The key results in this study were: (1) An impaired antioxidant status was observed in seminal plasma of infertile men. (2) The fertile group showed a significant decrease in seminal lipid peroxidation levels compared to the three abnormal groups. (3) Positive correlations exist between enhanced seminal antioxidant status and good semen quality. Total sperm motility was significantly correlated to the seminal trace elements and different forms of GSH. The sperm concentration was positively associated to the seminal Zn and seminal GSHr amounts. However, the percentage of atypical sperm forms showed a significantly negative relationship to seminal GSSG. (4) Inversely to the non-enzymatic antioxidants, seminal MDA levels were negatively correlated to the sperm parameters. These findings indicated that some idiopathic infertile men may be poorly equipped to deal with oxidative stress due to the impaired seminal antioxidant defences. As a consequence, we suggest such a possibility and call for more systematic research on the role of oxidative stress in idiopathic male infertility.
The detailed biochemical mechanisms underlying the physiopathology of male infertility are not clearly understood. There has been evidence supporting the notion that major changes in seminal antioxidants could be related to abnormal spermatozoa function and fertilization capacity [9
]. Hence, seminal plasma is considered to be the central source of antioxidants that protect sperm cells against oxidative damages. The antioxidant system eliminates ROS to maintain a reduced environment in cells through enzymatic and non-enzymatic approaches. The most studied antioxidants are the SOD, GPX and catalase enzymes. Information about seminal non-enzymatic antioxidants and male fertility potential is quite inadequate till date. Moreover, it is important to underline the contradictions and the controversial outcomes found in the literature. In fact, these differences can be due to several variables among which are inclusion and exclusion criteria for patient selection, analytical methodologies, sperm anomalies (in our study, each patient group has a single sperm anomaly; whether asthenozoospermia, or oligozoospermia or teratozoospermia), lifestyle or dietary pattern and the patient's origin.
In our study, we tried to explore the concentrations of Zn, Se and GSH in the seminal plasma of our patient groups and controls in order to estimate their contribution to maintain a good quality of sperm.
The mean seminal Zn concentrations in control and abnormal groups were between 120 and 144 mg/l. These values were comparable with a few similar studies reported by Lewis Jones et al. (118 mg/l) [17
] and Kruse et al. (123 mg/l) [18
]. However, the reported Zn levels in other studies were considerably higher than our findings; Omu et al. (171 mg/l) [19
] and Chia et al. (183 mg/l) [7
]. These differences may be due to the slightly different techniques used in the measurement of Zn concentrations and the difference in cigarette-smoking habits. In comparison with the control group, our infertile patients showed decreased seminal Zn concentrations; however these differences were not significant. Inflammatory conditions, prostatitis, accumulation of toxic heavy metals and frequent ejaculation can negatively influence the secretory function of the prostate and lead to a drop in seminal Zn content [7
]. Nevertheless, these factors were excluded from our investigation and reduced Zn amounts of infertile patients can be explained by other mechanisms. Increased sperm ROS in infertile men explain the decrease of seminal Zn concentrations, arising the harmful effects of ROS to sperm cells which are associated with abnormal sperm parameters [8
]. Powell [21
] supports this hypothesis, noting that reduction in Zn concentration can lead to an increase in oxidation of membrane lipids, DNA and proteins; which can provoke the loss of membrane integrity. In our study, the negative and not significant correlation between Zn and MDA confirmed that reduction in seminal Zn levels is associated to the decline of its antioxidant capacity and the increase of lipid peroxidation and low sperm quality.
Additionally, seminal Zn concentrations demonstrated significant differences between fertile and infertile men, which corroborated with the findings of Colagar et al. [6
]. Furthermore, like Zaho et al. [22
] we observed positive relationships between good sperm production, motility and increased seminal Zn content. With these findings we can support the extensive evidence defending the antioxidant capacity of seminal Zn to yield various benefits in sperm including reduction of MDA and decreasing of DNA fragmentation [10
]. Therefore, Zn may be useful in idiopathic oligoasthenoteratozoospermia in reducing oxidative stress and the associated sperm membrane and DNA damage [10
Se is also an essential trace element that plays an important role in a number of physiological processes including human reproduction [22
]. This element becomes crucial for maintaining normal spermatogenesis and male fertility [23
]. In our study, we reported a slight increase of seminal Se in controls compared to infertile groups. This was in agreement with the observations of Saaranen et al. [24
] and Akinloye et al. [9
] who showed a significant decrease of seminal Se levels in asthenozoospermics and oligozoospermics. Takasaki et al. [25
] did not find any difference in seminal Se amounts between fertile and infertile men. Meanwhile, a follow-up study 4.5-5 years after the initial assay revealed that low Se levels (< 35 ng/ml) were associated with male infertility.
