IL-1ɑ C376A Transversion Variant and Risk of Idiopathic
Male Infertility in Iranian Men: A Genetic Association Study
Materials and Methods
In this case-control study, 2 ml of blood was collected from 230 fertile and 230 infertile men. After DNA extraction, the C376A variant was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). In addition, the molecular effects of the C376A transversion were analysed using bioinformatics tools.
A significant association was observed between the homozygous genotype CC with male infertility [odds ratio (OR)=1.97, 95% confidence interval (CI)=1.14-3.41, P=0.016)]. Carriers of C (AC+CC) showed a similar risk for male infertility (OR=1.78, 95% CI=1.06-2.99, P=0.030). Also, allelic analysis showed that the C allele is associated with male infertility (OR=1.43, 95% CI=1.09-1.88, P=0.011). In sub-group analysis, we found that the AC genotype is associated with asthenozoospermia (OR=2.38, 95% CI=1.03-5.53, P=0.043). In addition, carriers of C were at high risk for asthenozoospermia (OR=2.25, 95% CI=1.01-4.10, P=0.047). Also, C allele was significantly associated with oligozoospermia (OR=1.44, 95% CI=1.01-2.06, P=0.049) and non-obstructive azoospermia (OR=1.67, 95% CI =1.04-2.68, P=0.034). Finally, in silico analysis showed that the C376A polymorphism could alter splicing especially in the acceptor site.
This is the preliminary report on the association of
Male infertility is a multifactorial syndrome that affects up to 12% of men (1). Male factors are responsible for 40-50% of total infertility cases (2). In more than 70% of cases, there is a conclusive reason including varicocele, aneuploidies, infectious diseases and post-testicular obstruction, however, in less than 30% of infertile males, the cause of their infertility is unknown and are thus diagnosed as idiopathic (3, 4).
Environmental, lifestyle, physiological and genetic factors are involved in male infertility (5-7). From numerous genetic factors that are essential for normal spermatogenesis, cytokines play an important role (8). These are regulatory peptides which regulate testicular and glandular function (9).
Human seminal plasma contains several cytokines in.
cluding IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-11,
IL-12, IL-13, IL-17, IL-18, IL-23, TNFa, IFN-., TGFa,
TGFß (8). One of the most important gene sets involved
in fertility is the interleukin-1 (
Single nucleotide polymorphisms (SNPs), by altering
the structure of genes involved in spermatogenesis, may
affect gene expression, mRNA structure and protein function,
and may therefore lead to male infertility (14-16).
Therefore, evaluating SNPs in the
Materials and Methods
Subjects and inclusion criteria
In this cross-sectional study, a total of 460 samples comprising 230 infertile men (with mean age of 30.93 ± 5.47) and 230 fertile men (with mean age of 32.12 ± 5.52) selected among individuals attending the Kashan Infertility Centre (Shahid Beheshti Hospital, Kashan, Iran). Infertile patients were defined as ‘idiopathic’ and selected based on andrological examination. Patients with previous testis trauma, obstruction of the vas deferens, infectious and chronic diseases, hypogonadotropic hypogonadism, abnormal hormonal profile (Luteinizing, Follicle Stimulating, and testosterone hormones) and abnormal karyotype or Y chromosome microdeletions were excluded from the study. According to the World Health Organization (WHO) 1999 criteria, the patient sub-groups were determined (23) and the subjects were categorized into non-obstructive azoospermia (n=51) without spermatozoa in the ejaculated semen, oligozoospermia (n=95) with sperm concentration less than 20 million/ml, and asthenozoospermia (n=84) with progressive sperm motility less than 50%.
The control group was randomly selected from healthly men referred to the Kashan Infertility Centre. They had normal sperm parameters, had no history of chronic and familial diseases and had at least one offspring. Finally, a total of 2 ml of whole blood was collected from all males into EDTA-K3 containing tubes and were stored in -20°C for further usage. Written informed consent was obtained from all case and control subjects. The study was approved by the Medical Research Ethics Committee of the Kashan University of Medical Sciences (IR.KAUMS.REC.1394.6).
