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[Sperm agglutination, sperm immobilization and sperm immunofluorescence test in married couples with unexplained infertility (author's transl)].

Thirty-four married couples with clinically unexplained infertility and normal spermiograms in all male partners were investigated for sperm antibodies by means of the sperm agglutination, sperm immobilization and sperm immunofluorescence test. In 50% of the couples, sperm antibodies were found in at least one of the partners and with at least one of the tests, e.g. ten times in the female partner alone, six times in the male partner alone, and once in both partners. The correspondance of the three tests was astonishingly low; it is assumed, therefore, that they indicate different types of antibodies. On the basis of the present investigations it is not possible to assess which type of antibody is responsible for the infertility of the female or male partner respectively. This seems only possible when the decrease of certain antibody types can be determined after a period of no antigen contact (avoidance of sperm contact) and when conception has taken place. The significance of autoantibodies against sperm in clinically healthy men with normal spermiograms remains open.

Antibodies

Effect of sperm-agglutinating antibodies on sperm capacitation and acrosome reaction.

OBJECTIVE: To study the effect of polyclonal/monospecific antisera on sperm agglutination versus capacitation as well as acrosome reaction. MATERIALS AND METHODS: Swim-up spermatozoa from cauda epididymides of fertile male hamsters were incubated under liquid paraffin with polyclonal/monospecific antisera obtained from immunized BALB/C mice, as well as with normal serum from control BALB/C mice, at various dilutions. RESULTS: The anti-sperm antibodies caused a significant (P < .05) sperm agglutination of various types of dilutions below 1:1000. Both capacitation and true acrosome reaction were inhibited significantly in the spermatozoa incubated with polyclonal/monospecific antisera. Capacitation in the spermatozoa with normal serum started earlier, i.e., at 2 hours of incubation compared to 3 hours of incubation in controls. CONCLUSION: The data differentiate the sperm agglutinating activity from anticapacitation and antiacrosome reaction activity of antisperm antisera at 1:1000 dilution.

Acrosome

Biochemical characterization of sperm agglutination antigen-1, a human sperm surface antigen implicated in gamete interactions.

The anti-sperm monoclonal antibody (mAb) S19 was previously demonstrated to agglutinate human spermatozoa, inhibit sperm penetration of cervical mucus, and inhibit sperm-zona pellucida binding. These results implicated the cognate S19 antigen, designated sperm agglutination antigen-1 (SAGA-1), in gamete interactions and identified SAGA-1 as an attractive candidate for immunocontraceptive development. In the present study, evaluation of sperm agglutination with video microscopy showed that the S19 mAb rapidly and completely agglutinated human spermatozoa in a "tangled" pattern of agglutination. One- and two-dimensional immunoblot analyses identified SAGA-1 as a highly acidic, polymorphic sperm protein with an apparent molecular mass of 15-25 kDa and an isoelectric point of 2.5-3.0. Periodate treatment abolished this immunoreactivity, demonstrating that the S19 mAb reacted with a carbohydrate epitope and indicating that SAGA-1 is a glycoprotein. Absence of S19 immunoreactivity in postvasectomy seminal fluid implicated the testis, epididymis, and/or proximal vas deferens in the expression of SAGA-1. In solubility and phase partitioning assays, SAGA-1 was extracted from spermatozoa in Triton X-114 and exhibited the hydrophobic characteristics of integral and glycosylphosphatidyl inositol-anchored membrane proteins. These results identify SAGA-1 as a hydrophobic, highly acidic sperm glycoprotein that is localized on the entire sperm surface and has potential significance as a target for antibodies that inhibit sperm function and gamete interactions.

Antibodies, Monoclonal

Isolation of human spermatozoa membrane antigens binding sperm-immobilizing and sperm-agglutinating antibodies.

Peptides of human spermatozoa were dissolved with Hyamine 2389 and Triton X-100 and separated by chromatography on Biogel P4 columns into seven fractions. The antigenic activities of the separated sperm-membrane fractions were tested according to their capacity to inhibit sperm agglutination and sperm immobilization (immune inhibition test) in human sera of sterile patients. Sperm-agglutinating and sperm-immobilizing activity was tested by the microtray agglutination and microtray immobilization test. A titer reduction was achieved only in sperm-immobilizing sera. Four sperm antigenic fractions revealed in the majority of the repeatedly tested sperm-immobilizing sera an inhibition of the antigen-antibody reaction. No reaction was observed after exhaustive absorption of the tested seven antigen fractions with sperm-agglutinating sera. Therefore the conclusion can be drawn that sperm-agglutinating and sperm-immobilizing antibodies react with different sperm antigens. Normal human sera without sperm antibodies served as control. As no sperm agglutination or sperm immobilization was obtained after absorption of these control sera our antigen fractions do not produce sperm agglutination or sperm immobilization.

