Introduction

Infertility is the inability to achieve pregnancy after 12 months of regular unprotected sexual intercourse, affecting approximately 10 to 15% of couples globally [1, 2]. Male infertility encompasses a complex pathophysiology with multivariate underlying factors, the most common being varicocele, cryptorchidism, hypogonadism, genital tract infection, endocrine abnormalities including hypothalamic, pituitary, thyroid, diabetes mellitus, adrenal gland, testicular cancer, environmental toxins, systemic disease, exogenous drugs, and genetic factors [3]. In most infertility and/or subfertility cases, the underlying causes remain elusive which may be referred to as idiopathic infertility [4].

Oxidative stress (OS) has been implicated in male infertility [5] and contributes 30 to 80% of male infertility cases worldwide [6, 7]. OS results when there is an imbalance of redox status producing high levels of oxidants and/or low levels of antioxidants resulting in cellular damage [8]. Reactive oxygen species (ROS) are highly reactive molecules produced as a byproduct of cellular metabolism and play important roles in cell signaling and homeostasis [5]. ROS are produced in small amounts in sperm cells to accomplish physiological functions such as regulation of sperm maturation, sperm capacitation and hyperactivation, and sperm-oocyte fusion [8,9,10]. Nevertheless, an imbalance of oxidative/antioxidant factors leads to OS, increased sperm DNA fragmentation, reduced sperm motility and damage to the sperm cell membrane, and reduced fertility [11, 12]. Spermatozoa are particularly susceptible to ROS-mediated damage due to the presence of structurally unstable polyunsaturated fatty acid in their plasma membrane [8, 13, 14].

Several studies have shown beneficial effects for antioxidant therapy in idiopathic male infertility [15, 16]. Coenzyme Q10 (CoQ10), non-enzymatic antioxidants [17], has been also reported to improve semen parameters in several studies [1, 18]. It is an isoprenylated benzoquinone that transports electrons from complexes I and II to complex III in the mitochondrial respiratory chain–regulating cytoplasmic redox potential-protecting cell membrane against lipid peroxidation-induced damage and regulating the mitochondrial permeability transition pores [19]. CoQ10 has two forms: a reduced form ubiquinol and an oxidized form ubiquinone [20]. Safarinejad and colleagues have reported an increase in sperm concentration and motility after CoQ10 therapy [21]. Observations by Nadjarzadeh et al. also support the above findings which reported a reduction in the sperm OS markers after CoQ10 therapy [20]. Other studies, however, reported no improvement in one or more of seminal fluid parameters following CoQ10 therapy [22, 23].

Selenium is an integral part of selenoproteins and cofactor of the thioredoxin reductase and the antioxidant enzyme glutathione peroxidase that regulate spermatogenesis, chromatin condensation and protection of sperm outer membrane and DNA against oxidative stress [2, 24]. In addition to antioxidant effect, selenium plays essential roles in inflammation, cell growth, cytotoxicity, and transformation, probably via activator protein 1 (AP1) [24]. Deficiencies of serum and seminal plasma selenium have been shown to positively correlate with abnormal semen parameters, due to depletion of glutathione peroxidase or inactivation of AP1 [24]. Several studies have proved the beneficial effect of selenium in improving sperm parameters and reducing OS [25,26,27]. In contrast, a clinical trial on 42 men demonstrated that administration of selenium (300 μg/day) for 48 weeks increased plasma and seminal fluid selenium concentrations but without improvement in any of semen parameters [28].

Antioxidant treatment including CoQ10 and selenium may improve seminal fluid parameters, live births, and clinical pregnancy rates [29, 30], but there is lack of agreement on the type of antioxidant, dose, and duration of treatment and whether to use monotherapy or combination of antioxidants [18]. Therefore, the present study aims to evaluate the effects of CoQ10 and selenium treatment on seminal fluid parameters and antioxidant status in men with idiopathic oligoasthenoteratospermia.

