Effects of sperm immobilizing antibodies on pregnancy outcome in infertile women treated with IVF-ET.

PROBLEM: Since it was found that anti-sperm antibodies could impair in vitro development of fertilized eggs in the presence of complement in rats, the effects of sperm immobilizing antibodies on human pregnancy were examined in infertile women treated with IVF-ET. METHODS: The pregnancy outcomes of 143 ET cycles in 58 infertile women with sperm immobilizing antibodies and 363 ET cycles in patients with tubal infertility as control were compared. Diagnosis of chemical pregnancy was done when the urinary hCG level had risen over 50 IU/L but a gestational sac could not be demonstrated later. Antibody titers of sperm immobilizing antibodies (SI50 units) were measured by a quantitative sperm immobilization test. RESULTS: 33 (23.1%) of 143 cycles in the patients with sperm immobilizing antibodies and 56 (15.4%) of 363 cycles in the control patients were diagnosed as pregnancy. The pregnancy rates were significantly higher in the former than in the latter (P < 0.05). In the patients with sperm immobilizing antibodies, 12 (36.4%) were chemical pregnancies, 5 (15.2%) were clinical abortions, and 16 (48.5%) had deliveries. In the control group, 18 (32.1%) were chemical pregnancies, 10 (17.9%) were clinical abortions including ectopic pregnancies and 28 (50.0%) had deliveries. There was no significant differences in each category. When the SI50 titers at the time of conception were considered, chemical pregnancy rates were 22.2% (4/18) in patients with SI50 titers below 10 units, but those in patients with SI50 titers above 10 were 50.0% (5/10) and above 100 were 60.0% (3/5), respectively, (P > 0.05). In four of five patients who had both chemical and clinical pregnancies, the SI50 titers at the time of conception were higher in the chemical pregnancy cycles than in the clinical pregnancy cycles. CONCLUSIONS: Though the pregnancy rates were significantly higher in the patients with sperm immobilizing antibodies as compared to those with tubal infertility, chemical pregnancy rates were also higher in the patients with higher sperm immobilizing antibody titers. These results suggest that sperm immobilizing antibodies may cause the damage of early development of human embryos in vivo in the small number of patients with a high titer of the antibodies.

Direct production of the Fab fragment derived from the sperm immobilizing antibody using polymerase chain reaction and cDNA expression vectors.

PROBLEM: Sperm immobilizing antibodies cause infertility mainly through complement dependent sperm immobilization. To analyze any effect of sperm immobilizing antibody on fertilization, we had already established cell lines that secrete IgM monoclonal antibody (MAb H6-3C4) and IgG monoclonal antibody (MAb EnBCMGS). The latter was a class-switched recombinant IgG antibody that shares the same variable region as MAb H6-3C4. The biological effects of the IgG antibody were also reported previously to eliminate sperm immobilizing or sperm agglutinating activities. However, the method of chemical digestion of IgG had some disadvantage to prepare the purified Fab fragment stably and in large quantities. This time we report a unique method to obtain the recombinant Fab fragments (Fab EnBCMGS) using polymerase chain reaction (PCR) and cDNA expression vectors. METHOD: Two kinds of PCR primers were designed to make a truncated heavy chain (Fd) gene of MAb EnBCMGS. The amplified Fd gene and light chain gene were ligated into cDNA expression vectors and then transfected into mammalian cells. RESULTS: Expression of the Fd gene and light chain gene were confirmed by Northern blotting. Secretion of the recombinant Fab fragment from mammalian cells was also confirmed by Western blotting. The Fab fragment showed biological activity as is expected by FACS analysis. CONCLUSION: This method enables the stable production of genuine Fab fragments of IgG in mammalian cells without any chemical treatment that may be time consuming and affect the quality of the Fab fragments.

Techniques for sperm immobilization test.

