Background

Statistics show that the prevalence of infertility in the world is about 10 to 15 %, of which approximately 45% is due to male factors [1]. More than 25% of infertile men show abnormal semen profiles with an unknown cause called idiopathic infertility [2]. However, oxidative stress has been introduced as one of the main reasons for idiopathic infertility [3] and it has been reported that the levels of reactive oxygen species (ROS) were elevated among 25% of infertile patients [4, 5].

Spermatozoa and white blood cells are the sources of ROS production in the semen [4, 5]. However, there is a balance between ROS production and elimination under physiological conditions and therefore its level is strictly controlled in sperm and seminal plasma. In oxidative stress, the balance is lost and a large amount of ROS accumulates in semen [6]. Oxidative stress can negatively affect sperm motility, acrosome reaction, and oocyte fusion and subsequently cause infertility. To prevent oxidative stress, there are enzymatic and non-enzymatic antioxidant defense systems in semen such as superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase, vitamins E and A, ascorbic acid, and glutathione [7].

The GPX, as one of the most important antioxidant enzymes, converts hydrogen peroxide (H2O2) into H2O and O2 [8]. Studies demonstrated that GPX 1, 3, 4, and 5 isoforms are present in the human reproductive system. Recent studies have indicated that GPX activity is associated with sperm DNA integrity as well as motility and count [9, 10]. The GPX3 isoform, also known as extracellular GPX, has been found in human seminal fluid. More interestingly, Giannattasio et al. [11] showed that GPX3 activity was ten-time higher in the seminal fluid of fertile than in infertile men.

Polymorphism of the genes which are involved in antioxidant defense or spermatogenesis could induce oxidative stress and male infertility [12, 13]. One of the factors that may affect the expression and activity of GPX3 in semen is the functional polymorphisms of its gene [14]. One of the most important polymorphisms of GPX3 which is located in the promoter region is rs8177404 (T to C). Studies showed that this polymorphism could reduce the expression of GPX3 and increase the risk of metabolic syndrome [15]. Another functional polymorphism in the promoter region of the GPX3 gene is rs3828599 (A to G). Studies indicated that the G allele could increase the expression of the GPX3 gene [14] and therefore the GG genotype could act as a protective factor against cancers [16].

Given the role of GPX3 in protecting sperm against oxidative stress in seminal fluid, and also the existence of functional polymorphisms in the GPX3 gene which may be involved in male fertility, in this study, we investigated the possible association between rs3828599 and rs8177404 polymorphisms with the risk of male infertility. Moreover, we evaluated the relationship between these polymorphisms and GPX3 levels in seminal fluid and semen parameters in fertile and infertile men.

Methods

Study population and sample collection

In this study, a total of 200 men (100 idiopathic infertile and 100 fertile men) were recruited. The infertile men were selected from the patients who were referred to the infertility department of Al-Zahra Hospital or the Milad Infertility Clinic of Tabriz. Infertile men had no children after 1 year (or more) of unprotected sexual intercourse and had an abnormal semen profile according to World Health Organization (WHO) [2010] guidelines. Moreover, the cause of their infertility was unknown and patients with known infertility causes such as varicocele were excluded. Furthermore, men with severe oligozoospermia (<5 million/ml sperm) and azoospermia were not enrolled in this study. Fertile men were recruited from age-matched individuals who had children in the last 3 years without benefit from assisted reproductive technologies (ART). The persons who were taking medication, smoking cigarettes, or had diabetes, liver, kidney, and thyroid diseases were excluded from the study.

After 3 days of sexual abstinence, the semen samples were collected from all participants. After 40-min liquefaction, the semen parameters were analyzed according to the WHO criteria [WHO 2010] under a light microscope by a well-trained technician. Then, the samples were divided into sperm (pellet) and seminal fluid (supernatant) parts using centrifugation. The supernatant was kept at −20°C for measurement of GPX3 levels and the sperm were stored at −80°C for DNA isolation and gene analysis.

