Seminal Antioxidant Capacity in Pre- and Postoperative Varicocele
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《男科医学杂志》
the Institutes of Endocrinology and Biochemistry and Clinical Biochemistry and the Center for Study and Research on Natural Fertility Regulation, Catholic University of the Sacred Heart, Rome, Italy.
Abstract
In order to explore the impact of surgical treatment on antioxidant defense system in varicocele (VAR), we evaluated seminal total antioxidant capacity (TAC) in 25 patients affected by VAR, in 14 patients studied 10-24 months after varicocelectomy (post-VAR) and separated into normo- and oligospermic groups, and in 24 non-VAR control patients with seminal parameters matched to patients with VAR in the oligo- and normospermic groups (7 subjects with idiopathic oligospermia and 17 normal fertile subjects). TAC was measured in seminal plasma with the system H2O2-metamyoglobin as a source of radicals, which interact with a chromogen 2,2',-azinobis (3-ethylbenzothiazoline-6-sulphonate) (ABTS), generating a radical cation spectroscopically detectable. The presence of antioxidants induces a lag time in the production of ABTS cation proportional to the concentration of antioxidant compounds. When whole groups of patients were analyzed, lag values were significantly higher in VAR vs non-VAR controls (mean ± SEM, 106.6 ± 8.8 seconds vs 78.7 ± 8.8 seconds) but were not modified by surgery (mean ± SEM, 105.8 ± 8.6 seconds). In groups separated according to seminal parameters, oligospermic VAR presented significantly higher lag values than oligospermic controls. Finally, when exploring a possible association of TAC with seminal parameters, we found a significant correlation between lag and sperm motility only in patients with VAR who were in the normospermic group (r = 0.65, P < .01). This correlation was not yet manifest post-VAR. In conclusion, surgical treatment does not seem to modify absolute values of TAC but influences its fine regulation and relationships with sperm motility.
Key words: Infertility, reactive oxygen species, seminal plasma, spermatozoa
Human sperm cells produce reactive oxygen species (ROS), which are implicated in hyperactivated motility and acrosome reaction, but they are also sensitive to ROS-induced damage (Aitken et al, 1989). Therefore, they possess a major antioxidant defense against ROS, including catalase, superoxide dismutase, and glutathione peroxidase, whose effectiveness is limited by their low concentration and cellular distribution (Aitken and Fishel, 1994). Seminal plasma, on the contrary, is well endowed with antioxidant buffer capacity.
Patients with varicocele (VAR) represent an interesting model because they exhibit an augmented ROS generation and high levels of nitric oxide, which is related to ROS generation (Hendin et al, 1999).
In our previous study, we assessed the status of the total nonenzymatic antioxidant defenses of human seminal plasma in patients with VAR, showing augmented values in patients with VAR vs control subjects (Mancini et al, 2001; Meucci et al, 2003). This datum could indicate an ineffective utilization of antioxidant system in patients with VAR, which can be added to other known biochemical abnormalities in patients with VAR (Marmar, 2001).
Even if it is debated whether a surgical repair of VAR is indicated in the presence of infertility, with or without an alteration of seminal parameters, it is undoubted that a long-term exposure to the oxidative damage in high-grade VAR could explain the impaired fertilization ability of these patients.
To further explore a possible molecular defect and the impact of surgical treatment of VAR, we performed this descriptive study to 1) evaluate total antioxidant capacity (TAC) in preoperative patients with VAR (normal and oligospermic groups), in patients who were studied 10-24 months after varicocelectomy (post-VAR) and also separated into normal and oligospermic groups, and in patients in a control non-VAR group that was matched according to seminal parameters (idiopathic oligospermia and normal fertile subjects); and 2) explore a possible association of TAC and other seminal parameters.
Methods
From the outpatient clinic of the male infertility factor, 25 patients aged 19-40 years and affected by VAR and 14 patients aged 22-45 years who were studied 10-24 months post-VAR were recruited.
Patients were affected by primary infertility. All subjects participated in this study after they had given informed consent in respect of guidelines of the Helsinki Declaration.
All patients were given a general physical and genital examination; the scrotal contents were examined with patients in a standing position. Volume, position, and consistency of testes and epidydimes as well as presence of vasa deferentia were noted. Palpation of the pampiniform plexus was done with and without the patients performing a Valsalva maneuver. The clinical diagnosis of VAR was confirmed by Doppler technique.
