Article Seminal plasma total antioxidant capacity and semen parameters in patients with varicocele

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1 RBMOnline - Vol 18 No Reproductive BioMedicine Online; on web 20 March 2009 Article Seminal plasma total antioxidant capacity and semen parameters in patients with varicocele Simone Giulini graduated in medicine in 1995 and then in 2000 specialized in obstetrics and gynecology at the University of Modena and Reggio Emilia, Italy. Since then he has worked at the Centre of Reproductive Medicine of the University Hospital of Modena. His fields of interest are male and female infertility, assisted procreation, and fertility preservation. Since 2005 he has been the coordinator of the Centre of Reproductive Medicine of University Hospital of Modena. Dr Simone Giulini Simone Giulini 1 3, Valeriana Sblendorio 2,Susanna Xella 1, Antonio La Marca 1, Beniamino Palmieri 2, Annibale Volpe 1 1 Mother-Infant Department, Institute of Obstetrics and Gynecology, University of Modena and Reggio Emilia, Largo del Pozzo71, 41100, Modena, Italy; 2 Department of General Surgery and Surgical Specialties, Surgical Clinic, University of Modena and Reggio Emilia, Largo del Pozzo 71, Modena, Italy 3 Correspondence: Tel: ; Fax: ; sgiulini@unimore.it Abstract Total antioxidant capacity (TAC) was evaluated in the seminal plasma of infertile patients with varicocele in relation to their semen parameters. The study recruited 60 patients affected by varicocele and 10 fertile non-varicocele subjects as controls. Controls had normal semen parameters and proven fertility. On the basis of semen parameters, patients with varicocele were grouped into normozoospermic (n = 12), asthenozoospermic (n = 8), oligoasthenozoospermic (n = 40). The group with oligosthenozoospermia was divided into mild (< /ml; /ml), moderate (< /ml; /ml), and severe (< /ml), based on sperm count. Antioxidant activity was measured in seminal plasma and peripheral blood using the free oxygen radicals defence test. No significant differences were observed in peripheral blood TAC concentrations between controls and groups. In patients with varicocele and moderate oligoasthenozoospermia or severe oligoasthenozoospermia, seminal plasma TAC concentrations were significantly lower (P < 0.05) than in controls and normozoospermic patients with varicocele. Moreover, in patients with severe oligosthenozoospermia, seminal plasma TAC concentrations were also significantly lower (P < 0.05) than in asthenozoozpermic patients with varicocele. In all subjects, concentrations of TAC showed a positive correlation with sperm concentration (r = 0.93, P < 0.05) and motility (r = 0.92, P < 0.05). Keywords: antioxidants, oxidative stress, semen parameters, varicocele Introduction In the aetiology of male infertility, there is growing evidence that a key role is played by damage to spermatozoa by reactive oxygen species (ROS) (Agarwal and Saleh, 2002; Agarwal et al., 2003). Spermatozoa contain large quantities of polyunsaturated fatty acids and are, therefore, susceptible to ROS-induced damage. It has been suggested that ROS induce membrane lipid peroxidation in spermatozoa (Esfandiari et al., 2003; Agarwal et al., 2004; Sanocka and Kurpisz, 2004). ROS are highly reactive oxidizing agents belonging to the class of free radicals. Oxygen is required to support life of spermatozoa, but its metabolites, such as ROS, can modify cell functions, endanger cell survival, or both (de Lamirande and Gagnon, 1995). Hence, ROS must be inactivated continuously in order to maintain only the small amount necessary for maintenance of normal cell function. A battery of different antioxidants normally protects against oxidants. Seminal plasma is well endowed with an array of antioxidants which act as free radical scavengers to protect spermatozoa against oxidative stress. Seminal plasma contains a number of enzymatic antioxidants such as superoxide dismutase (SOD) and catalase. In addition, it contains a variety of nonenzymatic antioxidants (Agarwal and Prabakaran, 2005; Baker and Aitken, 2005; Agarwal et al., 2006a, b). Findings on seminal plasma enzymatic, and non-enzymatic total antioxidants capacity (TAC) are controversial (Sanocka Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK

2 618 et al., 1996; Sanocka et al., 1997; Zini et al., 2000; Siciliano et al., 2001). Recently it has been demonstrated that seminal plasma TAC concentrations are lower in asthenozoospermic, asthenoteratozoospermic and oligoasthenoteratozoospermic samples compared with those in normozoospermic men, and also TAC showed a positive correlation with sperm motility and morphology (Koca et al., 2003; Khosrowbeygi and Zarghami, 2007). In the aetiology of male infertility, varicocele is one of the leading causes, and the relationship between oxidative stress, varicocele and infertility has been investigated. A recent metaanalysis (Agarwal et al., 2006) concluded that in varicocele patients ROS production is enhanced irrespective of fertility status, and that TAC concentrations are significantly lower in the infertile varicocele