CL IN I CAL CORNER : COMMUN ICAT ION

Association of interleukin-1beta C + 3953T gene polymorphism withhuman male infertility

Deepika Jaiswal1, Sameer Trivedi2, Neeraj K Agrawal3, Rajendra Singh4, and Kiran Singh1∗

1Department of Molecular and Human Genetics, 2Department of Urology, 3Department of Endocrinology and Metabolism,Institute of Medical Sciences, Banaras Hindu University, Varanasi, India, 4Division of Endocrinology, Central Drug ResearchInstitute, Lucknow, India

Cytokines are involved in the regulation of spermatogenesislikely mediating the crosstalk among Sertoli and germ cellsto facilitate germ cell movement across the seminiferous epi-thelium during cellular events such as germ cell differentiation.Members of the Interleukin-1 (IL-1) family are pleiotropic cyto-kines that are involved in inflammation, immunoregulation,and other homeostatic functions. Interleukin-1 alpha (IL-1α),IL-1β, and the IL-1 antagonistic molecule (IL-1 Ra) are presentin the testis under normal homeostasis and they furtherincrease upon infection/inflammation. In the present studywe have examined the association of C + 3953T polymorphismof the human IL-1B gene with human male infertility. The casecontrol study comprised of two groups: 222 infertile patientsand 230 fertile healthy control men. Genotyping for SNP C +3953T IL-1B was carried out by polymerase chain reaction fol-lowed by analysis with specific endonucleases (PCR-RFLP).DNA sequencing was used to validate the PCR-RFLP results.The genotype frequencies of the IL-1B Taq C/T polymorphismwere compared between infertile men and controls. The fre-quency was significantly higher in asthenozoospermic patientscompared to fertile control men (odds ratio = 10.4, CI: 2.50-43.96, p = 0.001). The C + 3953T of the IL-1B gene is associatedwith male infertility risk in the asthenozoospermic patientsfrom an Indian population.

Keywords infertility, interleukin, spermatogenesis, testis

Introduction

Spermatogenesis involves a series of processes that isinitiated with the differentiation of the primordial germcell through meiosis, spermiogenesis, and spermiation,within the unique microenvironment of the testis [Singhand Jaiswal 2011]. The testicular microenvironment alsorequires an immune system that acts as a safeguard for im-munogenic male germ cells and at the same time permitsnormal inflammatory response against invading pathogens

[Rozwadowska et al. 2007]. Sertoli cells produce many cyto-kines including the TGF-β superfamily, interleukins, tumornecrosis factor as well as others. These cytokines are keyplayers in regulating junction dynamics [Xia et al. 2005].Thus altered expression of cytokines during injury, illness,or infection may contribute to the disruption of testicularfunction and fertility. Elevated levels of these cytokines inthe seminal plasma of men with fertility problems havebeen well documented [Comhaire et al. 1999; Rajasekaranet al. 1995; Huleihel et al. 1996].

The interleukin-1 (IL-1) is a classic pro-inflammatory cy-tokine which occurs as two isoforms α and β [Dinarello1996]. The IL-1α is mainly produced in seminiferoustubules and the IL-1 β is secreted in interstitial spaceswhere it is known to affect the process of steroidogenesis[Hedger and Meinhardt 2003]. Many studies have shownthat IL-1 inhibits both basal and luteinizing hormone (LH)and human chorionic gonadotrophin (hCG) -stimulated tes-tosterone production in in vitro cultures. Interleukin-1 hasdirect inhibitory effects on gonadotrophin-stimulated andro-gen production by the Leydig cells thereby affecting fertility[Hutson 2006; Svechnikov et al. 2001; Lin et al. 1991]. Cyto-kines affect capacitation during fertilization [Masayuki et al.2008 ]. Interleukin-1 receptor antagonist (Ra) is a natural re-ceptor antagonist for the IL-1 gene and regulates IL-1 (α andβ) production by inhibiting a positive feedback loop inwhich IL-1 induces its own production thus affecting itsfunction [Granowitz et al. 1992]. Recently, our group hasshown association of Interleukin 1 receptor antagonistgene Variable Number Tandem Repeats (IL1RN VNTR)polymorphism with human male infertility in a NorthIndian population [Jaiswal et al. 2012]. The IL-1B gene islocated on chromosome 2q14.2, and a polymorphism inexon 5 of IL-1B (C + 3953T) was found to correspond withincreased IL-1β expression in monocyte cells in vitro[Pociot et al. 1992]. Based on the biological and pathologicimportance of C + 3953T polymorphism of the human

∗Address correspondence to Kiran Singh, Department of Molecular & Human Genetics, Banaras Hindu University, Varanasi-221005, India E-mail:singhk4@rediffmail.com, skiran @bhu.ac.in

Received 29 March 2013; revised 15 June 2013; accepted 17 June 2013.

