Gilbert G. Haas

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 husbands 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 partners leukocytes did not incite or increase the antisperm antibody titer, with any of the assay techniques.

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.

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.

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.

The effect of fixatives and/or air-drying on the plasma and acrosomal membranes of human sperm

It has been hypothesized that some fixatives and conditions of slide preparation expose internal sperm antigens and thus are not suitable for demonstrating surface-specific antigens by immunocytochemical assays. This study examined the ability of five fixatives (glutaraldehyde, acetone, methanol, paraformaldehyde, and periodate-lysine-paraformaldehyde [PLP]) and two conditions of slide preparation (air-drying or maintaining sperm in a liquid phase) to maintain the integrity of human spermatozoal membranes at the ultrastructural level as monitored by transmission electron microscopy. Regardless of the fixative employed, air-drying was detrimental to plasma and acrosomal membrane integrity. Acetone and methanol completely disrupted the plasma and acrosomal membranes over the entire sperm surface whether air-drying or liquid phase conditions were employed. Fixation with glutaraldehyde and paraformaldehyde (to a lesser degree) maintained the morphologic integrity of acrosomal and plasma membranes provided the sperm were maintained in a liquid phase. However, glutaraldehyde and paraformaldehyde fixation increased the postfixation binding of immunoglobulins to the sperm surface. We concluded that immunocytochemistry at the light microscopy level may imply erroneous interpretations regarding antigen/antibody interactions on the sperm plasma membrane surface unless care is taken to verify that the antibodies of interest are binding to the antigenically intact plasma or acrosomal membrane.

Flow cytometric quantitation of the expression of membrane cofactor protein as a marker for the human sperm acrosome reaction

A mAb (TRA-2-10, IgG1) to an embryonal carcinoma cell line (2102ep) that recognizes an antigen termed membrane cofactor protein (CD 46, TLX antigen) binds to human sperm after chemical induction of acrosomal loss by Ca2+ ionophore. An indirect immunofluorescence assay was developed in which sperm membrane cofactor protein was detected by flow cytometry on acrosome-reacted living human sperm. The expression of the membrane cofactor protein antigen on acrosome-reacted sperm may represent a marker that can be used in a rapid, quantitative, and reproducible flow cytometric assay for the evaluation of human sperm acrosomal loss.

Lack of complement activation in the seminal plasma of men with antisperm antibodies associated in vivo on their sperm

ABSTRACT: A specific and sensitive “sandwich”‐type radiolabeled antiglobulin assay (RAA) using monoclonal anti‐human C5b‐9 neoantigen and polyclonal anti‐human C5b‐9 was used to evaluate the presence of the in vivo product of human complement (C) activation (SC5b‐9) in the seminal plasma (SP) of 19 fertile and 61 infertile men. SP SC5b‐9 was detectable in 7 (8.7%; 1 fertile and 6 infertile men) of the 80 men with a range of 10 to 175 ng/ml. Levels of SP SC5b‐9 in other men were below the limit of detection (less than 10 ng/ml). Of the 33 infertile men with sperm‐associated immunoglobulin (Ig) G and/or IgA, 27 (82%) had undetectable levels of SP SC5b‐9 immunoreactivity. There was no correlation between the SP SC5b‐9 levels and the degree of sperm‐associated IgG (r = 0.086) or IgA (r = 0.23) activity. However, significant deposition of sperm‐bound C5b‐9 due to autologous C activation was demonstrated by flow cytometry of donor sperm treated with sera from autoimmune men with ASA in their sera and on their sperm. These findings suggest that sperm‐bound Ig cannot activate C in SP.

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.

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.

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.