Assessment was done with the patients in the upright and supine positions during the Valsalva maneuver while the patients breathed normally. Color Doppler scrotal ultrasound was performed with a 7.5 MHz high-resolution linear array transducer with pulsed and color Doppler capabilities. Veins that form pampiniform plexus were analyzed bilaterally. Doppler samples of the spermatic vein were obtained during Valsalva maneuver and while the patients breathed normally. The direction of blood flow and the changes in direction with breathing and the Valsalva maneuver were evaluated with Doppler-wave spectral analysis along with color Doppler images. Hirsh et al (1980) classified VAR in 3 grades according to Valsalva-induced reflux: grade I with no spontaneous venous activity, grade II with intermittent reflux, and grade III with continuous reflux. Reflux, whether Valsalva-induced or spontaneous, defined as caudate flow in the internal spermatic vein, was considered abnormal if it lasted longer than 1 second. Patients had undergone ligation of the left internal spermatic vein. Postoperative Doppler examination excluded VAR relapsing. Among the group of patients with VAR, 17 were affected by monolateral VAR and 8 were affected by bilateral VAR. Among the group of post-VAR patients, 10 were affected before the surgical procedure by monolateral VAR and 4 were affected by bilateral VAR.
In all patients, standard semen analysis was performed assessing semen parameters according to the World Health Organization (WHO) (1999), including volume (2.0 mL or more), sperm concentration (20 x 106 spermatozoa/mL or more), percent motility (50% or more motile of grade "a + b" or 25% or more with progressive motility of grade "a" within 60 minutes of ejaculation), and sperm morphology (50% or more normal morphology). On the basis of sperm concentration, patients with VAR were classified as oligozoospermic (n = 9) or normozoospermic (n = 16), and post-VAR patients were likewise classified as oligozoospermic (n = 6) or normozoospermic (n = 8). The leukocyte concentration of each sample was less than 1 x 106 white blood cells/mL, which is considered acceptable according to WHO criteria.
Regarding controls, we studied a group of 24 subjects who were non-VAR: 7 (aged 22-44 years) were affected by idiopathic oligozoospermia and 17 (23-40 years) were normal fertile subjects. These different control-group subjects were recruited in order to perform a comparison with patients with VAR, matching with the corresponding seminal picture.
To perform TAC evaluation, semen specimens were analyzed within 1 hour of collection. Liquefied semen samples were centrifuged at 700 x g for 10 minutes. The seminal plasma was aliquoted and kept frozen at -80°C until assayed (within 5 months, equaling storage time in all samples). Repeated assays on a reference seminal plasma sample showed that sample storage in these conditions did not significantly influence the evaluated parameters.
Semen antioxidant capacity was determined with a method developed for the evaluation of this parameter in blood plasma (Rice-Evans and Miller, 1994). The method is based on the antioxidants' inhibition of the absorbance of the radical cation chromogen 2,2',-azinobis (3-ethylbenzothiazoline-6-sulphonate) (ABTS) formed by the interaction between ABTS (150 μm) and ferrylmyoglobin radical species, generated by activation of metamyoglobin (2.5 μm) with H2O2 (75 μm). Aliquots of the frozen seminal plasma were thawed at room temperature, and 10 μL of the samples were tested immediately. The manual procedure was used with only minor modifications (ie, temperature was at 37°C to be in more physiological conditions and each sample was assayed alone to carefully control timing and temperature). The reaction was started directly in cuvette through H2O2 addition after 1 minute of equilibration of all other reagents (temperature control by a thermocouple probe, model 1408 K thermometer, Digitron Instrumentation Ltd, United Kingdom) and followed for 10 minutes under continuous stirring, monitoring absorbance at 734 nm typical of the spectroscopically detectable ABTS. The presence of chain-breaking antioxidants induces a lag time (the lag phase) in the accumulation of ABTS, whose duration is proportional to the concentration of this type of antioxidants. The length of lag phase is expressed in seconds. Trolox, a water-soluble tocopherol analogue, was used as a reference standard and assayed in all experiments to control the system. Absorbance was measured with a Hewlett-Packard 8450A UV/V spectrophotometer (Palo Alto, Calif) equipped with a cuvette stirring apparatus and a constant-temperature cell holder. Measurements of pH were made with a PHM84 Research pHmeter (radiometer) (Copenhagen, Denmark); the electrode response was corrected for temperature. Unless stated differently, experiments were repeated 2-3 times; qualitatively similar results were obtained with individual values varying less than 8%.
In the lag mode, the assay mainly measures nonprotein and nonenzymatic antioxidants, which are primarily extracellular chain-breaking antioxidants, such as ascorbate, urate, and glutathione.
Statistics
The distribution of the data was evaluated by the Kolmogorov-Smirnov test. Because the data were not normally distributed, the comparison among groups was performed by the Kruskal-Wallis test. Moreover, logarithmic transformation was applied to perform linear regression analysis. Arcus Quickstat Software (Software Publishing, Biomedical Version 1.2, United Kingdom) was used for statistical analysis.
