International Journal of

Introduction

    One in six couples of reproductive age presents with infertility (Hull et al., 1995). Poor sperm motility is the major underlying cause of male infertility, the etiology of which is not completely elucidated. One of factors that potentially can cause asthenozoospermia is oxidative stress (Gagnon and de Lamirande 1999). Oxidative stress arises as a consequence of excessive production of Reactive oxygen species (ROS) and impaired antioxidant defense mechanisms.    
ROS play a dual role in male  infertility  (Sanocka and Kurpisz, 2004).On one hand, ROS plays a key-role in processes such as capacitation, the acrosome reaction, and fertilization. On the other hand, excessive production of ROS can inflict severe damage to spermatozoa (Ichikawa et al., 1999; Pasqualotto et al., 2000; Agarwal et al., 2003). Spermatozoa contain large quantities of polyunsaturated fatty acids (PUFA); therefore, they are susceptible to ROS-induced damage (Sanocka and Kurpisz 2004). It has been suggested that ROS induce membrane lipid peroxidation in sperm. The toxicity of generated fatty acid peroxides are important causes of sperm malfunction (Fraczek et al., 2001; Agarwal and Saleh 2002). The most widely used   assay    for  lipid   peroxidation   involves the measurement of MDA-TBA  adducts    due    to     its simplicity (Gomez et al., 1998; Antoine et al., 2002).



Recently, it has been shown that 15-F_2t-isoprostane (8-iso-PGF_2α) is a specific, chemically stable, and quantitative marker of oxidative stress in vivo (Lawson et al., 1999; Roberts and Morrow 2000; Roberts and Morrow 2002). 15-F_2t-isoprostane is a prostaglandin isomer synthesised in vivo, independently of the activity of cyclo-oxygenase (Meagher and FitzGerald 2000; Pratico et al., 2001).
    Homocysteine is a thiol-containing amino acid produced by the intracellular demethylation of methionine in the methylation processes. Total homocysteine level (reffered to as tHcy) is the sum of all homocysteine species. tHcy is prominently present in the oxidized form; mixed disulfides with proteins (Voutilainen et al., 1999). Homocysteine is metabolized to either cysteine or methionine. In any condition which homocysteine production is high or its metabolism is impaired, homocysteine accumulates in the cell and is exported to the extracellular fluids (Jacobsen 2000). Homocysteine is receiving a lot of attention these days as a new risk factor for a variety of disease. One mechanism by which increased homocysteine has been imposed to influence its pathological effects is by promoting increased oxidative stress (Voutilainen et al., 1999; Jacobsen, 2000; Zappacosta et al., 2001; Powers et al., 2002; Perna et al., 2003).
     Available data on the impact of oxidative stress on sperm motility are based on the measurement of seminal plasma and sperm MDA (Suleiman et al., 1996; Gomez et al., 1998; Fraczek et al., 2001; Rhemrev et al., 2001; Dandekar et al., 2002; Nakamura et al., 2002; Keskes-Ammar et al., 2003).  The results of these findings are controversial. Suleiman et al., (1996) did not observe any correlation between seminal plasma levels of MDA and sperm motility. Study of Frazcek et al. (2001) showed that seminal plasma level of MDA is higher   
 
in asthenozoospermic males than this level in normozoospermic    men.    Nakamura  et  al.   (2002) investigation showed that there is not significant difference in seminal plasma level of MDA between asthenozoospermic and normozoospermic males. Keskes-Ammar et al., (2003) observed that there is an indirect correlation between seminal plasma levels of MDA and sperm motility. To the best of our knowledge no information is available on the relationship between sperm parameters and seminal plasma 15-F_2t-isoprostane or homocysteine levels and also the findings on the relation of seminal plasma levels of MDA in normozoospermic vs. asthenozoospermic males are controversial. Therefore, we designed a case-control study with simple random sampling to (i) compare seminal plasma MDA, 15-F_2t-isoprostane, and tHcy levels in normozoospermic vs. asthenozoospermic males and to examine their association with sperm motility and also to (ii) investigate the relationship between seminal plasma tHcy levels and lipid peroxidation, as measured by MDA and 15-F_2t-isoprostane.
 
