Background IL-1α produced by Sertoli cells is considered to act as a growth factor for spermatogonia. In this study, we investigated the association of the C376A polymorphism in IL-1α with male infertility in men referring to the Kashan IVF Center. 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. Results 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. Conclusion This is the preliminary report on the association of IL-1α C376A polymorphism with male infertility in the Kashan population. This association shows that the IL-1α gene may be a biomarker for male infertility, and therefore needs additional investigations in future studies to validate this.
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 (IL-1) gene family which
encodes regulatory cytokines playing multifaceted roles
in the male reproductive system. For example, they may
act as growth factors and are involved in physiological
protection, germ cell proliferation and differentiation,
regulation of junctions and steroidogenesis (10, 11).
The IL-1 gene family members include IL-1α(OMIM:
147760),IL-1ß(OMIM: 147720) and IL-1RA(OMIM:
147679), all of which are located on chromosome 2q14
(12). IL-1α is secreted from seminiferous epithelium and
is known as a growth factor for immature Sertoli cells and
spermatogonia (13).
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 IL-1 gene family could
be considered as an interesting research topic. A SNP
(C376A; rs2071376) has been found to have a high frequency
in the IL-1α gene. The association of this SNP with
some disorders has been investigated in different studies
including cancers (17, 18), systemic sclerosis (19), periodontitis
(20), endometriosis (21) and keratoconus (22).
The association between the C376A SNP and idiopathic
male infertility has, however, not been reported. In this
study, we investigated the association between the IL-1α
C376A SNP and idiopathic male infertility in an Iranian
population as a preliminary project. Also, we evaluated
the functional effects of C376A on IL-1α using bioinformatics
tools.
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 IL-1α
C376A SNP was genotyped by the polymerase chain reaction-
restriction fragment length polymorphism (PCR-
RFLP) method. For this purpose, forward and reverse
primers flanking the SNP were designed based on the
complete sequence of IL-1α by the Oligo7 software (Molecular
Biology Insights, Inc., Cascade, CO, USA).
The sequences of the primers were:
5´-ATGCTAAAATTACCGTGATTCT-3´
5´-AGATCAATGGAATAAATGGATG-3´ respectively.
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 BstYI restriction enzyme (CinnaGen,
Iran). For this purpose, approximately 0.1 µg of the
PCR product was incubated with 5 units of BstYI at 37°C
for 16 hours. Finally, BstYI was inactivated by incubation
at 65°C for 20 minutes. The digested fragments were
separated on a 1% agarose gel stained with DNA Green
Viewer (CinnaGen, Iran) and visualised under the UV
light. To verify PCR-RFLP results, 2% of samples were
sequenced randomly. PCR product recovery kit (Roche
Applied Science, Mannheim, Germany) was used to purify
the PCR product (368 bp in length). Direct sequencing
of the purified PCR products was undertaken by Bioneer
(Daejeon, Korea). Chromas (version 2.33) was used to
check the chromatograms.
Statistical analysis
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
of the IL-1α C376A intronic SNP on RNA structure and
splicing pattern. The effect on RNA structure and splicing
was assessed with RNAsnp online server (24) and NetGene2
(25) respectively. Finally, reported interactions of
IL-1α with other molecules were obtained from the BioGRID
interactome database (26).
Results
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).
Polymerase chain reaction-restriction fragment length polymorphism
(PCR-RFLP) and DNA sequencing results. A. The M, 1, 2 and 3 lanes
show the 100 bp DNA ladder, and the AA, AC and CC genotypes, respectively
and B. Partial sequence of IL-1. flanking the single nucleotide polymorphism
(SNP) (red box).
IL-1α C376A distribution
In this study, the genotype and allele frequencies of the
IL-1α C376A SNP were compared between the infertile
and healthy groups (Table 1). We observed a significant association
between the homozygous genotype CC with male
infertility (OR=1.97, 95% CI=1.14-3.41, P=0.016). Carriers
of C (AC+CC) were at 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 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, there was a significant association
between carriers of C and 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).
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 IL-1α splice site pattern on the
direct strand (+ strand) especially for the acceptor splice
pattern (Fig .2). The BioGRID interactome showed that
IL-1α has 17 gene-gene interactions (Fig .3).
Results of NetGene2 and RNAsnp. A. Splice sites prediction by NetGene2
when nucleotide A is present at the C376A position, A’. Splice sites
pattern after the C substitution at the C376A position. Some changes were
observed after the substitution especially in the acceptor site (the differences
between the splice patterns are shown by the blue box), and B.
The presumptions of variant and ancestral sequences are introduced in
lower and upper triangle of the plots respectively. The single nucleotide
polymorphism (SNP) is highlighted by yellow color.
