Inhibition of bacterial growth in vitro by human ejaculate seminal plasma fraction 1 and its possible practical use in male infertility phenomenon.

PURPOSE: The in vitro bacteriostatic effect on bacteria by a fraction of human ejaculate (HuSePl-Fr1) was studied. METHOD: As bacterial indicators, Pseudomonas aeruginosa (Pa), total coliforms (Tc), fecal coliforms (Fc), Escherichia coli (Ec), and Staphylococcus aureus (Sa) were used. These bacterial strains are known as agents that cause urinary and sexually transmitted infections (STI). RESULTS: Preliminary results suggest that HuSePl-Fr1 has a significant in vitro bacteriostatic effect on Pa and Sa at 42 degrees C. In addition, Pa was inhibited at 37 degrees C after 48 hr incubation. The remaining bacterial indicators were not affected. CONCLUSION: The results indicate a possible protective role of HuSePl-Fr1 in treatment of infertile men due to genital infections.

Multiple malignancies in immunocompetent patients.

We have reported two patients with multiple primary cancers in the presence of normal tests of cellular immune function, including normal natural killer cell activity. Immunodeficiency has been associated with an increased incidence of neoplastic disorders, but the resulting malignancies are unique, consisting of non-Hodgkin's lymphomas and a limited number of carcinomas. Immunosuppressive therapy and AIDS have been associated with aggressive sarcomas. Immunocompetence is of major importance against certain tumors. On the other hand, in spite of the limitations of the clinical evaluation of immunologic function, immunocompetence is insufficient to protect against neoplasia.

Relationship of blood groups and bladder cancer. A retrospective study.

A retrospective study to evaluate the relationship of blood groups of 96 patients with transitional cell carcinoma of the bladder in Israeli population, reports that patients with blood group O had higher grade tumors and higher recurrence rates than the other blood groups, without a difference in the mortality rates.

Immunosuppressant material in human seminal fluid. Inhibition of blast transformation and of NK activity by seminal fluid patients of a male infertility clinic.

Human seminal plasma from normal or patients with abnormal parameters of the ejaculates contains an inhibitory material that expresses potent in vitro inhibitory activity on PHA-M-induced blast transformation and NK activity. Using the test of inhibition of NK activity, the semen samples from individuals with higher concentrations of fructose had higher inhibitory activity. The results described herein suggest that inhibitory activity for blast transformation may be present in the prostatic fluid while the NK inhibition aspects are correlated with the vesicle-marker (fructose). Inhibition of the immune responses by human seminal plasma of the effector functions indicates the interesting implication that soluble factors may indirectly protect against or promote human autoimmune infertility disease.

Effect of the inhibitory material from male genital tract on natural killing activities.

A male inhibitory material (HuSePl-Frl) was separated from normal human seminal plasma by gel filtration on Sephadex G-100. This material has high avidity for cell membranes of human red blood cells (RBC). In vitro, PHA-M-induced blast transformation of human lymphocytes is inhibited by small quantities of HuSePl-Frl. This inhibition was also evident when RBC coated with HuSePl-Frl is added to mitogen stimulated lymphocytes in cultures. Small quantities of HuSePl-Frl are inhibitory to natural killer (NK) cell activation. The modulating role of the HuSePl-Frl in preventing the spermatozoal rejection during fertilization is discussed.

Successful long-term immunotherapy for human seminal plasma anaphylaxis.

Anaphylactic sensitivity to her husband's seminal fluid occurred in a 46-year-old woman six weeks after a hysterectomy. Her husband was azoospermic as a result of an elective vasectomy. A pooled sample of his seminal fluid was used for skin and laboratory tests. Fractionating (Sephadex G100) chromatography was also performed on his seminal fluid. The patient showed positive intracutaneous skin tests to his unfractionated seminal plasma and a Sephadex G100 fraction designated No. 3. This fraction was also passively transferable to a nonallergic volunteer and it stimulated histamine release from the patient's leukocytes. The dose-response effects of this fraction compared with unfractionated seminal plasma in these assays showed greater bioactivity. After informed consent was obtained, the patient was immunized to fraction 3 in a classic desensitization protocol. Two weeks after the final dose of immunotherapy, intercourse was performed without condoms in a medical facility and under the supervision of a physician. No adverse symptoms were experienced by the patient on this occasion. Regular and frequent sexual activity is required to maintain her tolerant state.

