A radiolabeled antiglobulin assay to identify human cervical mucus immunoglobulin (Ig) A and IgG antisperm antibodies

Antisperm immunoglobulin (Ig) A and IgG antibodies in human cervical mucus (CM) were identified by a radiolabeled antiglobulin assay. Cervical mucus samples from fertile and infertile women were exposed to a 1:3,200 dilution of 2-mercaptoethanol (2-ME), and 5 micrograms of the solubilized CM protein were assayed for the presence of IgA and IgG antisperm and anti-Candida activity by the radiolabeled antiglobulin assay. Purified human secretory IgA and IgG exposed to 2-ME retained the molecular integrity and functional activity of the untreated antibody molecules. CM aliquots collected after high-performance liquid chromatography (HPLC) fractionation were assessed for antisperm antibody activity; antisperm antibody activity was retained in the appropriate IgA or IgG CM fractions. The incidence of CM antisperm antibodies was minimally affected when the radiolabeled antiglobulin assay was performed with a motile sperm population. Approximately 70% of the CM IgA antisperm antibodies were of the IgA1 subclass; CM IgG was primarily of the IgG4 subclass. When Candida antigen was substituted for sperm in the radiolabeled antiglobulin assay, the CM antisperm antibodies were found to be exclusively sperm-specific. These data indicate that the radiolabeled antiglobulin assay using 2-ME to extract CM antibodies is a specific method for the assay of antisperm antibodies in CM.     

Comparison of the indirect immunobead, radiolabeled, and immunofluorescence assays for immunoglobulin G serum antibodies to human sperm

The relative sensitivities of the indirect immunobead test, the indirect flo cytometric immunofluorescence assay, and an indirect radiolabeled antiglobulin assay were compared. Eighteen immunobead test positive sera and 18 negative sera were used as the standard for the other two assays. Of the 18 positive sera, 14 (77%) and 5 (27%) were positive in the immunofluorescence assay and the radiolabeled antiglobulin assay, respectively. Four (22%) of the low titer immunobead test positive sera were negative by both the immunofluorescence assay and the radiolabeled antiglobulin assay. However, there was a significant positive correlation between the results of the immunofluorescence assay and the radiolabeled antiglobulin assay (r = 0.73) and between the results of the radiolabeled antiglobulin assay and the titer of the immunobead test (r = 0.82). The use of an unselected sperm population in the radiolabeled antiglobulin assay and the classical indirect immunofluorescence method using methanol-fixed sperm gave false-positive results in the radiolabeled antiglobulin assay and the immunofluorescence assay. These results suggested that immunoglobulin G antisperm antibody positive sera may be reactive both to sperm surface and internalized sperm antigens.     

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.     

精浆内抗精子抗体和免疫复合物的研究——对男性不育患者及输精管粘堵术者的观察

本文用ELISA法研究了174名男性不育患者及43名输精管粘堵术后精浆中的抗精子抗体及其种类。结果表明,精浆中抗精子抗体的检出率,在男性不育患者中为24.1%,输精管粘堵术后者为48.8%。在这些患者的精浆中可检出IgA、IgG和IgM型抗精子抗体,其中主要是IgA。同组人群血清和精浆中的抗精子抗体缺乏相关性。精浆中抗精子抗体的存在与精子活动率的下降有一定的关系。应用PEG法测定精浆中免疫复合物,在66例精子抗体阳性的患者中,阳性率为56.1%,表明局部抗精子的自身免疫反应,在一定条件下可导致免疫复合物的形成。     

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.     

Identification of antibody-laden sperm by cytofluorometry

A direct assay for measuring sperm-associated immunoglobulin G (IgG) and an indirect assay for measuring IgG plasma antisperm antibodies were devised utilizing fluorescein-labeled antihuman IgG and a fluorescein-activated flow cytometer. The method singularly analyzed Ig attached to the surface of sperm because sperm and nonsperm cells were separately assessed. The direct assay technique results correlated with results from a previously reported direct radiolabeled antiglobulin assay. As few as 100,000 sperm are required in the indirect method; the sensitivity of the indirect technique corresponded with that of a previously reported indirect radiolabeled antiglobulin assay.     

