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From the * Department of Obstetrics and
Gynecology, the Department of Urology, and the
Department of Clinical Biochemistry, Harran
University Faculty of Medicine, Sanliurfa, Turkey; and the
Department of Urology, Sanliurfa State
Hospital, Sanliurfa, Turkey.
| Correspondence to: Dr Fatma Ferda Verit, Department of Obstetrics and Gynecology, Harran University, Faculty of Medicine, Tr-63100 Sanliurfa, Turkey (e-mail: fverit{at}harran.edu.tr; fverit{at}gmail.com). |
| Received for publication January 8, 2008; accepted for publication July 1, 2008. |
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Abstract |
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Key words: Oxidative stress, spermatozoa, male infertility
All living aerobic cells are normally exposed to reactive oxygen species (ROS), and oxidative stress arises as a consequence of excessive production of ROS and impaired antioxidant defense mechanisms (Sikka, 2001). It is proposed that oxidative stress precipitates the range of pathologies that currently are thought to afflict the reproductive function (Sikka, 2001). Increased levels of seminal oxidative stress have been correlated with sperm dysfunction through different mechanisms that include lipid peroxidation of sperm plasma membrane and impairment of sperm metabolism, motility, and fertilizing capacity (Saleh et al, 2003). It has been reported that asthenozoospermic, asthenoteratozoospermic, and oligoasthenoteratozoospermic subfertile males had significantly lower values of catalase activity and total antioxidant capacity when compared with a normozoospermic group (Khosrowbeygi and Zarghami, 2007). Studies have demonstrated that 25%–88% of nonselected subfertile patients have high levels of seminal ROS (Lewis et al, 1995).
Paraoxonase-1 (PON-1) is a high-density lipoprotein (HDL)–associated antioxidant enzyme with paraoxonase, arylesterase, and dyazoxonase activities (Aslan et al, 2007). PON-1 activity has been suggested to be inversely associated with oxidative stress (Rozenberg et al, 2003). It is responsible for the antioxidant effect of HDL (Durrington et al, 2001). PON-1 has been shown to prevent low-density lipoprotein (LDL) and HDL oxidation and has also been proposed to stimulate cholesterol efflux, the first step in reverse cholesterol transport (Mackness et al, 1993). Reduced PON-1 activities have been reported in several groups of patients with diabetes, hypercholesterolemia, and cardiovascular disease who are under increased oxidative stress (Mackness et al, 1993; Ayub et al, 1999). Concerning the role of oxidative stress in male subfertility, the aims of the study were to investigate 1) seminal PON-1 activity as an antioxidant enzyme in subfertile men with normal and abnormal semen parameters with other markers of oxidative stress and 2) whether seminal PON-1 activity has any relationship to semen parameters.
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Materials and Methods |
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Hyperlipidemia was defined as follows: serum LDL cholesterol 
160 mg/dL,
total cholesterol 240 mg/dL, triglyceride 200 mg/dL, HDL cholesterol
<40 mg/dL (Expert Panel on Detection,
Evaluation, and Treatment of High Blood Cholesterol in Adults,
2001). A patient was considered as diabetic with a fasting plasma
glucose level 126 mg/dL.
Semen Analysis
Semen samples were collected by masturbation in a clean specimen container
after sexual abstinence for 3–5 days, were allowed to liquefy at
37°C, and were evaluated immediately according to World Health
Organization recommendations (ejaculate volume, pH, time to liquefaction,
sperm concentration, motility, and morphology). Morphology smears were scored
using the Kruger strict criteria (Kruger
et al, 1986). Sperm concentration was expressed as sperm per
milliliter of semen, and motility and morphology were expressed as
percentages. Sperm parameters were considered normal when sperm concentration
was 20 x 106/mL semen, motility was 50%, and normal
sperm forms were >14% by the Kruger strict criteria
(Kruger et al, 1986). Seminal
leukocytes were quantified by a myeloperoxidase staining test, and values were
considered to be normal at concentrations of 
1 x 106
peroxidase-positive leukocytes per milliliter of semen. The remaining semen
samples were centrifuged at 1500 x g for 10 minutes to obtain
the seminal plasma. The separated seminal plasma was then stored at
–80°C until further analysis of TOS, total antioxidant status (TAS),
and PON-1 measurement.
Measurement of TOS in Seminal Plasma
The TOS of semen samples was determined by using a new automated
colorimetric measurement method (Erel,
2005). The assay is based on the oxidation of ferrous ion to
ferric ion in the presence of various oxidant species in acidic medium and the
measurement of the ferric ion by xylenol orange. Within- and between-batch
precision values were lower than 3%. The results were expressed as µmol
hydrogen peroxide (H2O2) equivalent per liter
(Verit et al, 2006).
Measurement of TAS in Seminal Plasma
TAS of semen samples was determined by using a novel automated measurement
method developed by Erel
(2004). In this method, the
hydroxyl radical, the most potent radical, is produced via Fenton reaction and
consequently the colored dianisidinyl radical cations, which are also potent
radicals, are produced in the reaction medium of the assay. Antioxidant
capacity of the added sample against these colored potent free radical
reactions measured the total antioxidant capacity. The assay has excellent
precision values; within- and between-laboratory precision values are lower
than 3%. The results were expressed as millimoles of Trolox equivalent per
liter (Verit et al, 2006).