Results
When analyzing groups who were separated according to sperm concentration (Figure 1), we found that oligospermic VAR presented significantly higher lag values than did oligospermic controls.
There was no significant difference when comparing pre- and post-VAR in either oligospermic or normospermic groups. Oligospermic post-VAR maintained higher lag values than did controls (Figure 1).
When exploring a possible association of TAC with seminal parameters, we found a significant correlation between lag and sperm motility only in patients who were preoperative with normospermic VAR (r = 0.65; P < .01). This correlation disappeared in post-VAR patients (Figure 2). No correlation was present when plotting lag values against other parameters, such as sperm density or sperm morphology (Table 2).
Discussion
During the past decade, oxidative stress has been implicated as a mediator of sperm dysfunction. VAR is a common cause of male infertility, with a possible involvement of oxidative balance. In fact, testicular VAR results in defective spermatogenesis and spermiogenesis, with the subsequent decrease in sperm production and the release of immature ROS-producing spermatozoa into the seminiferous tubules.
The spermatozoon has a high content of polyunsaturated fatty acids within the plasma membrane and a low concentration of scavenging enzymes within the cytoplasm. Therefore, it is susceptible to the peroxidation in the presence of elevated ROS seminal levels (Aitken et al, 1989), and oxidative stress induces DNA damage in both the mitochondrial and nuclear genomes (Aitken and Krausz, 2001).
Seminal plasma possess major antioxidant defenses, including enzymatic and nonenzymatic antioxidants. Chain-breaking antioxidants trap ROS directly to prevent amplification of radical formation and subsequent damage to sperm.
Enzymatic antioxidants consist of superoxide dismutase (SOD), catalase, glutathione peroxidase, and glutathione reductase. Nonenzymatic defense consists of scavenger molecules (eg, ascorbate, urate, thiol groups), antioxidants that interrupt the propagation of peroxidation (eg, alpha-tocopherol), and iron-binding molecules (eg, transferrin and lactoferrin) (Aitken and Fishel, 1994). The high levels of seminal plasma antioxidants indicate an important role in preservation of spermatozoa.
Different studies have tried to clarify the origin of redox enzymes but have not obtained conclusive results. Bauche et al (1994) sustained a testicular origin of enzymatic antioxidant and suggested that SOD is an important antioxidant enzyme protecting the testis from ROS. The authors demonstrated a differential distribution of SOD and reduced glutothione (GSH) among rat testicular cell types: Sertoli peritubular cells had elevated SOD and GSH-dependent enzyme activities associated with high GSH content, whereas pachytene spermatocytes and spermatids presented higher SOD and GSH content but very low GSH-dependent enzyme activity; spermatozoa had the same enzymatic system but were devoid of GSH. The authors concluded that the different categories of testicular cells probably display a highly variable susceptibility to oxidative stress.
Other studies suggested that enzymatic antioxidants did not originate substantially from the testis or epididymis but are primarily posttesticular (probably with main contribution of prostatic origin) and serve to protect ejaculated spermatozoa from oxidative stress such as that which occurs in the female reproductive tract (Yeung et al, 1998; Zini et al, 2002).
Total antioxidant capacity of seminal plasma receives an important contribution from the epididymis, which possesses region-specific antioxidant activity. It may potentially protect spermatozoa from oxidative attack during storage at this site. Potts et al (1999) reported that seminal plasma from men who have had vasectomies contains less total antioxidant capacity, lower thiol group concentrations, and higher amounts of lipid peroxidation compared with men with an intact ductal system.
Additionally, low molecular weight nonenzymatic scavengers from seminal plasma appeared more important than high enzymatic molecular weight components (Kovalski et al, 1992). Previous studies assessed the capacity of human seminal plasma to protect sperm from oxidative stress and demonstrated that addition of antioxidants significantly decreased the amount of spermatozoal DNA damage and lipid peroxidation induced by ROS in vitro (Lopes et al, 1998; Potts et al, 2000).
In previous studies, an inverse relationship between nonenzymatic antioxidant capacity and lipid peroxidation potential strongly suggests that impaired antioxidant defenses play an important role in infertile disorders (Smith et al, 1996; Sharma et al, 1999).
This antioxidant nonenzymatic buffer capacity has recently been quantified by various techniques and has been found to have an impaired nonenzymatic antioxidant capacity in infertile men (Lewis et al, 1995; Smith et al, 1996). Although sperm are exposed to seminal plasma in vivo briefly, human spermatozoa are naturally protected from oxidative injury by the antioxidant properties of seminal plasma.
