Document Type : Original Article
1 Medical Biotechnology Research Center, Ashkezar Branch, Islamic Azad University, Ashkezar, Yazd, Iran
2 Genetic Unit, Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
It is estimated that about 15% of couples globally
suffer from infertility. Male infertility constitutes
50% of causes among which genetic factors
are mainly responsible (
In this study, given that TNFα is an important regulator of steroidogenesis and may affect spermatogenesis, we investigated the association of the TNFα -308 G/A SNP with different kinds of sperm abnormality in infertile males of Iranian origin.
This case-control study included 180 infertile
males as the case group and 100 healthy normospermic
individuals as the control group. The
case individuals were recruited from Yazd Research
and Clinical Center for Infertility from September
2012 until August 2013. They were divided
based on sperm abnormality into azoospermia
(n=91, AZ group), oligospermia (n=26, OL group),
teratospermia (n=30, T group) and asthenoteratospermia
(n=33, AT group) groups. This study was
approved by the Ethics Committee of Shahid Sadoughi
University of Medical Sciences, Yazd, Iran.
Written informed consent was obtained from each
individual. All semen analysis and clinical examinations
were done according to the World Health
Organization guidelines (
Genomic DNA was extracted from whole blood samples using the salting out method. We used the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for genotyping of TNFα -308G/A. The F: 5'-AGGCAATAGGTTTTGAGGGCCAT-3' and R: 5'-TCCTCCCTGCTCCGATTCCG-3' primers were used to amplify a107 bp fragment of the TNFα promoter that included this SNP. PCR was carried out in a total volume of 25 μl containing 3-5 μl genomic DNA, 1 μl of each primer (10 μM) and 12.5 μl of PCR Master Mix (Cinnagen, Iran) and dH2O. The condition of DNA amplification was an initial denaturation step at 94°C for 5 minutes, followed by 35 cycles of 94°C for 40 seconds, 60°C for 1 minute and 72°C for 40 seconds, and a final extension step at 72°C for 5 minutes and hold at 4°C. Subsequently, the PCR products were digested with NcoI restriction enzyme (14 hours at 37°C) and the specific bands were identified using 2% agarose gel electrophoresis in 1X Tris/Borate/EDTA (TBE) buffer and visualized under the ultraviolet (UV) light. When digested, the PCR fragment was cleaved into two fragments with sizes 87 bp and 20 bp.
The frequency of alleles and genotypes were compared with a 2×2 contingency table using Chisquared and Fisher’s exact test. Fisher’s exact test was used when sample sizes were small in each category. We considered P<0.05 as a statistically significant and 95% confidence interval (CI) for calculating odds ratios (OR).We used the SPSS statistical software (version 20, SPSS Inc., Chicago, IL, USA) for all statistical analyses.
In this study, PCR-RFLP was able to identify both
alleles efficiently at position -308 in the promoter
The results of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of TNFα -308 polymorphism. Lane M shows the molecular weight marker, Lanes 1, 3 and 4 show the AA genotype. Lanes 2 and 5 show the GA genotype. Lane 6 shows the GG genotype.
Table 1 shows the related parameters of each group. The frequencies of alleles and genotypes and their association with the studied group are listed in Table 2. Ancestral genotype GG and allele G were taken as reference. Statistical analysis showed that there is a significant association between this SNP and the AZ, O and T patient groups but not with the AT group. The frequency of the AA genotype was 13% in the healthy normospermic (N) group, 27.4% in the AZ group [OR (95% CI)=2.535], 26.9% in the O group [OR (95% CI) =2.97], 30% in the T group [OR (95% CI)=2.86] and 15.2% in the AT group. The AG genotype was identified in 22% of the N group, 32.9% of the AZ group [OR (95% CI)=1.74], 34.6% of the O group [OR (95% CI)=1.87], 43.3% of the T group [OR (95% CI)=2.71] and 24.2% of the AT group.
The sperm parameters in the case group
|Group||Sperm parameter||Mean ± SD|
|Azoospermia||Sperm count (106/mL)||0|
|Oligospermia||Sperm count (106/mL)||6.6 ± 2.3|
|Asthenoteratospermia||Motility (grades a+b)%/Morphology (% normal forms)||10.7 ± 5.8/4.7 ± 2.8|
|Teratospermia||Morphology (% normal forms)||6.9 ± 3.4|
The frequencies of alleles and genotypes of the -308 G/A SNP in the TNFα promoter in the azoospermic, teratospermic, Asthenoteratospermic and Oligospermic groups
|Genotype-allele/group||Normospermic (%) n=100||Azoospermic (%) n=91||P; OR (95% CI)||Oligospermic (%) n=26||P; OR (95% CI)|
|AA||13 (13%)||25 (27.4%)||0.018; 2.535 (1.2-5.3)*||7 (26.9%)||0.040; 2.97 (1.076-8.22)*|
|AG||22 (22%)||30 (32.9%)||0.010; 1.74 (0.916-3.20)*||9 (34.6%)||0.206; 1.87 (0.736-4.787)|
|GG*||65 (65%)||36 (39.5%)||Ref.||10 (38.5%)||Ref.|
|AA+AG||35 (35%)||55 (60%)||0.001; 2.837 (1.57-5.107)*||16 (61%)||0.024; 2.971 (1.22-7.240)*|
|A||48 (24%)||80 (43.9%)||<0.001; 2.484 (1.604-3.845)*||23 (44.2%)||0.005; 2.51 (1.32-4.7)*|
|G**||152 (76%)||102 (56%)||Ref.||29 (55.8%)||Ref.|
|AA||13 (13%)||9 (30%)||0.049; 2.86 (1.083-7.599)*||5 (15.2%)||0.772; 1.195 (0.392-3.648)|
|AG||22 (22%)||13 (43.3%)||0.033; 2.71 (1.143-6.428)*||8 (24.2%)||0.812; 1.135 (0.449-2.864)|
|GG*||65 (65%)||8 (26.7%)||Ref.||20 (60.6%)||Ref.|
|AA+AG||35 (35%)||22 (73%)||<0.001, 5.107 (2.061-12.657)||13 (39%)||0.679; 1.207 (0.537-2.714)|
|A||48 (24%)||31 (51.7%)||<0.001; 3.385 (1.85-6.17)*||18 (27.3%)||0.623; 1.18 (0.632-2.23)|
|G**||152 (76%)||29 (48.3%)||Ref.||48 (72.7%)||Ref.|
TNFα; Tumor necrosis factor alpha, SNP; Single nucleotide polymorphism, OR; Odds ratios, CI; Confidence interval, *; Significant P<0.05. Ancestral genotypes GG* and alleles G** were taken as reference.
Genetic variation such as SNPs in TNFα promoter region may affect its expression. There are several studies that have investigated the association of
In this study, we analyzed the TNFα -308 G/A SNP to identify its possible association with sperm abnormality in Iranian males. To the best of our knowledge, this study has not been undertaken in Iranian males. Our findings indicate that this SNP is significantly associated with azoospermia, oligospermia and teratospermia. In other words, this SNP is among many genetic factors that may lead to a decreased count of sperm and abnormal morphology in our cases.
Similar to our study, Tronchon et al. (
Based on the results of previous studies, TNFα is known to affect spermatogenesis by changing the structure of the blood-testis barrier and apical ectoplasmic specialization of Sertoli cells, which may lead to abnormal spermatogenesis (
Our study shows that there is a positive association between TNFα -308 G/A SNP and different sperm abnormalities in the Iranian population. Given that the A allele leads to increased expression of TNFα, anti-TNFα agents could be a useful treatment for male infertility.