Methods

Patients

In this prospective randomized study, seventy patients (mean age 25.4 ± 7.71 years) with idiopathic oligoasthenoteratospermia were recruited at Fertility Clinic, Babyl, Iraq (EC/2018/8866) from June to October 2018 and enrolled in the study (four patients did not complete the study). All patients underwent medical assessment including history, physical examination and laboratory and radiological investigations. The study was conducted as a prospective study with 3 months of follow-up. The selected patients who fulfilled the selection criteria were randomly assigned (using simple randomization) to two treatment groups: Group 1 (n = 35) received 200 mg of oral CoQ10 (reduced form ubiquinol) (America Medic and Science AMS, WA, USA) single dose daily for 3 months. Group 2 (n = 35) received 200 μg of selenium orally (21st Century, AZ, USA) single dose daily for 3 months. The second group served as an active control. We have used doses used in previous studies [26, 31]. Semen analysis, seminal total antioxidant capacity (TAC), catalase (CAT), and superoxide dismutase (SOD) were measured and compared before and after therapy (follow-up period from September 2018 to January 2019). The sample size was calculated to have 80% power and 5% level of significance with (1:1) enrolment ratio and was estimated to be 30 for each group. The study protocol was approved by local ethical committee at the University of Sumer, Iraq (EC/2018/8866) and all participants consented for participation in the study.

Eligibility Criteria

Inclusion criteria comprised a history of infertility of at least 12 months despite regular unprotected intercourse. Oligoasthenoteratospermia was diagnosed according to the WHO guidelines (5th edition) [32] by semen analysis showing abnormal sperm concentration (< 15 million/ml), progressive motility (< 32%), and total motility (< 40%). Abnormal morphology (< 30% normal morphology) was assessed by the WHO guidelines (4th edition) [33]. Exclusion criteria comprised azoospermia, varicocele, genital tract infection, cryptorchidism, testicular trauma or scrotal surgery, endocrine disorders like hypothalamic, pituitary, thyroid, diabetes mellitus, adrenal gland and exogenous medications, systemic illness, recent antioxidants intake, smoking, alcohol, relevant medications, and the presence of female factors. All participants consented for participation in the study.

Semen Analysis

The semen samples were obtained by masturbation after sexual abstinence for 2 to 3 days, collected into a sterile wide-mouth plastic container, and held at 37 °C until liquefied and then analyzed within 1 h of collection. Semen analysis was performed according to the WHO guidelines (5th edition) [32] and morphology was assessed using the WHO guidelines (4th edition) [33] to determine volume, sperm concentration, motility, and morphology. All semen analysis was performed by the same investigator for the sake of data consistency and all patients underwent two semen analyses before and after therapy and average scores were used.

Seminal Total Antioxidant Capacity

Semen samples were centrifuged at 3000 rpm for 5 min and seminal plasma was aspirated and stored frozen for further biochemical analysis. TAC was measured in seminal plasma by a colorimetric method using the Total Antioxidant Capacity Assay Kit (#E-BC-K136, Elabscience, TX, USA). The test is based on the principle that antioxidants in the body can reduce Fe3+ to Fe2+ and Fe2+ can form stable complexes with phenanthroline substance. TAC was calculated by measuring the absorbance at 520 nm using a standard formula.

Seminal Superoxide Dismutase Activity

SOD activity was determined in seminal plasma by a colorimetric method as described by Magnani [34]. The principle of this method is based on the competition between the pyrogallol autoxidation by O2•− and the dismutation of this radical by SOD. The activity of SOD was calculated by measuring the absorbance at 420 nm using the standard formula.

Seminal Catalase Activity

Seminal catalase (CAT) activity was determined in seminal plasma by a colorimetric method using Catalase (CAT) Assay Kit (#E-BC-K031, Elabscience, TX, USA). The test is based on the principle that catalase (CAT) decomposes H2O2 and the reaction can be stopped by ammonium molybdate. The residual H2O2 reacts with ammonium molybdate to generate a yellowish complex. CAT activity was calculated by measuring the absorbance of the yellowish complex at 405 nm using a standard protocol.

Statistical Analysis

Statistical Package for Social Sciences (SPSS, v.24) was used for the statistical analysis. Results were expressed as mean ± SD. The Kolmogorov-Smirnov test was used to assess data normality. Paired t test was used to compare means before and after treatments. Pearson correlation coefficient (r) was used for correlations between seminal parameters and TAC, CAT, and SOD. p value of less than 0.05 was considered significant.

