Endometrial antigens involved in the autoimmunity of endometriosis

Serum and peritoneal fluid from five fertile women without endometriosis and serum (n = 23) and peritoneal fluid (n = 12) from infertile women with endometriosis were tested for the presence of antibodies against endometrial tissue antigens by a Western blot analysis. Antigens with molecular weights (MW) of 19, 31, 38, and 42 kd reacted with antibodies in the serum and peritoneal fluid from both fertile and infertile women. Antibodies in 20 of 23 (87%) sera and all 12 (100%) peritoneal fluid samples from endometriosis patients reacted against endometrial antigens with molecular weights (MW) of 26 kd and/or 34 kd. Serum from 10 patients (43%) and peritoneal fluid from 6 patients (50%) also had antibodies to an endometrial antigen with MW of 21.5 kd. Reactivity to other endometrial antigens with MW 16, 24, 48, and 75 kd was also noted in patients with endometriosis. Antibodies in the serum and peritoneal fluid from fertile women failed to react against these antigens. It is concluded that the humoral and local endometrial autoimmunity detected in patients with endometriosis is primarily directed against antigens with MW of 26 and 34 kd.     

Evaluation of immunofluorescence on pig zona pellucida for detection of anti-zona antibodies in human sera

Using immunofluorescence tests, antibodies reacting with pig zona pellucida could be detected in 31% of 103 sera from patients in different clinically defined categories, mainly fertile males and females and patients from couples with unexplained infertility. The antibodies could in all cases be absorbed by means of pig erythrocytes — in most sera with all erythrocyte suspensions tested, but in some cases only with erythrocytes from certain animals — indicating that antibodies both to generally occurring pig antigens and alloantigens may be present in human sera. Staining of pig zonae was recorded equally frequently with unabsorbed sera from infertile and fertile males and females, respectively, but titres of 1 : 16 or more occurred more frequently among infertile than among fertile females (P = 0.053).     

Occurrence of serum antisperm antibodies in patients with cystic fibrosis

OBJECTIVE: To determine if acquired obstruction of the vas deferens in men with cystic fibrosis (CF) induced the development of antisperm antibodies with genital tract obstruction similar to other men. DESIGN: Serum antisperm antibodies were assayed by an indirect immunobead test and an indirect immunofluorescence assay. Both homologous (human sperm/human zona) and heterologous (human sperm/zona-free hamster ova) sperm/egg interactions were evaluated in the presence of serum antisperm antibodies from patients with CF. SETTING: Cystic Fibrosis Clinic at the University of Oklahoma Health Sciences Center, a tertiary care referral center. PATIENTS: Fifteen CF patients (10 male and 5 female), 3 non-CF antisperm antibody-positive infertile patients (2 male and 1 female), 20 fertile controls (7 males and 13 females), and 9 fertile sperm donors were used. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Serum antisperm antibody levels in patients with CF. In those patients with antisperm antibodies, determine effect of these sperm antibodies on sperm/egg interactions and complement-mediated events. RESULTS: Sera from 3 (30%) of 10 men with CF demonstrated immunoglobulin (Ig)G, IgA, and/or IgM antisperm antibodies, whereas sera from all 5 CF women and the 20 control sera were negative for antisperm antibodies. The maximal titers for IgG, IgA, and IgM antisperm antibody were 1:8, 192, 1:256, and 1:64, respectively. The immunobead binding, which was restricted to the sperm head and tail-tip or the midpiece and tail-tip, correlated with the indirect immunofluorescence pattern. Antisperm antibody-positive sera from men with CF impaired both the binding and penetration of human zonae and the penetration of hamster ova by human sperm. CONCLUSIONS: Similar to other men with congenital or acquired obstruction of their genital tract, antisperm antibodies may occur in some men with CF. Antisperm antibodies may contribute to immune sperm dysfunction in some men with CF by activated complement-mediated events and interfering with sperm/egg interactions.     

Antisperm antibodies to sperm surface antigens in women with genital tract infection

Antisperm antibodies to sperm surface antigens in nulligravid women with primary upper genital tract infections were measured by the sperm mixed agglutination reaction assay. As many as 56% of women with a primary episode of pelvic inflammatory disease had antisperm antibodies. In addition, 69% of those women with no history of genital tract infection but with laparoscopic evidence of past pelvic infection had significant levels of circulating antisperm antibodies. Electroimmunoblots of sperm preparations probed with the sera of women who had either known or presumed upper genital tract infection revealed a uniformly recognized 69 kd antigen. In contrast, women with circulating antisperm antibodies before primary upper genital tract infection recognized up to five distinct sperm antigen determinants of 27, 54, 131, 146, and 174 kd. It is a distinct possibility that genital tract infections may lead to immunopotentiation of antisperm antibodies that could affect fertility.     

