[Role of sperm antibodies and cellular autoimmunity to sperm in the pathogenesis of male infertility]. / Role protilátkové a bunecné autoimunity proti spermiím v patogenezi muzské neplodnosti.

OBJECTIVE: To test the hypothesis of relationship between sperm pathology and elevated humoral and/or cell-mediated antisperm autoimmunity in male partners from infertile couples. DESIGN: Analytic study. SETTING: Department of Immunobiology, Institute for the Care of Mother and Child, Prague. METHODS: Sperm samples were evaluated according to WHO rules. Sperm-bound antisperm autoantibodies (ASA) were determined by SpermMar Test (FertiPro N. V., Sint-Martens-Latem, Belgium). For evaluation of cell-mediated antisperm autoimmunity (CMAA) the authors used their own modification of migration-inhibition test (Dimitrov et al., J. Immunol. Methods 154: 147, 1992). RESULTS: The pool of men was divided into groups according to the result of sperm examination: normozoospermia (740 men), asthenozoospermia (244), teratozoospermia (191), oligoasthenozoospermia levis (61), oligoasthenozoospermia gravis (29), oligoteratozoospermia (82), and azoospermia (54). Subgroup of fertile men (32) consisted of normozoospermic men--fathers of child younger than 3 years. Percentage of sperm-bound ASA-positive samples was significantly higher in asthenozoospermia in comparison with normozoospermia in both IgA (20.8% versus 10.6%) and IgG classes (13.8% vs 6.8%). Positivity of CMAA was significantly more frequent in group of asthenozoospermic (52%) than in normozoospermic (28.5%) and fertile (12.5%) men. CONCLUSION: Antisperm autoimmunity, namely its cell-mediated form, appears to play a significant role in impairment of spermiogenesis. Sperm-bound autoantibodies were found more frequently in asthenozoospermia, but also in some men with normozoospermia they may impair fertility.

In vivo immunosuppressive effects of recombinant ovine interferon-tau (trophoblastin): r.oTP (r.oIFN-tau) inhibits local GVH reaction in mice (PLN assay), prevents fetal resorptions, and favors embryo survival and implantation in the CBA/J x DBA/2 mice combination.

PROBLEM: Ovine trophoblastin protein, be it natural or recombinant (oTP,r.oTP), a member of the tau interferon family (r.oIFN-tau), has been shown to possess immunosuppressive properties in vitro. It acts as a cytostatic agent across species. Indeed, it was immunosuppressive when tested on human and murine lymphocytes in a variety of in vitro immune assays, as it is also on syngenic (ovine) lymphocytes. METHODS: In the present paper, we first verified that this property to act across species also occurred in vivo assays; r.oTP was able to down regulate a local GVH reaction assay (PLN assay) in mice. We then took advantage of these properties of r.oTP to investigate its in vivo effects during murine pregnancy as there is no ovine equivalent of the murine CBA/J x DBA/2 resorption prone mating combination. RESULTS: When given in the postimplantation period, r.oTP drastically boosted resorptions in the CBA/J x DBA/2 matings, as did murine recombinant gamma interferon. However, the same r.oTP treatment in the peri-implantation period resulted in a reduction in resorptions in this spontaneous abortion system. CONCLUSION: The data suggested that r.oTP might have acted more by favouring implantation and embryo survival than by preventing the resorption process itself. The mechanisms possibly underlying these effects, as well as the putative uses of r.oTP evolving from these data, are discussed.

Alloimmunization against well defined polymorphic major histocompatibility or class I MHC transfected L cells antigens can prevent poly IC induced fetal death in mice.

