Detection of Antispermatozoal Antibodies of IgG,IgA, and IgM Immunoglobulin Classes in Cervical Mucus

ABSTRACT: The immunobead test (IBT) was applied to bromelin-treated cervical mucus (CM) samples from 78 infertile women. Seven (8.9%) of the patients were positive for IgG and/or IgA class antibodies, whilst none (0/35) were found positive for IgM class antibodies. We found that 57% of the positive samples contained IgG and IgA classes, whilst the remaining samples contained IgA alone. The implications of these results were discussed.     

Comparison of the Immunobead Binding Test (IBT) and Immunospheres (IS) Assay for Detecting Serum Antisperm Antibodies

PROBLEM: The IBT is considered the gold standard of sperm antibody assays. This test uses polyacrylamide beads labeled with antiglobulins (anti-IgG, anti-IgA, and anti-IgM), which bind to the corresponding antibody on the sperm surface. The IS uses color-coded latex beads of uniform 3.0 μm size coated with the antiglobulins which can be viewed with brightfield light microscopy. The purpose of the present study was to compare the IBT and IS in an indirect test using human serum. METHOD: Serum specimens (n=42) were tested for the presence of antibody isotypes IgG, IgA, and IgM to sperm using the standard protocol for IBT and IS. Donor sperm was washed in BWW with 5% BSA and diluted to a final concentration of 50 times 10⁶ motile sperm/ml. The sperm were incubated with a 1:10 dilution of test serum for 30 min to 1 h at 37°C and then washed by three cycles of centrifugation. The sperm and beads (IBT, IS) were mixed on a glass slide, covered with a coverslip, and observed within 5 min. At least 100 motile sperm were counted and scored for bead binding. A specimen was considered positive if 20% or more of the sperm were coated with one or more beads. The data were analyzed using calculation of the non-parametric kappa statistic with correction for chance expected agreement, and by calculating the proportion of specific agreement between the two methods. RESULTS: The results are summarized in the following table: The IS was able to detect 94% of IgG antibodies, 91% of IgA antibodies, and 100% of Ig M antibodies. One serum specimen was IgG negative by IS (14% binding), but positive by IBT (20%). A second serum specimen was IgA negative by IS (16%) yet positive by IBT (29%). There were no false positives with the IS assay. Of the IgM positives (five of six) occurred alone and not with IgG or IgA, suggesting the necessity for testing all specimens also for IgM. CONCLUSION: Antisperm antibody test results obtained by the IS assay are in agreement with the results obtained with the IBT test. The Immunospheres are monodispersed, color coded, and can be visualized with brightfield microscopy.     

Sperm Antibodies in Normal Men: Association with a History of Nongonococcal Urethritis (NGU)

ABSTRACT: The incidence and specificity of sperm antibodies was examined, retrospectively, in a group of 80 normal men (64 sperm donors and 16 prevasectomy patients). Seven (8.5%) were positive for sperm-bound antibodies by immunobead test (IBT), but the antibodies were restricted to the sperm tail-tip. There was a higher (p < 0.001) incidence of nongonococcal urethritis (NGU) in the IBT-positive men, suggesting a possible causal relationship.     

Sperm-cervical mucus interaction, in particular in the presence of antispermatozoal antibodies

Disturbances of the interaction between spermatozoa and cervicalmucus can cause subfertility or infertility. The diagnosis ofsuch a disturbed interaction is possible with simple laboratorytests. Oligomucorrhoea and dysmucorrhoea are the most frequentcauses of a disturbed sperm-cervical mucus interaction. AntispermatozoalIgA plays a quantitatively limited, but qualitatively importantrole. Sophisticated time-consuming laboratory investigationshave mostly only additional value for the diagnosis.     

Reproductive Immunology and Microbiology News*

ABSTRACT: A fertile man had sperm-agglutinating activity in his serum (titers 1:16-1:128) and in his seminal plasma (titers 1:128-1:2048). The antibodies in the seminal plasma could be absorbed with anti-IgA antiserum but not with anti-IgG antiserum. A fresh ejaculate showed strong auto-agglutination of the spermatozoa. With mixed antiglobulin reaction tests (MART) and/or immunobead tests (IBT), IgA and IgG were detected on almost all motile spermatozoa; the erythrocytes, in the MART, and the latex spheres, in the IBT, adhered mainly to the tip of the tail. After mixing the fresh semen with cervical mucus, only 40% of the spermatozoa were locally shaking. The spermatozoa showed excellent penetration of cervical mucus in vitro. This case shows that IgA coating of the tails of the spermatozoa does not necessarily lead to adherence of these spermatozoa to the micelles of the cervical mucus and that the sperm cervical mucus contact test has a better predictive value than the sperm agglutination titer in the seminal plasma.     