Systems Biology in Reproductive Medicine, 2013, Early Online: 1–5Copyright © 2013 Informa Healthcare USA, Inc.ISSN 1939-6368 print/1939-6376 onlineDOI: 10.3109/19396368.2013.830234

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IL-1B gene, it is possible that this variation may contribute tothe clinical outcomes of male infertility. Therefore, we eval-uated the association of C + 3953T functional variant of IL-1B in cases of idiopathic male infertility compared tocontrols.

Results and Discussion

A total of 452 individuals (222 infertile patients and 230fertile controls) were examined by PCR RFLP (Fig. 1).DNA sequencing was used to ascertain PCR-RFLP (Fig. 2).The genotype frequencies for C + 3953T polymorphism inthe IL-1B gene were compared between the differentgroups using a recessive model. The distributions of geno-types of the SNP IL-1B (C + 3953T) followed Hardy–Wein-berg equilibrium in both the patients (azoospermia andasthenozoospermic men) and control group ( p = 0.68 andp = 0.33, respectively). The sensitivity and specificity of thestudy were 0.82 and 0.18, respectively. Also, significantdifference was observed in the distribution of both C andT allele between asthenozoospermic men as compared tocontrols ( p = 0.001; Table 1).

Cytokines and immunological factors are found inseminal plasma of men that play an important role innormal fertility [Fijak and Meinhardt 2006]. The testis isan immune privileged site, but is not isolated from theimmune system [Holstein et al. 2003]. Cytokines secretedin the seminal plasma exert an effect on steroidogenesis,spermatogenesis, and sperm functions and thus affect ferti-lization and pregnancy [Gremlich et al. 2008]. Anti-inflam-matory cytokines are known to effect testicular development[Soder et al. 2000]. Altered expression of cytokines duringinjury, illness, or infection may contribute to the disruptionof testicular function and fertility [Li et al. 2009]. The IL-1family of cytokines has an inhibitory affect on Leydig cellsteroidogenesis by blocking testosterone formation and cyto-chrome p450 side-chain cleavage in Leydig cells, thereby af-fecting spermatogenesis [Mathur et al. 2011]. In light ofavailable data, we have investigated the association of

polymorphism of the human interleukin-1 gene with maleinfertility. A polymorphism in exon 5 of IL-1B (IL-1B TaqC/T) was found to correspond with increased IL-1βexpression in vitro [Pociot et al. 1992]. Bentz et al. [2007]showed that homozygous mutant genotype carriers of theIL-1B Taq C/T polymorphism have a fourfold-increasedrisk of sperm pathology compared with a control populationand, thus, carriage of this polymorphism may predisposemen to develop oligoasthenoteratozoospermia syndrome( p = 0.001). Gopichandran et al. [2006] studied the role ofnumerous cytokines present within the seminal plasma inimmunomodulation, uterine leukocyte recruitment, andtrafficking. Elheija et al. [2011] have shown IL-1β levelswere higher in Leydig/interstitial cells of normal biopsiescompared with Sertoli and germ cells. However, themRNA levels of IL-1β were significantly lower in cases of im-paired spermatogenesis as compared to normal. In thepresent study the C + 3953T IL-1B polymorphism associatedwith asthenozoospermia phenotype ( p = 0.004). Interleu-kin-1B interferes with endocrine regulation to influencehormone production and as a result affects spermatogenesis[Huleihel and Lunenfeld 2004]. Altered levels of IL-1Balong with Tumor Necrosis factor alpha (TNFα) affectsperm membrane lipid per-oxidation and increase ROS pro-duction thereby affecting the level of sperm DNA damage[Martinez et al. 2007]. Moreover, increased levels ofseminal reactive oxygen species (ROS) and higher DNAfragmentation in the sperm results in decreased sperm mo-tility [Dada et al. 2011]. This suggests negative effects of theIL-1 on the spermatogenesis and sperm motility. This is the