METHOD: It is possible to induce increased fetal resorption in a number of inbred murine matings by injecting Poly (I) Poly (C12U) 3.5 days postconception, a maneuver associated with natural killer-mediated damage to the feto placental unit such as occurs in spontaneous fetal resorptions. RESULTS: We show here that alloimmunization can block this effect. In addition, maternal immune responses induced by alloimmunization against isolated mutant class I or class II, as well as by immunization with class I MHC alloantigens (Kd) transfected L cells are sufficient to restore normal fetal viability. It is not necessary that the maternal immune response be specifically directed against paternal alloantigens fr the fetal protection to ensue, since the effect occurs in inbred matings when the mother is immunized against unrelated class I or class II alloantigens. As in previous studies conducted in the murine species, not all MHC alloimmunizations are protective. In addition, as control, immunization with a monomorphic class I MHC molecular (37), transfected L cells, sheep red blood cells or hen egg lysozyme is without effect. CONCLUSION: These results indicate that defined MHC antigens can mediate fetal protection from induced fetal resorption, and suggest that one driving force in promoting MHC antigen polymorphism in mammals is their capacity to confer protection from NK mediated fetal demise.

Effects of immunization with sperm specific lactate dehydrogenase with & without muramyl dipeptide as adjuvant.

The effect of sperm specific lactate dehydrogenase-C4 (LDH-C4) alone or with muramyl dipeptide (MDP) and Freund's complete adjuvant (FCA) on immune responses and breeding capacity have been studied in isogeneic C57 BI/Ks (H-2d) mice. Results per se suggested that LDH-C4 generates isoantibodies even in absence of adjuvant. Though MDP could be substituted for FCA as adjuvant, amplification by MDP of the antibody levels is reduced to half that obtained with FCA. LDH-B4 from kidney did not produce any antibody response under similar conditions. Systemic immunization with LDH-C4 did not reduce the overall pregnancy rate but reduced the frequency of embryo resorption and increased litter size significantly. However, mothers with foetal non-resorption had in general, lower antibody titres than the mothers showing foetal resorptions. The popliteal lymph node (PLN) assay for local graft versus host (LGVH) reaction revealed that the maternal lymphocyte cell competence was depressed by the sperm specific isozyme during gestation; depression in stimulation index was associated with the low antibody titre group. MDP, like LDH-C4 alone did not modify LGVH reaction significantly although it exerted a similar effect as LDH-C4 in embryo protection.

Correlation of asthenozoospermia with increased antisperm cell-mediated immunity in men from infertile couples.

To investigate whether cell-mediated immunity (CMI) against sperm and/or antisperm circulating antibodies are associated with poor semen quality, a leukocyte migration inhibition factor (LMIF) assay and an enzyme-linked immunosorbent assay (ELISA) were performed in groups of men from infertile couples, men from fertile couples and sperm donors. Twenty-five of 102 men (25%) revealed positive CMI against sperm and 10 (10%) had positive antisperm antibody titers in their sera. Fifteen of 28 asthenozoospermic men (53%) from infertile couples revealed positive antisperm CMI. The incidence of antisperm CMI was significantly increased (P < 0.05) in the infertile men with asthenozoospermia compared with the men from the other two groups (men from fertile couples and sperm donors). No significant differences between migration indices were seen when such a comparison was done for oligoasthenoterato- and teratozoospermics. The results indicate that increased antisperm CMI is associated with asthenozoospermia in a significant number of men from infertile couples. The importance of these findings is discussed.

Suppression of local and systemic GVHR by supernatants from human choriocarcinoma cell lines.

Graft versus host reaction (GVHR) and host versus graft reactivity during pregnancy share at least some cellular pathways. Immunosuppression mediated by placental suppressor factors may actively contribute to the survival of the conceptus in the immunocompetent mother. We have previously reported that supernatants from human choriocarcinoma cell lines inhibit mitogen-activated and antigen-activated proliferation of lymphoid cells. From this perspective, we investigated the capacity of supernatants from human choriocarcinoma cell lines (HCS) to suppress in vivo graft versus host reactions in mice. HCS was tested for its ability to block both local as well as systemic GVHR induced by the injection of parental cells into F1 mice. HCS mediates profound suppression of acute and chronic GVHR. These results confirm that human choriocarcinoma cell lines secrete a potent immunosuppressive factor(s) which can efficiently modulate immune responses in vivo.

Modulation of allo-immune responses in vivo and in vitro by sperm specific lactate dehydrogenase-C4.