In a previous paper, we evaluated the status of TAC of human seminal plasma in patients who were either affected or unaffected by VAR, who we studied as controls (Mancini et al, 2001; Meucci et al, 2003). We found significantly higher TAC in patients with VAR vs patients without VAR. Moreover, when classifying patients according to seminal parameters, we showed similar lag values in normospermic and oligospermic VAR; however, lag phase showed significant difference when comparing oligospermic VAR with oligospermic controls.
Because antioxidant levels indicate a balance between production and utilization, the augmented lag values could indicate an ineffective utilization of antioxidant system in patients with VAR and oligospermia, not related to oligospermia per se because idiopathic oligospermia did not show a similar pattern.
Such an explanation has also been used to explain higher seminal plasma levels of specific antioxidants, such as Coenzyme Q10 (Mancini et al, 1994). Other explanations for augmented lag values, such as a better buffering mechanism or a low consumption due to a smaller quantity of ROS, are not supported by the well-documented higher oxidative stress found in VAR (Chen et al, 2001).
A peculiar finding was the correlation between lag and sperm motility observed only in the group with normospermic VAR. We hypothesized that such a correlation indicates the need of adequate amounts of antioxidants in VAR to protect sperm motility; the absence of such a correlation in normal subjects could indicate that even low TAC values are compatible with normal motility. In oligospermic VAR, the correlation is not present for the greater testicular damage reflected in the reduced spermatozoa production. Therefore, the physiopathology of patients with normo- and oligospermic VAR seems to be different, even if we cannot precise the factors involved in this difference. Variable effects in individual patients can be explained by disease-modifier genes or other unknown pathologic conditions (other VAR itself) that can actually be responsible for oligospermia and increased oxidative stress observed in those men.
In this study, we evaluated the impact of surgical treatment on total antioxidant capacity in patients with VAR and the possibility of a partial reversal of this molecular defect post-VAR.
A recent study (Mostafa et al, 2001) showed that surgical treatment induces a reduction of ROS (malonildialdehyde and hydrogen peroxide), ROS radical (nitric oxide), and an increase of some antioxidants (superoxide dismutase, catalase, glutathione peroxidase, and Vitamin C) in seminal plasma. Vitamin E, on the contrary, is reduced. Therefore, post-VAR seems to profoundly affect the balance between oxidant species and antioxidant activity. In this sense, ROS-TAC scores have been recently indicated as a more reliable index of oxidative stress.
Although we have not evaluated this datum, we observed some modification of plasma TAC after surgery without a full restoration of a pattern similar to control subjects. In fact, we found that lag values were not influenced in the whole group of patients; moreover, oligospermic post-VAR remained different from oligospermic controls. However, the peculiar correlation between TAC and motility was no more detectable in post-VAR patients; in this sense, such patients were similar to controls. The disappeared correlation could be a sign of a re-established equilibrium in the protective role of antioxidants toward sperm motility.
In conclusion, surgical treatment in patients with VAR does not seem to modify absolute values of TAC but influences its fine regulation and relationships with sperm motility.
References
Aitken RJ, Fishel H. Reactive oxygen species generation and human spermatozoa: the balance of benefit and risk. Bioessays. 1994; 16:259-267.
Aitken RJ, Irvine MD, Wu FC. Prospective analysis of sperm-oocyte fusion and reactive oxygen species generation as criteria for the diagnosis of infertility. Am J Obstet Gynecol. 1989; 164:542-551.
Aitken RJ, Krausz C. Oxidative stress, DNA damage and Y chromosome. Reproduction. 2001; 122:497-506.
Bauche F, Fouchard MH, Jegou B. Antioxidant system in rat testicular cells. FEBS Lett. 1994; 349:392-396.
Chen S, Chang LS, Wei Y. Oxidative damage to proteins and decrease of antioxidant capacity in patients with varicocele. Free Radic Biol Med. 2001;11:1328-1334.
Hendin BN, Kolettis PN, Sharma RK, Thomas AJ Jr, Agarwal A. Varicocele is associated with elevated spermatozoal reactive oxygen species production and diminished seminal plasma antioxidant capacity. J Urol. 1999;161:1831-1834.
Hirsh AV, Cameron KM, Tyler JP, Simpson J, Pryor JP. The Doppler assessment of varicoceles and internal spermatic vein reflux in infertile men. BJU Int. 1980;52:50-56.
Kovalski NN, de Lamirande E, Gagnon C. Reactive oxygen species generated by human neutrophils inhibit sperm motility: protective effect of seminal plasma and scavengers. Fertil Steril. 1992; 58:809-816.
Lewis SE, Boyle PM, McKinney KA, Young IS, Thompson W. Total antioxidant capacity of seminal plasma in fertile and infertile men. Fertil Steril. 1995; 64:868-870.
