Expression of CD15 (Lewisx) antigen on human sperm and its role in sperm-egg interaction.

PROBLEM: The carbohydrate epitope 3-fucosyl-N-acetyllactosamine (CD15) is a constituent of cell surface glycoconjugates that has been implicated in cell-cell adhesion mediated by carbohydrate-specific ligands. The present study was designed to investigate whether CD15 is present on human sperm and whether it plays a role in human sperm-egg interaction. METHODS: Fluorescent flow cytometry was used to quantitate the binding of monoclonal antibodies (mAb) to sperm-bound CD15 and CD46 antigens on acrosome-intact (AI) and acrosome-reacted (AR) sperm. The location of the binding site of these mAbs was assessed by fluorescence microscopy. The effects of anti-CD15 and anti-CD46 mAbs on gamete interaction were tested utilizing both homologous (human zona binding and penetration) and heterologous (zona-free hamster egg binding and penetration) systems. RESULTS: The mean percentage of capacitated sperm which bound anti-CD15 or anti-CD46 mAbs was low (4.8% and 5.1%, respectively). Exposure to calcium ionophore A23187 (CaI) resulted in an increase in anti-CD15 (38.6 +/- 4%) and anti-CD46 (83.4 +/- 2%) binding to sperm. Both anti-CD15 and anti-CD46 binding sites were localized by fluorescence microscopy on the sperm acrosomal region. In four experiments, the percent of zona-free hamster eggs penetrated by human sperm were medium control 93% (62/66), irrelevant mAb 74% (70/94), anti-CD46 0% (0/107), and anti-CD15 10% (9/90). One hundred percent (6/6) of human zona were penetrated by human sperm exposed to medium control, 88% (8/9) following exposure to irrelevant mAb, 0% (0/11) following exposure to anti-CD46, and 50% (5/10) following exposure to anti-CD15. The mean (+/- SD of tightly bound sperm to hamster eggs were medium control 57 +/- 18%, irrelevant mAb 64 +/- 16%, anti-CD46 37 +/- 13%, and anti-CD15 19 +/- 10%. The corresponding values for human zona were: medium control 118 +/- 14%, irrelevant mAb 61 +/- 11%, anti-CD46 39 +/- 18%, and anti-CD15 99 +/- 19%. CONCLUSION: CD15 antigen is expressed on human sperm that have undergone acrosomal loss. mAb to CD15 was shown to inhibit significantly sperm binding and penetration of zona-free hamster eggs and penetration of human zona pellucida. These findings suggested that sperm-egg interaction may be mediated in part by the CD15 antigen. Capsule: Acrosome-reacted human sperm bind monoclonal antibodies specific for CD15 (Lewis(x)) epitope. The binding sites were located on the sperm head. Anti-CD15 antibody impaired both the binding and penetration of zona-free hamster eggs and the penetration of human zona by human sperm.

Antibodies to carbonic anhydrase in endometriosis: prevalence, specificity, and relationship to clinical and laboratory parameters.

