{"paper_id":"94ee608e-3bd0-471d-a546-115b3564706d","body_text":"Male infertility is a multifactorial syndrome that affects\nup to 12% of men ( 1 ). Male factors are responsible for\n40-50% of total infertility cases ( 2 ). In more than 70% of\ncases, there is a conclusive reason including varicocele,\naneuploidies, infectious diseases and post-testicular obstruction,\nhowever, in less than 30% of infertile males,\nthe cause of their infertility is unknown and are thus diagnosed\nas idiopathic ( 3 ,  4 ).\nEnvironmental, lifestyle, physiological and genetic factors\nare involved in male infertility ( 5 - 7 ). From numerous\ngenetic factors that are essential for normal spermatogenesis,\ncytokines play an important role ( 8 ). These are regulatory\npeptides which regulate testicular and glandular\nfunction ( 9 ).\nHuman seminal plasma contains several cytokines in.\ncluding IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-11, \nIL-12, IL-13, IL-17, IL-18, IL-23, TNFa, IFN-., TGFa, \nTGFß ( 8 ). One of the most important gene sets involved \nin fertility is the interleukin-1 ( IL-1 ) gene family which \nencodes regulatory cytokines playing multifaceted roles \nin the male reproductive system. For example, they may \nact as growth factors and are involved in physiological \nprotection, germ cell proliferation and differentiation, \nregulation of junctions and steroidogenesis ( 10 ,  11 ).\nThe  IL-1  gene family members include  IL-1α (OMIM: \n147760),IL-1ß(OMIM: 147720) and IL-1RA(OMIM: \n147679), all of which are located on chromosome 2q14 \n( 12 ).  IL-1α  is secreted from seminiferous epithelium and \nis known as a growth factor for immature Sertoli cells and \nspermatogonia ( 13 ).\nSingle nucleotide polymorphisms (SNPs), by altering \nthe structure of genes involved in spermatogenesis, may \naffect gene expression, mRNA structure and protein function, \nand may therefore lead to male infertility ( 14 - 16 ).\nTherefore, evaluating SNPs in the  IL-1  gene family could \nbe considered as an interesting research topic. A SNP \n(C376A; rs2071376) has been found to have a high frequency \nin the  IL-1α  gene. The association of this SNP with \nsome disorders has been investigated in different studies \nincluding cancers ( 17 ,  18 ), systemic sclerosis ( 19 ), periodontitis \n( 20 ), endometriosis ( 21 ) and keratoconus ( 22 ). \nThe association between the C376A SNP and idiopathic \nmale infertility has, however, not been reported. In this \nstudy, we investigated the association between the  IL-1α  \nC376A SNP and idiopathic male infertility in an Iranian \npopulation as a preliminary project. Also, we evaluated \nthe functional effects of C376A on  IL-1α  using bioinformatics \ntools.\n\nIn this cross-sectional study, a total of 460 samples comprising \n230 infertile men (with mean age of 30.93 ± 5.47) \nand 230 fertile men (with mean age of 32.12 ± 5.52) selected \namong individuals attending the Kashan Infertility \nCentre (Shahid Beheshti Hospital, Kashan, Iran). Infertile \npatients were defined as ‘idiopathic’ and selected based \non andrological examination. Patients with previous testis \ntrauma, obstruction of the vas deferens, infectious and \nchronic diseases, hypogonadotropic hypogonadism, abnormal \nhormonal profile (Luteinizing, Follicle Stimulating, \nand testosterone hormones) and abnormal karyotype or Y \nchromosome microdeletions were excluded from the study. \nAccording to the World Health Organization (WHO) 1999 \ncriteria, the patient sub-groups were determined ( 23 ) and \nthe subjects were categorized into non-obstructive azoospermia \n(n=51) without spermatozoa in the ejaculated semen, \noligozoospermia (n=95) with sperm concentration \nless than 20 million/ml, and asthenozoospermia (n=84) \nwith progressive sperm motility less than 50%.\nThe control group was randomly selected from healthly \nmen referred to the Kashan Infertility Centre. They had \nnormal sperm parameters, had no history of chronic and \nfamilial diseases and had at least one offspring. Finally, a \ntotal of 2 ml of whole blood was collected from all males \ninto EDTA-K3 containing tubes and were stored in -20°C \nfor further usage. Written informed consent was obtained \nfrom all case and control subjects. The study was approved \nby the Medical Research Ethics Committee of the Kashan \nUniversity of Medical Sciences (IR.KAUMS.REC.1394.6).\nTotal genomic DNA was isolated from whole blood \nby using a DNA extraction kit (Bioneer, Korea). Purified \nDNA was stored at -20°C for further use. The  IL-1α  \nC376A SNP was genotyped by the polymerase chain reaction-\nrestriction fragment length polymorphism (PCR-\nRFLP) method. For this purpose, forward and reverse \nprimers flanking the SNP were designed based on the \ncomplete sequence of  IL-1α  by the Oligo7 software (Molecular \nBiology Insights, Inc., Cascade, CO, USA).