Measurement of PON-1 Activity in Seminal Plasma
PON-1 activity was determined by using paraoxon as a substrate and measured
by increases in the absorbance at 412 nm because of the formation of
4-nitrophenol as already described (Verit
et al, 2008). Briefly, the activity was measured at 25°C by
adding 50 µL of seminal plasma to 1 mL Tris-HCl buffer (100 mM at pH 8.0)
containing 2 mM CaCl2 and 5.5 mM of paraoxon. The rate of
generation of 4-nitrophenol was determined at 412 nm. Enzymatic activity was
calculated by using the molar extinction coefficient 17 100
M–1 cm–1.
Oxidative Stress Index
The percentage ratio of TOS to TAS gave the oxidative stress index (OSI),
an indicator of the degree of oxidative stress ([TOS/TAS] x 100;
Verit et al, 2006).
Statistical Analysis
Results were expressed as 
± SD for all continuous variables. Differences between control and male
infertility groups were assessed by using ANOVA followed by a Tukey test.
Associations between TOS, TAS, OSI, and PON-1 activity and semen parameters
were evaluated by Pearson's correlation test. The area under the receiver
operating characteristic curve (ROC) was used to assess the discriminative
ability of PON-1 activity in male-factor subfertility and in idiopathic
infertility.
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Results |
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There was no significant difference in terms of age among the groups. Seminal TOS was significantly higher and seminal TAS and PON-1 activity were significantly lower in the male-factor subfertility group as compared with idiopathic subfertile men and fertile donors (Table 1).
The relationship between seminal TOS, TAS, OSI, and PON-1 activity and semen parameters in the overall group (n = 90) is shown in Table 2. There were negative correlations between TOS and OSI and sperm parameters such as concentration, motility, and morphology (P < .0001 for all; Table 2). In addition, TAS and PON-1 activity had significant positive correlations with all sperm parameters in the study (P < .0001 for all; Table 2).
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PON-1 had a positive correlation with seminal TAS (r = .76, P < .0001) and was negatively correlated with seminal TOS (r = –.61, P < .0001) and OSI (r = –.73, P < .0001) in the study.
ROC analysis revealed a high diagnostic value for PON-1 activity with respect to male-factor subfertility, with an area under curve (AUC) of 0.95 (95% confidence interval = 0.89–1.01), sensitivity = 97%, and specificity = 88% with a cutoff value of 1.75 U/L (lower than that value was related to male-factor subfertility), shown in the Figure.
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However, PON-1 activity was not effective statistically in the diagnosis of idiopathic infertility (AUC < 0.5).
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Discussion |
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ROS have been found to have a dual effect on human spermatozoa. Sperm plasma membrane has a high concentration of polyunsaturated fatty acids, which can undergo lipid peroxidation initiated by ROS (Saleh et al, 2003). Such peroxidative damage to the sperm plasma membrane leads to a loss of membrane fluidity and integrity, as a result of which the spermatozoa lose their competence to participate in the membrane fusion events associated with fertilization (Aitken and Fischer, 1994; Alvarez and Storey, 1995; Storey, 1997). In addition, ROS are also known to attack DNA, inducing strand breaks and oxidative base damage in human spermatozoa (Hughes et al, 1996; Kodoma et al, 1997).
It has been indicated that levels of ROS are negatively correlated with the quality of sperm in the original semen (Gomez et al, 1998). High levels of ROS production in human ejaculates may originate from morphologically abnormal spermatozoa and/or seminal leukocytes (Aitken and West, 1990). Many studies have reported that spermatozoa from oligozoospermic or asthenozoospermic men showed a greater production of oxidative stress (Aitken et al, 1992; Sharma and Agarwal, 1996; Griveau and de Lannou, 1997; Pasqualotto et al, 2000). We also found that serum TOS was significantly higher in subfertile men with abnormal semen parameters in this study. Moreover, TOS was negatively correlated with semen parameters such as concentration, motility, and morphology in the study.
Previous reports have described that patients with idiopathic male infertility have elevated levels of ROS (Pasqualotto et al, 2000; Saleh et al, 2003). It has been also suggested that lipid peroxidation of sperm membrane may be one of the key mechanisms involved in the pathophysiology of idiopathic male infertility (Alkan et al, 1997). However, the correlation between oxidative stress and male idiopathic infertility is not clear, and there were some limitations in these studies. Saleh et al (2003) found significant correlations between abnormal sperm parameters including leukocytospermia and oxidative stress, but did not state the level of leukocytospermia in each group in the study population. Moreover, subfertile men with normal semen parameters had increased oxidative stress compared with normozoospermic fertile donors in this study, and it is a matter of debate how oxidative stress was increased in that group with normal semen parameters. In another study that was reported by Pasqualotto et al (2000), the study size was small, and the study was poorly designed because the normospermic group was not homogenous but also included varicocele patients. Many studies have demonstrated that oxidative stress is increased in varicocele even in the presence of normal semen parameters (Agarwal et al, 2006; Smith et al, 2006; Pasqualotto et al, 2008). In this study, we found that there was no difference in seminal oxidative stress between men with idiopathic subfertility and fertile donors, which supports our previous work (Verit et al, 2006). Moreover, Ochsendorf et al (1998) found that spermatozoa of oligozoospermic patients contained much lower concentrations of the endogenous antioxidant thiol glutathione than did those of normozoospermic men. Another study also demonstrated that oxidative stress was increased in asthenozoospermic patients compared with normozoospermic men (Tavilani et al, 2005). We suggest that oxidative stress is dependent on sperm parameters but is not directly related to the diagnosis of male-factor infertility.
