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 normal forms, as well as reducing sperm motility and viability, markedly reducing the likelihood of natural conception [1] . The development of ASA in the male depends on the release of sequestered antigens on germ cells following the disruption of the blood-testis barrier. A surgical operation such as a 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, but over the years there have been many reports suggesting putative health risks associated with the procedure. ASA develops after vasectomy, which can result in autoimmune male infertility. Moreover, concerns over the possible association of vasectomy with a number of medical difficulties 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 general population, 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 respect to autoimmunity and infection, and it is pivotal in 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) that play key roles in the immune system by presenting antigens to T cells [5,6] . It is demonstrated that people with certain HLA antigens are predisposed to developing certain autoimmune diseases, but to date correlation between HLA gene and ASA production has not been fully identified [8] . We evaluated the ASA level in Iranian men one year after vasectomy and examined the association of ASA production with HLA class I and II in 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 all men 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 and HLA-II antigens, respectively. Firstly, PBMCs were isolated from blood by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density gradient centrifugation, then isolated PBMCs were washed twice with PBS, placed on nylon wool columns (Biotest, Breieich, Germany) and incubated at 37uC for 30 minutes. Unabsorbed cells were removed by extensive washing with warm RPMI for 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 for purified cells (viability . 85%). HLA typing was carried out by 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-II antigens. Terasaki microtiter plates containing various HLA-I and II antibodies were seeded with 1 mL (2000 lymphocytes. After incubation 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 assessment of lysed and non-lysed lymphocytes was carried out using an inverse phase contrast microscope. The gelatin agglutination test showed that ASA was detectable in 95% of vasectomized men, with slightly