patients than in controls. The objective of this study was to evaluate seminal plasma total antioxidant capacity in infertile patients with varicocele in relationship to their seminal parameters. Materials and methods This study recruited 60 patients, aged years, affected by varicocele, and 10 fertile non-varicocele control subjects, aged years, from the study centre. All subjects participated in the study after informed consent was obtained. The study protocol was approved by the local ethics committee. All patients were clinically examined and the diagnosis of varicocele (grade II or III) confirmed by Doppler technique (Hirsh et al., 1980). Semen specimens were analysed within 1 h of collection. In all patients, standard semen analysis was performed, assessing semen parameters, including volume, sperm count, and total percentage sperm motility according to World Health Organization (1993) parameters. Sperm morphology was assessed using Kruger s strict criteria (Kruger et al., 1986). Semen smears were performed in order to detect white blood cells (WBC). Smears were air dried, stained by the Papanicolaou method (Papanicolaou, 1942) and examined with the use of oil immersion microscopy. The WBC concentration was calculated as the number of WBC counted, divided by the number of spermatozoa counted, multiplied by semen sperm concentrations (Menkveld and Kruger, 1998). Subjects whose ejaculates contained WBC concentration greater than /ml were excluded from the study, since leukocytes are a source of ROS. Aliquots of seminal plasma obtained after centrifugation (1000 g for 15 min) were stored at 20 C until antioxidant capacity evaluation was performed. On the same day as semen collection, a peripheral blood sample was obtained and after centrifugation (1000 g for 15 min) the serum was stored at 20 C until TAC evaluation was performed. All controls had normal semen parameters and proven fertility. On the basis of semen parameters, patients with varicocele were grouped into normozoospermic (n = 12), asthenozoospermic (n = 8), oligoasthenozoospermic (n = 40). According to World Health Organization parameters, oligozoospermia was defined as < /ml spermatozoa and asthenozoospermia as <50% of sperm motility. Moreover, with the aim of correlating the antioxidant concentrations with the sperm concentrations, the group with oligoasthenozoospermia was divided on the basis of sperm count into mild oligoasthenozoospermia (< /ml; /ml), moderate oligoasthenozoospermia (< /ml; /ml), and severe oligoasthenozoospermia (< / ml). This study also found the presence of teratozoospermia (<14% of normal spermatozoa, according to Kruger s strict criteria (Kruger et al., 1986) in patients with severe oligoasthenozoospermia. Total antioxidant capacity Multiple tests are available to measure TAC, such as oxygen radical absorbance capacity, the ferric-reducing ability of plasma and the trolox equivalent antioxidant capacity assay (Agarwal and Prabakaran, 2005). These assays used enhanced chemiluminescence or colourimetric techniques to measure the antioxidant status of semen (Said et al., 2003). Enhanced chemiluminescence technique for TAC measurements has been widely used and its results have been validated (Sharma et al., 1999; Pasqualotto et al., 2000). This study measured antioxidant (AO) activity in seminal plasma and in peripheral blood using the free oxygen radicals defence (FORD) test (Miller et al., 1993). FORD is a colourimetric test based on the ability of antioxidants present in plasma to reduce a preformed radical cation. The principle of the assay is that at an acidic ph (5.2) and in the presence of a suitable oxidant solution (FeCl 3 ), the FORD chromogen 4-amino-N,N-diethylaniline can form a stable and coloured radical cation. Antioxidant molecules (AOH) present in the sample that are able to transfer a hydrogen atom to the FORD chromogen radical cation reduce it, quenching the colour and producing a decolouration of the solution which is proportional to their concentration in the sample: Chromogen (uncoloured) + oxidant (Fe 3+ ) H + Chromogen.+ (purple) Chromogen.+ (purple) + AOH Chromogen + (uncoloured) + AO FORD colour quenching is determined in particular by the contribution of antioxidants such as proteins, reduced glutathione and vitamins. These antioxidants are among the most important contributors to antioxidant plasmatic barrier. Aliquots of the seminal plasma stored at 20 C were thawed at room temperature and immediately assessed for antioxidant capacity. Seminal plasma was diluted 1:100 with deionized water. Like many other methods (Miller et al., 1993; Sharma et al., 1999, Pasqualotto et al., 2000; Meucci et al., 2003; Said et al., 2003), FORD results are expressed as 6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid (Trolox) equivalents (mmol/l) using a calibration curve plotted with different amounts of standard Trolox which is stored on the dedicated instrument (FORM Plus, FORM ox and CR3000 series diagnostic analysers, Callegari SpA, Catellani Group, Parma, Italy). The linearity of the FORD test system was between mmol/l Trolox.