OBJECTIVE: To investigate the presence and clinical association of serum autoantibodies to carbonic anhydrase (CA) in women with and without endometriosis. DESIGN: Sera were tested in an ELISA against human and bovine CAI and/or CAII isoenzymes and by Western immunoblotting of trypsin-digested fragments of human CAII as antigens. The ELISA positivity was defined as mean + 2 SD of 100 control sera. Positive sera also were tested for the presence of antiendometrial antibodies and antinuclear antibodies (ANA) by indirect immunofluorescence assays (IFA) on endometrial (ECC) and HEp-2 cells, antibodies to single-stranded (ss) and double-stranded (ds) DNA by the Farr-type RIA and Crithidia IFA, and extractable nuclear antigens (Sm, nRNP, Ro, and La) by an ELISA. PATIENTS: Sera from 319 patients with laparoscopic diagnosed pelvic endometriosis (100 stage I, 95 stage II, 67 stage III, and 57 stage IV), 100 with other gynecologic disorders, and 100 control women were used. RESULTS: In the ELISA, 113 of 319 (35.4%) endometriosis sera had elevated immunoglobulin G antibodies against nondenatured CA isoenzymes. The reactivity of sera from the endometriosis group was significantly higher (35%) in all four subgroups of patients than each of the nonendometriosis sera (< 12% and < 6%, respectively). No stage-dependent variation of an autoantibody pattern was evident. However, anti-CA autoantibodies were present in 66.3% of women with endometriosis-associated infertility. The frequency of anti-CA autoantibodies was significantly higher (by 51.7%) in women with antiendometrial antibodies detectable by IFA. In addition, in sera positive for anti-CA antibodies, the frequency of ANA also was increased (20/113 [17.6%]) with titers of 1:40 to 1:1,080. The ANA-positive sera were negative for anti-ssDNA, anti-dsDNA, anti-Sm, anti-nRNP, and anti-La. However, three sera were positive for anti-Ro antibodies. Immunoblotting study of autoantibody reactivity with trypsin-digested subfragments of human CAII revealed consistent immunoreactivity with 14 to 6.2-kd range CAII peptides. CONCLUSIONS: [1] A subgroup of patients with endometriosis have autoantibodies directed to native and linear epitopes of the CA protein. [2] Prevalence of anti-CA antibodies was associated with antiendometrial antibodies and ANA. [3] Anti-CA antibodies were associated with a higher predictive value of the disease when all patient subgroups were considered together.

Recombinant soluble human complement receptor type 1 inhibits antisperm antibody- and neutrophil-mediated injury to human sperm.

The pathogenesis of antisperm antibody (ASA)-mediated infertility is postulated to be related in part to complement (C)-dependent sperm dysfunction in the female genital tract. We have previously demonstrated that C can be involved in ASA-mediated sperm injury by the deposition of activated C3 fragments and the assembly of terminal membrane attack complex (C5b-9) leading to C3-mediated sperm binding to neutrophils or C5b-9-mediated sperm motility loss. This study evaluated the protective effect of recombinant soluble C receptor type 1 (sCR1) on ASA-and C-mediated neutrophil/sperm interaction, neutrophil aggregation, and sperm motility loss. Motile sperm with or without neutrophils were incubated in the presence of 10% C-fixing ASA+ serum or ASA- control sera in the presence or absence of sCR1. After defined incubation periods, the following neutrophil and sperm parameters were evaluated: 1) neutrophil aggregation (by the flow cytometric pulse processing method), 2) sperm phagocytosis (by light microscopy), 3) the deposition of C3 cleavage fragments (C3b, iC3b, and C3d) on motile sperm (by immunofluorescence flow cytometry), and 4) the relation between sperm motility loss and sperm-bound C3d. Only the coincubation of neutrophils with sperm in the presence of C-fixing ASA+ sera resulted in marked neutrophil aggregation (20.5 +/- 0.26% vs. 2.4% +/- 1.6; p < 0.0001) and a concomitant increase in neutrophils containing ingested sperm (71 +/- 5.8% vs. 3.5%; p < 0.0001). Soluble CR1 inhibited ASA- and C-mediated neutrophil aggregation by 46% and sperm phagocytosis by 57%. Motile sperm incubated with C-fixing ASA- sera showed a time-dependent increase in the binding of C3 fragments as detected by flow cytometry using anti-iC3b neoantigen, anti-C3c, and anti-C3d monoclonal antibodies (mAbs). A negative correlation (r2 = -0.930; p < 0.001) was found between the increase in sperm-associated C3d fluorescence and the percentage motile sperm in the presence of ASA- sera. Soluble CR1 (200 micrograms/ml) maximally inhibited the binding of anti-C3b, anti-C3c, and anti-C3d mAbs to sperm by 96%, 83%, and 72%, respectively. Thus, sCR1 abrogated the binding of C3 fragments to human sperm and fully protected sperm from C5b-9-mediated sperm immobilization. These findings suggested the therapeutic potential of sCR1 as an intravaginal pharmacophore to prevent C-dependent sperm dysfunction and related inflammatory events in the female genital tract.