Enzyme-linked immunosorbent assay (ELISA)

The levels of GPX3 (ng/ml) were measured by a commercial human ELISA kit (Cat. Num. MBS765842 MyBioSource, San Diego, CA) as instructed by the company. This sandwich ELISA kit had a detection range of 1.56–100 ng/ml with a sensitivity of <0.938 ng/ml. The enzyme-substrate reaction was terminated by the provided stop solution and then the color change was spectrophotometrically measured at a wavelength of 450 nm. Finally, the enzyme levels in each sample were determined by comparing the O.D. of the samples to the standard curve.

Genotyping

The genomic DNA was isolated from the sperm using a DNA extraction kit (BIORON, Germany). To analyze the quality and integrity of the extracted DNA sample, a 1.5% agarose gel electrophoresis was used. Moreover, the ratio of A260/A280 absorbance was obtained using NanoDrop 1000 (NanoDrop, Wilmington, USA) to evaluate the DNA purity. Tetra-primer ARMS-PCR was carried out to investigate the genotypes of rs8177404 and rs3828599 polymorphisms. For this purpose, we used the PCR master mix (Ampliqon, Denmark) and specific primers (listed in Table 1). After performing the PCR, the PCR product was run on 1.5% agarose gel and the size of products was evaluated after visualization under ultraviolet (UV) light. In the rs8177404 polymorphism, the existence of two bands with a length of 540bp and 370bp was assumed as the TT genotype, while the existence of bands with the length of 540bp and 223bp was considered as the CC genotype. Furthermore, the presence of three fragments with the length of 540bp, 370bp, and 223bp was referred to the TC genotype (Fig. 1a). As shown in Fig. 1b, the presence of two bands with the length of 494bp and 235bp, and length of 494bp and 316bp and three fragments (495bp, 316bp, and 235bp) were referred as the AA, GG, and AG genotypes of rs3828599 polymorphism, respectively.

Table 1 The list of primers used for in tetra-primer ARMS-PCR method to detect the genotypes of GPX3 rs8177404 and rs3828599 polymorphisms
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Fig. 1
figure 1

The PCR products of rs8177404 and rs3828599 GPX3 gene polymorphisms on 1.5% agarose gel using tetra-primer ARMS-PCR. a The presence of two bands with a length of 540bp and 370bp, and length of 540bp and 223bp and three fragments (540bp, 370bp, and 223bp) were referred as the TT, CC, and TC genotypes of rs8177404 polymorphism, respectively. b The presence of two bands with a length of 494bp and 235bp, and length of 494bp and 316bp and three fragments (495bp, 316bp, and 235bp) were referred as the AA, GG, and AG genotypes of rs3828599 polymorphism, respectively. L, DNA ladder

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Statistical analysis

The results were expressed as mean ± standard deviation. The normal distribution of data was confirmed by the one-sample Kolmogorov-Smirnov test. The semen parameters and GPX3 levels were compared between the infertile and fertile groups and among the genotypes by t-test and One-way ANOVA analysis, respectively. Tukey post hoc test was also applied as a follow-up test to evaluate the quantitative data between two genotypes. The genotype distribution was checked for Hardy-Weinberg equilibrium. The frequencies of allele and genotype of the polymorphisms were compared between the groups using the chi-square test. To the relative risk of the genotypes/alleles on male infertility, logistic regression analysis was used. The p<0.05 level was considered as the statistical significance and the data were analyzed using SPSS V.16 software.

Results

There was no significant difference in age (24.25±6.19 vs. 33.69±5.24 years, p=0.491) and body mass index (BMI) (26.37±3.03 vs. 25.50±3.76 kg/m2, p=0.077) between fertile and infertile men. We found that the concentration of GPX3 in the seminal fluid of fertile men was significantly higher than in infertile patients (2.62±0.89 vs. 1.8±0.80 ng/ml, respectively; p= <0.01). The sperm parameters of fertile and infertile men are summarized in Table 2. The range of sperm concentration in fertile and infertile groups was 45–80 and 8–65 million/ml, respectively. We found that there were significant differences in concentration, motility, and morphology of sperm between the groups (p<0.05).

