HLA antigens and anti-sperm antibody production in Iranian vasectomized men

HLA antigens and anti-sperm antibody production in Iranian vasectomized men

Dear Editor:

Anti-sperm antibodies(ASAs)are composed of numerous antibodies interacting with multiple sperm antigens that play a role in fertility.In males,ASAs cause‘immune infertility’by decreasing sperm counts and normalforms,as wellas reducing sperm motility and viability,markedly reducing the likelihood ofnatural conception[1].The development of ASA in the male depends on the release of sequestered antigens on germ cellsfollowing the disruption ofthe blood-testis barrier. Asurgicaloperation such asa vasectomy could also lead to immunogenic sperm antigens being exposed to the immune system,thus initiating an immune response to produce ASAs[2].Vasectomy is widely regarded as a safe method of birth control,butover the years there have been many reports suggesting putative health risks associated with the procedure.ASA develops after vasectomy,which can resultin autoimmune male infertility.Moreover,concerns overthe possible association of vasectomy with a number of medicaldifficulties including cardiovascular disease,testicular cancer, prostate cancer,and a variety of immune complexmediated disease processes have been reported[3].There is a high correlation between vasectomy and ASA production;thus,the prevalence of ASA is higher in vasectomized men than in the generalpopulation,which causes limited success in regaining fertility,even after successful vasovasostomy[4].The human leukocyte antigen(HLA)is the most important region in the genome with respectto autoimmunity and infection, and itis pivotalin adaptive and innate immunity.The HLA region encodes several molecules composed of the two polymorphic classes HLA-I(A,B,and C)and HLA-II(DR,DQ,and DP)thatplay key roles in the immune system by presenting antigens to T cells[5,6].It is demonstrated thatpeople with certain HLA antigens are predisposed to developing certain autoimmune diseases,butto date correlation between HLA gene and ASA production has notbeen fully identified[8]. We evaluated the ASA levelin Iranian men one year after vasectomy and examined the association of ASA production with HLA class Iand IIin Iranian vasectomized men.

One hundred and ten vasectomized men(＜50 year) one-year post vasectomy and 130 healthy controls agreed to participate in this cross-sectional study. Written informed consent was obtained from allmen and the Ethics Committee of Tehran University of Medical Sciences approved the study.Blood sample was collected from each subject to isolate peripheral blood mononuclear cells(PBMCs)and serum.For quantification of ASA in serum samples,we performed the Gelatin Agglutination Test(Kibrick)and the results were confirmed by the indirect mixed antiglobulin reaction(MAR)test.In this study,we used T cells and purified B cells as the source of HLA-I an d HLA-II antigens,respectively.Firstly,PBMCs were isolated from blood by Ficoll-Hypaque(Pharmacia, Uppsala,Sweden)density gradientcentrifugation,then isolated PBMCs were washed twice with PBS,placed on nylon woolcolumns(Biotest,Breieich,Germany) and incubated at 37°C for 30 minutes.Unabsorbed cells were removed by extensive washing with warm RPMIfor HLA-A,-B and-C typing;B cells were also eluted from the column to use for HLA-DR and-DQ typing.Cell viability and cell counts were assessed by the Trypan blue exclusion method forpurified cells (viability＞85%).HLA typing was carried outby a standard complement dependent microlymphocytotoxicity or Terasaki test.Microtiter plates(Biotest AG,Dreieich,Germany)containing HLA antisera with known specificity were used for determination of HLA-I or HLA-IIantigens.Terasaki microtiter plates containing various HLA-I and II antibodies were seeded with 1μL(2000 lymphocytes.Afterincubation at room temperature(HLA-I:30 minutes and HLA-II: 60 minutes)and addition of rabbit complement,the results were visualized by adding 5%eosin.The assessmentof lysed and non-lysed lymphocytes was carried outusing an inverse phase contrastmicroscope.

The gelatin agglutination testshowed that ASA was detectable in 95%of vasectomized men,with slightlymore than halfofthem(53%)having an ASA titerof1/ 32 or1/64.By contrast,serum ASA was notdetectable in 86.2%of healthy controls and was found in only 13.8%of the healthy subjects with a maximum titer 1/8(Fig.1).

Fig.1ASA titer in vasectomized and control groups.As shown,the highest ASA titers in vasectomized men are 1/32 or 1/64,whereas the maximum titer of serum ASA in normal men is 1/8.