Lopes S, Jurisicova A, Sun JG, Casper RF. Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa. Hum Reprod. 1998;13:896-900.
Mancini A, De Marinis L, Oradei A, Hallgass ME, Conte G, Pozza D, Littarru GP. Coenzyme Q10 concentrations in normal and pathological human seminal fluid. J Androl. 1994; 6:591-594.
Mancini A, Meucci E, Milardi D, et al. Total antioxidant capacity in seminal fluid of varicocele patients: 1-correlation with sperm motility. In: Robaire B, Cemes H, Morales CR, eds. Andrology in the 21st Century. Engelwood, NJ: Medimond Publishing Co; 2001 : 505-510.
Marmar JL. The pathophysiology of varicoceles in the light of current molecular and genetic information. Hum Reprod Update. 2001;5:461-472.
Meucci E, Milardi D, Mordente A, Martorana GE, Giacchi E, De Marinis L, Mancini A. Total antioxidant capacity in patients with varicoceles. Fertil Steril. 2003; 79:1577-1583.
Mostafa T, Anis TH, El-Nashar A, Imam H, Othman IA. Varicocelectomy reduces reactive oxygen species levels and increases antioxidant activity of seminal plasma from infertile men with varicocele. Int J Androl. 2001;24:261-265.
Potts RJ, Jefferies TM, Notarianni LJ. Antioxidant capacity of the epididymis. Hum Reprod. 1999; 14:2513-2516.
Potts RJ, Notarianni LJ, Jefferies TM. Seminal plasma reduces exogenous oxidative damage to human sperm, determined by the measurement of DNA strand breaks and lipid peroxidation. Mutat Res. 2000; 14:249-256.
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Sharma RK, Pasqualotto FF, Nelson DR, Thomas AJ, Agarwal A. The reactive oxygen species-total antioxidant capacity score is a new measure of oxidative stress to predict male infertility. Hum Reprod. 1999;14:2801-2807.
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Zini A, Fischer MA, Mak V, Phang D, Jarvi K. Catalase-like and superoxide dismutase-like activities in human seminal plasma. Urol Res. 2002;30:321-323.(ANTONIO MANCINI, ELISABET)
Abstract
In order to explore the impact of surgical treatment on antioxidant defense system in varicocele (VAR), we evaluated seminal total antioxidant capacity (TAC) in 25 patients affected by VAR, in 14 patients studied 10-24 months after varicocelectomy (post-VAR) and separated into normo- and oligospermic groups, and in 24 non-VAR control patients with seminal parameters matched to patients with VAR in the oligo- and normospermic groups (7 subjects with idiopathic oligospermia and 17 normal fertile subjects). TAC was measured in seminal plasma with the system H2O2-metamyoglobin as a source of radicals, which interact with a chromogen 2,2',-azinobis (3-ethylbenzothiazoline-6-sulphonate) (ABTS), generating a radical cation spectroscopically detectable. The presence of antioxidants induces a lag time in the production of ABTS cation proportional to the concentration of antioxidant compounds. When whole groups of patients were analyzed, lag values were significantly higher in VAR vs non-VAR controls (mean ± SEM, 106.6 ± 8.8 seconds vs 78.7 ± 8.8 seconds) but were not modified by surgery (mean ± SEM, 105.8 ± 8.6 seconds). In groups separated according to seminal parameters, oligospermic VAR presented significantly higher lag values than oligospermic controls. Finally, when exploring a possible association of TAC with seminal parameters, we found a significant correlation between lag and sperm motility only in patients with VAR who were in the normospermic group (r = 0.65, P < .01). This correlation was not yet manifest post-VAR. In conclusion, surgical treatment does not seem to modify absolute values of TAC but influences its fine regulation and relationships with sperm motility.
Key words: Infertility, reactive oxygen species, seminal plasma, spermatozoa
Human sperm cells produce reactive oxygen species (ROS), which are implicated in hyperactivated motility and acrosome reaction, but they are also sensitive to ROS-induced damage (Aitken et al, 1989). Therefore, they possess a major antioxidant defense against ROS, including catalase, superoxide dismutase, and glutathione peroxidase, whose effectiveness is limited by their low concentration and cellular distribution (Aitken and Fishel, 1994). Seminal plasma, on the contrary, is well endowed with antioxidant buffer capacity.
Patients with varicocele (VAR) represent an interesting model because they exhibit an augmented ROS generation and high levels of nitric oxide, which is related to ROS generation (Hendin et al, 1999).
In our previous study, we assessed the status of the total nonenzymatic antioxidant defenses of human seminal plasma in patients with VAR, showing augmented values in patients with VAR vs control subjects (Mancini et al, 2001; Meucci et al, 2003). This datum could indicate an ineffective utilization of antioxidant system in patients with VAR, which can be added to other known biochemical abnormalities in patients with VAR (Marmar, 2001).
