Introduction
Male infertility is negatively influenced by reactive oxygen species and by other oxidant radicals [1]. In this regard, the relevant role of Coenzyme Q10 (CoQ10) in contributing to the total antioxidant buffer capacity of semen has been extensively investigated [1]. Two distinct studies conducted by our group, showed that in idiopathic asthenozoospermic patients undergoing CoQ10 therapy a significant increase in spermatozoa motility was observed [1, 2], suggesting its potential therapeutic role. On the other hand, Aspartic acid (d-Asp) supplementation was found to increase the concentration and motility of spermatozoa in oligo-asthenozoospermic and asthenozoospermic subjects [3]. Some evidence suggests a protective effect against oxidative stress for this molecule [4].
Only a few interventional studies have assessed oxidative stress in sperm of idiopathic infertile men treated with CoQ10 and d-Asp [5–9]. However, they just examined some of the markers of oxidative stress, focusing, in the great majority of cases, only on subjects who were administered CoQ10 [6–9]. Given these premises, we conducted a study in infertile idiopathic subjects who underwent co-administration of CoQ10 and d-Asp in order to evaluate some previously untested parameters of sperm oxidative stress, like superoxide dismutase (SOD) activity, nitric oxide (NO) and peroxynitrite levels, and analyze the effects of these substances on sperm DNA damage markers.
Materials and methods
In this observational study, twenty patients (age: 31.5 ± 4.08 years) affected by idiopathic asthenozoospermia [10] were considered. Subjects were administered oral dietary supplement including CoQ10 (200 mg) and d-Asp (2,660 mg) on the basis of clinical reasons (Genadis, MerckSerono S.p.A., Italy). Subjects took one soluble granular preparation per os daily of this dietary supplement for 3 months. Evaluations were carried out at baseline and after 3 months. Institutional ethics committee approved the study. Informed consent was obtained from each individual.
Analysis of semen
Samples were evaluated according to the World Health Organization guidelines [10]. Motility was evaluated using a computer-assisted semen analysis system.
Determination of CoQ10
Coenzyme Q10 levels and its oxidative state were assayed in seminal fluid and in sperm cells using a dedicated HPLC system with electrochemical detector [11]. Extracellular and intracellular CoQ10 was expressed as μg/ml of seminal fluid and ng/106 sperm cells, respectively.
Determination of d-Asp in human seminal plasma
The procedure of purification of d-Asp from seminal plasma and its quantification was determined by HPLC according to the method previously described [12].
Sperm markers of oxidative stress
SOD activity
Superoxide dismutase activity was evaluated by colorimetric assay kit (Enzo Life Sciences, Pennsylvania, USA) as previously described [13].
NO levels
Nitric oxide production was evaluated by the Total NO Assay Kit (Enzo Life Sciences, Pennsylvania, USA). NO levels were determined using a standard curve generated and expressed as μmol NO/mg protein. Protein concentration was determined by the Bradford assay, using serum albumin as a standard [14].
Peroxynitrite levels
Peroxynitrite production was determined by the hydroxyphenyl fluorescein fluorometric assay (Cell Technology Inc., Mountain View, CA, USA) as previously described [15].
Comet assay
DNA integrity was analyzed following procedure by Donnelly et al. [16]. Before analysis, DNA was stained with ethidium bromide and the comets were analyzed as previously reported [17]. For each comet, data relative to tail intensity were calculated.
Statistical analysis
Data are expressed as median (interquartile range) when not-normally distributed, and as mean ± SD when normally distributed. Parameter variations between the two phases (∆−) were calculated as the value present “after” minus the value present “before” the therapy; statistical comparison between the two phases was made using Student t test for paired data or the Wilcoxon test as appropriate. Pearson or Spearman correlations between the significant variations of parameters were performed. Significance was set at p < 0.05.
Results
A significant increase of the administered substances, i.e., CoQ10 and d-Asp, in sperm cells and seminal fluid, respectively, was evident (Table 1). Conversely, CoQ10 seminal plasma levels did not increase significantly in this experimental setting (Table 1). A significant improvement of sperm kinetic parameters was observed (Table 1). On the contrary, sperm count and atypical sperm cells were not affected by oral supplementation (Table 1).