Our findings sustain the data supporting the negative influence of low seminal Se levels on the number of spermatozoa and sperm motility [9
]. This evidence can be confirmed by the correlation of the seminal Se and sperm motility observed in our investigation. The association of Se with a protein present in the tail of spermatozoa isolated from bulls and rats [26
] and its localization in the mitochondria capsule protein of the midpiece [27
] of spermatozoa are possible indications of its importance in sperm motility and male infertility.
Decreased Se concentrations in seminal plasma of infertile men were accompanied by increased levels of seminal MDA of the same patients. In this respect, the diminution of Se activity as an antioxidant can explain the elevated lipid peroxidation in infertile patients. In effect, the only biochemical role of Se in mammals depends on its presence as a seleno-cysteine residue at each of the four catalytic site of the enzyme "GPX" [9
]. GPX plays a crucial role in the antioxidant defences of the epididymis and the ejaculated spermatozoa [27
In addition to the impaired Zn and Se concentrations in seminal plasma of infertile men, we reported also decreased seminal GSHt, GSSG and GSHr levels in the three abnormal groups. Oshendorf et al. [28
] found a moderate reduction of GSH in oligozoospermic compared to normozoospermic men, while other studies found GSH levels below the limit of detection (< 2.5 μM) in seminal plasma of oligozoospermics, or GSH levels to be significantly reduced in seminal plasma of infertile males compared to those of fertile ones [1
]. Even GSH therapy was found to improve the semen quality [29
]. Our results provide evidence that higher levels of GSH in seminal plasma seem to play a role in protection against oxidative damage and to improve the sperm motility and morphology. While stating that, other studies could not observe any difference in GSH concentration between fertile and infertile men [8
] and this may be due to the contribution of spermatozoal ROS leading to the up-regulation of thiol synthesis in order to protect sperm from oxidative damage. Raijmakers et al. [30
] also reported that median levels of seminal GSH were significantly lower in infertile males as compared to normozoospermics, and found a positive association between seminal GSH level and sperm morphology and motility. In our investigation, we found that higher GSHt, GSSG and GSHr levels in seminal plasma were associated with a higher quality of sperm motility and count, however lower GSSG levels were associated with a higher degree of spermatozoa with abnormal morphology and immobility. These findings were compatible with that observed by Bhardwaj A et al. [12
] and Eskiocak S et al. [31
Accordingly; we can suggest that the loss of sperm motility in asthenozoospermic samples may result from the over-oxidation of sperm sulphydryl. GSH displays maximal staining in the mid-piece and tail region, which are important regions for motility of spermatozoa [31
]. These results imply that seminal plasma GSH levels may play a role in the protection against oxidative damage by reducing lipid peroxidation on sperm membrane. It could therefore be proposed that the concentration of GSH could be used as a chemical parameter to assess male fertility. The beneficial role of GSH to minimize oxidative damage to the spermatozoa can make it a suitable candidate for therapeutic usage in the treatment of male infertility.
Altered antioxidant status was observed in the seminal plasma, which was collected during our study from infertile men. This might cause oxidative damage and makes the sperm highly susceptible to lipid peroxidation. MDA production reflects the peroxidation of membrane polyunsaturated phospholipids [32
]. Our results established a significant increase in the amount of seminal MDA in abnormal groups compared to normozoospermics. Accordingly, lipid peroxidative degradation of sperm membrane integrity may be held responsible for abnormal sperm motility, concentration and morphology. Our results were corroborative with Hesham et al. [3
] and Ben Abdallah F et al. [34
] who reported that MDA content was elevated in oligozoospermic and asthenozoospermic groups. However, there was controversy about seminal MDA activity and sperm quality. We showed that higher seminal MDA levels were negatively associated with sperm motility and sperm count. Nevertheless, a positive relationship was observed between increased lipid peroxidation and the abnormal sperm morphology, which was compatible with the findings of Suleiman et al. [35
]. Increased MDA levels in seminal plasma of abnormal groups could represent the pathological effects lipid peroxidation has on the spermatozoa membrane and consequently on sperm motility and viability.