1. Introduction
Despite advances in male reproductive health, idiopathic male infertility remains a challenging condition in diagnosis and treatment. Increasing data suggest that oxidative stress plays a major role in the pathophysiology of male infertility, with 30% to 80% of infertile men having elevated levels of free radicals in sperm [
1]. Therefore, a comprehensive assessment of male reproductive capacity should include an assessment of sperm oxidative stress. Human semen contains several antioxidant systems to remove oxygen free radicals and prevent oxidative tissue damage. They are classified as enzymatic and non-enzymatic antioxidants [
2]. The formation of the enzymatic antioxidant system, the elements of which vary between organisms, is a crucial breakthrough in spermatogenesis to ensure the protection of sperm against oxidative stress [
3]. Under normal conditions, reactive oxygen species (ROS) are neutralized by enzymes such as peroxide dismutase, catalase, glutathione peroxidase, and glutathione transferase [
4].
Glutathione S-transferases (GSTs) comprise a superfamily of ubiquitously expressed multifunctional enzymes that play a pivotal role in protecting cells against oxidative stress. Among them, the
GSTM1 gene, encoding the glutathione S-transferase Mu-1, is mutated and is devoid of any specific enzymatic activity (
GSTM1-null genotype). Interestingly, half of the Caucasian population carry the null genotype. Therefore,
GSTM1 gene deletion might be correlated with an increased susceptibility to diseases associated with oxidative stress. There are also studies reporting that
GSTM1 might be a critical isozyme in the detoxification of oxidative stress products [
2,
5,
6].
An association has been demonstrated between
GSTM1 polymorphism, markers of oxidative stress, and damage in spermatozoa and seminal plasma in subjects with idiopathic male infertility. Infertile individuals with the
GSTM1-null genotype are more susceptible to oxidative stress than
GSTM1-positive infertile males. Furthermore, the
GSTM1-null genotype has been associated with higher ROS, protein carbonyl, and malondialdehyde (MDA) levels [
2].
The aim of the present study is to investigate the effect of GSTM1 polymorphism and static redox potential (sORP) to the basic seminal parameters of fertile and infertile men. Furthermore, our study’s aim is to examine whether testing for GSTM1 polymorphism could be a potential biomarker in male infertility investigation.
2. Materials and Methods
2.1. Sample Collection
The case–control study was conducted at “Alexandra” University Hospital in collaboration with the sperm cryopreservation bank “Cryogonia” and a written informed consent was obtained from all the involved patients. The study included 90 Caucasian males divided into two groups, namely the study group and the control group. The study group consisted of semen samples from 51 men identified as infertile according to the WHO 2010 guidelines (study group). Control group consisted of 39 fertile men with normal seminal parameters according to the WHO 2010 guidelines, and at least one successful pregnancy with their partner without assisted reproductive technologies. Exclusion criteria for both groups include evidence of any other fertility-related disease, such as prostate cancer, cryptorchidism, varicocele, diabetes, seminal infections, or karyotype abnormalities. Moreover, individuals with obesity (body mass index greater than 30 kg/m2), systematic alcohol consumption, or active nicotine abuse were also excluded. Each subject donated 1 mL (patient cohort) or 0.5 mL (donor cohort) of ejaculated semen obtained by masturbation after a minimum of 4 days of abstinence.
2.2. Semen Analysis
Each sample was subjected to conventional semen analysis according to the recommendations, semen evaluation protocols, and standards of the World Health Organization (WHO). More specifically, seminal volume, sperm concentration, motility, and morphology were analyzed 30 min after liquefaction.
2.3. DNA Extraction and Detection of GSTM1 Polymorphism
DNA was isolated from all sperm samples using a commercial PureLink™ Genomic DNA Mini Kit (Thermo Fisher Scientific, Waltham, MA, USA). DNA quantification was performed via spectrophotometer, and DNA integrity was verified by agarose electrophoresis. Polymerase Chain Reaction was performed to detect
GSTM1 polymorphism. A Taq DNA polymerase kit (New England Biolabs, Ipswich, MA USA) was used, and the primers were the following: GSTM1F 5′-GAACTCCCTGAAAAGCTAAAGC-3′ and GSTM1R 5′-GTTGGGCTCAAATATACGGTGG-3′ [
7]. The conditions of the PCR were as follows: 94 °C for 10 min, 94 °C for 1 min, 58 °C for 1 min, 72 °C for 1 min. This was repeated for 35 cycles, with a final elongation step at 72 °C for 10 min. Subsequently, the PCR products were electrophoresed on 2% agarose gel and visualized under UV light. A single band at 219 bp corresponded to the presence of the
GSTM1.
2.4. Evaluation of Oxidative Stress Using the MiOXSYSTM System
The MiOXSYSTM system (GryNumber Health Group, Vilnius, Lithuania) provides an evaluation of the static redox potential (sORP), which represents an integrated measure of the balance between total levels of oxidants and antioxidants in a biological system or sample, such as human semen. The MiOXSYSTM system requires a small semen volume (30 μL) and produces results in less than 5 minutes. The measurement of sORP offers the andrology and embryology laboratory an efficient tool for the measurement of oxidative stress. The assay provides the possibility to directly assess the oxidative potential in semen samples in contrast to oxidative stress markers such as ROS, TAC or MDA. Its advantages compared to other methods of oxidative stress evaluation include short duration, technical simplicity, as well as small sample volume required for the measurement.