Antioxidants are important in maintaining the oxidant-antioxidant balance in tissues. Among the well-known biological antioxidants, superoxide dismutase, catalase, and the glutathione peroxidase/reductase system have a significant role in protecting the sperm against peroxidative damage (De Lamirande and Gagnon, 1993; Sharma and Agarwal, 1996). Depressed seminal antioxidant capacity has been implicated in male subfertility. TAS levels have been shown to be lower in the semen of subfertile men as compared with fertile men (Lewis et al, 1995, 1997; Smith et al, 1996). More specifically, Raijmakers et al (2003) reported significantly higher seminal plasma thiol glutathione concentrations in fertile men compared with subfertile men. In accordance with this finding, it has been reported that ascorbate levels were significantly reduced in seminal plasma of asthenozoospermic subfertile men (Lewis et al, 1997). Furthermore, studies have suggested that subfertile men empirically treated with antioxidants have demonstrated improved semen characteristics, fertilization in vitro, and higher pregnancy rates in the treatment group (Lenzi et al, 1993; Geva et al, 1996). In our study, TAS was significantly decreased in subfertile men with abnormal semen parameters, but not in the idiopathic subfertile group.
PON-1 is an antioxidant enzyme that is highly effective in preventing lipid peroxidation of LDL (Mackness et al, 1993). It is principally responsible for the breakdown of lipid peroxides before they accumulate on LDL (Mackness et al, 1993). PON-1 can also destroy H2O2, a major ROS produced under oxidative stress during atherogenesis (Aviram et al, 1998), and increase the LDL clearance (Shih et al, 2000).
PON-1 also protects HDL against lipid peroxidation (Mackness et al, 1993; Aviram et al, 1998; Rozenberg et al, 2003). Inhibition of HDL oxidation by PON-1 preserves the antiatherogenic effects of HDL in reverse cholesterol transport (Aviram et al, 1998). The antioxidant effect of HDL is also assumed by PON-1 (Aviram and Rosenblat, 2004).
The association between PON-1 activity and male infertility is unknown. PON-1 activity was significantly lower in male-factor subfertile patients compared with idiopathic subfertile men and fertile donors in the present study. There were also significant positive correlations between PON-1 activity and semen parameters such as concentration, motility, and morphology. We suggest that decreased PON-1 activity must be related to enhanced production of ROS. In addition, it has been previously shown that PON-1 activity was decreased in some diseases because of ROS pathogenesis under oxidative stress and inflammation conditions such as diabetes, coronary artery disease, and endometriosis (Ayub et al, 1999; Durrington et al, 2001; Verit et al, 2008).
The most widely used methods for measuring ROS are colorimetry, fluorescence, chemiluminescence, and electron spin resonance (ESR) spectroscopy (Tarpey et al, 2004). We measured total oxidant levels in seminal plasma by a colorimetric method that was developed by Erel (2005) in this study. This technique has many advantages. Various other methods that have been developed for measuring TOS had no accepted reference method. In addition, this method led to a certain decision concerning the standardizations, the terms, and the units (Erel, 2005). Moreover, the fluorescence, chemiluminescence, and ESR methods need sophisticated techniques, and in most routine clinical biochemistry laboratories these improved systems are not available.
Measurement of TAS within semen can be conducted in a variety of ways. The ability of seminal plasma to inhibit chemiluminescence elicited by a constant source of ROS (horseradish peroxidase) is a commonly used technique. The TAS is usually quantified against a Vitamin E analogue (Trolox) and expressed as a ROS-TAS score (Sharma et al, 1999). However, colorimetry techniques based on the color change of 2,2'-azinobis3-ethylbenzo-thiazoline-6-sulphate (ABTS) are now becoming more popular because they are cheaper and easier to perform (Said et al, 2003; Erel, 2004). The reduced ABTS molecule is oxidized to ABTS+ using H2O2 and a peroxidase to form a relatively stable blue-green color measured at 600 nm with a standard spectrophotometer. Antioxidants present within seminal plasma suppress this color change to a degree that is proportional to their concentrations. Again the antioxidant activity is quantified using Trolox.
In conclusion, our results showed that TOS was significantly higher and TAS and PON-1 activity were significantly lower in patients with male-factor subfertility, but not in an idiopathic subfertile group. Reduced PON-1 activity may play a role in the pathogenesis of male subfertility. Therefore, both protection from oxidative stress and increases in PON-1 activity could be used as a powerful tool for the prevention of subfertility.
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