Fig.2Frequency of HLA class I antigens in vasectomized and controlmen.HLA typing in vasectomized and normalmen were carried out by a standard complement dependentmicrolymphocytotoxicity.There is a statistically significant correlation in the low responder group(ASA≤1/8) with frequency of HLA-A2 and B5 class Iantigens.(*=X2＞3.84).A:Frequency of HLA-A antigens in vasectomized and controls.B:Frequency of HLA-B antigens in vasectomized and control men.C:Frequency of HLA-C antigens in vasectomized and control men.

Fig.3 Frequency of HLA class II antigens in vasectomized and control men.The HLA-DRw53 and DQw2 class II antigens have a statistically significant correlation with low responder group(ASA≤1/8).*=X2＞3.84

We further categorized the vasectomized subjects into the low responder group(n=18;titer≤1/8), the middle responsder group(n=92;titer between 1/16 and 1/64),and the high responder group(n= 44;titer≥1/64).HLA-A,-B,-C,-DR and-DQ typing of the above three groups and controlgroup revealed no statistically significantcorrelation between the high responder group and frequency of HLA antigens, whereas there was a statistically significantcorrelation (X2＞3.84,P＜0.05)between the low responder group and the frequency of HLA-A2(X2=8.85,P =0.0029)and B5(X2=10.17,P=0.0014)class I antigens(Fig.2)as well as HLA-DRw53(X2= 6.88,P=0.008 7)and DQw2(X2=5.37,P= 0.02)class II antigens(Fig.3).

Itis widely accepted thatimmune infertility due to ASA is one of the majorcauses of unexplained infertility in humans.More than 50-70%of men develop ASA after vasectomy and there is limited success in the reestablishment of fertility,even after successful surgical reanastomosis in vasovasostomy.A 2008 study by Nowroozi et al.showed that 2.5%of men had ASA at the time of vasectomy,whereas 53.5% of the study population subsequently develo ped ASA.The number of men with circulating ASA was increased significantly for the firstthree months after vasectomy,but the highest incidence of titers was one year after vasectomy.Our findings showed that ASA wasproduced in 95%vasectomized men one year aftervasectomy.There has been lack ofinformation on the relation between HLA antigens and ASA production in vasectomized men.Law et al.found that the HLA-A28 was strongly associated with production of ASA after vasectomy.While we found no significant association between the high responders and frequency of HLA antigens,there was a significantassociation in the low responders and frequency of HLAA2,B5,-DRw53 and-DQw2.Men who had these HLA phenotypes produced a lower titer of ASA after vasectomy.

In conclusion,the predisposition to production of ASA appears to be correlated with HLA antigens. Men who have HLA-A2,-B5,-DRw53 and-DQw2 produce lower levels of ASA than other populations although a better understanding of the relation between HLA and ASA production requires further study.

Yours Sincerely,✉Dr.Gholamreza Azizi, Imam Hassan Mojtaba Hospital, Alborz University of Medical Sciences, Karaj, Iran,

Fax:+98-21-88211188,

E-mail:drnamaki@yahoo.com;

Dr.Saeed Namaki, Department of Pathobiology, Faculty of Paramedical Sciences, Shahid Beheshti University of Medical Sciences, Tehran,Box:6446, Iran;

Dr.Abbas Mirshafiey and Dr.Kabir Magaji Hamid, Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.

The authors reported no conflict of interests.

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[3]K o¨hler TS1,Fazili AA,Brannigan RE.Putative health risks associated with vasectomy.Urol Clin North Am 2009;36(3):337-345.

[4]Heidenreich A,Bonfig R,Wilbert DM,et al.Risk factors for antisperm antibodies in infertile men.Am J Reprod Immunol 1994;31(2-3):69-76.

[5]Goug h SC,Simmond s MJ.The HLA Regio n and Autoimmune Disease:Associations and Mechanisms of Action.Curr Genomics 2007;8(7):453-465.

[6]Chen X,PE Jensen.MHC class IIantigen presentation and immunologicalabnormalities due to deficiency of MHC class IIand its associated genes.Exp MolPathol2008;85(1):40-44.

©2015 by the Journal of Biomedical Research.All rights reserved.

10.7555/JBR.29.20140113