Detection of Sperm Antibodies in Semen Using the Immunobead Test: A Survey of 813 Consecutive Patients

ABSTRACT: The aims of this investigation were to determine the incidence of sperm-bound antibodies in an unselected infertile population and also to further evaluate the immunobead test (IBT) with respect to specificity and reproducibility. The results of the survey showed that 7.8% of 813 men had antibodies of IgG and/ or IgA class bound to the surface of at least 20% of their motile spermatozoa. The results of crossed-inhibition tests with purified human immunoglobulins and comparison of the IBT results with the sperm-immobilization test (SIT) in serum and sperm agglutination in semen suggested that the IBT is an immunologically specific test for sperm antibodies. Comparison of repeat tests on 123 patients showed that the IBT is reproducible in 97.5% of cases. There was no difference in mean count, percentage motile, or morphology between the groups of patients with positive or negative IBT results. The incidence of sperm agglutination was significantly (Chi-squared, p < 0.001) higher in the positive IBT group. The results of this investigation therefore suggest that the IBT is an excellent test for routine screening of men for sperm antibodies.     

Comparison of human cervical mucus and artificial sperm penetration media.

The cervical mucus penetration tests aid research and determine the clinical importance of positive sperm antibody tests. Limited availability and variability of human cervical mucus have instigated the search for mucus substitutes for these tests. This study compares sperm migration in cervical mucus with that in artificial media including hyaluronate solution, egg white and albumin Tyrode solution. Results were quantified by measuring the migration distance (the maximum distance of capillary migration from a semen reservoir by spermatozoa after 1 h) and the sperm concentration at half the migration distance. The mean of both measures for cervical mucus and hyaluronate solution were equivalent [4.4 +/- 1.1 (SD) versus 4.3 +/- 1.0 cm and 118 +/- 51 versus 111 +/- 44x10(3)/ml], and higher than in egg white and albumin Tyrode solution. Antisperm antibodies impaired sperm penetration in cervical mucus and hyaluronate solution in a similar manner (r = 0.92). These results suggest that hyaluronate solution sufficiently resembles human cervical mucus in terms of penetrability that it may be used as a substitute for mucus in capillary tube tests of sperm function. The higher penetrability of cervical mucus and hyaluronate solution is probably related to a channelling effect due to their polymeric structure.     

Human and bovine cervical mucus penetration as a test of sperm function for in-vitro fertilization

Human and bovine cervical mucus penetration tests (n = 57) wereperformed preceding IVF to test their prognostic value as spermfunction tests for IVF. This evaluation also induded resultsfrom conventional semen analysis and from a computerized spermanalysis system. The bovine cervical mucus penetration testwas shown to be at least as valuable as the human cervical mucuspenetration test in evaluating sperm function. The migrationdistance of the vanguard sperm (P < 0.001) and the spermdensity at a fixed migration distance in the mucus column (P< 0.05) correlated most closely with the IVF results. A clearparallelism with the out come of the ‘swim up’ techniquewas also found. Of the sperm parameters examined, only spermmotility In the ejaculate (P < 0.05) correlated significantlywith the results of IVF. It is concluded that the outcome ofa bovine cervical mucus penetration test depends on the samesperm functions as re quired for IVF. Therefore, this test maybe of predictive value in an IVF programme.     

Development of a novel home sperm test

BACKGROUND: The majority of men find the production of a semen sample an embarrassing and stressful experience. Consequently, the availability of an over-the-counter home sperm test, which would reliably and accurately allow the patient to obtain an assessment of fertility potential at their convenience, would be a major benefit. Our objective was to develop and evaluate a home sperm test that provides a visual estimate of the concentration of progressively motile sperm in a semen sample. METHODS: Three particular challenges are described (i) developing a visualization system; (ii) optimization of the detection limit; and (iii) controlling variation due to changes in ambient temperature. The accuracy of the device was tested against two reference methods: computer-assisted sperm analysis (CASA) and a hyaluronate migration test (HMT). RESULTS: In 129 semen samples, where both reference methods agreed (positive or negative), the accuracy of the device was 95%. The observed likelihood ratio of 8.8 indicated that a sample showing a red line in the device was over eight times more likely to have a positive (normal) result in CASA and HMT than a sample without a red line. CONCLUSIONS: The final device provides a visual estimate of the concentration of progressively motile sperm in a semen sample using a test that is completed within approximately 1 h of production of the sample and can be used by the man in the comfort of his own home.     