Materials and Methods

   Semen Samples

    The semen samples were collected from males undergoing infertility screening. All specimens were collected into sterile plastic containers by masturbation at the clinical andrology laboratory at Tabriz Medical School after an abstinence period of 48-72 hrs, and were analyzed within 1h of collection. After allowing at least 30 min for liquefaction, semen analysis was performed to measure sperm concentration, normal sperm morphology, percentage sperm motility, and semen volume in accordance with the recommendations of the World Health Organization (WHO 1999). Samples with a leukocyte concentration >10⁶/mlof ejaculate were excluded from   this   study.   Myeloperoxidase   staining   was performed to evaluate the leukocyte concentration in specimen   (LeucoScreen;   Fertipro).   After    semen analysis, subjects were determined to be either normozoospermic, as the control group, (n=15) or asthenozoospermic, as the case group, (n=15). Liquefied semen samples were centrifuged at 700g for 10 minutes. The supernatant seminal plasma was then carefully removed and transferred to Eppendrof tubes. The seminal plasma was frozen at -80ºC until examination.


of variation was <10%. The sensitivity of the tHcy assay was 2.0 µM.
   c) Malodialdehyde (MDA). The amount of MDA was determined by the TBA assay (Dandekar et al., 2002). All reagents that were used in this assay were obtained from Merck (Darmstadt Germany). Briefly, 0.50 ml of seminal plasma was added to 3.00 ml of 1.00% phosphoric acid, 1.00 ml of 0.60% TBA, and 0.15 ml of 0.20% butylated hydroxytoluene (BHT) in 95% methanol. The samples were heated in a boiling water bath for 45 minutes, cooled and 4.00 ml of 1-butanol was added. The butanol phase was separated by centrifugation at 3000 rpm for 10 minutes and absorbance was measured at 532 nm. The concentration of MDA was expressed as µM. 

    Statistical analysis

    Based on a pilot study, using an α value of 0.05 and a β value of 0.2 (80% power), the minimum sample size required was 15 samples per group. The Mann-Whitney U test was used to compare two groups. Coefficients of correlation were calculated using Spearman’s correlation analysis. All hypothesis tests were two-tailed with statistical significance assessed at the p value <0.05 level with 95% confidence intervals (CI). The data are expressed as the mean ± SEM. Statistical computations were calculated using SPSS 10 for windows software (SPSS Inc, Chicago, IL, USA).

Results

     A total of 30 samples were tested. Of these, 15 were normozoospermic and 15 were asthenozoospermic. The profile of sperm quality parameters of these samples is shown in Table I. The means percentages of progressively motile sperms, rapid motile sperms (WHO 1999), and normal sperm morphology showed   a   significant   difference   and tHcy levels did not show a significant difference between two groups. Seminal plasma MDA, 15-F_2t-isoprostane, and tHcy levels of the samples are shown in Table II. The means of 15-F_2t- isoprostane between the two groups (p>0.05). While, the mean values for MDA between the two groups were significantly different (p<0.05).

Seminal plasma 15-F_2t-isoprostane levels showed an inverse significant correlation with progressively motile sperms (r= -0.41, p<0.05) (Fig. 1) and also with normal sperm morphology (r= -0.42, p<0.05). The correlation between seminal plasma 15-F_2t-isoprostane levels and rapid motile sperm was significant only in asthenozoospermic group (r=-0.60, p<0.05). Seminal plasma levels of malondialdehyde showed an indirect correlation with progressively motile sperms (r= -0.53, p<0.05) (Fig. 2), but this correlation was not observed in each group. We also observed an indirect correlation between the malondialdehyde levels and the percentages of rapid motile sperms (r= -0.58, p<0.05) (Fig. 3). However, this relationship was not found with    15-F_2t-isoprostane and tHcy levels. No relationship was found between malondialdehyde and normal sperm morphology.
    Finally, the correlation between tHys and lipid peroxidation, as measured by MDA and 15-F_2t-isoprostane, was examined. Seminal plasma levels of tHcy showed no correlation with lipid peroxidation.