Blot-immunobinding test for the detection of anti-sperm antibodies

A blot-immunobinding test was used to detect anti-sperm antibodies in human sera and to identify the corresponding auto- or iso-antigens on human sperm. A high proportion of sera at a 1:100 dilution from fertile persons, as well as infertile patients, contains antibodies reactive with sperm. This phenomenon might be physiological. At 1:2,000 dilution, a higher binding capacity was detected in the sera from infertile groups, but a few fertile persons were also positive. Antibodies to a single antigenic determinant with Mr of approximately 14,000 were found in a significantly higher proportion among males with unexplained infertility.     

Human sperm protein encyclopedia and alloantigen index: mining novel allo-antigens using sera from ASA-positive infertile patients and vasectomized men

Anti-sperm antibodies (ASA) are an important cause of immunological infertility. The objective of this study was to identify immunodominant sperm antigens recognized by anti-sperm antibodies (ASA) in serum samples of infertile men, women and vasectomized men. High-resolution two-dimensional gel electrophoresis was employed to separate human sperm proteins using isoelectric focusing (IEF) or nonequilibrium pH gradient electrophoresis (NEPHGE), followed by PAGE and Western blotting. Serum samples from five infertile male and five infertile female subjects that contained ASA as assayed by the immunobead binding test (IBT), were analyzed by Western blotting using NEPHGE gels followed by enhanced chemiluminescence (ECL) to identify the basic sperm antigens reactive to the sera. Serum samples from five fertile male and five fertile female subjects that were ASA-negative by IBT were used as controls. Serum samples from six vasectomized men collected before vasectomy and at different time intervals until 6 months after vasectomy were analyzed by Western blotting using IEF gels. The ECL blots were analyzed to compare immunoreactivity between serum samples from fertile and infertile subjects and identify antigens unique to sera of the infertile subjects. Similarly, immunoreactivity between serum samples from pre- and post-vasectomy was compared to identify antigens unique to sera collected following vasectomy. Five allo-antigenic basic protein spots were recognized by sera from infertile males but not from fertile subjects. Five sperm iso-antigenic basic spots were recognized by infertile female subjects. Two among six of the vasectomized men's sera showed a difference in the Western blot profile 6 months after vasectomy, recognizing at least one new protein spot in each case when compared to pre-vasectomy sera. The acrosomal protein SP-10 was identified as an alloantigen recognized by a post-vasectomy serum. Molecular identities of the known allo- and iso-antigens identified in this study and in previous studies from this laboratory are reviewed and discussed.     

Antibodies to microbial, leukocyte and organ antigens

Hemagglutinating antibodies to Mycoplasma hominis were present in 30 of 83 infertile, 15 of 40 pregnant and 5 of 20 post-partum females and 20 of 82 infertile males in contrast to only 2 of 21 fertile females and 5 of 25 fertile males. Their presence correlated with sperm antibody detection by TAT in Lab. 4, the immunobead-binding assay of Lab. 1 and the SIT of Lab. 11, but not with other sperm antibody assays. Immunofluorescent antibodies to Chlamydia trachomatis, on the other hand, did not correlate with the incidence of sperm antibodies. Among 305 serum samples tested, 12 were positive for testicular antibodies, 8 had antibodies to kidney, 7 to ovary and 15 to endometrium. A majority of serum samples positive for antibodies to testis and ovary, but not endometrium, reacted against sperm in different assays. Eight of 135 samples tested had antibodies to human leukocyte antigenic HLA-Aw19 (Aw19, A28, A29, A30 and A32) and/or B35 (B35, B5 and B15) complexes. Six of these samples were also positive for sperm antibodies by one or more antibody assays. Cross-reactive antigens may be present in sperm, M. hominis, testis, ovary and leukocytes.     