Fluorescence-labeled fucolectins are superior markers for flow cytometric quantitation of the human sperm acrosome reaction.

OBJECTIVE: To evaluate the binding of three fluorescein (FITC)-labeled fucose-specific lectins, Anguilla anguilla agglutinin (AAA), Tetragonolobus purpureas agglutinin (TPA), and Ulex europaeus-1 agglutinin (UEA-1), to unfixed, acrosome-intact and acrosome-reacted (AR) human sperm by flow cytometry and to compare the results with those found using FITC-labeled Pisum sativum agglutinin (PSA) and Arachis hypogaea agglutinin (PNA). DESIGN: The binding of five FITC-labeled lectins (PSA, PNA, AAA, TPA, and UEA-1) to capacitated, calcium ionophore A23187 (CaI)-treated, or solvent-treated human sperm was quantitated by fluorescence flow cytometry. The binding of FITC-labeled lectins was compared with the binding of anti-CD46 monoclonal antibody (mAb), a marker for the human sperm AR. The effect of fucose alpha-1->2-, alpha-1->3-, and alpha-1->4-linked oligosaccharides to inhibit the binding of FITC-fucolectins to AR sperm also was tested. SETTING: University of Oklahoma Health Sciences Center, a tertiary care referral center. RESULTS: The average percentage of fluorescing, solvent-treated sperm labeled with PSA, PNA, AAA, TPA, or UEA-1 was 98 percent, 97 percent, 15 percent, 13 percent, and 17 percent, respectively. The corresponding values for CaI-treated, lectin-labeled sperm were 98 percent, 98 percent, 89 percent, 91 percent, and 92 percent, respectively. The increase in mean fluorescence intensity for the binding of the five lectins to CaI-treated versus solvent-treated sperm was 2.9-, 6.4-, 34.5-, 22-, and 36.7-fold, respectively. The binding site of the fucolectins was confined to the equatorial segment of the AR sperm. High positive correlations were observed between the percentage of AR sperm detected using three FITC-labeled fucolectins (AAA, TPA, and UEA-1) and anti-CD46 mAb (r2 = 0.87, 0.99, and 0.99, respectively). Fucolectin binding to AR sperm was sensitive to competitive inhibition by fucose alpha-1->2-linked lacto-N-fucopentaose I and fucoidan. CONCLUSIONS: Fucosylated glycans are expressed on AR human sperm. Fluorescence-labeled fucolectins markedly improved the signal:noise ratio in the detection of acrosomal loss of human sperm when compared with FITC-PSA or FITC-PNA. Fluorescence-labeled fucolectins can be used as specific markers for flow cytometric quantitation of unfixed AR sperm in suspension.

Beta 2-integrin (CD11b/CD18) is the primary adhesive glycoprotein complex involved in neutrophil-mediated immune injury to human sperm.

