The inhibitory effect of sperm-associated immunoglobulins on cervical mucus penetration.
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Cervix Mucus↗
Biomedical subjects
G G Haas
Publications and source records attributed to G G Haas.
Immunologic influences on reproductive biology: sperm gametogenesis and maturation in the male and female genital tracts.
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Animals↗
Failure to identify HLA ABC and Dr antigens on human sperm.
An indirect radiolabeled antiglobulin assay (IRAA), adsorption procedures, and an indirect immunofluorescent assay using cytofluorometry (IFAA) failed to identify HLA ABC or D/Dr antigens associated with untreated human sperm or human sperm exposed to capacitating conditions. Two monoclonal clones against HLA ABC and two clones against Dr antigens were utilized. Test results in the IRAA were not different regardless of whether sperm from fertile men or human red blood cells were employed in the IRAA. However, there were highly significant differences in the IRAA between the HLA antigen expression of human sperm and leukocytes. If the antihuman HLA monoclonal antibody was preadsorbed with 25 X 10(6) sperm, there was no diminution in the ability of the adsorbed antibody to identify HLA antigens on leukocytes; however, preadsorption with 2.5 X 10(6) leukocytes reduced the activity of the monoclonal antihuman HLA antibody to levels equivalent to background activity.
Adult↗
Antibodies to spermatozoa and seminal plasma antigens detected by various enzyme-linked immunosorbent (ELISA) assays.
Various ELISA methods have been applied by different research centers to test the efficiency of this approach for the diagnosis of sperm-immune infertility cases. The antigens used were either whole spermatozoa or solubilized spermatozoal membrane preparations and were immobilized on microtiter plates, except in one case where plastic beads were employed. Polyvalent second antibodies or protein-A labelled with enzymes served as tracers. A high frequency of positive sera was found in all groups including fertile controls with tests using whole spermatozoa as antigen. The methods using solubilized antigen preparations showed fewer positives on the whole and correlated better with the various clinical categories of the WHO sera. Whilst there was some agreement in the results between the various laboratories on a few sera, most of the positive sera found by one laboratory were reported as negative by others. More investigative work is needed to improve reproducibility between different laboratories and to reduce non-specific reactions with normal controls. A more precise definition of the proper cut-off levels for positives and negatives is also needed. Despite these short-comings, the development of an ELISA for the diagnosis of sperm-immune infertility cases seems to be justified in the long term.
Antibodies↗
The effect of immunoglobulin occurring on human sperm in vivo on the human sperm/hamster ova penetration assay.
We selected 91 infertile men who were tested for increased sperm-associated immunoglobulin and also tested in the human sperm/hamster ova penetration assay. There was a statistically significant association between the presence of increased sperm-associated IgG alone (p = 0.0218) and both sperm-associated IgG and A (p = 0.0187) when correlated with the failure to penetrate any hamster ova. There was a trend but no statistical significance when sperm-associated immunoglobulin A alone was present. There was a trend but no statistical relationship between the presence of sperm-associated immunoglobulin and the sperm penetration assay when the criteria for normality of the sperm penetration assay was a 15% or greater ovum penetration rate.
Animals↗
Clarifying antibody-mediated infertility.
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Antibodies↗
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.
Flow Cytometry↗
The effect of freezing on sperm-associated immunoglobulin G (IgG).
Previously frozen and fresh sperm from infertile and fertile men were assayed for sperm-associated immunoglobulin G (IgG) to determine the effect of a simple freezing technique on the assay results. A direct radiolabeled antiglobulin assay (DRAA) and a direct fluorescein-labeled antiglobulin assay (DFAA) utilizing a fluorescein-activated flow cytometer were employed. The amount of IgG detected by the DRAA and the percentage of sperm that fluoresced in the DFAA generally declined when frozen sperm were used, but only the DFAA showed a statistically significant decrease. However, the degree of decline was predictable (r = 0.91 in both assays), and whether the test remained positive or negative when frozen sperm were used was not significantly affected.
Flow Cytometry↗
The incidence of sperm-associated immunoglobulin and C3, the third component of complement, in infertile men.