\nThe sequences of the primers were:\n5´-ATGCTAAAATTACCGTGATTCT-3´\n5´-AGATCAATGGAATAAATGGATG-3´ respectively.\nThe PCR was carried out in a total volume of 20 µl containing \n10µl pre-mix (CinnaGen, Iran), 0.35 µM of each \nforward and reverse primers, and 3 µl of template DNA. \nPCR cycling conditions were an initial denaturation step \nat 94°C for 5 minutes followed by 35 cycles of denaturation \nat 94°C for 45 seconds, annealing at 56.9°C for 1 \nminute and extension at 72°C for 1 minute along with a \nfinal extension at 72°C for 5 minutes. PCR products were \nthen digested with the  BstYI  restriction enzyme (CinnaGen, \nIran). For this purpose, approximately 0.1 µg of the \nPCR product was incubated with 5 units of  BstYI  at 37°C \nfor 16 hours. Finally,  BstYI  was inactivated by incubation \nat 65°C for 20 minutes. The digested fragments were \nseparated on a 1% agarose gel stained with DNA Green \nViewer (CinnaGen, Iran) and visualised under the UV \nlight. To verify PCR-RFLP results, 2% of samples were \nsequenced randomly. PCR product recovery kit (Roche \nApplied Science, Mannheim, Germany) was used to purify \nthe PCR product (368 bp in length). Direct sequencing \nof the purified PCR products was undertaken by Bioneer \n(Daejeon, Korea). Chromas (version 2.33) was used to \ncheck the chromatograms.\nThe difference in frequencies of genotypes and alleles \nbetween the case and control groups was analyzed by \nChi-square test. For association analysis, the odds ratios \n(ORs) and 95% confidence intervals (95% CI) were estimated \nby a binary regression logistic test. A two-tailed p-\nvalue less than 0.05 (P<0.05) was considered significant. \nAll analyses were conducted in the SPSS software (SSPS \nInc., IBM Corp, Armonk, NY, USA) version 19.\nBioinformatics tools were used to analyze the influence \nof the  IL-1α  C376A intronic SNP on RNA structure and \nsplicing pattern. The effect on RNA structure and splicing \nwas assessed with RNAsnp online server ( 24 ) and NetGene2 \n( 25 ) respectively. Finally, reported interactions of \n IL-1α  with other molecules were obtained from the BioGRID \ninteractome database ( 26 ).\n\nResults of PCR-RFLP showed that 368 bp fragment was \nfully digested into 114 bp and 254 bp fragments in some \nsamples, showing the efficiency of the method used. The \nsamples with two, three and one fragments were identified \nas CC, AC, and AA genotypes respectively ( Fig .1A ). \nThe data from direct sequencing also confirmed the results \nof PCR-RFLP ( Fig .1B ).\nPolymerase chain reaction-restriction fragment length polymorphism \n(PCR-RFLP) and DNA sequencing results. A. The M, 1, 2 and 3 lanes \nshow the 100 bp DNA ladder, and the AA, AC and CC genotypes, respectively \nand B. Partial sequence of IL-1. flanking the single nucleotide polymorphism \n(SNP) (red box).\nIn this study, the genotype and allele frequencies of the \n IL-1α  C376A SNP were compared between the infertile \nand healthy groups ( Table 1 ). We observed a significant association \nbetween the homozygous genotype CC with male \ninfertility (OR=1.97, 95% CI=1.14-3.41, P=0.016). Carriers \nof C (AC+CC) were at a similar risk for male infertility \n(OR=1.78, 95% CI=1.06-2.99, P=0.030). Also, allelic \nanalysis showed that the C allele is associated with infertility \n(OR=1.43, 95% CI=1.09-1.88, P=0.011). In sub-group \nanalysis, we found that the AC genotype is associated \nwith asthenozoospermia (OR=2.38, 95% CI=1.03-5.53, \nP=0.043). In addition, there was a significant association \nbetween carriers of C and asthenozoospermia (OR=2.25, \n95% CI=1.01-4.10, P=0.047). Also, C allele was significantly \nassociated with oligozoospermia (OR=1.44, 95% \nCI=1.01-2.06, P=0.049) and non-obstructive azoospermia \n(OR=1.67, 95% CI=1.04-2.68, P=0.034).\nFunctional consequence of the C376A transversion on \nRNA structure was evaluated. However, no significant \neffect on RN (distance: 0.0191, P=0.686) was observed \n( Fig .2 ). Minimum free energy of normal RNA was equal \nto -81.80 kcal/mol but increased to -80.50 kcal/mol for \nthe variant allele. The data from NetGene2 revealed that \nthe C370A SNP alters the  IL-1α  splice site pattern on the \ndirect strand (+ strand) especially for the acceptor splice \npattern ( Fig .2 ). The BioGRID interactome showed that\n IL-1α  has 17 gene-gene interactions ( Fig .3 ).