The pathogenesis of antisperm antibody (ASA)-mediated infertility is postulated to be related in part to complement (C)-dependent neutrophil-mediated injury to sperm in the female genital tract. We have reported that sperm-bound IgG activated human C and deposited C3 fragments on motile sperm. We also demonstrated that IgG and C3-bound motile sperm adhered to human neutrophils in vitro, and that this adhesion potentiated the localized release of oxygen radicals at the site of neutrophil/sperm membrane contact. The goal of the present study was to identify the neutrophil surface receptor(s) involved in neutrophil/sperm adhesion and to evaluate their relevance to the pathogenesis of neutrophil-mediated immune injury to sperm. Neutrophils were coincubated with motile sperm in the presence of C-fixing ASA+ sera or control sera. After defined incubation periods, the following neutrophil variables were evaluated: 1) surface expression of Fc (Fc gamma RII and Fc gamma RIII) and C receptors (CR1[CD35], CR3 [CD11b/CD18], and CR4 [CD11c/CD18]) by flow cytometry, 2) neutrophil aggregation by flow cytometry, 3) tyrosine phosphorylation of neutrophil proteins by flow cytometry, and 4) the immune adherence and ingestion of sperm by light and scanning electron microscopy (SEM). The functionality of adhesion receptors was studied by use of a panel of anti-leukocyte monoclonal antibodies (mAbs) for their ability to block neutrophil/sperm adhesion and neutrophil aggregation. Only the incubation of neutrophils and sperm in the presence of C-fixing ASA+ sera resulted in marked (> 70%) sperm binding to neutrophils. Consistent with this pattern, a significant (p < 0.0001) increase in surface expression of neutrophil CD11b and neutrophil aggregation was evident. According to SEM, most of the sperm were linked to the neutrophil by the acrosomal region of sperm head. Maximum expression of CD11b antigen was obtained when neutrophils were coincubated with sperm in the presence of C-fixing ASA+ sera. CD11b up-regulation correlated with a significant (p < 0.05) increase in tyrosine phosphorylation of neutrophil proteins during sperm phagocytosis. Only the combination of mAbs directed to the beta 2-integrin, CD11b (D12/SHCL-3), or CD18 (MHM23) subunits maximally inhibited ASA- and C-mediated sperm adhesion to neutrophils (by 70%) or sperm phagocytosis (by 75%), as well as neutrophil aggregation (by 96%). These findings strongly implicate the CD11b/CD18 glycoprotein complex (CR3) in the adhesive events involved in ASA- and C-mediated immune destruction of motile sperm by neutrophils.

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.

Protection of sperm from isoimmune attack in vivo by pretreatment with antisperm Fab: fertility trials in the immune rabbit model.

We tested the hypothesis that binding of rabbit isoimmune antisperm Fab fragments to rabbit sperm prior to artificial insemination could protect the sperm from isoimmune attack in vivo and lead to normal pregnancy and healthy offspring in isoimmune does. Twenty-four female rabbits and 4 males were used. Twelve does were immunized with rabbit sperm to induce isoimmunity and 12 does immunized with adjuvant/saline served as controls. Following 5 immunizations, the immune serum IgG fraction and its Fab fragments were prepared. The does from the control group and isoimmune group were artificially inseminated following the 6th and 7th immunization with untreated sperm or sperm pretreated with antisperm IgG or Fab in quadruplicates and allowed to complete a pregnancy. Three fertility trials were performed to investigate the therapeutic efficacy of Fab-coated sperm to restore fertility in isoimmune does. In the 3 fertility trials, none of the isoimmune does inseminated with untreated sperm had a successful pregnancy. By contrast, in 3 cases, rabbit sperm pretreated with antisperm Fab were able to fertilize in vivo with successful pregnancy in a control and isoimmune doe resulting in normal offspring. These results demonstrate that in vitro treatment of sperm with antisperm Fab fragments had a protective effect from isoimmune attack in vivo. The successful use of Fab fragments for reversal of antisperm antibody-mediated infertility observed in the isoimmune rabbit model offers the prospect of a new means of restoring fertility in some isoimmune women.

Autoantibodies to decondensed sperm nuclear deoxyribonucleic acid in patients with antisperm antibodies and systemic lupus erythematosus detected by immunofluorescence flow cytometry.