Circulating antisperm antibodies in recurrently aborting women

One hundred seventy-three women with a history of three or more recurrent consecutive abortions were analyzed for circulating antisperm antibodies with a radiolabeled antiglobulin assay (RAA), a modified enzyme-linked immunosorbent assay (ELISA), a tray agglutination test (TAT), and a sperm immobilization test (SIT). No pregnancies were subsequently gestated to term in women who were antisperm antibody-positive unless they were inoculated with their husband's leukocytes as treatment for an immune basis (not related to antisperm antibodies) for their recurrent abortions. In women with an immune basis for their recurrent abortions, immunization with leukocytes from their male partners increased the ability of these women previously aborting their fetuses to carry their fetuses to term, even if they had positive results in the ELISA, TAT, and SIT; women with positive results in the RAA continued to abort subsequent pregnancies, despite leukocyte immunization. Immunization of antisperm antibody-positive women with their partner's leukocytes did not incite or increase the antisperm antibody titer, with any of the assay techniques.     

The MAR test as immunologic screening of male infertility

The mixed antiglobulin reaction was developed for detection of platelet antibody and then modified to demonstrate antibodies on spermatozoa. We have used the test as a simple method of screening for antibodies in the male, performing the test during the analysis of semen samples. We evaluated 320 samples from partners of sterile couples attending our Sterility centers. The IgG MAR test was not applicable to 82 samples because the sperm concentration and sperm motility was not sufficient. In all uncertain, positive and firmly positive cases of MAR test and in 50 negative cases of IgG MAR test, as control group we performed MAR test for IgA. We correlate our results with the sperm test penetration, SCMC test and identification of antisperm antibodies test and Isojima test for sperm immobilizing antibodies. Our results reconfirm the utility of IgG MAR test as screening test for the presence of antibodies but its positiveness requires the performance of IgA MAR test and, if necessary, of other tests in order to diagnose sterility depending on immunologic factors.     

Antisperm antibodies and fertility after vasovasostomy: a follow-up study of 216 men

A group of 216 vasovasostomized men were tested with the mixed antiglobulin reaction for immunoglobulin (Ig)G, IgA, and secretory IgA antisperm antibodies bound to the sperm membrane. Free antisperm antibodies in serum and seminal plasma were detected with the gelatin agglutination test and the tray agglutination test. The results were related to the conception rates. In a subgroup with a pure IgG response, the conception rate reached 85.7%, whereas only 42.9% of the men who also had IgA on the sperm induced pregnancy. When 100% of the spermatozoa were covered with IgA, the conception rate was reduced to 21.7%. The combination of IgA on all sperm and a strong immune response (titer in serum greater than or equal to 256) was associated with a conception rate of zero.     

An immunosorption assay for antibodies to spermatozoa: comparison with agglutination and immobilization tests

We have developed an enzyme-linked immunosorption assay (ELISA) with an antigen extract made by treatment of freeze-thawed sperm pools with 0.3 M lithium 3,5-diiodosalicylate (LIS). This procedure is quick, safe, and simple to perform in a clinical laboratory. When the LIS antigen preparation was compared by ELISA with whole sperm and several other antigen extracts, it was found to be superior in detecting human antisperm antibodies related to infertility; it gave less variability, better precision, and fewer false-positive reactions than whole sperm fixed to the wells as antigen. The antigen was stable when stored at -20 degrees C. In one experimental series, serum samples from 18 normal control subjects and 159 infertility patients with positive sperm-immobilizing titers, positive sperm-agglutinating titers, or both, were tested by ELISA with LIS antigen; the correlation yielded a P less than or equal to 0.0001. Based on another series of 156 samples from patients with positive antibody titers by the sperm agglutination test and 30 sera from normal humans, the predictive value of the test was 96%. In a third series of 162 serum samples obtained from 82 women and 80 men, we found no difference in the test results based on sex. The assay is specific for the antibody binding portion of the immunoglobulin. Absorption of positive sera with sperm but not red blood cells markedly reduces the response. In addition to being more efficient and precise than standard methods, the ELISA eliminates the need for fresh sperm and provides data that can be objectively interpreted.     