Nitric oxide and peroxynitrite levels decreased, whereas SOD activity increased significantly after treatment (Table 1). Furthermore, the percentage of damaged DNA, quantified by the index tail intensity, decreased significantly after CoQ10 and d-Asp administration (Table 1).
Correlation analysis revealed a negative relationship between the increase of CoQ10 and the decrease of NO and DNA damage and a positive one between the increase of CoQ10 and the rise of SOD activity (Table 2). On the contrary, no significant correlation was found between the increment of d-Asp and the changes of any of the considered markers (Table 2).
Increase of SOD activity and decrease of NO levels were negatively (r:−0.456; p:0.044) and positively (r:0.458; p:0.042) correlated with the decrease of DNA damage index, respectively, whereas no significant correlation was found between ∆-peroxynitrite and ∆-tail intensity (r: −0.266; p > 0.05).
Discussion
In the present study, CoQ10 and d-Asp administration improved antioxidant defences (SOD activity), lowered NO-related oxidant species and improved oxidative DNA damage. Correlation analysis revealed the antioxidant effect of CoQ10 and, at the same time, the lack of influence of d-Asp on those parameters. The absence of a relationship of d-Asp with SOD activity, NO, and peroxynitrite is an unexpected result. In fact, the relationship between d-Asp and oxidative stress is controversial. Talevi et al. found that the in vitro effect of a mixture containing zinc, d-Asp, and CoQ10 prevents the increase of the percentage of peroxidized spermatozoa [5]. Rathore and Gupta used lenses of goat to evaluate the effect of d-Asp against oxidative stress produced by 1 mM hydrogen peroxide solution and they found that, after 24 h of incubation at 37 °C, the lenses challenged with hydrogen peroxide in the presence of d-Asp showed an higher content in reduced glutathione compared to control exposed to hydrogen peroxide alone [4]. Conversely, Chandrashekar and Muralidhara administered d-Asp in the testis of prepubertal rats, confirmed a modulation of reduced glutathione levels and showed elevated activities of glutathione-dependent enzymes and catalase. Nonetheless, they also demonstrated a significant elevation of oxidation products, malondialdehyde and hydroperoxide in cytosol, and mitochondria of testis [18].
Infertile men are reported to have substantially increased DNA damage in the spermatozoa, which has been associated with compromised reproductive outcomes [5]. In this respect, CoQ10 antioxidant effects have been shown to produce a protective effect in vivo in peripheral blood lymphocytes of supplemented subjects [19]. However, very little is known about the relationships and mechanisms that link CoQ10 and d-Asp with sperm DNA integrity. Talevi et al. found that in vitro effects of d-Asp and CoQ10 prevented the increase of spermatozoa with impaired DNA [5]. In addition, Abad et al. in their study, administered antioxidant treatment, including CoQ10, to twenty infertile men with asthenoteratozoospermia and showed an improvement in sperm DNA integrity [20]. In our sample, sperm DNA damage significantly improved after treatment and, from the results of correlations, only CoQ10, and not d-Asp, administration seems to be involved in this protective effect, by exerting an influence on SOD activity and NO levels.
In conclusion, only CoQ10 seems to play a protective role against oxidative stress and DNA damage, thus contradicting some previous findings, which suggested these effects also for d-Asp.
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Acknowledgments
No external funding, apart from the support of the Polytechnic University of Marche, was available for this study. The authors wish to thank Dr. Anna Tangorra for skillful technical assistance.
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The Authors have no conflict to disclose.
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Tirabassi, G., Vignini, A., Tiano, L. et al. Protective effects of coenzyme Q10 and aspartic acid on oxidative stress and DNA damage in subjects affected by idiopathic asthenozoospermia. Endocrine 49, 549–552 (2015). https://doi.org/10.1007/s12020-014-0432-6
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DOI: https://doi.org/10.1007/s12020-014-0432-6