One hundred four consecutive men who underwent a semen analysis as part of their infertility evaluation were assayed for sperm-associated immunoglobulin (Ig) and the third component of complement (C3). Sixteen (15%) of the 104 men had increased IgG, and 10 (12%) of 86 men tested had increased IgA on their sperm; however, only 4 (5%) of the 87 men tested were positive for plasma IgG antisperm antibody. Only 2 (13%) of the 16 men with increased sperm-associated IgG had detectable IgG antisperm antibodies in their seminal plasma. Five (10%) of 50 men tested had increased sperm-associated C3; none had seminal plasma that deposited C3 on sperm. These data suggest that of the three compartments that can be tested--circulating plasma, seminal plasma, and the sperm surface--the direct measurement of sperm surface Ig may be most important in the evaluation of infertile men. The presence of sperm-associated IgG or IgA was not associated with semen abnormality. In contrast, the presence of sperm-associated IgG was associated with decreased mucus penetration at the time of postcoital testing despite normal-appearing mucus.
Complement C3↗
Identification of antisperm antibodies on sperm of infertile men.
An indirect radiolabeled antiglobulin test for plasma antisperm antibodies was modified so that sperm-associated antibodies could be identified. Ten of 16 (63%) infertile men with plasma IgG antisperm antibodies were found to have an elevated sperm-associated IgG. Two of these men with an elevated sperm-associated IgG also had an elevated sperm-associated IGA; one other man with plasma IgG antisperm antibodies, but no detectable sperm-associated IgG, was noted to have an elevated sperm-associated IgA. Eighteen infertile men with a history that suggested antibody-mediated infertility did not have plasma IgG antisperm antibodies; one (6%) of these men was found to have an elevated sperm-associated IgG; none of these 18 men were found to have an elevated sperm-associated IgA. No patient was found to have sperm-associated IgM.
Antibodies↗
Antibodies to sperm-associated antigens detected by solid phase assays.
Solid phase assays using radiolabeled (RIA) or enzyme-linked (ELISA) antibodies are described for the quantitation of antibodies to sperm-associated antigens. Antibodies were detected in serum from rabbits immunized with human sperm emulsified in Freund's complete adjuvant and in the plasmas or sera of vasectomized males and patients with suspected antibody-mediated infertility. The RIA and ELISA assays are comparable to each other in sensitivity and specificity, rapid in execution, and inexpensive.
Animals↗
Immunologic infertility: identification of patients with antisperm antibody.
Immunologic causes of infertility have been postulated for many years, but a precise means for identification of men or women with antibodies against sperm has not been available. We studied 614 patients, including 257 couples, with unexplained in fertility, to detect the presence of circulating antisperm antibody. Using a specific radiolabeled-antiglobulin test, we identified a subset of patients (10 per cent) with IgG antisperm antibody; this antibody was present in 13 per cent of the women and 7 per cent of the men. We followed the course of six infertile couples in whom one partner had sperm antibody. In four couples, pregnancy was achieved; pregnancy correlated with resolution of antibody activity. In two of these four couples the partner (one man and one woman) with antisperm activity had been pretreated with corticosteroids. We conclude that this objective test may be used to identify and then to help manage infertility in patients with suspected antibody-mediated infertility.
Adult↗
The interfering effect of human IgG antisperm antibodies on human sperm penetration of zona-free hamster eggs.
A liquid-phase radioimmunoassay was used to quantitate sperm-associated IgG in a population of patients with unexplained infertility. Plasma IgG antisperm antibody activity was identified in a subpopulation of these patients. Using the human sperm/hamster egg assay, a unique functional test of human sperm penetrating capacity, we identified a subset of these positive patients whose plasma significantly and reproducibly inhibited penetration. This study implies that antigen-antibody reactions may prevent sperm responses that are critical to sperm-egg fusion.
Animals↗
Effect of repeated washing on sperm-bound immunoglobulin G.