3 Statistical analysis Data are presented as mean SD, unless otherwise indicated. Differences between groups were assessed using the Wilcoxon or the Mann Whitney U-test as appropriate. The relationship between parameters was assessed by stepwise multiple linear regression. A probability of 0.05 was considered statistically significant. Results Semen parameters of the subjects are reported in Table 1. Table 2 shows comparison of peripheral blood and seminal plasma TAC concentrations between patients and control group. No significant differences were observed in peripheral blood TAC concentrations between the control and varicocele groups. In all of the study population with varicocele, the seminal plasma TAC concentrations ( ) were significantly (P < 0.05) lower than in controls ( ). However, when analysing subgroups of patients, no significant differences in seminal plasma TAC concentrations were observed between controls without varicocele, normozoospermic and asthenozoospermic and mild oligoasthenozoospermic patients with varicocele. TAC concentrations in patients with varicocele and moderate oligoasthenozoospermia ( ) or severe oligoasthenozoospermia ( ) were significantly lower (P < 0.05) than controls ( ), and normozoospermic patients with varicocele ( ). Moreover, in patients with severe oligoasthenozoospermia ( ), TAC concentrations were significantly lower (P < 0.05) than in asthenozoospermic patients ( ) with varicocele. Subsequently, all subjects were examined for the correlation between seminal parameters and seminal plasma TAC concentrations. Concentrations of TAC showed a positive correlation with sperm concentration (r = 0.93, P < 0.05) (Figure 1). A direct correlation between concentrations of TAC and sperm motility was also observed (r = 0.92, P < 0.05) (Figure 2). Table 1. Semen parameters in control and subgroups of patients with varicocele. Diagnosis Concentrations Motility Morphology (10 6 /ml) (%) (%) Normozoospermia (control) Normozoospermia Asthenozoospermia Mild oligoasthenozoospermia Moderate oligoasthenozoospermia Severe oligoasthenoteratozooospermia Data are reported as mean SEM. Table 2. Peripheral blood and seminal plasma total antioxidant capacity (TAC) in controls and patients. Subjects Peripheral Seminal blood TAC plasma (Trolox, TAC (Trolox, mmol/l) mmol/l) Controls (n = 10) Patients (varicocele) Total (n = 60) a Normozoospermia (n = 12) Asthenozoospermia (n = 8) Mild oligoasthenozoospermia (n = 6) Moderate oligoasthenozoospermia (n = 22) b Severe oligoasthenoteratozooospermia (n = 12) c Data are reported as mean SEM. TAC = total antioxidant capacity. a P <0.05 in comparison with controls and patients (varicocele) with normozoospermia. b P <0.05 in comparison with controls and patients (varicocele) with normozoospermia. c P <0.05 in comparison with controls, patients (varicocele) with normozoospermia and asthenozoospermia. 619

4 Figure 1. Correlation between sperm concentrations and seminal plasma total antioxidant capacity. r = 0.93; P = Figure 2. Correlation between sperm motility and seminal plasma total antioxidant capacity. r = 0.92; P = Discussion In male infertility, the findings on seminal plasma enzymatic and non-enzymatic total antioxidants capacity (TAC) are controversial (Sanocka et al., 1996, 1997; Zini et al., 2000; Siciliano et al., 2001; Koca et al., 2003; Khosrowbeygi and Zarghami, 2007). However, in focusing attention on seminal plasma non-enzymatic antioxidant capacity (TAC), some studies have shown that infertile men have an impaired total antioxidant capacity compared with fertile ones, suggesting an association between decreased TAC and male infertility. Siciliano et al. (2001) evaluated antioxidant capacity of seminal plasma in asthenozoospermic and oligoasthenozoospermic specimens with normal viscosity and hyperviscosity. Their study showed that in semen with normal viscosity seminal plasma enzymatic (catalase and SOD) and non-enzymatic (TAC) antioxidant capacities do not alter in asthenozoospermic