OBJECTIVE: To evaluate a flow cytometric method to detect and quantitate serum anti-DNA antibodies using unfixed, swollen and decondensed human sperm nuclei and to examine the relationship between antibodies against sperm surface antigens to the presence of antibodies against nuclear antigens. DESIGN: Serum IgG and IgG subclass antibodies to decondensed sperm nuclei were detected by indirect immunofluorescence (IIF) flow cytometry. Sera were screened by IIF for anti-double-stranded DNA antibodies using the protozoan Crithidia luciliae as the substrate and for antinuclear antibodies using human epithelial (HEp 2) cells, respectively. All sera were assessed for antibodies against the sperm plasma membrane by an indirect immunobead test. SETTING: Infertility laboratory at the University of Oklahoma Health Sciences Center and rheumatology laboratory at the Oklahoma Medical Research Foundation. PATIENTS: Sera from 33 antisperm antibody-positive patients (5 subgroups), 33 patients with systemic lupus erythematosus (SLE; 6 subgroups), and 20 normal controls were selected. RESULTS: IgG antibodies against decondensed sperm nuclear DNA were detected in 11 (33.3%) of 33 antisperm antibody-positive patients versus 14 (42.4%) of 33 patients with SLE. Anti-DNA antibodies were most prevalent in vasectomized men and in antisperm antibody positive women with SLE. In the sera from patients with SLE, the presence of the anti-nuclear ribonucleoprotein antibody was associated with the presence of sperm head-directed antisperm antibodies. Anti-double-stranded DNA antibodies were found in 6 (18.1%) of 33 sera from patients with antisperm antibody and 17 (51.5%) of 33 sera from patients with SLE. Antinuclear antibodies were found in only 9 (27.2%) of 33 sera from patients with antisperm antibody and 30 (90.9%) of 33 sera from patients with SLE. All 20 of the control sera gave negative results in the three tests. Serum IgG reactivity to sperm nuclei was predominantly of the IgG1 and IgG3 subclasses. CONCLUSION: Anti-DNA is frequently found in either patients with antisperm antibodies or patients with SLE. Our results indicated that decondensed sperm nuclei can provide a specific substrate for screening serum anti-DNA antibodies.

Preferential binding of cross-reactive group A streptococcal anti-DNA monoclonal antibodies to decondensed human sperm DNA.

Four murine anti-streptococcal monoclonal antibodies (mAbs) that cross-react with human DNA were evaluated by immunofluorescence flow cytometry for their reactivity with condensed and decondensed human sperm nuclei. All 4 anti-DNA mAbs reacted with condensed sperm nuclei. The reactivity of 3 of these mAbs with decondensed sperm nuclei was 13 to 177 times higher than that found with condensed nuclei. Under identical conditions, mAbs to cytoskeletal/cytocontractile proteins lacked reactivity with decondensed sperm nuclei. Binding of monoclonal anti-DNA antibodies to decondensed sperm nuclei was abolished by preincubation with double-stranded DNA. The preferential binding of anti-DNA antibodies to decondensed sperm DNA suggests the utility of decondensed sperm nuclei as the antigenic substrate for screening anti-DNA antibodies by flow cytometry.

Heterogeneity of human sperm surface antigens identified by indirect immunoprecipitation of antisperm antibody bound to biotinylated sperm.

Target Ag recognized by clinically important C-fixing anti-sperm antibodies (ASA) were identified by indirect immunoprecipitation after incubation of biotinylated, motile sperm with ASA-positive sera from autoimmune, isoimmune, and vasectomized patients. This method assessed native protein thus permitting analysis of conformation-dependent membrane Ag that would be missed by previously reported Ag identification techniques. Sera from 35 ASA-positive and 10 ASA-negative patients were exposed to capacitated, surface-biotinylated motile sperm. After detergent-extraction of the membrane fraction, the immune complexes were precipitated with protein G-agarose. The Ag were eluted under reducing conditions, electrophoresed on SDS-10% PAGE, electroblotted onto nitrocellulose membranes, and visualized by the biotin/avidin-peroxidase detection method. By this method, only high titered ASA-positive sera demonstrated variable reactivity restricted to a total of eight to nine protein bands. Individual sera revealed a differential reactivity to a constant set of protein bands (63/61, 58/56, 29, and 21/19 kDa) and a preferential reactivity to either 45-, 43-, or 41-kDa protein Ag that were patient specific. None of these bands were precipitable by ASA-negative sera. ASA directed against the 63/61-kDa and 58/56-kDa polypeptides were found in 85% of immunoprecipitable sera; the 29-kDa was detectable with 70% of sera. Immunoreactivity to the 45-kDa to 41-kDa bands was detectable with 30% of the immunoprecipitable sera. Although the pattern of Ag recognized by ASA from autoimmune, isoimmune, and vasectomized patients was similar, our results indicate that in addition to the recognition of common human sperm surface determinants, distinct sperm surface epitopes are also recognized as foreign by the patient's immune system. Sera that immunoprecipitated the predominant monomeric 29-kDa or the dimeric 58/56-kDa Ag when preincubated with capacitated sperm markedly inhibited sperm binding and penetration to the human zona when compared with ASA-negative serum controls. It is suggested that C-fixing ASA directed to these surface determinants may account for some of the clinical manifestations of ASA-mediated infertility.