Table 2 Semen profile of infertile and fertile males
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As demonstrated in Table 3, there was a moderate positive correlation between GPX3 concentration in seminal fluid and the percentage of progressively motile sperm in both groups (p<0.05). Moreover, we observed a moderate positive correlation between GPX concentration and the number of sperm with normal morphology in the fertile group (r=0.428 and p=0.047).

Table 3 Correlation between semen profile and levels of glutathione peroxidase 3 (GPX3) in the seminal fluid of fertile (n=100) and infertile (n=100) men
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Our results indicated that the frequency of rs8177404 and rs3828599 genotypes and alleles was significantly different between the fertile and infertile groups as shown in Tables 4 and 5, respectively. In this regard, we found a higher frequency of TT genotype (or T allele) of rs8177404 polymorphism in fertile men compared to infertile individuals (p=0.034). In rs3828599 polymorphism, infertile men carried a higher frequency of the wild-type genotype (AA) when compared with fertile persons (p=0.025). Logistic regression analysis showed that having the rs8177404 polymorphism (TC and CC genotypes) could increase the risk of idiopathic infertility more than 2-fold (Table 4). On the other hand, the genotype GG (rs3828599) showed a protective effect against infertility that can decrease the risk of male infertility about 3 (1/0.338) times (Table 5).

Table 4 The frequency of GPX3 rs8177404 (T to C) genotypes, alleles, and odds ratios in fertile (n = 100) and infertile (n = 100) males using χ2 test and regression logistics analysis
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Table 5 The frequency of GPX3 rs3828599 (A to G) genotypes, alleles, and odds ratios in fertile (n = 100) and infertile (n = 100) males using χ2 test and regression logistics analysis
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Our results demonstrated that the fertile men carrying CC genotype had significantly lower progressively motile sperm and higher immotile sperm compared with subjects carrying TT and TC genotypes (Table 6). Moreover, men with the CC genotype had statistically lower sperm with normal morphology in comparison with TT carriers. In the infertile group, the individuals with TT genotype had higher levels of progressively motile sperm and sperm concentration compared to the CC carriers. The rs3828599 polymorphism did not show a significant association with sperm parameters. However, GG carriers had significantly higher progressively motile sperm than AA carriers in the fertile group. In the infertile group, individuals with the GG genotype had lower immotile sperm than AA genotype carriers.

Table 6 Semen parameters according to the various genotypes of rs8177404 and rs3828599 polymorphisms in fertile (n=100) and infertile group (n=100)
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As shown in Table 7, the fertile men with genotypes of CC (rs8177404) and GG (rs3828599) had significantly lower and higher levels of GPX3 in the seminal fluid, respectively. In the infertile group, the CC genotype was also associated with lower GPX3 levels.

Table 7 Seminal fluid levels of glutathione peroxidase 3 (ng/ml) according to the various genotypes of rs8177404 and rs3828599 polymorphisms in fertile (n=100) and infertile group (n=100)
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Discussion

Given the role of oxidative stress in abnormal sperm function [6, 7] and the importance of the local antioxidant system particularly GPX3 in the protection of male fertility [9, 10], in the current study, we evaluated the association between rs3828599 and rs8177404 polymorphisms of GPX3 gene with its levels in seminal fluid and subsequently with the risk of male infertility.