Even if it is debated whether a surgical repair of VAR is indicated in the presence of infertility, with or without an alteration of seminal parameters, it is undoubted that a long-term exposure to the oxidative damage in high-grade VAR could explain the impaired fertilization ability of these patients.
To further explore a possible molecular defect and the impact of surgical treatment of VAR, we performed this descriptive study to 1) evaluate total antioxidant capacity (TAC) in preoperative patients with VAR (normal and oligospermic groups), in patients who were studied 10-24 months after varicocelectomy (post-VAR) and also separated into normal and oligospermic groups, and in patients in a control non-VAR group that was matched according to seminal parameters (idiopathic oligospermia and normal fertile subjects); and 2) explore a possible association of TAC and other seminal parameters.
Methods
From the outpatient clinic of the male infertility factor, 25 patients aged 19-40 years and affected by VAR and 14 patients aged 22-45 years who were studied 10-24 months post-VAR were recruited.
Patients were affected by primary infertility. All subjects participated in this study after they had given informed consent in respect of guidelines of the Helsinki Declaration.
All patients were given a general physical and genital examination; the scrotal contents were examined with patients in a standing position. Volume, position, and consistency of testes and epidydimes as well as presence of vasa deferentia were noted. Palpation of the pampiniform plexus was done with and without the patients performing a Valsalva maneuver. The clinical diagnosis of VAR was confirmed by Doppler technique.
Assessment was done with the patients in the upright and supine positions during the Valsalva maneuver while the patients breathed normally. Color Doppler scrotal ultrasound was performed with a 7.5 MHz high-resolution linear array transducer with pulsed and color Doppler capabilities. Veins that form pampiniform plexus were analyzed bilaterally. Doppler samples of the spermatic vein were obtained during Valsalva maneuver and while the patients breathed normally. The direction of blood flow and the changes in direction with breathing and the Valsalva maneuver were evaluated with Doppler-wave spectral analysis along with color Doppler images. Hirsh et al (1980) classified VAR in 3 grades according to Valsalva-induced reflux: grade I with no spontaneous venous activity, grade II with intermittent reflux, and grade III with continuous reflux. Reflux, whether Valsalva-induced or spontaneous, defined as caudate flow in the internal spermatic vein, was considered abnormal if it lasted longer than 1 second. Patients had undergone ligation of the left internal spermatic vein. Postoperative Doppler examination excluded VAR relapsing. Among the group of patients with VAR, 17 were affected by monolateral VAR and 8 were affected by bilateral VAR. Among the group of post-VAR patients, 10 were affected before the surgical procedure by monolateral VAR and 4 were affected by bilateral VAR.
In all patients, standard semen analysis was performed assessing semen parameters according to the World Health Organization (WHO) (1999), including volume (2.0 mL or more), sperm concentration (20 x 106 spermatozoa/mL or more), percent motility (50% or more motile of grade "a + b" or 25% or more with progressive motility of grade "a" within 60 minutes of ejaculation), and sperm morphology (50% or more normal morphology). On the basis of sperm concentration, patients with VAR were classified as oligozoospermic (n = 9) or normozoospermic (n = 16), and post-VAR patients were likewise classified as oligozoospermic (n = 6) or normozoospermic (n = 8). The leukocyte concentration of each sample was less than 1 x 106 white blood cells/mL, which is considered acceptable according to WHO criteria.
Regarding controls, we studied a group of 24 subjects who were non-VAR: 7 (aged 22-44 years) were affected by idiopathic oligozoospermia and 17 (23-40 years) were normal fertile subjects. These different control-group subjects were recruited in order to perform a comparison with patients with VAR, matching with the corresponding seminal picture.
To perform TAC evaluation, semen specimens were analyzed within 1 hour of collection. Liquefied semen samples were centrifuged at 700 x g for 10 minutes. The seminal plasma was aliquoted and kept frozen at -80°C until assayed (within 5 months, equaling storage time in all samples). Repeated assays on a reference seminal plasma sample showed that sample storage in these conditions did not significantly influence the evaluated parameters.