Are Sperm Immobilizing Antibodies in Cervical Mucus an Explanation for a Poor Postcoital Test?

ABSTRACT: Previous authors reported high incidences of complement dependent sperm immobilizing antibodies in cervical mucus (CM) and found a correlation with poor postcoital tests. In all those investigations the CM samples were tested after dilution with a salt solution. In the present study we investigated 50 CM samples from infertile couples who had during the routine fertility investigation poor postcoital tests. All CM samples were tested after dilution with an equal volume of normal human serum; only one sample was found positive. On the other hand, if the CM samples were diluted with an equal volume of a buffered salt solution, many positive results were obtained. With some guinea pig serum batches, however, all tests with buffer-diluted CM samples were negative, despite good reaction of the positive control serum. We concluded that the positive results with buffer-diluted CM samples were due to a factor in the animal serum used as complement preparation and that complement-dependent sperm immobilizing antibodies in CM are an unlikely cause of a poor postcoital test.     

Critical evaluation of methylcellulose as an alternative medium in sperm migration tests.

BACKGROUND: The aim of this study was to evaluate the ability of human spermatozoa to penetrate methylcellulose (MC) and to compare this with penetration in hyaluronic acid. METHODS: Spermatozoa from normal (>or=20 x 10(6) sperm/ml, >or=50% progressive motility, >or=5% normal forms) and oligozoospermic (<20 x 10(6) sperm/ml) semen samples were allowed to swim into glass capillary tubes containing methylcellulose with a viscosity of 15 centipoise (cp) (MC15) and 4000 cp (MC4000), hyaluronic acid (rooster comb) or Sperm Select. Penetration of the spermatozoa at 1, 2, 3 and 4 cm were correlated with basic semen parameters (concentration, motility and morphology). The effects of temperature on penetration into MC4000 were explored at 17-37 degrees C. RESULTS: Higher numbers of spermatozoa penetrated MC4000 (10 mg/ml) compared with MC15 and the hyaluronic acid preparations. There was a highly significant correlation between the number of spermatozoa at all migration distances in MC4000 (10 mg/ml) and semen parameters. Increases in temperature from 17-37 degrees C were accompanied by significantly higher numbers of spermatozoa at each penetration distance. MC4000 at 10 mg/ml was at least as favourable to sperm penetration as human cervical mucus. Effective discrimination between normal and abnormal samples was achieved using MC4000 (10 mg/ml). CONCLUSION: Our results suggest the potential use of methylcellulose (MC4000, 10 mg/ml) as a reproducible and effective alternative to hyaluronic acid in sperm migration tests.     

The effect of three anti-oestrogen drugs on cervical mucus quality and in-vitro sperm--cervical mucus interaction in ovulatory women

The anti-oestrogens, clomiphene citrate, tamoxifen and cyclofenilare commonly used in the treatment of female infertility. Theirrole in the management of anovulation is well established butthere is continuing controversy about their relevance to otherareas of management. We have studied the effects of each ofthese drugs on cervical mucus and sperm—cervical mucusinteraction among 23 patients with unexplained infertility.Each patient received all three drugs in an alternative monthtreatment regime and in addition acted as her own control. Thestarting point in each patient was randomized. Luteinizing hormone(LH) and oestradiol were measured daily from day 10, and folliclescanning was also undertaken. Cervical mucus quality and sperm—cervical mucus interaction were studied on the day ofonset of the LH surge. The use of clomiphene and tamoxifen resultedin a significant reduction in cervical mucus score and sperm— cervical mucus interaction as judged by the distancetravelled by the vanguard spermatozoa. Cyclofenil had no effecton these parameters.     

Immunoglobulin Class and Regional Specificity of Antispermatozoal Autoantibodies Blocking Cervical Mucus Penetration by Human Spermatozoa

ABSTRACT: The immunoglobulin class and regional specificity of sperm antibodies was analyzed in relation to in vitro cervical mucus penetration using a capillary test. There was a highly significant (X², P < 0.001) association of strong (≥ 95% sperm coated) immunobead test (IBT) results for sperm-bound antibodies of IgA immunoglobulin class with poor (< 3.0 cm/2 h) mucus penetration. There was also a significant (P < 0.01) association between poor penetration and the presence of antibodies on the sperm tail mainpiece. In contrast, IgG class antibodies did not show any significant association with poor penetration. These results confirm and extend previous work and imply that development of a male antispermatozoal vaccine that inhibits mucus penetration must involve stimulation of local IgA response rather than systemic antibody levels.     