84KD精子膜蛋白与抗精子抗体

为了进一步研究精子抗原及其相应抗体在不育中的作用,本文用SDS-PAGE和免疫印迹法检查了一对长期不育夫妇之间的抗精子免疫应答。该夫妇双方均含高滴度抗精子抗体。丈夫精子膜蛋白提取液中分子量84KD蛋白含量显著增加,为正常男性的3～5倍。该成份在免疫印迹中可被夫妇双方血清特异性识别。提示不育男性精子抗原成份与生育力正常男子有量或质的差异,可能是产生抗精子抗体并造成不育的一个原因。     

A reverse (antibody capture) enzyme-linked immunosorbent assay for detection of antisperm antibodies in sera and genital tract secretions

A reverse (antibody capture) enzyme-linked immunosorbent assay (ELISA) for detection of antisperm antibodies has been developed. The assay enables detection of immunoglobulin (Ig) M, IgG, IgA, or IgM, IgG, and IgA--antisperm antibodies in serum, cervical mucus, and seminal plasma samples. The reverse ELISA is more specific and sensitive than conventional ELISA in detecting human antisperm antibodies of different isotypes. Using this assay, statistically significant differences in levels of antibodies between infertile and fertile individuals were demonstrated in sera and in genital tract secretions. Studies with 143 infertile couples revealed that the presence of antibodies in sera was not necessarily reflected in individual's genital tract secretion and vice versa. These data emphasize the importance of detecting antisperm antibodies in sera as well as in genital tract secretions for correct evaluation of sperm immunity.     

ELISA法检测不育男子精浆中抗精子IgG和IgA

将精子经Tritonx－100处理，冷冻高速离心后，经抗人全血清-SephadexG－75亲和柱层析分离，提取分子量为59KD人精子膜蛋白作为抗原，经ELISA间接法对20例生育男子和50例不育男子精浆中抗精子IgG和IgA进行了测定。结果显示：生育男子精浆中抗精子IgG为阴性；抗精子IgA阳性率为5％。不育男子精浆中抗精子IgG和IgA阳性率分别为10％、30％；抗精子IgG和IgA均为阳性者3例，均为阴性者33例；抗精子IgG阳性而IgA阴性者2例；抗精子IgA阳性而IgG阴性者12例。不育组与生育组间抗精子IgG阳性率无显著性差异（P＞0．05）；而IgA阳性率间则有显著性差异（P＜0．05）。不育组抗精子IgG和IgA阳性率间有极显著性差异（P＜0．01）。本文利用ELISA法对精浆中抗精子抗体的分类及可能来源进行了讨论。     

Presence of antisperm antibodies in fertile and infertile persons.

The sera of 70 infertile and 30 fertile couples were tested for the presence of sperm agglutinins with Kibrick's macroagglutination technique and Boyden's haemagglutination test. 15.7% of the couples studied showed the presence of sperm agglutinins (by Kibrick's method) of which 5.7% were from males and 10.0% were from females. By the haemagglutination test, 13% of the couples studied were found to possess sperm agglutinins, of which 3% were from males and 10% were from females. 30 fertile men and women studied for sperm agglutinins were found to be negative by both methods. It was also observed that these two tests detected different types of sperm agglutinins. The cervical mucus samples from 45 females (15 fertile and 30 infertile) were tested for sperm agglutinins with a mucus penetration test. 23.1, 16.5, and 57.4% of the samples from infertile females, showed 0-degree, 1-degree and 2-degree penetration respectively. In case of samples from fertile females, 6.6, 13.2 and 79.2% showed 0-degree, 1-degree and 2-degree penetration respectively. Addition of serum from infertile females to cervical mucus from the infertile female increased the 0-degree penetration percent cases to 50%, as compared with 23.1 when only cervical mucus was used. Addition of serum from fertile females, or horse serum or pure albumin or globulins did not increase the 0-degree penetration cases.     

Macrophages and infertility: enhancement of human macrophage-mediated sperm killing by antisperm antibodies

The mechanism by which antisperm antibodies inhibit fertility is not completely understood. Macrophages may play a role in mediating infertility by interacting with sperm and destroying gametes. Experiments were conducted evaluating the effect of antisperm antibody on the phagocytosis and lysis of sperm by human peritoneal macrophages in vitro. Sperm from a fertile man treated with sera from normal men and women or medium alone had 5 to 280 molecules of IgG/sperm, as determined by a 125I-labeled anti-human IgG monoclonal antibody assay. By contrast, sperm treated with sera containing antisperm antibodies had 310 to 1240 molecules of IgG/sperm. Peritoneal macrophages harvested from infertile women with tubal/adhesive problems mediated phagocytosis and lysis of 111In-labeled sperm which was enhanced by treatment of the sperm with sera containing antisperm antibodies (39.0% +/- 1.5% versus 76.3% +/- 3.2% phagocytosis, and 3.3% +/- 0.3% versus 23.3% +/- 2.3% lysis of sperm [control versus antibody-treated]). The likelihood of fertilization in couples with antisperm antibody may be determined not only by the antibody but also by the presence of genital tract macrophages capable of destroying the antibody-coated sperm.     

