Association of nitric oxide synthase 3 gene rs1799983 G/T polymorphism with idiopathic asthenozoospermia in Iranian Azeri males: a case-control study

Introduction

Infertility is a prevalent disorder, which affects approximately 10–15% of couples in the world. Men factors are responsible for approximately half of the infertility cases [1], [2]. Sperm dysfunction in effects of genetic variants or environmental toxins are important causes of men infertility [3], [4]. Male spermatogenesis disorders such as idiopathic asthenozoospermia (AZS) is found in approximately 18% of infertile male [5]. Idiopathic AZS, reduced sperm motility, is one of the important cause of male infertility, which most of its causes are still unknown [6], [7].

Recently, oxidative stress (OS) is a main cause of sperm dysfunction, impaired sperm motility, and AZS, which associated with men infertility [8]. Nitrogen monoxide (NO) is a reactive free radical gas with various functions, which act as a messenger in several biologic processes [9], [10]. Previously demonstrated that NO is synthesized by human male gamete and involved in reproductive and sexual functions in human [11]. Although, high concentrations NO lead to dysfunction, whereas low concentrations of NO play an important role in sperm physiology [12].

Nitric oxide synthase (NOS) enzymes can produce NO through catalyzing 5-electron oxidation of a guanidino nitrogen of L-Arg [13]. In human three genes encode NOS enzymes include cytokine induced (iNOS or NOS2), endothelial (eNOS or NOS3), and neuronal (nNOS or NOS1) [14]. The endothelial NOS (NOS3) play an important role in cellular signaling pathways in a calcium/calmodulin dependent manner, and activated by agonists that elevate intracellular free Ca²⁺ [15]. Previous studies reported that NOS3 is a cytoplasmic protein in Sertoli and Leydig cells, spermatogenesis process, and epithelium of vas deferens and epididymis, as well as involved in sperm motility, capacitation, and acrosome reaction [16], [17].

A SNP on NOS3 gene within exon 7 is a G>T mutation at 894 nucleotide position, which causes to substitution glutamic acid by aspartic acid at 298 codon position (rs1799983 Glu298Asp) protein translation. So far, association of this SNP and AZS was not investigated in Iranian Azeri infertile men. Therefore, we investigated association of NOS3 gene rs1799983 G/T polymorphism in infertile Azeri Iranian male with idiopathic AZS.

Materials and methods

DNA extraction and genotyping

The sperm samples (3 mL) were drawn from all subjects, and genomic DNA extraction from sperm samples was performed using a kit (FAVORGEN, Taiwan) according to manufacturer’s instructions. The quantity and quality of extracted DNA were investigated using nanodrop instrument and electrophoresis on 1% agarose gel. The genotype determination was performed by tetra-primer amplification refractory mutation system-polymerase chain reaction (Tetra-ARMS PCR) method. A 25 μL total volume was used as following: PCR buffer (2.5 μL), 1 μg template DNA, dNTP (0.1 mmol), 0.5 μL each primer (25 pmol), Mgcl2 (1.5 mmol/L), and Taq DNA polymerase (1.5 unit). PCR condition was as following: 1 cycle initial denaturation (94 °C for 4 min), 40 cycles denaturation (94 °C for 40 s), annealing (50 °C for 30 s), and extension (72 °C for 25 s), and 1 cycle final extension (72 °C for 5 min). The amplified PCR products were electrophoresed on 1% agarose gel with 50 bp size marker. A gel documentation instrument was used to visualize the bands of PCR products. Product sizes were 305 bp for the G allele and 188 bp for the T allele (Figure 1).

Figure 1:

The PCR products of NOS3 gene rs1799983 G/T polymorphism electrophoresis on 1% agarose gel.

Results

The obtained results showed that there was a significant difference between AZS patients and healthy controls in terms of semen parameters, family history, and alcohol drinking; whereas there was no significant difference in term of age, BMI, and tobacco smoking (Table 1).