\nResults of NetGene2 and RNAsnp. A. Splice sites prediction by NetGene2 \nwhen nucleotide A is present at the C376A position, A’. Splice sites \npattern after the C substitution at the C376A position. Some changes were \nobserved after the substitution especially in the acceptor site (the differences \nbetween the splice patterns are shown by the blue box), and B. \nThe presumptions of variant and ancestral sequences are introduced in \nlower and upper triangle of the plots respectively. The single nucleotide \npolymorphism (SNP) is highlighted by yellow color.\nAllelic and genotypic distribution of the  IL-1α C376A  SNP\nSNP; Single nucleotide polymorphism, OR; Odds ratio, Oligo; Oligozoospermia, Asteno; Asthenozoospermia, and NOA; Non-obstructive azoospermia.\nSignificant differences between the case and control groups are shown in bold type.\nNetwork of human  IL-1α  interactions based on BioGRID.  IL-1α  interacts \nwith 17 other molecules. Purple and yellow lines show interactions \ndetected by genetic and physical experiments respectively.\n\nIn this study, we examined the association of the  IL-1α  \nC376A SNP with male infertility in an Iranian population \n(Kashan, Iran) as a pilot study. Our study revealed that not \nonly the CC genotype was associated with male infertility, \nbut also the C allele showed significant association. In \naddition, carriers of the C allele were at almost two-fold \nrisk for male infertility. Sub-group analysis revealed that \nAC genotype and carriers of C were associated with asthenozoospermia. \nAlso, the C allele was significantly associated \nwith oligozoospermia and non-obstructive azoospermia. \nTherefore,  IL-1α  C376A is a potential genetic \nrisk factor for male infertility, although further studies \nof different ethnicities in Iran and other populations are \nrequired to obtain a more accurate picture. After Hardy-\nWeinberg equilibrium (HWE) calculation in the control \ngroup, we found a highly significant deviation. However, \nthe case group showed no deviation. even though it does \nnot necessarily need to follow HWE due to the inherent \nsampling bias in cases. The deviation from HWE in the \ncontrol group (normozoospermic men) could also be due \nto the selection bias ( 27 ) given that not all men in the general \npopulation will be fertile.\nSpermatogenesis is a dynamic process in which many \nfactors are necessary for creating and regulating balance \nin this process. For example, growth factors and cytokines \nare essential for development of functional spermatozoa \n( 28 ,  29 ). Interleukin-1 is produced by epithelia of seminiferous \ntubules and acts as a physiological paracrine/\nautocrine factor on testicular cells and required for immunological \nprotection ( 30 ). There is a probable mechanism \nthat in the absence of testosterone, followed by increased \ncell apoptosis, spermatogenesis is finally reduced ( 31 , \n 32 ). The second probable mechanism is excess reactive \noxygen species (ROS).\nThe presence of the associated SNP and the consequent \nchange in the amount of interleukin along with excess \nproduction of ROS may reduce sperm motility. One of \nthe reasons for reduced sperm motility may be DNA damage \nand lipid peroxidation of sperm membrane ( 33 ). Also, \nincreased ROS with oxidizing DNA or proteins, enzyme \ninhibition, cell death and apoptosis of sperm may cause \nthe oligozoospermia phenotype ( 34 ,  35 ). Due to these \npossible mechanisms, the association of the  IL-1α  SNP \nwith some abnormalities in sperm parameter may be explained. \nSNPs could change the gene expression pattern \n( 14 ), mRNA structure ( 36 ,  37 ), splicing pattern ( 38 ) and \nprotein function ( 39 ,  40 ). In silico tools, which can predict \nthe damaging effects of SNPs, were therefore used \nespecially that  IL-1α  C376A is an intronic SNP and may \naffect RNA structure and splicing. Although we found no \nevidence for C376A to affect RNA structure, we observed \na predicted effect on splicing alteration. Therefore, the association \nof this SNP may be due to this effect. In this \nstudy, there were various limitations including gene-environment \nand gene-gene interactions that must be considered in subsequent studies. Also, lack of  in vitro  studies\nsuch as investigating the effect of the SNP on  IL-1α  gene \nexpression and isoform formations due to splicing alterations \nis another limitation of this study.\n\nOur study suggests that the  IL-1α  C376A SNP may increase \nthe risk of male infertility up to two-fold. Since this is \nthe first study, future studies with larger sample sizes in different \nethnicities and populations is warranted given the variable \nenvironmental factors in different geographic regions.","source_license":"CC-BY-4.0","license_restricted":false}