Several semiquantitative, quantitative, and microassay techniques had been developed to detect antibodies bound to human spermatozoa: sperm agglutination test (SAT), sperm immobilization test (SIT), immunofluorescence test, radioantiglobulin test, enzyme-linked immunosorbent assay (ELISA), mixed erythrocyte-spermatozoa antiglobulin reaction (MAR), "Panning" test, and immunobead test (IBT). Clinical application of these techniques include (a) detection of sperm immobilizing antibodies in sera of sterile women, (b) follow-up study of sperm immobilizing antibodies, and (c) detection of sperm immobilizing antibodies in cervical mucus and other secretions. The chemical structure of antigen epitope corresponding to Mab H6-3C4 may recognize the internally located repetitive unbranched N-acetyllactosamine structure, regardless of terminal substitution at Gal (i.e., sialyl-i as well as i structure). The majority of sperm-immobilization (SI) positive women's sera were absorbed with carbohydrate components on ejaculated sperm, but only one serum competed with Mab H6-3C4 on binding to sperm except a serum from whom lymphocytes were donated to make Mab H6-3C4. The SI agglutinating antibodies (Abs) in women's sera were raised to the carbohydrate epitopes of glycoprotein in HSP, but epitopes might have several different conformational structures. Studies are in progress to find whether or not SI-Abs could be generated to peptide epitope of human seminal plasma (HSP) or sperm.

Monoclonal sperm antibodies: their potential for investigation of sperms as target of immunological contraception.

We generated 149 hybridoma cell lines secreting antibodies against human spermatozoal antigens of which antibodies from 136 hybridoma lines also reacted with seminal plasma constituents. The occurrence of common antigeneic determinants on spermatozoa and seminal plasma was further demonstrated by competitive inhibition ELISA tests. We found that seven hybridoma clones secreted antibodies reactive with spermatozoa but nonreactive with seminal plasma. Antibodies from 5 clones were sperm-agglutinating and from 15 clones sperm-immobilizing. Localization of sperm antigens reactive with the monoclonal antibodies was demonstrated by indirect immunoperoxidase staining. A synthetic decapeptide, earlier shown to be reactive with naturally occurring human iso- and autosperm antibodies, was shown to be reactive with the monoclonal antibody VII-5 in ELISA tests.

Vasectomy and non-fatal myocardial infarction.

The incidence of non-fatal myocardial infarction among 4830 vasectomised men was 0.9 cases per 1000 man-years during 24 420 man-years of observation. This was slightly lower than the rate in 24 150 non-vasectomised men, matched with a vasectomised man for calendar year of birth and duration of observation. Review of medical records for a matched sample of study subjects indicated no measurable confounding by important cardiac risk factors.

PIP: This study was undertaken to investigate the risk of nonfatal myocardial infarction among vasectomized men. Among a group of 4830 vasectoized men there were 23 first time infarctions, giving a rate of 0.90 infarction/1000 man-years at risk. There were 120 first time infarctions in a control group of nonvasectomized men, giving a rate of 1.0 infarctions/1000 man-years at risk. Incidence rate rose with time in both groups as the men became older, and obesity and smoking were both associated with infarction in both groups. The lack of a vasectomy-related effect was present throughout an observation period of 7 years after vasectomy. Some laboratory data gathered on monkeys suggest that there may be long-term risks not yet manifested in human populations. Long-term studies are needed to obtain more information on the subject.

Spermatozoal hapten gained via autolysis.

Using autolysis different spermatozoa surface peptides were solubilized. The resulting low molecular weight compounds were further separated by gel-chromatography on Biogel P-4, thin-layer chromatography on Cel 400 and thin-layer-electrophoresis. After the conjugation of the compound with cytochrome C a conformation-independent determinant was detected with immuno-electrophoresis. Out of the 17 fractions tested only fraction 3, with a molecular weight of 1800 daltons revealed a precipitation reaction with human sperm-immobilizing sera and rabbit antihuman sperm-serum. All 17 fractions showed no precipitation reaction against human sperm-agglutinating sera.