Reconstruction of humoral response to sperm antigens in scid mice

OBJECTIVE AND DESIGN: Production of specific human antisperm antibodies by using human-SCID mice model with deposited peripheral blood lymphocytes. MATERIALS AND METHODS: Human peripheral blood lymphocytes (PBL's; CD8(+)-negative cell fraction) were grafted to the peritoneal cavity of severely-combined immunodeficient (SCID) mice at concentration of 20-35 x 10(6) cells per mouse. Lymphocytes were obtained from non-sensitized individual (to sperm antigen) and from in vivo primed males (vasectomized). Two sets of experiments were carried out, with 'native' (glycosylated) and enzymatically deglycosylated sperm antigenic extracts. In all applied variants, sperm antigens were administered with Complete and then with Incomplete Freund adjuvant to improve an immune response. RESULTS AND CONCLUSION: This approach allowed us to obtain better pronounced humoral antisperm response, specific to sperm deglycosylated antigens when PBL's were obtained from individuals in vivo sensitized to sperm (after vasectomy).     

Evaluation of the antisperm antibodies in infertile couples afflicted with a cervical factor

OBJECTIVES: The purpose of this study was to evaluate methods used for antisperm antibodies detection in infertility. METHODS: The studied cohort comprised 38 infertile couples with a distinct cervical factor. Presence of antisperm antibodies and their levels in circulation were evaluated in sera samples of both partners and also in the cervical mucus and semen with the Latex Agglutination test. Western Blotting was applied as an additional method in antibody detection. We also assessed: the number of sexual partners, potentially allergizing sexual behaviour and other potentially sensitising factors. RESULTS: The positive antisperm antibodies were detected merely twice and only in one case there was evidence of insemination-impeding antisperm humoral response. The Western Blotting method enabled us to obtain a reaction to a range of sperm proteins which reacted with antibodies both in serum and in seminal plasma. CONCLUSIONS: Determination of infertility on immunological grounds on the basis of a single determinant on sperm presents little diagnostic value. In our view, the combination of patient's clinical status with immune-system response to a spectrum of sperm antigens provides means of infertility evaluation. We propose Western Blotting as an useful technique for detection of antisperm antibodies.     

Characterisation of a sperm coating auto-antigen reacting with antisperm antibodies of infertile males using monoclonal antibodies

Objective In a previous study a number of sperm-specific antigens were identified which reacted with antisperm antibodies from both infertile and vasovasostomised males. To investigate the localisation and distribution of these antigens and their role in male fertility, monoclonal antibodies were raised against them; immunoblotting techniques were used to select only those antibodies which competed with human antisperm antibodies for these human auto-antigens.
Design One antibody, NW21, reacted with an 18 kDa auto-antigen present on epididymal sperm but absent from testicular sperm. Immunohistochemical studies showed that the antigen is produced in small basal cells between the columnar epithelium of the corpus epididymis, passes up into the tubule and then coats sperm passing along the epididymis. Sperm stored in the cauda epididymis and ductus deferens stain strongly for this sperm coating glycoprotein.
Conclusions The localisation of this antigen supports the suggestion that auto-immune infertility may represent a response to epididymal rather than testicular sperm. Monoclonal antibodies raised to unique and immunologically accessible sperm coating proteins, produced in the epididymis rather than in the testis, would seem to present an excellent theoretical solution to male contraception.     

Etiology of sperm immunity in women

Sperm immunity in females can reduce the likelihood of natural conception, and sperm antibodies from female sera have been shown to inhibit IVF in humans and in several animal models. The etiology of sperm immunity in human females is unknown, but several possible mechanisms have been proposed, including cross-reactivity with microbial antigens and interferon gamma-mediated potentiation of the antisperm immune response in women whose male partners have sperm autoantibodies in their semen. This article reviews these ideas and postulates a novel hypothesis based on the potential for the generation of anti-idiotype antibodies in women whose partners have sperm antibodies in their semen.     

The detection in human sera of antisperm antibodies reactive with FA-1, an evolutionarily conserved antigen, and with murine spermatozoa

Evolutionarily conserved antigens are present on spermatozoa of several mammalian species. We tested sera from infertile men and women containing antisperm antibodies (ASAs) for their reactivity with FA-1, an antigen known to be present on murine and human spermatozoa. Fifty percent of male sera and 63% of female sera contained anti-FA-1 antibodies, as judged by enzyme linked immunosorbent assay (ELISA). Fourteen percent of male sera and 50% of female sera were also shown to possess ASAs reactive with living mouse spermatozoa, and murine in vitro fertilization was inhibited by human antibodies. These results suggest that the transfer of immunoglobulins from human sera to spermatozoa of other species may provide a model to study how ASAs effect sperm function.     