Antigens, Surface

Contraceptive effect of sperm-agglutinating monoclonal antibodies in rabbits.

Immune infertility in humans correlates clinically with the presence of anti-sperm antibodies that trap (agglutinate) sperm in semen and cervical mucus. To test whether sperm-agglutinating antibodies can be effective contraceptive agents, several mouse anti-rabbit sperm (MARS) sperm-agglutinating monoclonal antibodies (mAbs) were developed that rapidly and completely agglutinate sperm: MARS-M3 (IgM), MARS-G16 (IgG3), and MARS-G17 (IgG3). Contraceptive efficacy of these mAbs was tested by mixing the mAb with 0.1 ml semen (approximately 1/5 of a whole ejaculate) immediately before artificially inseminating rabbits paracervically. This paracervical dose of semen provided a rigorous test since it delivered several thousand times more fertilizing doses than does a human ejaculate. All of the mAbs were contraceptively effective, and MARS-G16 reduced the number of fetuses per animal by 88% and 95% with doses of 150 microg and 2 mg, respectively. The contraceptive efficacy of the MARS mAbs in the rabbit suggests that human sperm-agglutinating mAbs may be effective agents for vaginal contraception in humans.

Animals

Sperm immunofluorescence in infertile men: Its relationship with sperm agglutination and gel agglutination.

The spermatozoa from 10 infertile male patients all with sperm agglutination were subjected to direct immunofluorescence using antisera raised against IgA, IgM, and IgG. Sera from these patients were subjected to the Gel Agglutination Test. Although the Gel Agglutination Test related closely with sperm agglutination, none of the spermatozoa from these patients produced a repeatable positive response to direct immunofluorescence. This study suggests that neither sperm agglutination nor the Gel Agglutination Test demonstrate the presence of sperm specific antibodies in the semen of these infertile men.

Agglutination Tests

[Sperm agglutination antibodies studied in infertile couples at the Women's Clinic in Plzen 1980-1988].

The authors assessed sperm-agglutination antibodies in infertile couples in 1980-1988, using the Kibrick test: in sera from 699 female patients, a positive result was found in 142 (20%) women, in sera from 352 men, in 6 (1.7%) sperm-agglutination antibodies were detected. Using the microagglutination test, they examined sera of 3501 sterile women; in 273 (6.8%) they found sperm agglutination up to 1:16, above titre 1:32 in 12 (0.4%) of the patients. Using the same test, they examined sera of 2803 men and found sperm-agglutination up to 1:16 in 21 (0.7%) and above 1:32 in 6 (0.3%). The most frequent type of sperm agglutination is aggregation of the heads; aggregation by tails or mixed agglutination are less frequent. In 3252 sterile women where no sperm-agglutination antibodies were found Kremer's test was negative in 1145 patients. The authors analyzed in 196 sterile women the ovulation mucus by the microagglutination test and found a high anti-spermatozoidal activity in 24 (12%) women. Concurrently an indirect MAR test was made. Antibodies against sperm cells were found in 24 (12.2%) of specimens in sIgA, in 8 (4.1%) in IgG, in 2 (2%) in IgM and in one sample (0.5%) in IgE. The indirect MAR-test in sera of 325 sterile women revealed a predominance of anti-spermatozoidal antibodies in IgG, in men in IgA. In seminal plasma the authors detected antibodies against sperm cells in sIgA. In seminal plasma the authors detected antibodies against sperm cells in sIgA and IgE.

Antibodies

Relationship between the direct mixed antiglobulin reaction (MAR) test and spontaneous sperm agglutination in men from infertile couples.

To determine the relationship between the direct MAR test and spontaneous sperm agglutination in men from infertile couples, semen samples from 160 men were studied. Spontaneous sperm agglutination was present in 80 samples, and absent in the others. Routine semen analysis and the direct MAR test were performed on all samples. The incidence of positive MAR tests in samples with and without spontaneous sperm agglutination were 15 and 0 per cent respectively (p < 0.05). There were no significant differences in mean sperm concentration, motility, and morphology between the samples with and without spontaneous sperm agglutination, and between the positive and negative MAR test groups. It can be concluded that spontaneous sperm agglutination relates significantly to antisperm antibodies detected by the direct MAR test, and this factor could be used as indication for antisperm antibody testing of infertile men.

Coombs Test

Difficulty of obtaining reproducibility in the Franklin and Dukes test for the detection of sperm-agglutinating antibodies in human sera.