Isolation of a human spermatozoal hapten "II2.2' and its reaction with naturally occurring human sperm-immobilizing sera from infertile patients.

An extract of human spermatozoa was prepared using Hyamine 2389 and Triton X-100. With gel and ion-exchange chromatography several fractions were obtained of which are reacted specifically with sperm-immobilizing antibodies of infertile females and males in an immune inhibition test. This fraction showed haptenic properties, ahd a molecular weight of 1600 and was excluded as an anticomplement factor. After conjugation with cytochrome c the hapten II2.2 formed precipitation reactions with 3 out of 4 sperm-immobilizing sera. The titer reduction in sperm-immobilizing sera after adsorption with II2.2 may represent an in vitro model for a possible treatment of infertility in cases of a humoral sensitization against spermatozoa. On the other hand, the hapten might easily be synthesized and could, after conjugation to an appropriate carrier, serve as a contraceptive vaccine.

The presence of complement in human cervical mucus and its possible relevance to infertility in women with complement-dependent sperm-immobilizing antibodies.

Full-complement component lytic activity was measured in human midcycle cervical mucus, using a sensitive 51Cr release hemolytic assay. The level measured was 11.5% of the activity of complement in an equal volume of undiluted human serum. The relevance of this level of complement to complement-dependent sperm-immobilizing antibody activity was studied. After 1 hour's incubation with mucus levels of complement, immobilization of about 50% of spermatozoa occurred and after 3 hours' incubation, immobilization of about 70% of spermatozoa occurred.

Sperm/cervical-mucus crossed hostility testing and antisperm antibodies in the husband.

The behaviour of sperms has been investigated in preovulatory cervical mucus in 44 infertile couples. In 22 couples, immobilising and agglutinating autoantibodies were detected in the husband's sera in high titres. In 10 couples, antisperm antibodies were detected in the husbands by indirect immunofluorescent testing. In 12 couples, no evidence of antisperm antibodies was found in either husbands or wives. The results obtained with husband and wife were compared with the behaviour of the husband's sperms in cervical mucus from fertile donors, and with the behaviour of sperms from fertile donors in the wives' mucus. This crossed hostility test indicated that high tires of immobilising and agglutinating antisperm antibodies in the husband effectively prevented the sperms from penetrating the cervical mucus, even though the sperms appeared normal on seminal analysis. Antibodies detected by indirect immunofluorescence did not have this effect. Poor penetration was also observed with low sperm motility or poor cervical mucus. It is concluded that this test, taken with the postcoital test, could provide a useful screen for immunological causes of infertility and an accurate test for the clinical relevance of antisperm antibody tests.

Specificity of human sperm antigens solubilized by N-cetylpyridinium chloride.

PIP: To test the specificity of sperm fractions solubilized by N-acetylpyridinium chloride, human spermatozoa were washed 3 times with phosphate-buffered saline and resuspended in 5 ml of the solubilizing solution. The mixture was ultrasonated for 5 minutes in ice water and then centrifuged for 20 minutes. The supernatant was used for specificity tests. Details of the techniques used are given. Using N-acetylpyridinium chloride, a medium polar cationic detergent, 8 antigenic sperm fractions were extracted from ultrasonated spermatozoa run against human sperm-agglutinating sera in 2-dimensional immuno-electrophoresis. In runs against sperm-immobilizing sera, 9 sperm fractions were visualized. In runs against rabbit antihuman spermatozoa serum 11 fractions were found. Most of the antigenic fractions that were separated showed cross-reactivity with allogenic or xenogenic tissues. Cross-reactivity with human and animal organs was observed in many instances. Sperm as well as species specificities were found in some fractions. None of the fractions found could be detected on the surface membranes of intact human spermatozoa. Specific reactivity against a sperm-immobilizing serum was detected but no specific activity against a sperm-agglutinating serum was found. The fraction designated Gi is considered to be the "immobilizing" antigen. The origin of the antigens detectable on the surface membranes of intact spermatozoa was not determined.^ieng