The effect of sperm washing on the stability of sperm-bound immunoglobulin G (IgG) antibody derived from plasma from four patients and also IgG bound in vivo on the spermatozoa of four other men was quantitatively evaluated. In the first series of experiments, human spermatozoa were incubated with an IgG antibody-containing plasma and subjected to 18 cycles of sperm washing. In the second set of experiments, spermatozoa from men positive for sperm-bound IgG were subjected to four cycles of sperm washing. The amount of residual antibody bound to a constant number of spermatozoa was quantitated by a radiolabeled antiglobulin assay during and following the washing procedures. There was no significant loss of sperm-bound antibody due to the washing procedures. The results of these studies undermine the utility of sperm washing as an effective treatment of antibody-mediated infertility in men.
Fluorescent Antibody Technique↗
Comparison of the direct radiolabeled antiglobulin assay and the direct immunobead binding test for detection of sperm-associated antibodies.
Thirty-seven semen samples were assayed for sperm-associated IgG and IgA using the immunobead test. Portions of these sperm samples were sent for testing with a direct radiolabeled antiglobulin assay and the testing results were compared. If the results of the immunobead test when only tail-tip bead binding was noted are regarded as negative, there was close correlation between the two assay methodologies.
Globulins↗
Effect of sperm-associated antibodies on the acrosomal status of human sperm.
The acrosomal status of human sperm was assessed by the specific binding of Pisum sativum lectin to the acrosomal matrix. Immunoglobulin G (IgG) fractions of plasmas that were positive for IgG antisperm antibodies inhibited acrosomal loss, initiated acrosomal loss, or had no effect on acrosomal loss. Two of five sperm samples associated in vitro with only IgG, zero of one sample associated with only sperm-associated immunoglobulin A (IgA), and six of eight samples associated with both IgA and IgG underwent acrosomal loss prior to exposure to calcium ionophore. Two sperm samples associated with IgG or IgA or both were inhibited from undergoing acrosome loss after exposure to calcium ionophore. None of the seven antibody-negative sperm samples underwent an increased spontaneous acrosomal loss or were inhibited from undergoing acrosomal loss after exposure to calcium ionophore.
Acrosome↗
Sperm immobilizing activity of a synthetic bioactive peptide 20-44 of 37-kDa cationic antimicrobial protein (CAP37) of human neutrophils.
We have previously reported that human sperm coincubated with human peripheral blood neutrophils in the presence of complement (C)-fixing antisperm antibody (ASA)-positive sera are rapidly internalized and degraded within the neutrophil phagolysosome. However, the mechanism by which motile sperm are processed within the phagolysosome is unknown. Various spermicidal/antimicrobial proteins contained in azurophilic granules that can be secreted into the phagolysosome may play a role in sperm disposal. In this study, we examined the expression of a 37-kDa cationic antimicrobial protein (CAP37) during sperm phagocytosis and the effect of its synthetic bioactive fragment, Peptide 20-44 (P20-44), on sperm motility, acrosomal integrity, and mitochondrial functionality. CAP37 expression by neutrophils undergoing ASA- and C-dependent sperm phagocytosis was increased as measured by flow cytometry. Exposure of motile sperm to a cationic P20-44, the bioactive antimicrobial fragment of CAP37, resulted in the loss of sperm motility without disruption of the acrosomal membrane. The sperm immobilizing activity (SIA) of P20-44 was modulated by the length of incubation, the concentration of the peptide, and the pH of the assay medium. SIA induced by P20-44 was partially reversible and was unaffected by the presence of anionic heparin or seminal plasma. Similar to the antimicrobial activity of P20-44, the SIA was also dependent on the presence of a disulfide bond between cysteine residues at positions 26 and 42 and was inhibited by Lipid A. However, the mechanism of action of P20-44 on sperm is not totally dependent on the molecule's cationicity, because five other cationic antimicrobial peptides had no detectable effect on sperm viability. Thus, the mechanism of action of P20-44 on human sperm is different from its cationic antibactericidal effect. These findings established that motile human sperm are sensitive to CAP37 or its synthetic bioactive peptide and suggested that this protein could play a role in neutrophil-mediated immune destruction of sperm in the female genital tract. P20-44 of CAP37 may be useful in investigating the regulation of human sperm motility and to construct "hybrid peptides" with enhanced potency as a component of vaginal contraceptive that could doubly be effective by killing infectious agents and inhibiting sperm transport.
Antimicrobial Cationic Peptides↗