The role of sperm specific lactate dehydrogenase-C4 (LDH-C4) in allo-immune responses using mixed lymphocyte cultures (MLC) and cytotoxic T cell (CTL) generation in vitro and local graft versus host (LGVH) reaction and allograft enhancement in vivo has been ascertained. LDH was purified from testes (LDH-C4) and kidney (LDH-B4) of C57Bl/Ks mice. MLC and CTL were performed using C57Bl/Ks-anti A/J lymphocytes in presence of 10(-3)-1 micrograms LDH-B4 or LDH-C4 per culture. The MLC and CTL responses showed biphasic action depending on the dose of LDH-C4. Early MLC culture gave significantly low stimulation index at 10(-2)-10(-1) micrograms LDH-C4 as compared to non-treated control cultures. However, the MLC response in presence of LDH-C4 was not different from the LDH-B4 treated one which showed a similar biphasic trend. On the other hand, 51Cr release from YAC-222 target cells was practically abolished by LDH-C4 at 10(-3)-1(-1) micrograms, and this was strikingly different from LDH-B4 or non-treated cultures. LGVH reactivity as performed by using C57Bl/Ks lymphocytes along with LDH-C4 in (C57Bl/Ks x A/J) F1 hybrids indicated a suppression of stimulation index in primary and secondary (i.e. preimmunized in presence of LDH-C4 or LDH-B4) LGVH. Allograft enhancement of Sa I (A/J) in C57Bl/Ks mice in presence of LDH-C4, was delayed slightly but significantly during primary or secondary transplantation reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

[Cell-mediated immune reactivity to sperm in disorders of human reproduction]. / Imunitní reaktivita proti spermiím zprostredkovaná bunkami (BI) v poruchách lidské reprodukce.

Besides the pathological anti-sperm humoral immunity, pathological anti-sperm cell-mediated immunity is considered as a crucial facet of the disturbances of human reproduction (male and female infertility, recurrent abortions, endometriosis, late EPH gestosis, fetal hypotrophy). A precise and objective method is designed, based on a one-step agarose Leukocyte Migration Inhibition Factor assay. The migration areas are evaluated by a computer-assisted image analysis system. Optimal concentrations of leukocytes and sperm, as well as technical conditions are described. The Radius Migration Indexes and Area Migration Indexes are computed and expressed as a Migration Index percentage for each patient or control. Preliminary clinical results indicate a highly significant association between leukocyte migration inhibition and cases of "immunopathological" infertility and repeated fetal loss.

A quantitative objective method for the evaluation of anti-sperm cell-mediated immunity in humans.

Anti-sperm cell-mediated immunity (CMI) is considered as a crucial facet of infertility in patients of both sexes. A precise and objective method is designed, based on a one-step agarose leukocyte migration inhibition factor (LMIF) assay. The migration areas are evaluated by a computer-assisted image analysis system. Optimal concentrations of leukocytes and sperm, as well as technical conditions are described. The radial migration indexes (RMI) and area migration indexes (AMI) are computed and expressed as a migration index (MI) percentage for each patient or control. Preliminary clinical results indicate a highly significant association between migration inhibition and cases of 'immunopathological infertility'. The method described is considered a promising tool for a rapid and quantitative evaluation of a suspected anti-sperm CMI in infertile and recurrently aborting patients.

Development and distribution of the white blood cells within various structures of the human menstrual corpus luteum examined using an image analysis system.

PROBLEM: Emerging evidences suggest that immunoendocrine interactions play definitive roles during development and regression of the human menstrual corpus luteum (hmCL). We have studied the distribution of immune cells within individual structures of hmCL during various stages of its development. METHOD: Immunoperoxidase-stained ultra-thin frozen sections were evaluated using light microscopy fitted with an image analysis system. RESULTS: The results suggest that monocytes/macrophages and MHC class II positive cells are the most prominent immune cells within the hmCL throughout its whole lifespan. Both cell types are concentrated within the trabeculae. In addition, MHC class II positive cells are abundant also within the granulosa-luteal layer. T helper/inductor (Th/i) and T cytotoxic/suppressor (Tc/s) cells were detected only in minor amounts within the thecal trabeculae of mature tissue. CONCLUSIONS: Possible links between the occurrence and functional roles of the immune cells studied are discussed.