Single nucleotide polymorphism genotyping
Total genomic DNA was isolated from whole blood
by using a DNA extraction kit (Bioneer, Korea). Purified
DNA was stored at -20°C for further use. The
The sequences of the primers were:
The PCR was carried out in a total volume of 20 µl containing
10µl pre-mix (CinnaGen, Iran), 0.35 µM of each
forward and reverse primers, and 3 µl of template DNA.
PCR cycling conditions were an initial denaturation step
at 94°C for 5 minutes followed by 35 cycles of denaturation
at 94°C for 45 seconds, annealing at 56.9°C for 1
minute and extension at 72°C for 1 minute along with a
final extension at 72°C for 5 minutes. PCR products were
then digested with the
The difference in frequencies of genotypes and alleles between the case and control groups was analyzed by Chi-square test. For association analysis, the odds ratios (ORs) and 95% confidence intervals (95% CI) were estimated by a binary regression logistic test. A two-tailed p- value less than 0.05 (P<0.05) was considered significant. All analyses were conducted in the SPSS software (SSPS Inc., IBM Corp, Armonk, NY, USA) version 19.
In silico analysis
Bioinformatics tools were used to analyze the influence
Polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing
Results of PCR-RFLP showed that 368 bp fragment was fully digested into 114 bp and 254 bp fragments in some samples, showing the efficiency of the method used. The samples with two, three and one fragments were identified as CC, AC, and AA genotypes respectively (Fig .1A,). The data from direct sequencing also confirmed the results of PCR-RFLP (Fig .1B,).
IL-1α C376A distribution
In this study, the genotype and allele frequencies of the
In silico analysis
Functional consequence of the C376A transversion on
RNA structure was evaluated. However, no significant
effect on RN (distance: 0.0191, P=0.686) was observed
(Fig .2,). Minimum free energy of normal RNA was equal
to -81.80 kcal/mol but increased to -80.50 kcal/mol for
the variant allele. The data from NetGene2 revealed that
the C370A SNP alters the
|Genotype/Allele||n (%)||OR (95% CI)||P value|
SNP; Single nucleotide polymorphism, OR; Odds ratio, Oligo; Oligozoospermia, Asteno; Asthenozoospermia, and NOA; Non-obstructive azoospermia. Significant differences between the case and control groups are shown in bold type.
In this study, we examined the association of the
Spermatogenesis is a dynamic process in which many factors are necessary for creating and regulating balance in this process. For example, growth factors and cytokines are essential for development of functional spermatozoa (28, 29). Interleukin-1 is produced by epithelia of seminiferous tubules and acts as a physiological paracrine/ autocrine factor on testicular cells and required for immunological protection (30). There is a probable mechanism that in the absence of testosterone, followed by increased cell apoptosis, spermatogenesis is finally reduced (31, 32). The second probable mechanism is excess reactive oxygen species (ROS).
The presence of the associated SNP and the consequent
change in the amount of interleukin along with excess
production of ROS may reduce sperm motility. One of
the reasons for reduced sperm motility may be DNA damage
and lipid peroxidation of sperm membrane (33). Also,
increased ROS with oxidizing DNA or proteins, enzyme
inhibition, cell death and apoptosis of sperm may cause
the oligozoospermia phenotype (34, 35). Due to these
possible mechanisms, the association of the
Our study suggests that the
We thank all the volunteers for providing blood samples. This work was supported by a grant from the Kashan University of Medical Sciences (Grant No. 94058). The authors have no conflict of interest.
M.K., H.N.; Planned and supervised the study upon which the current subset project was based. M.K., T.Z.-B.; Developed the outline for the current study and supervised the analysis of the samples. T.Z.-B.; Write the manuscript and M.K., H.N. revised the paper. M.K., H.N., T.Z.-B., A.A.T.; Contributed to data analysis and prepared the manuscript. All authors reviewed and approved the final manuscript.