging capacity of seminal plasma using oral antioxidant supplements. Vitamin E is a major chain-breaking antioxidant in sperm membranes and this effect appears to be dose dependent. Vitamin E scavenges the three major types of free reactive species, namely superoxide, hydrogen per- oxide, and hydroxyl radicals. In a randomized, double- blind, placebo-controlled trial (9), in vitro functional tests of human spermatozoa improved after 3 months of vitamin E (600 mg/day) therapy. While some studies suggest a potential role for vitamin E in the management of male infertility, another randomized trial failed to con- firm these findings (10). Vitamin C is another important chain-breaking antioxi- dant and is present at a higher concentration in seminal fluid than in plasma as well as being present in low but detectable amounts in sperm cells (11). Vitamin C neu- tralizes hydroxyl, superoxide, and hydrogen peroxide reactive species and prevents sperm agglutination, while preventing lipid peroxidation, recycling vitamin E, and protecting against DNA damage induced by hydrogen peroxide radicals (12). It has been suggested that oral administration of vitamin C with vitamin E significantly reduces hydroxyguanine levels in spermatozoa and also leads to an increased sperm count (8). CoQ10 is a component of the mitochondrial respiratory chain and plays a crucial role in energy metabolism and as a liposoluble chain-breaking antioxidant for cell mem- branes and lipoproteins (13). Recently, the role of CoQ10 as a gene inducer has also been investigated (14). CoQ10 biosynthesis is markedly active in the testis (15), and high levels of its reduced form, QH2, are present in sperm (16), suggesting a pro- tective antioxidant role. Levels of CoQ10 and QH2 in seminal plasma and sperm cells of infertile men with idiopathic and varicocele-associated asthenospermia were reduced significantly (17). On the basis of this find- ing, CoQ10 likely contributes to the total antioxidant buffer capacity of semen, and a decrease in levels is dele- terious in terms of dealing with oxidative stress. The mode of action of CoQ10 in male infertility is not clear but may be useful for vitalizing cells by providing greater energy to mitochondria, thereby improving motility and preventing oxidative damage through its actions as a free radical scavenger. CoQ10 recycles vitamin E and pre- vents its pro-oxidant activity (18). QH2 also acts as an antioxidant by preventing lipid peroxidation, whereas CoQ10 inhibits hydrogen peroxide formation in the seminal fluid and seminal plasma of infertile men (19). In a randomized, double-blind, placebo-controlled trial, the exogenous administration of CoQ10 increased the level of both CoQ10 and QH2 in semen and was effec- tive in improving sperm kinetics in patients with idio- pathic asthenozoospermia (17). When a molecule of vitamin E neutralizes a free radical it loses its antioxidant ability which is subsequently restored by the actions of other antioxidants such as vita- min C and CoQ10 (20). 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Correspondence Yoshitomo Kobori, MD (Corresponding Author) ykobori@dokkyomed.ac.jp Shigeyuki Ota, MD Ryo Sato, MD Hiroshi Yagi, MD Shigehiro Soh, MD Gaku Arai, MD Hiroshi Okada, MD Department of Urology, Dokkyo Medical University, Koshigaya Hospital 2-1-50, Minamikoshigaya, Koshigaya (Japan) 343-8555 Kobori_Stesura Seveso 26/03/14 10:10 Pagina 4