Impact of oxidative stress on semen parameters in normozoospermic infertile men: a case–control study

Oxidative stress has been implicated in male infertility through decrease in sperm quality. However, men with normal semen parameters (normozoospermia) may be unable to fertilize their partners even when they have normal sperm function. Thus, they would be considered infertile. The purpose of this study was to investigate the role of oxidative stress in the pathogenesis of unexplained male infertility. In this case–control study, infertile men with normozoospermia (n = 46) and fertile control group (n = 21) underwent seminal fluid analyses according to WHO 2010 criteria. Serum and seminal plasma levels of total antioxidant capacity (TAC), glutathione, malondialdehyde, uric acid and albumin were also measured using colorimetric methods. The level of total antioxidant capacity in both serum and seminal plasma was significantly lower in normozoospermic infertile men in comparison with fertile group (p < 0.0001). However, no significant differences were observed in serum and seminal plasma levels of glutathione, uric acid, albumin and malondialdehyde between infertile and fertile groups. Low TAC level induces oxidative stress and consequently causes sperm dysfunction and male infertility. Estimation of TAC can be a useful tool in the diagnosis of male infertility. Antioxidant supplementation should be considered in the treatment of oxidative stress-induced male infertility.


Background
Medical concern in infertility has increased recently due to the decrease in the reproduction capability of couples worldwide [1]. Male reproductive defects contribute to nearly 50% of all infertility issues. Male infertility has been attributed to deteriorated sperm number or function [2]. Sperm dysfunction has been linked to varicocele, cryptorchidism, hypogonadism, testicular cancer, urogenital tract infections, systemic diseases and genetic factors [3]. However, in nearly 25% of male infertility cases, men have demonstrated normal sperm parameters but still unable to father a child, which is termed unexplained infertility [4].
Oxidative stress (OS) has been involved in unexplained male infertility, and contemporary parameters have introduced OS as markers in the identification of potential causes of male infertility. OS is a state of imbalance between production and removal of reactive oxygen species (ROS) [5]. Antioxidants are compounds capable of scavenging and inhibiting the formation of ROS [6]. ROS have critical rule in male reproductive function, and these active molecules are required in small quantities for normal sperm function [7], while high quantities could decrease semen quality and impair sperm Palani and Alahmar Afr J Urol (2020) 26:50 fertilization capability [8]. Hence, antioxidants are critical to inactivate ROS continuously to maintain only the small amount required to normal sperm function [9].
Recently, there is growing evidence suggesting that oxidative stress is involved in many aspects of male infertility and antioxidants counteract the adverse effects of OS on male fertility potential [10]. Antioxidants in seminal fluid can be classified into two forms: enzymatic and nonenzymatic antioxidants. Low seminal plasma antioxidants have been found to be implicated in reducing sperm concentration, motility and morphology [11]. Total antioxidant capacity (TAC) has shown to be strongly correlated with semen quality [12]. Similarly, glutathione has a role in ROS detoxification and protection of sperm plasma membrane [13]. Uric acid protects sperm viability and enhances sperm motility through stabilizing of ascorbic acid and scavenging of ROS [14,15]. Albumin protects the spermatozoa against lipid peroxidation and prevents loss of sperm motility [16]. Malondialdehyde (MDA) is an index of lipid peroxidation; it is negatively correlated with sperm count, motility and morphology [17].
The aim of this study is to investigate the role oxidative stress in the pathogenesis of unexplained male infertility through estimation of semen and serum levels of TAC, glutathione, MDA, uric acid and albumin in normozoospermic infertile men.

Methods
This study was approved by ethical committee of university of Garmian (EC-G/191). Semen and serum samples were obtained from normozoospermic men after they were asked to fill consent form, during the period (March to November, 2017). A total of 64 subjects were included in this study, 42 infertile men with mean age (32.5 ± 6.3) years, and 22 fertile men with mean age (33.9 ± 7.2) years. Exclusion criteria for selection of subjects were: (1) smoking, (2) alcohol consumption, (3) abnormal BMI, (4) taking antioxidant medications, (5) etiologies related to physiological abnormality, such as varicocele, obstruction or small testicular volume and (6) having partner with low reproductive potential. Infertile men were attended to fertility clinics at Kalar city for infertility evaluation. Fertile men who had at least 1 child were enrolled in the study. Based on basic semen analysis results of infertile men, only participants with normal seminal fluid analysis were included in this study.
Semen collection and analysis Semen samples were obtained from patients and control group either by masturbation in the clinic or at home (delivered within 30 min) after 3-4 days of sexual abstinence. Basic semen analyses were performed after liquefaction following the WHO 2010 manual. Samples from different laboratories were reanalyzed at our laboratory and performed by the same investigator after exactly 60 min for the sake of data consistency.
Blood collection Along with semen sample collection, subjects were asked for venous blood sample. Blood samples were collected by venipuncture into plastic vacutainer tube, and then, sera were separated and transferred to different tubes and stored in deep freeze (− 45 °C) for further analyses of oxidative stress markers.
Estimation of total antioxidant capacity TAC was estimated using a colorimetric kit (Cell Biolabs, San Diego, CA, USA). The assay was based on the reduction of copper (II) to copper (I) by antioxidants and formation of a brown color with maximum absorbance at 490 nm. The absorbance of the samples was compared with a known uric acid standard curve. Absorbance values were proportional to the samples total antioxidant capacity.
Estimation of glutathione Glutathione was estimated using a method by Moron et al. The solution was incubated with 0.1 ml (10 mM DTNB) and 10 µl of 10% TCA at room temperature for 5 min. The optical density of the yellow color complex was measured at 412 nm. The concentration was calculated by plotting on glutathione standard curve [18].
Estimation of uric acid Uric acid was estimated using a colorimetric kit (Biolabo, Maizy, France). 20 µl of seminal plasma was added to 1.0 ml of uric acid reagent and incubated at 37° for 5 min. The developed color was read at 520 nm. Concentrations were calculated and expressed as mmol/ml.