Results

Following selenium and CoQ10 therapy, there was a significant improvement in sperm concentration in the CoQ10 group (p < 0.01) was noticed but not in the selenium group (p > 0.05). An increase in progressive motility was observed in both the groups, but higher in the CoQ10-treated subjects than in the selenium group (p < 0.01 and p < 0.05, respectively) (Table 1). Total motility was also significantly increased with both the treatments, but higher with CoQ10 than selenium (p < 0.01 and p < 0.05, respectively). There were no significant changes in morphology observed in either of the groups (Table 1).

Table 1 Patients characteristics and seminal fluid parameters before and after coenzyme Q10 and selenium treatment
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There was a significant improvement in the TAC level, which was more evident in the COQ10 treated group (p < 0.01 and p < 0.05, respectively) (Table 2). Seminal SOD activity was higher in the COQ10 group (p < 0.01 and p < 0.05, respectively), but CAT activity was found to be increased only in the CoQ10 group (p < 0.01 and p > 0.05, respectively).

Table 2 Serum total antioxidant capacity, superoxide dismutase, and catalase activity before and after coenzyme Q10 and selenium treatment
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There was a significant positive correlation recorded between TAC and sperm concentration (r = 0.52, p = 0.008), motility (r = 0.76, p = 0.001), and morphology (r = 0.37, p = 0.04) following the treatments (Table 3). A significant positive correlation also was found between SOD and sperm concentration (r = 0.46, p = 0.022), motility (r = 0.54, p = 0.006), and morphology (r = 0.4, p = 0.034). Regarding CAT, it showed a significant positive correlation with sperm concentration (r = 0.41, p = 0.028) and motility (r = 0.48, p = 0.014), and a non-significant positive correlation with morphology (r = 0.34, p = 0.08). The correlations of antioxidants were more pronounced with sperm concentration and motility than with sperm morphology.

Table 3 Correlations between serum total antioxidant capacity, superoxide dismutase, and catalase activity and seminal fluid parameters in patients post-therapy
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Discussion

The present study has shown an improvement in sperm concentration, motility, and semen antioxidant capacity in infertile men with oligoasthenoteratospermia after 3 months of treatment with CoQ10 and selenium treatment. It is noteworthy that greater improvements in semen parameters have been seen with CoQ10 therapy. Similar to several other studies, this study also supports that seminal fluid parameters positively correlate with antioxidant measures. These results suggest a beneficial effect of CoQ10 and selenium therapy in men with idiopathic OAT.

The results of our study on the effects of CoQ10 on improving semen parameters are consistent with previous studies [35, 36]. In a randomized placebo-controlled double-blind clinical trial by Boscaro et al., an oral administration of CoQ10 in idiopathic asthenospermic patients for 6 months resulted in a statistically significant improvement in all kinetics semen parameters in the treatment group as well as increased levels of CoQ10 in seminal plasma [35]. In a randomized placebo-controlled study by Safarinejad et al., treatment with CoQ10 for 26 weeks in infertile men with idiopathic OAT significantly improved the sperm count and motility but did not improve sperm morphology [36]. Furthermore, another study by Safarinejad in infertile men with idiopathic oligoasthenospermia treated with CoQ10 for 12 months demonstrated a significant improvement in concentration, sperm progressive motility, and morphology [1]. Kobori et al. also reported an improvement of semen concentration and motility, but no change in spermatozoa morphology, after 6 months of vitamin C, vitamin E, and CoQ10 treatment [37]. Two recent systematic reviews on the effect of antioxidants in men with idiopathic oligoasthenospermia reported beneficial effects for CoQ10 and other antioxidants on seminal fluid parameters [8, 38]. In another study, however; treatment with CoQ10 100 mg/day for 6 months did not improve sperm concentration in men with idiopathic infertility [22]. Furthermore, a meta-analysis of three randomized controlled trials of CoQ10 in infertile men demonstrated improvement in semen parameters but a lack of positive effect on live birth or pregnancy rates [39].

Sperm motility (total and progressive) showed greater improvement than morphology, probably due to the antioxidant effect on the mitochondrial respiratory chain. The lipophilic antioxidant effect of CoQ10 has been recognized in plasma lipoproteins [31]. Moreover, endogenous CoQ10 is significantly related to sperm concentration and kinetics due to its bioenergetic role in mitochondrial function [40]. Studies have also confirmed that exogenous administration of CoQ10 is associated with higher levels in seminal plasma [41]. Therefore, the increase in semen parameters in our study could be contributed to the role of CoQ10 in mitochondrial bioenergetics and its well-established antioxidant potential.