The expression of the complement regulators CD46, CD55, and CD59 by human sperm does not protect them from antisperm antibody- and complement-mediated immune injury.

OBJECTIVES: To investigate whether membrane-bound regulators of the initial (C3) and terminal (C5b-9) complement (C) pathway components are expressed on human sperm and to evaluate the protective effects of these regulators in restricting antisperm antibody and C-mediated injury. DESIGN: Sperm surface C inhibitors were quantitated by indirect immunofluorescence flow cytometry using murine monoclonal antibodies to detect C3 regulatory proteins, C3b/C4b receptor type (CR1, CD35), membrane cofactor protein (CD46), and decay-accelerating factor (CD55), and C5b-9 inhibitor, P18 (CD59) on acrosome-intact human sperm and sperm induced to undergo acrosomal loss. The susceptibility of sperm to antisperm antibody- and C-mediated immobilization was evaluated by sperm motility loss in the presence of antidecay-accelerating factor and/or anti-P18. SETTING: University of Oklahoma Health Sciences Center, a tertiary care referral center. RESULTS: Both decay-accelerating factor and P18 were detected on acrosome-intact and sperm induced to undergo acrosomal loss; neither expressed CR1. Membrane cofactor protein was detected on only acrosome-reacted sperm. The time course of sperm motility loss in antisperm antibody-negative sera in the presence of antidecay-accelerating factor, anti-P18, or both had a cumulative effect and brought about a time-dependent enhancement of sperm motility loss by serum C. However, sperm motility loss in C-fixing antisperm antibody-positive sera was unaffected by sperm membrane bound C3 and C5b-9 regulators. CONCLUSIONS: The susceptibility of human sperm to antisperm antibody- and C-induced motility loss may reflect a low potency of the sperm membrane C regulators membrane cofactor protein, decay-accelerating factor, and P18 in inhibiting serum C. Therefore, the use of serum C in the traditional assay for the diagnosis of cytotoxic antisperm antibody in infertile couples may have limitations. The inefficiency of these proteins in restricting serum C activity also suggested a secondary function in restricting localized proteolytic damage at the site of fertilization.

Phagocytosis of immunoglobulin G and C3-bound human sperm by human polymorphonuclear leukocytes is not associated with the release of oxidative radicals.

Antisperm antibody (ASA)- and complement (C)-mediated immune injury to human sperm is thought to be caused in part by phagocytic neutrophils. To investigate this process, we co-cultured purified human polymorphonuclear leukocytes (PMN) with swim-up sperm in the presence of ASA-positive and ASA-negative sera and assayed for PMN respiratory burst activity, monitored by the release of superoxide anion (O2-) and hydrogen peroxide (H2O2). Phorbol myristate acetate (PMA) and opsonized zymosan were used as positive controls. Phagocytosis of ASA-positive and C-bound sperm by PMN did not enhance O2- production when compared to incubation of sperm with ASA-negative sera. Phagocytosis of ASA-positive and C-bound sperm also resulted in minimal release of H2O2 when compared with ASA-positive and C-negative sperm that were not phagocytosed. In contrast, PMN were maximally stimulated to release O2- in response to either opsonized zymosan or PMA. The kinetics of PMA-induced O2- release was unaffected by the presence of ASA-positive and C-bound sperm. Cytocentrifuge preparations of PMN incubated with ASA-positive and C-bound sperm revealed limited O2- release at the site of PMN/sperm contact. These results indicated that 1) phagocytosis of motile sperm by PMN requires the binding of both ASA and C to the sperm surface; 2) phagocytosis of ASA-positive and C-positive sperm by PMN fails to release reactive oxygen species; and 3) metabolic processes associated with PMN respiratory burst activity may not be coupled to the ingestion of ASA-positive and C-bound sperm.