Effect of sperm antibodies on pregnancy outcome in a subfertile population

The relationship between sperm antibodies, conception, and miscarriage was examined in 109 infertile couples. Antibodies present on the surface of husbands' ejaculated sperm and antibodies in husbands' or wives' sera that reacted with a purified population of the husbands' motile spermatozoa were detected by an enzyme-linked immunosorbent assay. During an 18-month period, conception occurred in 33 (30.3%) of the couples; 16 (14.7%) women subsequently suffered a spontaneous miscarriage during the first trimester, whereas 17 (15.6%) women maintained their pregnancies past this time period. Antisperm antibodies were present in sera from only two of 17 (11.8%) women with successful pregnancies, whereas seven of 16 (43.8%) women who miscarried and 29 of 76 (38.2%) who did not conceive had these antibodies in their sera. IgG (22.4%) and IgM (21.1%) antisperm antibodies predominated in sera of women who did not conceive, whereas IgA (37.5%) and IgG (37.5%) antibodies were most prevalent in sera of women with miscarriages. In men, the presence of antisperm antibodies in sera was unrelated to fertility. However, there was a correlation between sperm surface antibodies and an inability to conceive. IgG was identified on ejaculated spermatozoa from eight of 76 (10.5%) men whose wives failed to conceive and in none of 33 men whose wives conceived. Similarly, IgA was present on spermatozoa from 16 (21.1%) infertile and two (6.1%) fertile men. Thus antisperm antibodies in female sera and on ejaculated spermatozoa were associated with a failure to conceive and first-trimester miscarriage.     

Identification of the human sperm protein that interacts with sperm-immobilizing antibodies in the sera of infertile women.

OBJECTIVE: To identify the target antigen of sperm-immobilizing antibodies present in the circulation of infertile women. DESIGN: Laboratory research. SETTING: Academic research laboratory. PATIENT(S): Twenty-nine infertile women with sperm-immobilizing antibodies, 22 infertile women with other disorders, and 20 fertile women. INTERVENTION(S): Titers of antibodies to the sperm protein, rSMP-B, were determined by ELISA using as substrate the synthetic peptide segment (rSMP-230) that corresponds with the hydrophilic domain of rSMP-B. Tests for sperm immobilization and zona pellucida penetration were performed using the human IVF system. MAIN OUTCOME MEASURE(S): Human sera with sperm-immobilizing activity were assayed for the presence of antibodies to rSMP-230. Polyclonal antibodies to rSMP-230 were assessed for the same biologic activities as sperm-immobilizing antibodies. RESULT(S): Antibodies to rSMP-230 were detected in 10 (34%) of 29 sera obtained from women with immunologic infertility. In contrast, only one serum sample (2%) from women without sperm-immobilizing activity had a low titer of antibodies to rSMP-230. Polyclonal antibodies to rSMP-230 completely immobilized human sperm in the presence of complement and blocked sperm penetration across the zona pellucida. CONCLUSION(S): The human sperm protein, rSMP-B, probably is the target antigen of sperm-immobilizing antibodies.     

Detection of autoimmunity to sperm: mixed antiglobulin reaction (MAR) test or sperm agglutination? A study on 537 men from infertile couples

Two different ways of testing for antisperm antibodies were compared: the mixed antiglobulin reaction (MAR) test for demonstration of antibodies of the IgG and IgA classes bound in vivo to the sperm membrane antigens and the gelatin agglutination test for detection of nonbound antisperm antibodies in serum and seminal plasma. Samples from 537 men from infertile couples were investigated. Antibodies bound to the sperm membrane were detected in 49 men (9.1%), IgG in 44 (8.2%), and IgA in 38 cases (7.1%). Sperm agglutinins were recorded in seminal plasma from 30 men (5.6%) and in serum (titer greater than or equal to 16) from 43 men (8.0%). The investigation revealed a very close correlation between the results of MAR testing and the occurrence of sperm agglutinins in serum and seminal plasma. However, if one focuses on antisperm antibodies of the IgA class, which seem to play the major role in male immune infertility, the MAR test offered the advantage that a minor group of patients with pure IgG responses could be distinguished, and rare cases with mainly or exclusively locally produced IgA antibodies could be detected.