Variation in Antisperm Antibody Results Using Different Assays

PROBLEM: The detection of various types of antisperm antibodies (ASA) in the serum varies among different assays. This variation may influence the diagnosis and management of infertile couples who are tested for such immunologic factors. This prospective study was conducted to determine the variation in the results of ASA as measured by the sperm immobilization (SI), sperm agglutination (SA), and the indirect immunobead (IB) assays. METHOD: The sera of 79 patients that tested positive for ASA by at least one of the assays listed above were concurrently tested with all three assays. RESULTS: Using an individual ASA assay, 66 (84%), 26 (33%) or 36 (46%) of sera tested positive by the SA, SI, or IB assays, respectively. However, using a combination of assays, 67 (85%), 78 (99%) or 40 (51%) of sera tested positive using either the SA+SI, SA+IB or SI+IB assays, respectively. CONCLUSION: These findings suggest that the utilization of different assays to detect ASA may detect sera that are positive for ASA with more reliability than single assay testing.     

Sperm Antigens and Their Use in the Development of an Immunocontraceptive

PROBLEM: Overpopulation is a global problem of significant magnitude, with grave implications for the future. World population is predicted to reach 10 billion people by the year 2050, an increase of more than 75% over the current population. Development of new contraceptives is necessary, since current forms of birth control are unavailable to many individuals due to sociological, financial, or educational limitations. METHODS: In an effort to identify new contraceptive alternatives, sperm-specific antigens are under investigation as the basis for immunological regulation of fertility through contraceptive immunization. Anti-sperm antibodies (ASA) inhibit sperm function in vitro, induce infertility in experimental models, and have been implicated in some cases of clinical infertility. Furthermore, the development of ASA in vasectomized men does not lead to physiological complications despite the persistence of these antibodies for years; thus, ASA induced by immunization of men and women may be similarly harmless. Immunization has several conceptual advantages as a method of fertility regulation: Employment of non-pharmacologically active agents, convenience of administration, low cost, and the potential for relatively long-lasting yet reversible effects. RESULTS: Various approaches have been taken to identify candidate sperm antigens for immunocontraceptive development. Studies that utilized monoclonal antibodies and polyclonal antisera generated against sperm preparations have identified such promising candidates as PH-20, fertilin, SP-17, and SP-10. In an animal model, 100% contraceptive effects were induced following PH-20 immunization and the effects were reversible. More recently, ASA from the sera of infertile patients were employed to identify several candidates as expressed by testis cDNA libraries. Perhaps the most extensively characterized and effective immunogen candidate identified thus far is lactate dehydrogenase-C₄ (LDH-C₄). Active immunization with LDH-C₄ suppressed fertility in a variety of mammalian species, including primates, and the reversibility of these effects was demonstrated. CONCLUSION: The successful results obtained thus far support the feasibility of an effective immunocontraceptive and indicate the importance for continued investigation of additional sperm antigens as contraceptive immunogen candidates.     

Cross-Reactivity of Sperm and T Lymphocyte Antigens*

ABSTRACT: Evidence is presented for cross-reactivity between antigens on human sperm and T lymphocytes. In 25 infertile couples in which both the males and females had significant antisperm immunity, antibody (Ab) titers to thymocytes (mean ± S.E.M. 159 ±4 and 72 ± 14, respectively, in males and females), T cell lines CCRF-CEM (69±5 and 48±8) and HSB-2 (56±15) and 41±8), suppressorenriched (T_G) cells (26±6 and 66±28) and helper-enriched (T_g—) cells (26±4 and 46±14) were significantly elevated, as compared wth Ab titers in 45 normal males and 45 normal females without antisperm immunity. Antibody titers to adult B cells, B cell line RAJI, and granulocytes were similar in the two groups. Antisperm Ab titers in sera, sperm extracts, and seminal plasma of the infertile subjects were significantly reduced after absorption with sperm, thymocytes, or T cell line CCRF-CEM but not with the B cell line RAJI. Antithymocyte Ab titers in the sera were significantly reduced (p < 0.001) after absorption with thymocytes, CCRF-CEM, or sperm, but not RAJI. Lymphocytes from the infertile patients, when stimulated with pokeweed mitogen in vitro, produced antisperm and anti-T-lymphocyte antibodies at significantly higher titers than normal controls.     