Semen antioxidant capacity was determined with a method developed for the evaluation of this parameter in blood plasma (Rice-Evans and Miller, 1994). The method is based on the antioxidants' inhibition of the absorbance of the radical cation chromogen 2,2',-azinobis (3-ethylbenzothiazoline-6-sulphonate) (ABTS) formed by the interaction between ABTS (150 μm) and ferrylmyoglobin radical species, generated by activation of metamyoglobin (2.5 μm) with H2O2 (75 μm). Aliquots of the frozen seminal plasma were thawed at room temperature, and 10 μL of the samples were tested immediately. The manual procedure was used with only minor modifications (ie, temperature was at 37°C to be in more physiological conditions and each sample was assayed alone to carefully control timing and temperature). The reaction was started directly in cuvette through H2O2 addition after 1 minute of equilibration of all other reagents (temperature control by a thermocouple probe, model 1408 K thermometer, Digitron Instrumentation Ltd, United Kingdom) and followed for 10 minutes under continuous stirring, monitoring absorbance at 734 nm typical of the spectroscopically detectable ABTS. The presence of chain-breaking antioxidants induces a lag time (the lag phase) in the accumulation of ABTS, whose duration is proportional to the concentration of this type of antioxidants. The length of lag phase is expressed in seconds. Trolox, a water-soluble tocopherol analogue, was used as a reference standard and assayed in all experiments to control the system. Absorbance was measured with a Hewlett-Packard 8450A UV/V spectrophotometer (Palo Alto, Calif) equipped with a cuvette stirring apparatus and a constant-temperature cell holder. Measurements of pH were made with a PHM84 Research pHmeter (radiometer) (Copenhagen, Denmark); the electrode response was corrected for temperature. Unless stated differently, experiments were repeated 2-3 times; qualitatively similar results were obtained with individual values varying less than 8%.
In the lag mode, the assay mainly measures nonprotein and nonenzymatic antioxidants, which are primarily extracellular chain-breaking antioxidants, such as ascorbate, urate, and glutathione.
Statistics
The distribution of the data was evaluated by the Kolmogorov-Smirnov test. Because the data were not normally distributed, the comparison among groups was performed by the Kruskal-Wallis test. Moreover, logarithmic transformation was applied to perform linear regression analysis. Arcus Quickstat Software (Software Publishing, Biomedical Version 1.2, United Kingdom) was used for statistical analysis.
Results
When analyzing groups who were separated according to sperm concentration (Figure 1), we found that oligospermic VAR presented significantly higher lag values than did oligospermic controls.
There was no significant difference when comparing pre- and post-VAR in either oligospermic or normospermic groups. Oligospermic post-VAR maintained higher lag values than did controls (Figure 1).
When exploring a possible association of TAC with seminal parameters, we found a significant correlation between lag and sperm motility only in patients who were preoperative with normospermic VAR (r = 0.65; P < .01). This correlation disappeared in post-VAR patients (Figure 2). No correlation was present when plotting lag values against other parameters, such as sperm density or sperm morphology (Table 2).
Discussion
During the past decade, oxidative stress has been implicated as a mediator of sperm dysfunction. VAR is a common cause of male infertility, with a possible involvement of oxidative balance. In fact, testicular VAR results in defective spermatogenesis and spermiogenesis, with the subsequent decrease in sperm production and the release of immature ROS-producing spermatozoa into the seminiferous tubules.
The spermatozoon has a high content of polyunsaturated fatty acids within the plasma membrane and a low concentration of scavenging enzymes within the cytoplasm. Therefore, it is susceptible to the peroxidation in the presence of elevated ROS seminal levels (Aitken et al, 1989), and oxidative stress induces DNA damage in both the mitochondrial and nuclear genomes (Aitken and Krausz, 2001).
Seminal plasma possess major antioxidant defenses, including enzymatic and nonenzymatic antioxidants. Chain-breaking antioxidants trap ROS directly to prevent amplification of radical formation and subsequent damage to sperm.
Enzymatic antioxidants consist of superoxide dismutase (SOD), catalase, glutathione peroxidase, and glutathione reductase. Nonenzymatic defense consists of scavenger molecules (eg, ascorbate, urate, thiol groups), antioxidants that interrupt the propagation of peroxidation (eg, alpha-tocopherol), and iron-binding molecules (eg, transferrin and lactoferrin) (Aitken and Fishel, 1994). The high levels of seminal plasma antioxidants indicate an important role in preservation of spermatozoa.
Different studies have tried to clarify the origin of redox enzymes but have not obtained conclusive results. Bauche et al (1994) sustained a testicular origin of enzymatic antioxidant and suggested that SOD is an important antioxidant enzyme protecting the testis from ROS. The authors demonstrated a differential distribution of SOD and reduced glutothione (GSH) among rat testicular cell types: Sertoli peritubular cells had elevated SOD and GSH-dependent enzyme activities associated with high GSH content, whereas pachytene spermatocytes and spermatids presented higher SOD and GSH content but very low GSH-dependent enzyme activity; spermatozoa had the same enzymatic system but were devoid of GSH. The authors concluded that the different categories of testicular cells probably display a highly variable susceptibility to oxidative stress.