Estimation of albumin
Albumin was estimated using a colorimetric kit (Biolabo, Maizy, France) at pH (4.3). Albumin binds to the BCG to form a BCG-albumin complex. The resulted green complex absorbed light at 630 nm, and it was directly proportional to the concentration of albumin in the sample.
Estimation of malondialdehyde (MDA) MDA was measured by the reaction with thiobarbituric acid (TBA). Sample was added to 0.9 ml D.W and 0.5 ml of TBA reagent. The mixture was heated for 1 h in a boiling water bath. The developed pink color was read at 532 nm. The MDA level was expressed as (nmol/mL) [19].

Statistical analysis
MedCalc for Windows, version 14.8.1 (MedCalc Software, Ostend, Belgium) was used for statistical analysis.
Semen parameters were expressed as Mean ± SD. Parameters were tested for normality using Kolmogorov-Smirnov test. Significant difference between infertile and fertile groups was calculated using Mann-Whitney independent sample test. Differences at p value ≤ 0.05 were considered as significant.

Results
Semen basic parameters of infertile and fertile men are presented in Table 1. No significant differences were observed in semen volume and sperm concentration, total count, motility, progressive motility, non-progressive motility and morphology.
TAC was significantly (p < 0.0001) lower in seminal plasma of infertile men, while no significant differences in glutathione, uric acid, albumin and MDA levels were observed (Fig. 1). In the comparison of oxidative stress parameters in blood serum, serum TAC level in normozoospermic infertile men was significantly (p < 0.01) lower than of fertile men, and no significant differences in glutathione, uric acid, albumin and MDA levels were found (Fig. 2).

Discussion
Normal semen parameters do not guarantee fertility; many factors may be related to male infertility rather than sperm count, motility and morphology which are not included in basic semen analysis [20]. However, new biochemical parameters have been studied widely over the last decades including oxidative stress to help in identifying the underlying cause of unexplained male infertility [21,22].
Recently, growing evidences have indicated the role of TAC in male infertility. Decreasing seminal plasma antioxidant status may have a significant role in the etiology of sperm abnormality [23]. In our study, TAC was lower in the seminal plasma of infertile men (Fig. 1); this finding agrees with previously reported results [24,25]. The negative correlation between ROS and antioxidants has been confirmed; low seminal antioxidant potential allows ROS overproduction in infertile men [26]. The exposure to ROS is extremely destructive to sperm plasma membrane [12]. Sperm-oocyte fusion is a membrane-dependent processes; oxidation of sperm membrane impairs the penetration ability of the spermatozoa and causes infertility in normozoospermic men [27].
Along with seminal antioxidants assessment, serum TAC determination has been recently proposed as a valuable marker to evaluate the sperm quality [28]. However, serum oxidative stress marker in relation to male infertility is less investigated and poorly understood [29]. Serum levels of TAC were significantly different (p < 0.01) between infertile and fertile men (Fig. 2). This finding is in agreement with the results of Benedetti et al. [29], and with our previous report [30], both were reported a decrease in serum TAC in infertile compared to fertile men. In contrast, our results disagree with Eroglu et al. [31]; they reported no significant difference in serum TAC between normozoospermic infertile and fertile men. Glutathione showed to be influencing sperm motility by way of prevention of lipid peroxidation of sperm cell membrane [32]. However, no significant difference in semen and serum glutathione level was found between the different groups. These results agree with previous reports by Guz et al. [33] and Feng et al. [34]; they reported no significant differences in semen and serum uric acid concentration between fertile and infertile men [33,34]. However, in a disagreement, Bhardwaj et al. [35] reported a significant decrease in semen glutathione level in infertile men, and Thiele et al. [14] reported significant decrease in uric acid concentration in normozoospermic infertile men comparing to fertile men. Few studies have investigated the possible relationship between seminal plasma albumin and sperm quality; a weak positive correlation between albumin and sperm concentration was reported [36]. In the present study, no significant difference in albumin concentration was found. MDA has related to glutathione concentration, and both have shown to influence sperm quality [37]. MDA level has been reported to negatively correlate with sperm concentration and motility [38]; MDA levels do not significantly differ between the studied infertile and fertile groups. However, it was previously reported that MDA has negatively correlated with sperm quality and significantly higher in seminal plasma of infertile men [39,40]. One explanation for the discrepancy of the findings of the aforementioned studies is the heterogeneity of studies in selection of patients, methodologies, methods used to assess OS as well as genetic and racial factors. Our

Conclusion
Our study demonstrates that total antioxidant capacity (TAC) affects male fecundity. TAC level induces oxidative stress and consequently declines sperm fertilization capability even in men with normal basic sperm parameters. Estimation of TAC can be used as an investigative