According to the χ2 tests, the rs1799983 G/T polymorphism was in HWE in AZS patients and healthy controls (p>0.05). We observed that the genotypes frequencies in AZS patients were as following: homozygous GG (20%), heterozygous GT (30%), and homozygous TT (0%). Also, genotypes frequencies in healthy controls were as following: homozygous GG (30%), heterozygous GT (15%), and homozygous TT (5%). The statistical analyze showed that the frequency of GT genotype in AZS patients was significantly more than healthy controls (p=0.002; OR=1.627; 95% CI=0.532–1.812). However, we did not find any significant difference in dominant (p=0.217; OR=0.436; 95% CI=0.211–1.665), recessive (p=0.675; OR=1.198; 95% CI=0.697–2.312), and overdominant (p=0.097; OR=1.554; 95% CI=0.767–2.122) inheritance models (Table 2).

The frequency of G allele in patients and healthy controls were 70 and 75%, respectively. The frequency of T allele in patients and healthy controls were 30 and 25%, respectively. The statistical analysis of alleles frequencies showed no significant differences between AZS patients and healthy controls (p>0.05).

Discussion

Infertility described as inability of a couple to pregnancy after one year unprotected sexual intercourse, which male infertility factors is present in approximately 50% of infertile couples [18]. Evidence suggested that infertility has substantial genetic basis with high prevalence of single gene mutations and chromosome aberrations [19]. Despite great advances in genetic and clinical diagnostics, infertility cause in many patients remains unidentified, which classifying as idiopathic infertility [20]. Thus, this study was performed to investigate the associations between NOS3 gene rs1799983 G/T polymorphism and AZS in infertile Iranian male with idiopathic asthenozoospermia. Previous studies reported that high seminal NO levels effects on sperm motility in men with asthenozoospermia [21], [22]. Evidence suggested that high concentrations of NO in semen of patients with AZS presents a negative effect on spermatogenesis, and thus a negative effect on sperm motility [10].

Many studies evaluated association of NOS3 gene SNPs and male infertility with contradictory results. Moreover, many studies evaluated association of NOS3 gene rs1799983 G/T polymorphism and AZS in various populations. Some studies reported no association between NOS3 gene rs1799983 G/T polymorphism and AZS [23], [24]. On the other hands, several studies demonstrated a significant association between this polymorphism and AZS [25], [26]. In a study by Yun et al. reported that NOS3 gene rs1799983 G>T polymorphism was associated with infertile Korean population with sperm morphology disorders [23]. In another study by Vučić et al. reported that the NOS3 gene rs1799983 G/T polymorphism was not associated with AZS in infertile male in a Serbian population [24]. On the other hands, Safarinejad et al. reported that NOS3 gene rs1799983 G/T polymorphism were significantly associated with semen parameters in Iranian infertile male [25]. Moreover, Yan et al. showed that NOS3 gene rs1799983 G/T polymorphism associated with male infertility in a Chinese population [26]. According to results of this study which was performed on 50 AZS patients and 50 healthy controls, there was a significant association between NOS3 gene rs1799983 G/T polymorphism and AZS in Iranian infertile male. The reasons for the difference of reported results among the above studies could be due to several other related genes and environmental factors, and difference in studied samples size, ethnicity, race, and geographical area [27], [28], [29].

In NOS3 gene rs1799983 G/T polymorphism glutamic acid is substituted with aspartic acid in codon 298 region. Evidence showed that that this mutation effects on protein-protein interactions and localization of NOS3 protein, which affect on protein function [30].

Conclusion

Generally, our study demonstrated a more understanding of AZS as a multifactorial disease and suggests that NOS3 gene rs1799983 G/T polymorphism is associated with the AZS in Iranian Azeri infertile male. However, the exact role and effects of this polymorphism in AZS are not fully identified. Therefore, for a better understanding of the association of this polymorphism with AZS, further studies are recommended on other populations and races with larger sample sizes.