In vitro studies in reproductive immunology: 4. Mechanisms of immune response control in human male genital tract.

Fractionation of human seminal plasma by high-speed centrifugation and by gel filtration on Sephadex G-100 produces a high molecular weight component which displays immunosuppressive characteristics. Both materials inhibit mitogen-induced blast transformation of normal human peripheral monocytes, the component isolated by gel filtration being more effective in this regard. Prior treatment with trypsin of the inhibitory material isolated by centrifugation virtually abolishes its inhibitory potency suggesting that an intact protein is essential for biological function. The results also suggest that suppression of the mitogen-induced blast transformation response of cultured monocytes, T- and B-cells can be reversed by the addition of freshly isolated T- or B-cells. The relevance of these experimental observations to problems of human infertility is discussed.

Localized and systemic hypersensitivity reactions to human seminal fluid.

Reaginic humoral antibodies to human seminal plasma were present in two women with systemic reactions. In one patient, IgE antibodies were demonstrated by direct skin tests, leukocyte histamine release, passive transfer to a nonallergic human recipient, the radioallergosorbent test, inhibition of the radioallergosorbent test and neutralization of passive transfer antibodies. A similar mechanism was established in the other case by direct skin tests and antigen-induced leukocyte histamine release. Sephadex G-100 fraction 2, derived from human seminal plasma, showed greater reaginic activity than other chromatographic fractions. Two other patients with histories of localized reactions did not develop humoral antibodies but did evince cell-mediated immunity to seminal fluid antigens. One patient with localized reactions also had significant titers of IgM and IgG sperm agglutinating antibodies to seminal plasma. Both reaginic and cell-mediated sensitization to human sperm extract were also observed in this series of patients. Histocompatibility leukocyte antigen (HLA) typing of all patients and their sexual partners showed a marked degree of shared histocompatibility focus antigens in members of each of two couples.

In vitro studies in reproductive immunology. 3. Restoration of mitogenic activity of lymphocytes inhibited by seminal plasma in man.

Human seminal plasma, (HuSePl) is inhibitory to mitogen (PHA-M, Con-A, and PWM) lymphocyte activation. The inhibition does not require the continuous presence of HuSePl. Lymphocyte cultures remain inhibited after 24-hr contact with HuSePl and subsequent washings. Addition of fresh autochthonous lymphocytes or T cells but not B cells can restore the mitogenic activity of HuSePl-inhibited lymphocyte cultures. These observations suggest that T-lymphocyte in the male genital tract are surveilled by HuSePl and lack of inhibitory material could induce an immunologic response by spermatozoal antigens.

In vitro studies in reproductive immunology. - 2. Demonstration of the inhibitory effect of male genital tract constituents on PHA-stimulated mitogenesis and E-rosette formation of human lymphocytes.

The effect of male genital tract components (human spermatozoa, intact and chromatographed seminal plasma fractions) on in vitro cell-mediated immune reactions was examined. Their addition to PHA-containing lymphocyte cultures resulted in a marked degree of inhibition of DNA synthesis, the response varying with the fractions employed. The interference by such components with a terminal cell event during blast transformation was suggested by the failure of stimulated cultures to incorporate thymidine irrespective of the time during the incubation period at which the constituents were added. Seminal plasma and sperm extracts were also shown to inhibit T-cell associated E-rosette formation. The inhibitory properties of genital fractions remained unaltered after repetitive freeze-thaw cycling when tested in the transformation and E-rosette assays. Heating to 100 degrees C modified the response with some of the fractions.

The effect of human spermatozoa on antigen and mitogen induced blastogenesis.

In an attempt to identify factors capable of specifically or nonspecifically modulating results in an in vitro blastogenic assay system, studies were performed evaluating cell mediated immune response to human spermatozoa. Spermatozoa were capable, on a dose dependent basis, of both inhibiting and stimulating normal lymphocyte DNA synthesis as well as suppressing mitogen-induced response. Pretreatment of spermatozoa with neuraminidase and alpha-methyl-D-mannoside abrogated suppressive properties on spontaneous and/or mitogen-induced stimulation. Such suppressive activity on mitogen-induced response was observed using B and T-cell enriched populations. Inhibitory properties were not evident using intact cell populations stimulated by specific antigen. It is suggested that: (1) receptors on spermatozoa bind lectin; and (2) such receptors can be inactivated by enzymes having glycoprotein specificity.