The proportion of symmetric and asymmetric IgG antibody molecules synthesized by a cellular clone (hybridoma) can be regulated by placental culture supernatants.

The purpose of the present work was to establish whether the placenta is producing factors favoring an increased synthesis of asymmetric IgG antibodies which are known to assume a protective effect upon paternal antigens to which they largely are specific. In this way they can contribute to fetal survival in the maternal uterine environment. The hybridoma cell lines OKT8 (anti-CD8) and 112B4 (anti-DNP) were used in this respect since they synthesized both symmetric and asymmetric molecules of the IgG2a and IgG1 subclasses, respectively, murine isotypes in which anti-paternal antibodies have been detected. The cells were cultured in RPMI 1640 medium supplemented with 10% BCS and different amounts (5, 10, and 20%) of human placental supernatant. After incubation for 3 days at 37 degrees C in a humid chamber containing 5% CO2 the cells were centrifuged and the antibodies were obtained from the culture medium by a purification procedure involving precipitation at 50% ammonium sulfate saturation followed by DEAE-cellulose chromatography. Symmetric and asymmetric antibodies were separated by Con A-Sepharose affinity chromatography, the latter lectin retaining selectively only asymmetric IgG molecules. Both OKT8 and 112B4 hybridomas presenting a stable background synthesis of 15-17% of asymmetric antibodies have shown an increased level reaching 27-28% of these molecules in the presence of 5-10% placental supernatant added to the RPMI 1640 culture medium. These results clearly show that placental factors can up-regulate efficiently the synthesis of asymmetric IgG molecules of different isotypes secreted by plasma cells.

Modulation of T cell functions by sperm-specific lactate dehydrogenase: fluorescence analysis of immune competent cells and local graft versus host reaction.

Immune responses to a well-defined sperm-specific isogenic lactate dehydrogenase-C4 (LDH-C4) have been studied in C57Bl/Ks (H-2d) mice after immunization through intra-rectal route. Presence of anti-LDH-C4-antibodies in the sera of females immunized in presence or absence of adjuvant suggested that the immune system of mice becomes exposed to sperm antigens following intrarectal insemination. LDH-C4 primed lymphocytes from both males and females, when transferred in F1 hybrids, suppressed stimulation index of local graft versus host reaction. However, contrary to females, male counterparts which did not elicit measurable anti-LDH-C4-antibody titer, showed the presence of a higher proportion of Ly2+ and Ia+ fluorescence labelled cells in the spleen of LDH-C4 administered mice. Results suggest that males are more susceptible for immune suppression of T cell functions through generation of T suppressor cells. Sex differences in relation to immune deviation by intra-rectal administration of sperm-specific LDH-C4 in mice and their consequences in AIDS and AIDS-related complex diseases are described.

Immunology and immunopathology of reproduction.

This review is dealing with the roles of the immune system in the development and functions of the male and female reproductive systems. Further, it describes the topical views on the roles of immunopathologic events and mechanisms involved in the male and female infertility, recurrent abortions, endometriosis, EPH gestosis and disorders of fetal development. Particular attention is paid to the complicated immunological cross-talk and interplay between the mother and its offspring, including the active role played by the placenta and mainly the trophoblast tissue, in the course of gestation. In the light of the "immunotrophic theory", maternal immune responses to foreign fetal components, occurring in normal pregnancies, within the limits of "tolerated" or even beneficial levels, are described. Emphasis is given on the possible deregulation of materno-fetal immunological balance, leading to immunopathological events and putting in danger the overall reproductive capacity of the couple. The contemporary therapeutic--mainly immunological--approaches to the main reproductive failures are also mentioned.