Giannattasio et al. [11] evaluated the GPX3 enzyme in seminal fluid and showed that its activity was ten-times higher in fertile men compared to the infertile patients. In accordance, we also found a higher concentration of GPX3 in the seminal fluid of fertile men than of infertile patients. Although, Giannattasio et al. [11] did not measure the levels of the enzyme, given the current study findings, part of the elevated activity in fertile men might be due to higher levels of the GPX3 enzyme in the seminal fluid of fertile individuals than the infertile group. In supporting the role of seminal fluid GPX3 in sperm function, we observed positive correlations between GPX3 concentration with sperm motility and morphology. Spermatozoa is very susceptible to the oxidative stress due to abundant polyunsaturated fatty acids in the membrane and also being transcriptionally inactive (reviewed in Ref. [3]). Therefore, low levels of GPX3 in the seminal plasma could result in excessive hydrogen peroxide and consequently oxidative stress. More interestingly, it has been shown that hydrogen peroxide is the major ROS responsible for impairing the motility of spermatozoa [17]. Crisol et al. [18] also reported lower activity of GPX (the total activity of isoforms) in samples with severe asthenozoospermia, oligozoospermia, and teratozoospermia compared with normal samples. Moreover, it has been indicated that the intracellular levels of GSH were lower when sperm morphology was severely impaired [19]. In contrast, Macanovic and colleagues [20] reported negative correlations between GPX activity with sperm morphology and motility. Such a contradiction could be due to, firstly, assessment of total GPX instead of isoform 3 alone, which is the most important isoform in the seminal fluid; secondly, the low sample size of Macanovic’s study; and thirdly, recruiting patients who were normozoospermic but applied for reproductive technology procedures for fertility treatment.

To investigate one of the possible reasons underlying low levels of GPX3 in the seminal fluid of infertile patients, we analyzed the frequency of rs8177404 and rs3828599 polymorphisms which are located at the promoter region of the GPX3 gene and can potentially affect the gene expression and consequently GPX3 levels [14, 15]. Our results represented that the frequency of the TT genotype (rs8177404) was higher in fertile men than infertile patients and having the C allele (both TC and CC genotypes) could increase the risk of male infertility. As shown in the current study, the CC genotype was negatively associated with sperm motility and morphology. On the other hand, the association between male infertility with abnormal morphology and motility of sperm has been well-documented [21,22,23]. A previous study showed that T to C substitution at promotor of GPX3 gene in rs8177404 polymorphism could reduce the gene expression [15] that can result in low levels of GPX3 enzyme; we also found lower levels of GPX3 in the seminal fluid of individuals with CC genotype compared to other genotypes. Therefore, it can be hypothesized that rs8177404 polymorphism can negatively affect sperm motility and morphology by reducing the levels of GPX3 and consequently increasing ROS in the seminal fluid. Huang et al. [24] also reported an association between this oxidative stress-related genetic variant and arterial ischemic stroke.

In rs3828599 polymorphism, the frequency of wild genotype (AA) was higher among infertile than fertile groups and the GG genotype demonstrated a protective effect against male infertility. Moreover, we found a positive association between the GG genotype and sperm motility. Previous studies showed that the GG genotype could increase the expression of the GPX3 gene and act as a protective factor [14, 16]. In parallel with previous findings, we also found higher levels of GPX3 in the seminal fluid of men carrying the GG genotype compared to other genotypes (AA and GA). Therefore, the GG genotype could increase the gene expression and levels of GPX3 enzyme in the seminal fluid and consequently protect sperm from oxidative stress.

For the first time, this study evaluated GPX3 gene polymorphisms in fertile and infertile men and their association with levels of GPX3 enzyme and sperm parameters. However, several limitations should be noted with this study, including relatively low sample size and lack of data regarding ROS status in the semen or sperm. Therefore, further studies are required to investigate the association between GPX3 polymorphisms and oxidative stress-related damages of sperm.

Conclusions

In conclusion, our results showed that GPX3 levels were significantly lower in the seminal fluid of infertile men compared to fertile individuals and its levels were significantly associated with sperm parameters, particularly motility and morphology. Moreover, we found positive and negative associations between rs8177404 and rs3828599 GPX3 gene polymorphisms and male infertility. It can be noted that these polymorphisms can affect GPX3 levels in seminal fluid and subsequently sperm parameters.