Studies of sperm antigenicity. 6. In vivo and in vitro cellular reactivity in guinea pigs sensitized with fractions of guinea pig spermatozoa.

Normal guinea pig spermatozoa cells were homogenized by a French pressure cell. Three soluble and three insoluble fractions were obtained by ultrascentrifugation and (emulsified in CFA) were used for guinea pig sensitization. The following were observed: 1) all fractions were immunogenic except one; 2) in vivo and in vitro delayed hypersensitivity was elicited in animals immunized with these fractions; 3) two distinctive histopathologic lesions were observed in the testes of sensitized animals: lesions of orchitis type developed in animals injected with some fractions. Other fractions induced lesions of aspermatogenic type. These results correlated well with delayed hypersensitivity results obtained by in vivo and in vitro tests. Although some other spermatozoal fractions did not cause severe changes in the testes. The lack of sperm accumulation in the epididymis was obvious.

[Leukocyte reactions "in vitro" to human spermatozoa in infertile females]. / Réactions leucocytaires "in vitro" aux spermatozoïdes humains chez des femmes infertiles

Leucocyte migration test was done in 55 couples facing fertility problems for detection of antispermatozoal cellular hypersensitivity. In 29 cases out of 55, the sterility was unexplained and in 26 cases out of 55, various infertility factors were found. In this study, wife's leucocytes separated on Ficoll-Urografin gradient were incubated (4.10(6) per capillary) with washed fresh husband's spermatozoa suspended at 7.10(6)/ml in M 199 and 20 p. 100 fetal bovine serum. The results were read after 18h. Two kinds of migration inhibition were found: 1) Specific wives leucocyte migration inhibition in 4 out of 55 cases, all of them unexplained infertility cases. 2) Leucocyte migration inhibition not related to wife's immunity state and possibly due to a direct effect of a toxic or inhibitory property of some husband's spermatozoa in 22 out of 55 cases, 11/19 cases with known male factors, and 11/36 cases without any male factor. In the follow up, only 1 woman out of the 22 couples with husband's spermatozoal inhibitory factor was pregnant later, while in the group of 29 women in which normal leucocyte spreading was found, 8 women conceived later. No woman out of the 4 suspected of antispermatozoal delayed hypersensitivity have conceived. Attention must be paid to the possible role of delayed hypersensitivity to sperm in unexplained infertility and also to the occurrence of spermatozoal toxic factor and its possible implications on human infertility.

Studies on sperm antigenicity, 5. In vivo and in vitro cellular reactivity in guinea pigs sensitized with fractions of human spermatozoa.

In vitro delayed type hypersensitivity was demonstrated with peritoneal exudate cells from guinea pigs immunized with different preparations of human and guinea pig seminal components emulsified with complete Freund's adjuvant-H37Ra. The seminal components were intact human spermatozoa (HuSp); intact guinea pig spermatozoa (GPSp); human seminal plasma (HuSePlFr); and fractions of human spermatozoa obtained by centrifugation of the homogenate at 5,000 X g (5S30 and 5p30), at 20,000 X g (20S30 and 20p30), and at 144,000 X g (144p120). Cellular sensitivity was demonstrated in vivo by skin testing and in vitro by the macrophage inhibition technique. Peritoneal exudate cells from guinea pigs sensitized with fractions 5p30 and 20p30 elicited a delayed hypersensitivity reaction which could be detected only with intact human spermatozoa. Other human spermatozoal fractions (5S30, 20S30, and 144p120) were weak immunogens. Sensitization of guinea pigs with fractions of human spermatozoa, in addition to causing delayed hypersensitivity reactions, elicited low titers of spermatoxic antibodies. Antibodies to human spermatozoal fractions 5S30, 5p30, 20S30, and 20p30 cross-reacted with intact human spermatozoa and intact guinea pig spermatozoa. It is postulated that the existence of "spermatozoa-specific" coating antigen(s) derived from other components of the reproductive tract might be responsible for human spermatozoal antigenicity.