Infertility: Characterization of Tru-Trax in-vitro penetration testing of cervical mucus

To determine the distribution of results of an in-vitro cervicalmucus penetration system employing both human and bovine mucus(Tru-Trax) in a general infertility population, 133 couplesprospectively underwent in-vitro and post-coital testing ofcervical mucus. The distribution of Tru-Trax results in coupleswith normal semen analyses and Insler scores showed significantlygreater penetration in bovine (22.3 ± 6.0 mm) than inhuman mucus (20.3 ± 5.4 mm) (P < 0.001). However,the lower limit of the 95% confidence interval of the normalpopulation with either type of mucus was 10 mm. This limit wassignificantly lower than that described by the kit manufacturer.The predictive value for post-coital tests using human mucusin the Tru-Trax system was good (>90%) in all groups. Theoverall penetration into either human or bovine mucus was significantlycorrelated with the percentage of motile spermatozoa in thesemen sample (P < 0.001). In conclusion, in-vitro cervicalmucus penetration tests with the Tru-Trax system are relatedto in-vivo post-coital tests, although the lower limit of therange of anticipated results in the normal population was significantlylower than that described by the manufacturer. The ultimatevalue of this type of testing will await clinical trials whichevaluate clinical end-points such as pregnancy rates.     

Ten-Year Experience with Antispermatozoal Activity in Ovulatory Cervical Mucus and Local Hydrocortisone Treatment

PROBLEM: During 1987–1996, cervical mucus spermagglutinating antibodies of secretory immunoglobulin A (sIgA) or IgG detected by the tray agglutination test (TAT) and the indirect mixed antiglobulin reaction (MAR) test were found in 234 women aged 23–39 years with previously unexplained infertility. METHOD OF STUDY: Hydrocortisone was applied to the ectocervix. RESULTS: Spermagglutinating antibodies of sIgA disappeared totally in 102 patients (91 resulting in deliveries of healthy babies, 3 in ectopic pregnancies, and 8 in spontaneous miscarriages). A decrease of spermagglutinating antibodies in ovulatory mucus during hydrocortisone application without pregnancy was registered in 60 infertile women. This group was referred to treatment by in vitro fertilization. No hydrocortisone effect on immunocompetent cells producing antispermatozoal IgG alone or combined with sIgA was seen in 72 patients. CONCLUSIONS: Vaginal mycosis, a side effect of hydrocortisone treatment, was seen in seven cases. Hydrocortisone for local immunosuppression becomes a valuable method of therapy in cervical immunologic infertility caused predominantly by antispermatozoal sIgA.     

Cervical Mucus Anti-Sperm Antibodies: Treatment With Intrauterine Insemination

PROBLEM: To determine (1) the incidence of cervical mucus anti-sperm antibodies in infertile women, and (2) the results of treatment by intrauterine insemination. METHOD: Cervical mucus was collected the morning after urinary LH surge occurred from 153 consecutive women being treated for unexplained infertility with intrauterine insemination. Immunobead testing for IgG, IgA, IgA1, and IgA2 was performed with only actively motile sperm being counted. RESULTS: Overall, 23/153 (15.0%) of cervical mucus samples were positive for anti-sperm antibodies: 9/23 (39.1%) were only IgA-positive (62% IgA1-positive, 38% IgA2-positive), 11/23 (47.8%) were only IgG-positive, and 3/23 (13.0%) were positive for both IgA and IgG. Insemination resulted in a pregnancy in 6/23 (26.1%) of women with cervical mucus anti-sperm antibodies after 1–3 cycles. CONCLUSIONS: Testing for cervical mucus anti-sperm antibodies should be performed in cases of “unexplained” infertility, and intrauterine insemination may be an effective treatment, resulting in pregnancies in over one-fourth of couples.     

宫颈粘液对人精子甘露糖受体表达的影响

目的：研究人宫颈粘液是否影响人精子甘露糖受体的表达。方法：正常人精子穿透宫颈粘液2h后．用金霉素（CTC）荧光染色法鉴定其获能及顶体状态,并用异硫氰酸荧光素标记的甘露糖化牛血清白蛋白（FITC-DMA）检测精子甘露糖受体的表达。对照组分别用获能培养基BWW培养0、2、6h后用相同方法检测。结果：人精子穿透宫颈粘液后“获能”型精子百分率较穿透前增高,而“顶体反应”型精子百分率及甘露糖受体的标记阳性率与穿透前无差别。结论：宫颈粘液促进精子获能,但不诱导顶体反应,且不影响其甘露糖受体的表达。