Figure 1. Interleukin-1B (C + 3953T) polymorphism was detected byPCR-RFLP. 249 bp PCR products were digested withTaq1. The allele Cis cut by the enzyme and gives 135 bp and 114 bp, whereas the T alleleyields 249 bp products. The lanes (lane 1-4) were indicated by M(marker) and genotypes (CC, CT, TT) from left to right.

Figure 2. Sequencing chromatogram showing the 3 genotypes ofsingle-nucleotide polymorphism (SNP) interleukin-1B (IL-1B) (C +3953T). Forward primer was used for sequencing on the genetic ana-lyzer 3130 automated sequencer. The SNP at position C + 3953T ofIL-1B is represented by star. (A) homozygous CC, (B) heterozygousCT, and (C) homozygous TT.

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first report on the association of the IL-1B (C + 3953T)polymorphism with asthenozoospermic phenotype ininfertile men from an Asian population. The IL-1betaassociation with male infertility provides a better under-standing of these cytokines in normal and infertile menthat may add to the factors modulating the risk of maleinfertility.

Material and Methods

SubjectsIn the present case control study subjects were recruitedfrom Varanasi and Lucknow regions of India. The studyconsisted of 222 infertile males (32 ± 4.8 y) and 230 fertilemales (36 ± 5.6 y) as controls. Both cases and controlswere from North India and had the same ethnicity. Patientsunderwent a standardized clinical and laboratory evaluation.Patients married for a minimum of two years, having unpro-tected intercourse, and absence of any obvious fertilityproblem in the female partner were considered for thepresent study. Three semen analyses were carried out after3-4 d of sexual abstinence to ascertain infertility status.The patients were categorized into two sub-groups as perWHO [1999] criteria. Asthenozoospermic men (n = 81)had a sperm count ≥ 20 million/ml, motility (‘a’+ ‘b’) ≤50% (Grade a: rapid progressive motility and Grade b: slowor sluggish progressive motility) and ≥ 30% normal mor-phology and non-obstructive azoospermic men (n = 141)had no sperm in the ejaculate. A questionnaire was main-tained for each patient to record details of their lifestyle,habits, and family history. Informed consent was obtainedfrom every participant of each group. Patients with obstruc-tive azoospermia, hypogonadism, hypoandrogenism,chronic diseases, history of pelvic/spinal injuries, karyotypeabnormalities, and AZF microdeletions were excluded. Thecontrol group consisted of healthy fertile males who had atleast one child and no history of chronic illness. Approvalof the University’s ethical committee for research onhuman material was obtained.

IL-1B (C + 3953T) polymorphismGenomic DNA was extracted from peripheral blood, using astandard salting-out procedure. A total of 5 ml of hepara-nised blood was collected in a 50 ml polypropylene tubeand three times the volume 0.85 % NaCl was added andmixed well. Tubes were centrifuged at 7,000 rpm in aREMI-Centrifuge (Refrigerated, Model No CPR-24, REMIINDIA) for 7 min at 25°C. Supernatant was discarded andloose cells were mixed with three volumes of hemolytic sol-ution (solution A: sucrose 109.5gm, 1M MgCl2 5 ml, TritonX 100 10 ml to 1 liter water, autoclaved and stored at 40C)for 5 min and centrifuged at 7,000 rpm for 7 min at 25°C.The supernatant was again discarded and white blood cellpellet was resuspended in solution B (1M Tris-Cl 40 ml,0.5M EDTA 12 ml, 1M NaCl 15 ml, water added to makeup to 95 ml, autoclaved, and after cooling 5 ml of 20%SDS added to make up the final volume to 100 ml, storedat room temperature) and mixed gently for 5 min andthen solution C (5M sodium per chlorate, autoclaved, andstored at room temperature) was added to it and againgentle mixing was done for 5 min. Chilled chloroform wasadded and mixed well. Tubes were recentrifuged at 7,000rpm for 7 min at 25°C. The aqueous layer was carefullytaken out in a separate test tube and approximately equalamount of isopropanol was added to precipitate out theDNA. DNA was transferred to another tube and 70%alcohol wash was given. Alcohol was discarded and it wasleft to dry for a few minutes. Finally the DNA pellet was dis-solved in TE according to the amount of DNA precipitated.