Selenium therapy also improved seminal fluid parameters in our study and these results were congruent with previous studies. In an experimental study by Ghafarizadeh et al., semen from asthenoteratospermic men were incubated in vitro with selenium, resulting in an improvement of sperm motility, viability, mitochondrial membrane potential, lower levels of malondialdehyde, and reduced DNA fragmentation when compared with semen incubated without selenium [42]. In addition, carnitine and selenium levels in semen correlated with a better hypo-osmotic swelling test (HOS) as a measure of membrane integrity in low-grade varicocele patients [43]. In a longitudinal study on 125 men with infertility, a positive correlation between seminal plasma selenium levels and sperm count and motility were observed and low levels of semen selenium were associated with male infertility [27]. Similarly, a recent study explored the impact of selenium (50 μg/day) for 3 months showed an increment in sperm concentration, motility, and morphology [44]. Administration of selenium to rats with varicocelectomy also improved the activity of antioxidant enzymes and decreased MAD levels [25]. Another study, however, reported that selenium (300 mg/day) administration for 48 weeks was not associated with increased sperm selenium, concentration, or motility [28].

Selenium plays a crucial role in spermatogenesis via two selenoproteins, namely phospholipid hydroperoxide glutathione peroxidase (PHGPx) and selenoprotein P [26]. Sperm capsular selenoprotein is also an integral part of sperm glutathione peroxidase (GSH-Px) and GSH-Px activity increases with selenium supplementation [45]. Therefore, the observed improvement in seminal fluid parameters in our study could be attributed to increment in seminal plasma antioxidant capacity mediated by selenoproteins and GSH-Px activity. Our findings are also corroborated by studies that have reported lower selenium levels [46] and a correlation between seminal plasma selenium levels and sperm concentration and motility in infertile men [47]. A study on 100 infertile men who received Se (200 μg) in combination with vitamin E (400 units) for at least 100 days showed improvement in sperm motility and morphology but not in sperm concentration [26]. Furthermore, other studies have failed to demonstrate improvement in semen parameters following selenium therapy [28, 40]. Once the positive effects of CoQ10 and selenium are confirmed, it remains necessary to optimize the dose and duration of treatment and to identify which seminal parameters benefit the most from certain antioxidants.

The present study showed an increase in TAC, CAT, and SOD following CoQ10 and selenium therapy. Moreover, a positive correlation between semen parameters and these antioxidants was observed. Previous studies have reported lower TAC [48], CAT [49], and SOD [50] activities in infertile men. Our results are consistent with those of Nadjarzadeh et al. [51] who found a significant positive correlation between TAC and sperm total motility after CoQ10 therapy. Eroglu et al. [47] also found a significant positive correlation between sperm parameters and seminal TAC in men with idiopathic infertility who received CoQ10 and selenium treatment. According to Agarwal et al., ROS in semen correlates negatively with all sperm parameters [13] so an increase in TAC and other antioxidant capacity measures may enhance sperm function and fertility potential. In a randomized placebo-controlled study by Nadjarzadeh et al., there was a significant positive correlation between CoQ10 concentration and normal sperm morphology, catalase activity, and SOD after 3 months in infertile men with OAT following CoQ10 supplementation [20].

Reduction of seminal plasma antioxidant capacity is associated with defective structural and functional integrity of spermatozoa [52], sperm DNA damage, and poor reproductive outcomes [53] due to limited intrinsic antioxidant mechanisms of sperm. Hence, our study findings of increased TAC, CAT, and SOD following CoQ10 and selenium treatment explain the observed improvement in seminal fluid parameters in patients due to enhanced seminal plasma antioxidant defense. Our study has some limitations including pregnancy or live birth rates post-therapy that were not measured but these were not the primary outcome measures of the study. Dietary regulation was also not measured in this study. Another limitation is the small sample size and lack of long follow-up, so further large-scale long-term clinical studies are warranted.

Conclusion

In conclusion, treatment with CoQ10 (200 mg/day) or selenium (200 μg) could improve sperm concentration, motility, and antioxidant status in infertile men with idiopathic oligoasthenoteratospermia, while CoQ10 treatments showed higher improvements.