specimens compared with normozoospermic men, whereas SOD activity is lower in oligoasthenozoospermic samples than normozoospermic males (Siciliano et al., 2001). Koca et al. (2003) evaluated TAC in infertile asthenozoospermic and asthenoteratozoospermic men compared with normozoospermic fertile men. Their study showed that asthenozoospermic and asthenoteratozoospermic males have significantly lower mean TAC value than the control group. Koca et al. (2003) also showed that TAC correlates positively to sperm motility. In a recent study, it has been demonstrated that seminal plasma TAC concentrations are lower in asthenozoospermic, asthenoteratozoospermic and oligoasthenoteratozoospermic samples compared with normozoospermic men, and TAC also showed a positive correlation with sperm motility and morphology (Khosrowbeygi and Zarghami, 2007). The inverse correlation between lipid peroxidation and sperm motility has been shown by Keskes-Ammar et al. (2003). The findings that TAC, defence against ROS, showed direct correlation with sperm motility might suggest that the higher concentration of antioxidant prevents lipid peroxidation in spermatozoa and therefore results in higher sperm motility. In the aetiology of male infertility, varicocele is one of leading causes. In animal models, studies on experimental varicocele have provided evidence for the detrimental effect of varicocele on testicular function with impaired sperm parameters and fertilizing capacity (Sofikitis et al., 1992, 1996). In rat models, it has been reported that the induced experimental left varicocele significantly increased the ROS (nitric oxide) concentrations in gonads which may represent the first sign of testicular distress (De Stefani et al., 2005). The relationship between seminal plasma oxidative stress, enzymatic, and non-enzymatic TAC has been investigated in patients with varicocele. A recent meta-analysis (Agarwal et al., 2007) concluded that in varicocele patients, ROS production is enhanced irrespective of fertility status and that TAC concentrations are significantly lower in the infertile varicocele patients than in controls. The selected studies of this meta-analysis (Hendin et al., 1999, Sharma et al., 1999, Pasqualotto et al., 2000, Saleh et al., 2003) included only those which measured the total antioxidant capacity by the enhanced chemiluminescence techniques; oligozoospermic as well as leukocytospermic samples were excluded from the analysis in both the varicocele patients and controls. Moreover, it has also been reported that varicocelectomy reduces ROS concentrations and increases antioxidant activity of seminal plasma (Mostafa et al., 2006; Hurtado de Catalfo et al., 2007; Chen et al., 2008). This study observed that the TAC seminal plasma concentrations are significantly decreased in varicocele patients in relationship with sperm parameters. Patients (varicocele) with normozoospermia, had similar TAC concentrations to controls without varicocele. Seminal plasma TAC concentrations were lower in patients (varicocele) with asthenozoospermia or mild oligoasthenozoospermia, but only subjects with significantly impaired semen parameters (moderate oligasthenozoospermia or severe oligoasthenoteratozoospermia) showed a significant reduction in seminal plasma TAC concentrations. Furthermore, in all of the study population, seminal plasma TAC concentrations showed a direct correlation with sperm concentrations and motility. In conclusion, in patients with varicocele, the reactive oxygen species (ROS) play a key role in infertility status. Seminal plasma is well endowed with an array of antioxidants which act as free radical scavengers to protect spermatozoa against oxidative stress. In varicocele patients, the decreasing seminal plasma TAC correlates with impaired semen parameters. However, further studies are necessary to confirm the relationship between TAC and semen parameters in infertile patients with varicocele

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