The predominance of IgG1 and IgG3 subclass antisperm antibodies in infertile patients with serum antisperm antibodies.

Mouse monoclonal antibodies (MAb) specific for each of the four human IgG subclasses and immunofluorescence flow cytometry were used to evaluate the subclass of the IgG antibody response to sperm in serum samples from 13 men and 6 women with a high titer (greater than 1:15,625) of IgG antisperm antibodies (ASA] determined by an indirect immunobead test. Five sera without ASA were also studied as a control. All 19 (100%) of the ASA-positive sera contained immunoglobulin (Ig)G ASA of the IgG1 and IgG3 subclasses. A 1:1 correlation was observed between the presence of IgG1 and IgG3 ASA. IgG2 was essentially undetectable, while IgG4 reactivity, although less intense than IgG1 and IgG3, was more prominent in the sera from the five vasectomized men. The ability of the IgG1 and IgG3 ASA-positive sera to deposit complement (C) on sperm was demonstrated by the concomitant binding to antibody-laden sperm of polyclonal antibodies to the membrane attack complex (C5b-9) of C. Both C-fixing and non-C-fixing ASA-positive sera were found to possess IgG1 and IgG3 antisperm antibodies. The predominance of IgG1 and IgG3 subclasses suggested a T-cell dependent immune response to sperm antigens.

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.

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.

Activation of human complement by IgG antisperm antibody and the demonstration of C3 and C5b-9-mediated immune injury to human sperm.

To investigate the role of C in the pathogenesis of antisperm antibody (ASA)-mediated infertility, we evaluated the binding and biologic effects of antisperm IgG and autologous C on human sperm. A flow cytometric assay using motile sperm as a target for IgG ASA+ (n = 30) and ASA- (n = 5) sera was developed for the concomitant detection of sperm-bound IgG and the initial (C3d) and terminal (C5b-9) C components on the surface of human sperm. Of the 30 IgG ASA+ sera evaluated by flow cytometry, 15 (50%) and 22 (73.3%) sera were also positive for sperm-bound C3d and C5b-9, respectively. Monomeric IgG purified from C-fixing ASA+ serum was able to bind to sperm and induced deposition of C3 on the sperm surface in the presence of human C. Incubation of motile sperm with C-fixing immune sera resulted in a significant loss (43 to 87%) of motility associated with characteristic C5b-9-induced alterations in sperm morphology leading ultimately to sperm lysis. When motile sperm were cocultured with purified polymorphonuclear leukocytes (PMN) in the presence of C-fixing immune sera, the binding of sperm heads to the PMN resulted in the formation of sperm rosettes, whereas non C-fixing or control sera had no such effect. Transmission electron microscopy of thin sections of the rosettes revealed ingestion of the sperm by the human PMN. These data suggested that 1) antibody bound to sperm is capable of activating autologous C by the classical pathway; 2) binding of both IgG and C proteins initiates C3-mediated sperm binding to PMN and sperm inactivation by deposition of membrane attack complex (MC5b-9) of C; and 3) concomitant detection of sperm-bound IgG, C3d, and C5b-9 may serve as an indicator of C-fixing cytotoxic ASA in the sera of infertile couples.

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.