Relationship between local and systemic autoantibodies to sperm, and evaluation of immunobead test for sperm surface antibodies

Immunobead test (IBT), mixed antiglobulin reaction test for IgG (IgG-MART) and tray agglutination test (TAT) were used to study sperm antibodies in semen and serum of 200 men in infertile partnerships. The value of the IBT was also assessed. Semen from 22 men had insufficient number of motile sperm and were unsuitable for IBT and IgG-MART. Twenty-four men (of 178 that had all tests done) had significant serum antibody titres; sperm surface IgG and IgA were detected by IBT in 23 of them. IgG-MART was also positive in all but 4 of these 23; these 4 had positive TAT in seminal plasma and serum, but the titres were never above 64. Semen from 3 men was found to have only IgA sperm antibodies; 2 of these 3 had negative serum TAT. All tests were negative in 30 fertile men used as controls. We conclude that IBT is a useful test and is more sensitive than IgG-MART at low antibody titres, local autoimmune response to sperm can occur without a systemic counterpart, IgA sperm antibodies can occur alone (i.e. without IgG) in semen.     

Sperm antibodies and human in vitro fertilization

In order to directly evaluate the effects of sperm antibodies in human in vitro fertilization (IVF), the authors preincubated donor sperm in female sera containing sperm antibodies and then inseminated supernumerary human oocytes from a gamete intrafallopian transfer (GIFT) program. The sperm were incubated for 30 minutes in medium containing 20% serum with antisperm activity (Test); or no antisperm activity (Control) as assessed by the immunobead test (IBT). Each oocyte was inseminated with 1 to 2 X 10(5)/ml of the preincubated motile sperm with Control or Test treatments allocated on a random basis. Six positive sera were tested in 17 experiments, resulting in a fertilization rate of 41% (25/61) versus 84% (36/43) for controls (P less than 0.001). When considered individually, three of six positive sera caused significant inhibition. The only serum that gave complete inhibition had the highest titer for IgG (10,000) and lower IgA (100). Absorption with protein A reduced the IgG titer to less than 10 and removed the fertilization inhibitory activity. These results confirm that sperm antibodies from female sera can inhibit human IVF.     

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.     

Assessment by fluorescence-activated cell sorting of whether sperm-associated immunoglobulin (Ig)G and IgA occur on the same sperm population.

Two-color fluorescence-activated cell sorting of antisperm antibody-positive sperm was used to detect simultaneously the presence of immunoglobulin (Ig)A and IgG antisperm antibodies associated in vivo on a man's sperm. Sperm positive for sperm-associated Ig were analyzed using phycoerythrin-conjugated antihuman IgA and fluorescein isothiocyanate-conjugated antihuman IgG; up to 87% of the same spermatozoa were stained with both labels. Sperm positive for only one of the antisperm antibody isotypes stained up to 90% of a man's sperm with only one fluorochrome. Immunocytochemistry studies revealed similar patterns of sperm binding for sperm-associated IgG and IgA. These results suggest that the sperm antigenic determinants reacting with antisperm IgA and IgG are present on the same sperm population at similar locations on the sperm surface.     

A comparison of sperm agglutination and immobilization assays with a quantitative ELISA for anti-sperm antibody in serum

An enzyme-linked immunosorbent assay (ELISA) that quantitates antisperm antibody in serum was compared with standard sperm agglutination and immobilization assays with the use of sera from 40 normal and 292 subfertile individuals. Quantitation of the assay was accomplished by standardizing assay parameters, including the incorporation of a standard reference curve, the number of whole target sperm, the optimal dilution of serum, the selection of microtiter plate, and the time and temperatures involved in the adsorption and incubation phases. With this method, the level of antisperm antibody binding to target sperm in 40 normal fertile individuals was found to be 2.3 (+/- 1.1 standard deviation [SD]) fg immunoglobulin (Ig)/sperm. An increased mean level of 7.4 +/- 3.7 fg Ig/sperm was determined in 84 infertile patients with positive agglutination and/or immobilization tests. In 208 individuals with negative agglutination and immobilization tests the mean concentration of antisperm antibody was 2.5 +/- 1.3 fg Ig/sperm. Postvasectomy patients assayed by this method had a mean Ig binding value of 7.1 +/- 2.4 fg Ig/sperm. The infertile group with positive agglutination and/or immobilization tests had a significantly higher mean antisperm antibody level than the normal fertile group, according to the Student's t-test for independent samples (P less than 0.001). This indirect serum-based assay reproducibly quantitates antisperm antibody binding to whole target sperm, suggests the normal and abnormal levels of antisperm antibody, and correlates with standard functional assays.     