The IL-1B gene polymorphism was queried by PCR-RFLP analysis [Genc et al. 2002] used the following reverseand forward primers (Forward Primer: 5′-GTTGTCATCAGACTTTGACC-3′; Reverse Primer: 5′-TTCAGTTCATATGGACCAGA-3’). The C > T change at +3953T positioncreates a restriction site for the Taq1 restriction endonu-clease. The polymorphic region was amplified by PCR: 30s at 94°C, 55 s at 62°C, and 55 s at 72°C. The first cyclewas preceded by a 5 min denaturation step and a singlestep of extension at 72°C for 10 min followed the lastcycle. PCR products were digested 3 h at 65°C with TaqI

Table 1. Interleukin-1B (+3953 T/C) gene polymorphism: genotype frequencies in infertile men and control group.

Odds Ratio (95% CI)# p-Value# Yates’ p-Value#

Genotype

Control(n =230)

Azoospermia(1) (n = 141)

Asthenozoospermia(2) (n = 81)

Total(1 + 2)(n = 222) (1) (2)

Total(1 + 2) (1) (2)

Total(1 + 2) (1) (2)

Total(1 + 2)

CC 151(65.7%)

96 (68.1%) 55 (68.1%) 151(68.1%)

1 - -

CT 76(33.0%)

42 (29.8%) 19 (21.8%) 61(27.1%)

TT 3 (1.3%) 3 (2.1%) 7 (10.1%) 10(4.8%)

1.67(0.31-8.80)

10.48(2.50-43.96)

3.13(1.04-9.44)

0.54 0.001∗∗ 0.04∗∗ 0.85 0.004∗∗ 0.07

OR: odds ratio; 95% CI = 95% confidence interval∗recessive genetic model: C/C + C/T vs. T/T∗∗ Significant p-value# Controls vs. (1), (2), (1 + 2)

IL1B C + 3953T polymorphism and human male infertility 3

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restriction enzyme and analyzed by agarose gel electrophor-esis. In C allele, the 249-bp gives two fragments of 114 and135 bp. In the presence of T allele the restriction site is abol-ished and thus a single band of 249 bp will be obtained. Toensure consistency of all assays, genotyping of both (IVS6 +55G/T and c.852C/T SNP) was repeated for 10% of thesamples and two samples of each genotype were randomlyselected and subjected to direct DNA sequencing to verifygenotype, confirming 100% concordance.

SequencingThe product of the PCR was sequenced with Taq-Dyedeoxy-terminator-cycle sequencing kit (Applied Biosystem,USA) using an automated genetic analyzer 3130 sequencerto ascertain the PCR-RFLP results. Forward primer wasused for IL-1B (C + 3953T) for cycle sequencing. The se-quences were aligned and analyzed using ABI DNA Sequen-cing Analysis software (version 5.2).

Statistical analysisAllele frequencies and genotype distribution among groupswere evaluated using a Chi-square test. The difference in fre-quencies between the case and control groups was analyzedfor statistical significance at the 95% confidence intervalusing χ2 test. The allele frequency IL-1B (C + 3953T) wasin Hardy-Weinberg equilibrium. Odds ratios (ORs) werecalculated and reported within the 95% confidence limits.Sensitivity and specificity was calculated using Graph PadPrism5. All statistical tests were two-sided, and P-value <0.05 was considered statistically significant.

Acknowledgments

We are thankful to all the patients and volunteers for provid-ing blood samples. Senior Research Fellowship by Council ofScientific & Industrial Research (CSIR), New Delhi, India tothe first author is duly acknowledged.

Declaration of interest: The authors declare no conflicts ofinterest.

Author contirbutions: Conceived and designed the exper-iments: DJ, KS; Performed the experiments: DJ; Analyzedthe data: KS, DJ; Contributed reagents/ materials/ analysistools: KS, ST, RS, NKA; Wrote the Manuscript: KS, DJ.

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