Discussion

     The most relevant finding from this study was a significant inverse correlation between seminal plasma levels of 15-F_2t-isoprostane and sperm motility. We observed that seminal plasma levels of 15-F_2t-isoprostane and tHcy did not differ significantly between normozoospermic and asthenozoospermic males. Our study also showed that there was not any correlation between seminal plasma levels of tHcy and lipid peroxidation.
    Suleiman SA et al., (1996) study showed that there is no relationship between seminal plasma MDA concentration and sperm motility. Investigation of Keskes-Ammar et al., (2003) showed that sperm motility inversely correlates with semen MDA levels. This correlation was also observed in our study. Frraczek M et al., (2001) observed that seminal plasma level of MDA is higher in asthenozoospermic males than in normozoospermic. This finding was also observed in our study. Nakamura et al., (2002) investigation showed that there is not a significant difference in seminal plasma MDA levels between normozoospermic and asthenozoospermic men. But in our study this difference was significant.
    We also measured seminal plasma level of 15-F_2t-
isoprostane. Recent studies have focused on 15-F_2t-isoprostane, as an index of lipid peroxidation. Quantification of 15-F_2t-isoprostane has been suggested to be a reliable measure of oxidative injury in vivo. Measurement of 15-F_2t-isoprostane may provide a reliable marker of lipid peroxidation in vivo, because, it is a stable compound. In addition, 15-F_2t-isoprostane is specific product of free radical-induced lipid peroxidation. 15-F_2t-isoprostane has also been found to be present in detectable quantities in all normal biological tissues and in free form in all normal biological fluids. This is important because it allows the definition of a normal range such that small increases in its formation can be detected in situations of mild oxidant stress. Finally, the levels of 15-F_2t-isoprostane is unaffected by lipid content of the diet (Pratico et al., 2001; Roberts & Morrow, 2002). Our study did not show a significant difference in seminal plasma levels of 15-F_2t-isoprostane between the two groups. 15-F_2t-isoprostane, like MDA, showed an inverse significant correlation with percentages of progressively motile sperm. It also correlated with percentages of normal sperm morphology. But, MDA showed no correlation with percentages of normal sperm morphology.
     We also measured total homocysteine. Homocysteine is receiving a lot of attention these days as a new risk factor for a variety of abnormality. One mechanism by which increased homocysteine has been proposed to influence its pathological effects is by promoting increased oxidative stress. One proposed mechanism is that because homocysteine is a thiol, it can undergo autooxidation and oxidation with other thiols. The resulting ROS-hydrogen peroxide and superoxide anion radical generate oxidative stress (Jacobsen 2000; Powers et al., 2002; Perna et al., 2003). Voutilainen et al., (1999) study showed that there is a significant correlation between tHcy and F2-isoprostane in hyperhomocysteinemic men. Our study did not show any correlation between seminal plasma tHcy levels and lipid peroxidation. This finding is related to our results that seminal plasma mean tHcy level showed no significant difference between the two groups in our study. Studies that found this correlation were carried out in hyperhomocysteinemic subjects.
   
     In summary, seminal plasma15-F_2t-isoprostane and tHcy levels between norozoospermic and asthenozoospermic men in current study were not significant, and no relationship was found between tHcy and lipid peroxidation. Our study also showed that sperm motility correlated significantly with seminal plasma levels of 15-F_2t-isoprostane and MDA. We also concluded that homocysteine metabolism may not impair in asthenozoospermic males.
A significant limitation of our study was a small sample size. Higher sample size is required to investigate the metabolism of 15-F_2t-isoprostane and tHcy in asthenizoospermic males and their relationship with sperm motility.

Acknowledgements

    This research was granted by Drug Applied Research Center of Tabriz University of Medical Sciences. The authors are grateful to Tabriz IVF center for their cooperation in this research.