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.     

Antisperm antibodies in infertile and homosexual men: relationship to serologic and clinical findings

The incidence and significance of antisperm antibodies in different groups of men were evaluated by a modified enzyme-linked immunosorbent assay. In serum, 4.0% of dermatologic patients (n = 223), 9.6% of andrologic patients (n = 178), and 28.6% of homosexual men (n = 42) were positive for IgG and/or IgM antibodies. In seminal fluids, 7.3% of the andrologic patients had IgA (and IgG) antibodies to spermatozoa. Only 1 of 29 positive men had antibodies both in serum and in seminal fluid. No correlation between antisperm antibodies and IgG/IgM concentrations was found in serum, whereas in seminal plasma men with antisperm antibodies showed higher IgG/IgA concentrations than men without (IgA, 3.2 versus 1.7 mg/dl; IgG, 9.8 versus 6.3 mg/dl). It is concluded that there is a high incidence of antisperm antibodies among homosexual men, probably because of contact of spermatozoa with the immune system by passive anal intercourse. There is little correlation between antisperm antibodies in serum and seminal plasma of infertile men because of a lack of relevant antibody transfer from the serum and the formation of local antibodies in seminal plasma. Antisperm antibodies in seminal fluid are associated with elevated local IgG and IgA concentrations.     

Evaluation of a simplified adenosine triphosphate release cytotoxicity test for the detection of sperm antibodies in human serum

The adenosine triphosphate release cytotoxicity test (ARCT) is an objective method for the detection of cytotoxic sperm antibodies in serum providing measurements without the bias of microscopic estimation. The procedure has been simplified by testing only one serum dilution and calculating the "sperm toxicity index". The latter is closely correlated with the sperm cytotoxic titer. Immunoglobulin class determination of 32 sera with sperm cytotoxic activity was performed by means of the indirect mixed antiglobulin reaction (MAR test) and the indirect immunobead (IB) test, and revealed that all sera were positive for IgG, 17% for IgM and 20% for IgA. Of 32 sera without sperm cytotoxic activity, 19% contained sperm specific IgG, 6% had IgM class antibodies belong mainly to the IgG class since the results of the ARCT and the indirect MAR test for IgG are closely correlated. The ARCT appears to be useful as a simple and objective method for the detection and quantitation of cytotoxic antisperm antibodies.     

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.     

Inhibition of sperm-zona pellucida tight binding by sperm immobilizing antibodies as assessed by the hemizona assay (HZA)

Researchers collected serum samples from 23 infertile patients with sperm immobilizing antibodies (SI-Ab) and 1 pregnant patient from the Department of Obstetrics and Gynecology at the Hyogo Medical College in Japan to screen sera to determine whether they contained factors to inhibit sperm-zona pellucida tight binding. They used the recently developed hemizona assay (HZA) to test for this binding. The HZA assay showed that all 23 serum samples inhibited sperm-zona pellucida tight binding. The hemizona index (HZI) ranged from 3-53 with a mean of 18.1 (standard deviation of = or - 12) compared to a normal (HZI) of 100. Serum samples with titers 10 of SI50 inhibited sperm-zona binding as well as those with titers -or= 10 of SI50 (HZIs=17.3 vs. 18.9; p.1). All 23 serum samples bound to the surface of sperm plasma membrane after 1 hour coincubation as evidenced by the fact that they all demonstrated 50% IgG beads bound. Further the results of the indirect immunobead test (I-IBT) showed that positive sera (+or= 20% IgG beads) significantly inhibited binding more than negative sera (20% IgG beads bound) (HZIs=12.4 vs. 24.4; p.05). Yet serum with positive I-IBT for IgM did not affect sperm-zona binding (HZIs=17.1 vs. 19.4; p.1). No association existed between HZI and site of IB binding. The researchers interpreted theses results to mean that sera with both SI-Ab and antibodies recognized I-IBT for IgG and IgA may play a significant role to inhibit the sperm-zona pellucida tight binding. In conclusion, physicians should expect patients with low HZI to have more problems conceiving than those with normal HZI. In vitro fertilization using heat inactivated human cord serum or donor serum may help them to conceive.