The in vivo antibody response to hen egg white lysozyme in H-2b-compatible responder and non-responder mice: is it regulated by its N-terminal peptide at the level of antigen-presenting cells?

We studied the in vivo antibody responses of three H-2b strains, BALB/b, C57BL/6 and BALB/B x C57BL/6 F1 to various lysozymes, REL and HEL, after priming with HEL, REL or the HEL N-terminal peptide. It was confirmed that C57BL/6 is a non-responder strain to HEL and that BALB/b is responder strain. The C57BL/6 non-responder trait was associated with HEL or peptide induction of suppressor cells, as shown by adoptive transfer experiments. We further demonstrated that the suppressor/non-responder trait is dominant in BALB/b x C57BL/6 F1 hybrids and that appropriately pulsed macrophages of BALB/b mice can bypass such suppression in these F1 mice. Possible mechanisms are discussed.

Immunoactive products of human placenta. IV. Immunoregulatory factors obtained from cultures of human placenta inhibit in vivo local and systemic allogeneic and graft versus-host reactions in mice.

We have shown in previous reports that human placental supernatants (HPS) display a variety of immunoregulatory properties in vitro. Here we report that these supernatants are able to suppress the ability of murine splenocytes to evoke an allogeneic reaction in the popliteal lymph node (PLN) assay, as well as their capacity to induce both local (popliteal lymph node swelling) as well as systemic chronic or lethal graft-versus-host reactions. Those results are discussed in the light of the potential use of HPS in the control of transplantation immune reactions as well as in the regulation of xenogeneic pregnancies, as the murine system that was used proved that the material exerted an effect across a species barrier.

Complete Freund adjuvant treatment of pregnant females influences resorption rates in CBA/J x DBA/2 matings via progesterone-mediated immunomodulation.

Treatment of pregnant CBA/J females with CFA at day 0.5 and 7.5 of pregnancy significantly reduced the fetal resorption rates from 45% to 29% (P less than 0.05). Supernatants of progesterone-treated spleen cells from CFA treated CBA/J females pregnant of DBA/2 males significantly reduced natural cytotoxicity, while those of untreated identically pregnant mice had no effect. Supernatants of CFA-treated virgin mice blocked natural cytotoxicity to the same extent as those of CFA-treated pregnant mice. These data suggest that nonspecific immunostimulation induces progesterone receptors in spleen cells of CBA mice and that these receptors allow a progesterone dependent suppressive pathway to exert an antiresorptive effect.

The effect of a progesterone-induced immunologic blocking factor on NK-mediated resorption.

Transfer of spleen cells from poly (I)-poly(C12U)-treated Balb/c mice to 6.5-days-pregnant Balb/c mice significantly increased the resorption rate (P less than 0.01) from 11% to 48%. The supernatant of progesterone-treated spleen cells from pregnant C3H mice abrogated the effect of spleen cell transfer. We conclude that by blocking NK activity in vivo, the progesterone-induced blocking factor favors the maintenance of pregnancy.

A progesterone-induced blocking factor corrects high resorption rates in mice treated with antiprogesterone.

Earlier we showed that because of the presence of functional progesterone receptors, lymphocytes of healthy pregnant women produced an immunomodulatory protein in the presence of progesterone, whereas those of nonpregnant persons did not. Progesterone-treated murine pregnancy lymphocytes release a similar factor. The present study reveals the biologic significance of this finding. Treatment of BALB/c mice that were 8 days pregnant with a progesterone receptor blocker (RU 486) resulted in 100% resorption of the fetuses. Simultaneous administration of the supernatant from progesterone-treated murine pregnancy spleen cells restored the resorption rate to the original 6% observed in untreated control animals. These data suggest that functional lymphocytic progesterone binding sites are needed for the maintenance of normal pregnancy. Because of the blockage of progesterone receptors and the consequent inability of the lymphocytes to produce the progesterone-induced blocking factor, abortion is initiated by immune factors. The fact that administration of the preformed blocking factor counteracted the effect of antiprogesterone treatment suggests that progesterone-mediated immunosuppression is needed for the maintenance of normal gestation.