Other studies suggested that enzymatic antioxidants did not originate substantially from the testis or epididymis but are primarily posttesticular (probably with main contribution of prostatic origin) and serve to protect ejaculated spermatozoa from oxidative stress such as that which occurs in the female reproductive tract (Yeung et al, 1998; Zini et al, 2002).
Total antioxidant capacity of seminal plasma receives an important contribution from the epididymis, which possesses region-specific antioxidant activity. It may potentially protect spermatozoa from oxidative attack during storage at this site. Potts et al (1999) reported that seminal plasma from men who have had vasectomies contains less total antioxidant capacity, lower thiol group concentrations, and higher amounts of lipid peroxidation compared with men with an intact ductal system.
Additionally, low molecular weight nonenzymatic scavengers from seminal plasma appeared more important than high enzymatic molecular weight components (Kovalski et al, 1992). Previous studies assessed the capacity of human seminal plasma to protect sperm from oxidative stress and demonstrated that addition of antioxidants significantly decreased the amount of spermatozoal DNA damage and lipid peroxidation induced by ROS in vitro (Lopes et al, 1998; Potts et al, 2000).
In previous studies, an inverse relationship between nonenzymatic antioxidant capacity and lipid peroxidation potential strongly suggests that impaired antioxidant defenses play an important role in infertile disorders (Smith et al, 1996; Sharma et al, 1999).
This antioxidant nonenzymatic buffer capacity has recently been quantified by various techniques and has been found to have an impaired nonenzymatic antioxidant capacity in infertile men (Lewis et al, 1995; Smith et al, 1996). Although sperm are exposed to seminal plasma in vivo briefly, human spermatozoa are naturally protected from oxidative injury by the antioxidant properties of seminal plasma.
In a previous paper, we evaluated the status of TAC of human seminal plasma in patients who were either affected or unaffected by VAR, who we studied as controls (Mancini et al, 2001; Meucci et al, 2003). We found significantly higher TAC in patients with VAR vs patients without VAR. Moreover, when classifying patients according to seminal parameters, we showed similar lag values in normospermic and oligospermic VAR; however, lag phase showed significant difference when comparing oligospermic VAR with oligospermic controls.
Because antioxidant levels indicate a balance between production and utilization, the augmented lag values could indicate an ineffective utilization of antioxidant system in patients with VAR and oligospermia, not related to oligospermia per se because idiopathic oligospermia did not show a similar pattern.
Such an explanation has also been used to explain higher seminal plasma levels of specific antioxidants, such as Coenzyme Q10 (Mancini et al, 1994). Other explanations for augmented lag values, such as a better buffering mechanism or a low consumption due to a smaller quantity of ROS, are not supported by the well-documented higher oxidative stress found in VAR (Chen et al, 2001).
A peculiar finding was the correlation between lag and sperm motility observed only in the group with normospermic VAR. We hypothesized that such a correlation indicates the need of adequate amounts of antioxidants in VAR to protect sperm motility; the absence of such a correlation in normal subjects could indicate that even low TAC values are compatible with normal motility. In oligospermic VAR, the correlation is not present for the greater testicular damage reflected in the reduced spermatozoa production. Therefore, the physiopathology of patients with normo- and oligospermic VAR seems to be different, even if we cannot precise the factors involved in this difference. Variable effects in individual patients can be explained by disease-modifier genes or other unknown pathologic conditions (other VAR itself) that can actually be responsible for oligospermia and increased oxidative stress observed in those men.
In this study, we evaluated the impact of surgical treatment on total antioxidant capacity in patients with VAR and the possibility of a partial reversal of this molecular defect post-VAR.
A recent study (Mostafa et al, 2001) showed that surgical treatment induces a reduction of ROS (malonildialdehyde and hydrogen peroxide), ROS radical (nitric oxide), and an increase of some antioxidants (superoxide dismutase, catalase, glutathione peroxidase, and Vitamin C) in seminal plasma. Vitamin E, on the contrary, is reduced. Therefore, post-VAR seems to profoundly affect the balance between oxidant species and antioxidant activity. In this sense, ROS-TAC scores have been recently indicated as a more reliable index of oxidative stress.
Although we have not evaluated this datum, we observed some modification of plasma TAC after surgery without a full restoration of a pattern similar to control subjects. In fact, we found that lag values were not influenced in the whole group of patients; moreover, oligospermic post-VAR remained different from oligospermic controls. However, the peculiar correlation between TAC and motility was no more detectable in post-VAR patients; in this sense, such patients were similar to controls. The disappeared correlation could be a sign of a re-established equilibrium in the protective role of antioxidants toward sperm motility.
In conclusion, surgical treatment in patients with VAR does not seem to modify absolute values of TAC but influences its fine regulation and relationships with sperm motility.
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