Allelic and genotypic distribution of the IL-1α C376A SNP
Genotype/Allele
n (%)
OR (95% CI)
P value
Controln=230
All casesn=230
Oligon=95
Astenon=84
NOAn=51
Total
Oligo
Asteno
Azo
Total
Oligo
Asteno
Azo
AA
44(19.13)
27(11.74)
14(14.74)
8(9.52)
5(9.80)
-
-
-
-
-
-
-
-
AC
90(39.13)
87(37.83)
30(31.58)
39(46.43)
18(35.29)
1.58(0.90-2.74)
1.05(0.51-2.17)
2.38(1.03-5.53)
1.76(0.61-5.05)
0.113
0.901
0.043
0.293
CC
96(41.74)
116(50.43)
51(53.68)
37(44.05)
28(54.90)
1.97(1.14-3.41)
1.67(0.84-3.33)
2.12(0.91-4.93)
2.57(0.93-7.09)
0.016
0.146
0.081
0.069
AC+CC
186(80.87)
203(88.26)
81(85.26)
76(90.48)
46(90.20)
1.78(1.06-2.99)
1.37(0.71-2.64)
2.25(1.01-4.10)
2.18(0.82-5.80)
0.030
0.348
0.047
0.127
A
178(38.70)
141(30.65)
58(30.53)
55(32.74)
28(27.45)
-
-
-
-
-
-
-
-
C
282(61.30)
319(69.35)
132(69.47)
113(67.26)
74(72.55)
1.43(1.09-1.88)
1.44(1.01-2.06)
1.30(0.89-1.88)
1.67(1.04-2.68)
0.011
0.049
0.172
0.034
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.
Network of human IL-1α interactions based on BioGRID. IL-1α interacts
with 17 other molecules. Purple and yellow lines show interactions
detected by genetic and physical experiments respectively.
Discussion
In this study, we examined the association of the IL-1α
C376A SNP with male infertility in an Iranian population
(Kashan, Iran) as a pilot study. Our study revealed that not
only the CC genotype was associated with male infertility,
but also the C allele showed significant association. In
addition, carriers of the C allele were at almost two-fold
risk for male infertility. Sub-group analysis revealed that
AC genotype and carriers of C were associated with asthenozoospermia.
Also, the C allele was significantly associated
with oligozoospermia and non-obstructive azoospermia.
Therefore, IL-1α C376A is a potential genetic
risk factor for male infertility, although further studies
of different ethnicities in Iran and other populations are
required to obtain a more accurate picture. After Hardy-
Weinberg equilibrium (HWE) calculation in the control
group, we found a highly significant deviation. However,
the case group showed no deviation. even though it does
not necessarily need to follow HWE due to the inherent
sampling bias in cases. The deviation from HWE in the
control group (normozoospermic men) could also be due
to the selection bias (27) given that not all men in the general
population will be fertile.
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 IL-1α SNP
with some abnormalities in sperm parameter may be explained.
SNPs could change the gene expression pattern
(14), mRNA structure (36, 37), splicing pattern (38) and
protein function (39, 40). In silico tools, which can predict
the damaging effects of SNPs, were therefore used
especially that IL-1α C376A is an intronic SNP and may
affect RNA structure and splicing. Although we found no
evidence for C376A to affect RNA structure, we observed
a predicted effect on splicing alteration. Therefore, the association
of this SNP may be due to this effect. In this
study, there were various limitations including gene-environment
and gene-gene interactions that must be considered in subsequent studies. Also, lack of in vitro studies
such as investigating the effect of the SNP on IL-1α gene
expression and isoform formations due to splicing alterations
is another limitation of this study.
Conclusion
Our study suggests that the IL-1α C376A SNP may increase
the risk of male infertility up to two-fold. Since this is
the first study, future studies with larger sample sizes in different
ethnicities and populations is warranted given the variable
environmental factors in different geographic regions.
Zamani-Badi, T., Karimian, M., Azami Tameh, A., & Nikzad, H. (2018). IL-1ɑ C376A Transversion Variant and Risk of Idiopathic Male Infertility in Iranian Men: A Genetic Association Study. International Journal of Fertility and Sterility, 12(3), 229-234. doi: 10.22074/ijfs.2018.5375
MLA
Tayyebeh Zamani-Badi; Mohammad Karimian; Abolfazl Azami Tameh; Hossein Nikzad. "IL-1ɑ C376A Transversion Variant and Risk of Idiopathic Male Infertility in Iranian Men: A Genetic Association Study". International Journal of Fertility and Sterility, 12, 3, 2018, 229-234. doi: 10.22074/ijfs.2018.5375
HARVARD
Zamani-Badi, T., Karimian, M., Azami Tameh, A., Nikzad, H. (2018). 'IL-1ɑ C376A Transversion Variant and Risk of Idiopathic Male Infertility in Iranian Men: A Genetic Association Study', International Journal of Fertility and Sterility, 12(3), pp. 229-234. doi: 10.22074/ijfs.2018.5375
VANCOUVER
Zamani-Badi, T., Karimian, M., Azami Tameh, A., Nikzad, H. IL-1ɑ C376A Transversion Variant and Risk of Idiopathic Male Infertility in Iranian Men: A Genetic Association Study. International Journal of Fertility and Sterility, 2018; 12(3): 229-234. doi: 10.22074/ijfs.2018.5375
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