The Franklin and Dukes (FD) test is used as a screening test for the determination of sperm-agglutinating antibodies and as a proof of therapeutic effects in cases of infertility, but various parameters of the FD test remain unclear and the application of different techniques has revealed highly different results. To test the statistical evidence for the FD technique, conditions regarded as optimal prerequisites were applied: a sperm concentration of 20 times 10(6)/ml was produced with Baker's buffer and serum dilutions were made with Baker's buffer, starting at 1:4. Only the standard deviation of parallel tests with one serum sample and one semen sample on the same day was found to be within an acceptable range. Using ejaculates of the same donor and the same serum sample on different days gave results that were not reproducible. The FD test should not, therefore, be used for quantification of sperm-agglutination antibodies except for a comparison in one test with one semen sample on the same day. Although the FD test only allowed a qualitative evaluation of sperm-agglutinating antibodies the % sperm agglutination was more informative than agglutination titres. For quantification of sperm-agglutinating antibodies, the FD test should be replaced by other techniques.

Agglutination Tests

Monoclonal antibody inducing human sperm agglutination.

A mouse fused splenic cell line producing antibody that induces agglutination of human sperm was cultivated. The antibody was localized to the midpiece and tail of human spermatozoa by an indirect immunofluorescence method. The interacting antigen was identified in a sperm membrane protein preparation by SDS gel/protein blot immunofluorescence method. The molecular weight of the antigen was in the vicinity of 84 Kdaltons.

Animals

Characterization of sperm agglutinating monoclonal antibody and purification of the human sperm antigen.

A monoclonal antibody capable of inducing tail-to-tail agglutination of human sperm was purified by ammonium sulfate fractionation and chromatography on DEAE-cellulose. A 75- to 100-fold purification was achieved. The antibody was of the IgG class and composed of gamma 1 heavy chain and lambda light chains. The purified preparation was effective in inducing sperm agglutination at a minimum concentration of 0.55 microgram/mL and was active after storage at 4 degrees C for over 3 months in the presence of sodium azide. The sperm antigen interacting with this YWK-1 antibody was purified by immunoaffinity chromatography on Sepharose 4B conjugated with YWK-1 antibodies. Two interacting proteins were isolated, with estimated Mr of 84 and 20 kD. The 84 kD protein had an isoelectric point in the range of pH 5.7 to 6.1, and probably lacks carbohydrate moieties. The 20 and 84 kD components may share common antigenic determinants.

Antibodies, Monoclonal

The significance of serum sperm-agglutinating antibodies in men with infertile marriages.

Three hundred consecutive male partners of infertile marriages had serum samples tested for sperm agglutinating and sperm immobilising antibodies, using a micro-technique: 30.3% were found to be positive. Out of a control group of 100 fertile men 10 had antibodies detected (10%) and these at low titre; this difference was significant. The results of these serum tests were found to correlate with the results of seminal plasma tests, the mixed antiglobulin reaction for IgG and sperm cervical mucus contact testing, although all of these tests were less discriminatory with regard to fertility. The serum micro-immobilisation test showed a statistical association with fertility when analysed in those couples where both husband's and wife's tests showed no other bar to fertility.

Antibodies

Postcoital tests and sperm-agglutinating antibodies in men.

Postcoital tests (PCTs) were examined from couples where the husbands had circulating head-to-head (H-H) or tail-to-tail (T-T) sperm-agglutinating antibodies. The results were compared with findings in couples without antibodies. The presence of H-H sperm-agglutinating antibodies did not interfere with the outcome of the PCT. High serum titers of T-T sperm-agglutinating antibodies, the presence of such antibodies in the seminal fluid, and/or a strong or complete autoagglutination in the husband's ejaculate markedly reduced the number of invading spermatozoa as well as their survival in the PCT. A moderate titer of T-T sperm-agglutinating antibodies was also reflected in the PCT but low titers did not have any influence on the results.

Autoantibodies

Characterization of a potent sperm-agglutinating monoclonal antibody and its cognate antigens.

OBJECTIVE: To identify sperm antigens that are capable of eliciting infertility-related sperm-agglutinating antibodies. DESIGN: In vitro laboratory experiments. SETTING: University research laboratory. PATIENT(S): Fertile semen donors. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Sperm agglutination, immunofluorescence localization, and flow cytometric analysis of surface expression of A36 antigens. Antigen analysis by Western immunoblotting. RESULT(S): Monoclonal antibody A36 induced intensive head-to-head, tail-to-tail, and head-to-tail agglutination of motile human spermatozoa. Antigens recognized by A36 were localized on the acrosomal cap and in the principal tail regions of motile, noncapacitated human sperm. Changes in subcellular levels and localization of the A36-recognized epitope occurred after capacitation and acrosomal loss. A36 reacted with a polymorphic series of proteins in Western blots of sperm extracts from humans and various other animal species, including mouse testis extracts. A common 53-kd antigen was recognized by the antibody in the different antigenic preparations. CONCLUSION(S): A mouse antibody to human sperm, monoclonal antibody A36, caused intensive agglutination of noncapacitated human spermatozoa and reacted with antigens on the acrosomal cap and in the principal tail regions. Of the multiple polypeptides that were reactive with the monoclonal antibody in sperm extracts from humans and other animal species, a common 53-kd antigen was recognized.

Agglutinins