2.5. Statistical Analysis
The χ2-test was performed to compare GSTM1-null genotype frequencies between groups. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to evaluate the association between variant genotype and group. T-test for two independent samples (or Mann–Whitney U, if this was deemed necessary) was performed to compare variant genotype and conventional semen parameters by group. Pearson’s correlation coefficient was used to determine the effect of sORP in semen parameters. The data were analyzed using the Statistical Package for Social Sciences software version 20.0 (SPSS Inc., Chicago, IL, USA), and p < 0.05 was considered statistically significant.
4. Discussion
Genetic variants in
GST genes may lead to detoxification system imbalance, thereby increasing susceptibility to oxidative stress damage and increased risk for male infertility [
8,
9]. In particular, several epidemiological studies have shown that the
GSTM1-null genotype, which results in total enzyme deficiency, is associated with increased susceptibility to oxidative-stress-related diseases. The possible relationship of
GSTM1 gene deficiency with male infertility has been studied extensively, but the results of the studies vary between different populations [
10].
The present case–control study focused on the potential impact of the GSTM1 polymorphism and redox potential on the risk for idiopathic male infertility. Our results demonstrated that the GSTM1-null genotype was present at a higher frequency in infertile men than in the fertile control group, with a 2.22-fold increase for the risk of male infertility. Thus, an increased risk of the GSTM1 polymorphism for developing male factor infertility is supported.
Our results are in agreement with several studies that reveal
GSTM1-null genotype as a potential risk factor for male idiopathic infertility by affecting semen quality [
11,
12,
13,
14,
15,
16]. Numerous population studies have suggested a negative effect of the
GSTM1-null genotype on male infertility, with patients carrying the
GSTM1-null genotype having a lower sperm concentration and sperm count. Fertile males with the
GSTM1-null genotype had a lower sperm concentration but normal sperm count [
17,
18]. More importantly, a similar relative risk for male factor infertility was observed in patients with the
GSTM1-null genotype. The same study showed that the combination of deletion genotypes of GST genes pose an even higher risk for infertility [
19].
Impaired semen quality is reported in infertile men regarding sperm concentration, count, motility, and morphology compared to fertile ones. In our study, fertile individuals carrying the
GSTM1-null genotype were found to have a lower percentage of typical spermatozoa regarding morphology and lower slow progressive motility, highlighting the negative effect of the presence of
GSTM1 polymorphism in conventional semen parameters in fertile males. Notably, Aydemir et al. showed that lower sperm concentrations and higher levels of oxidative stress and damage markers are presented in infertile males with the
GSTM1-null genotype compared to those with the
GSTM1-positive genotype. However, no significant difference in genotype distribution for the
GSTM1 variant between idiopathic infertile subjects and fertile ones was observed [
2]. Another study also demonstrated lower sperm concentration and motility in infertile men carrying the
GSTM1-null genotype compared to fertile ones with the
GSTM1-positive genotype. Similarly, the frequency of the
GSTM1-null genotype was significantly higher in infertile individuals than in fertile ones. Their findings are consistent with our research results [
20].
Moreover, an increased risk for male infertility of
GSTM1-null genotype was revealed in a previous meta-analysis. Interestingly, an even higher risk was reported upon a subgroup analysis of Caucasians [
21]. Similarly, a subsequent meta-analysis concluded that the
GSTM1-null polymorphism contributes to a significant increased risk for male infertility. Therein, significant associations were also observed in subgroups of Caucasian populations but not in Asian ones [
22].
On the contrary, in a meta-analysis conducted by Economopoulos et al., the
GSTM1-null genotype was not statistically associated with male infertility, underscoring the need for the accumulation of data regarding variants of GST genes [
23]. However, in a recent study, researchers proposed the
GSTM1-null genotype as a potential genetic risk factor for male infertility, interfering with certain oxidative stress markers (i.e., total antioxidant capacity and nitric oxide) in infertile men [
24].
With regard to oxidative stress variations, our results demonstrated an excess of oxidation-reduction potential in infertile men compared to fertile ones. Notably, poor semen quality, including low sperm concentration and count, higher percentage of slow progressive motility and immature forms, was reported upon redox potential elevation. Surprisingly, increases in sOPR had a negative impact on the semen motility characteristics of fertile males.
The measure of sORP is considered to be a better indicator of semen quality, providing reliable results for oxidative stress. Agarwal et al. standardized the sORP test in semen using the MiOXSYS System and reported that higher sORP levels were associated with poor sperm parameters, deteriorating the fertility status of subjects. Negative correlations emerged for conventional semen parameters, including concentration, total sperm count, motility, and morphology, indicating that oxidative stress impairs these parameters. The authors proposed testing sORP as an objective and accurate method, which in conjunction to routine semen analysis can reliably differentiate fertile from infertile men [
25,
26]. However, certain limitations should be taken into consideration regarding the difficulty to assess highly viscous semen using the MiOXSYS system, or conventional semen parameters, such as sperm morphology.