Journal of Reproductive Immunology, 2 (1980) 305-308 305 © Elsevier/North-Holland Biomedical Press

Report

WORKSHOP ON REPRODUCTIVE IMMUNOLOGY - KYOTO, JAPAN"

A small group of international scientists gathered in Kyoto for a two-day satellite workshop on reproductive immunology following the IXth World Congress of Obstetrics and Gynaecology held in Tokyo in October 1979. The main areas discussed were the antigenicity of spermatozoa, antibodies to zona pellucida and the feto-maternal relation- ship.

The session on sperm antigens was introduced by W.R. Jones (Adelaide, Australia) who identified several areas for priority research including the isolation and characterisa- tion of sperm membrane antigens, the clarification of the biological significance of semi- nal plasma antigens and their role in immunological infertility, and the need to develop new methodology for the quantitation of biologically relevant anti-sperm antibodies, based on pure and well characterised antigens.A.C Menge (Ann Arbor, U.S.A.)reviewed the current status of sperm antigens and antibodies and showed data supporting the use of the tray-agglutination test for the detection of antibody activity in women which cor- related with ongoing infertility.

A paper on seminal plasma antigens was presented by S. Isofima (Nishinomiya, Japan) who was the convener of the Workshop. Isojima described studies in which the comple- ment-dependent sperm immobilising activity in the sera of many infertile women could be absorbed out using lyophilised azoospermic semen. The completeness of the absorp- tion varied between different samples of azoospermic semen and some positive sera were only partly absorbed by semen but completely by washed sperm. Isojima also had in vitro evidence for cell-mediated immunity to seminal plasma antigens in infertile women. He concluded that at least part of the immune response to semen which produces infertility in women is directed against seminal plasma antigens as well as against intrinsic sperma- tozoal antigens.

Two different approaches to the investigation of the antigenic specificities of sperm agglutinating and immobilising antibodies in infertile and vasectomised individuals were described by T. H]ort (Aarhus, Denmark). One test system employed F(ab')2 fragments from sperm agglutinating and immobilising sera which were used to block auto- and iso-antigens in the sperm membrane. As these fragments had no immobilising activity, such F(ab')2-covered spermatozoa could be used in immobilisation tests with the various test sera. In cases in which the immobilisation of F(ab')2-covered spermatozoa was sig- nificantly reduced compared to the immobilisation obtained with untreated spermatozoa, it could be assumed that the F(ab')2 fragments and antibodies in serum were directed either against the very same antigenic determinant or against such closely associated anti- genic groups that steric hindrance could occur. By cross-testing a series of sera and their corresponding F(ab)2 fragments it appeared that essentially two patterns of reaction were observed, indicating that this test system could distinguish between two different sperm membrane antigens. An unexpected observation was that the patterns of reactivity showed no relation to the mode of agglutination observed in microscopic agglutination

306

tests. These results suggest that the different modes of agglutination are not necessarily a manifestation of reactions with different membrane antigens but could possibly reflect different qualities of antibodies.

The other approach employed tritium-labelled solubilised lectin-purified sperm mem- brane antigens which could be detected by indirect immuno-precipitation followed by polyacrylamide-gel electrophoresis (PAGE). Only sera with strong agglutinating and im- mobilising activity were able to pick up labelled membrane material. The results indi- cated that sera which in the F(ab')2 system had been found to react with one of the two antigens revealed identical PAGE patterns with three major peaks of molecular weights about 35 000, 45 000 and 77 000. Sera found to react with the other membrane antigen in the F(ab')2 system did not reveal any reaction with the lectin-purified solubilised mem- brane material. Consequently, the two approaches seemed to be in perfect agreement indicating that two different sperm membrane antigens may be involved in the various common sperm agglutination patterns.

Further studies on sperm immunity were presented by L. Mettler (Kiel, F.R.G.) who also described the preparation of low molecular weight haptens from solubllised human sperm and discussed their possible role in immunological infertility and fertility regula- tion.

There were two formal presentations on antibodies to zona pellucida. Y. Tsunoda (Chiba, Japan) discussed the biological aspects of anti-zona pellucida (ZP) antibodies. Anti-ZP antibodies were detected by four methods: (1) indirect immunofluorescence, (2) presence of immune precipitate on the outer surface of the zona pellucida, (3) block of in vitro fertflisation or sperm attachment to the ZP, and (4) inhibition of zona dissolu- tion by proteolytic enzymes. Immunofluorescence is the most sensitive method with the zona precipitate and block of fertilisation methods yielding similar results. Active immu- nisation of female mice with ZP material was not as effective in inhibiting fertility as was passive immunisation with rabbit anti-mouse ZP antiserum. A single injection of anti- serum inhibited fertility for over 30 days but by 60-70 days fertility returned to normal. The infertility was related to the presence of zona precipitate on the oocytes in the ovaries of the injected mice. Evidence suggested that to be effective the antibody must be bivalent since Fab preparations caused little inhibition of fertilisation either in vivo or in vitro. Sera from old mice tended to depress the fertilisation rate of treated ova from young mice. A large amount of cross reactivity exists for ZP antigens among animal species and with human. Goat anti-pig ZP serum effectively blocked fertility in the pig, mouse and cow but was less effective in the rat and rabbit. Also, 3 out of 10 sera from infertile women reacted with the zonae of mouse ova suggesting that the presence of anti- ZP antibodies may be related to human infertility.

S. Mori (Kyoto, Japan) discussed the clinical aspects of anti-ZP antibodies. He ob- served that immunofluorescent reactions seen with some human sera on porcine ZP were due to heteroagglutinins against porcine RBC antigens. After absorption of the sera with pRBCs, sera from approximately 10% of the women with unexplained infertility still reacted with pig ZP whereas only 0.6% of the absorbed control sera showed reaction. There was a trend for the incidence of positive sera to increase with patient's age. Positive sera were found to inhibit sperm attachment to human oocytes matured in vitro. The inhibition was closely related to the degree of zona precipitate produced by the differ- ent human sera.

307

The immunological aspects of the feto-maternal relationships were reviewed by A.E. Beer (Ann Arbor, U.S.A.) who contrasted the paradox of the survival of the feto-pla- cental unit as an allograft with the obviously protective and beneficial role mediated by maternal immunological recognition of the conceptus. Beer selectively emphasised certain areas of current interest. These included new concepts of the trophoblast as an immuno- logically privileged tissue. The high net electronegative charge generated at the surface of trophoblast cells may prevent similarly charged antigen-reactive lymphocytes as well as effector cells from interacting with the trophoblast. Evidence was quoted supporting the view that there is an intrinsic loss of antigenic determinants from the trophoblast plasma membrane resulting in a low antigen density thereby limiting the extent of potential reac- tivity of maternal lymphocytes, antibodies and complement. The possible role of pla- cental hormones in mediating immunosuppression was also considered in some detail. Beer quoted data relating to alpha fetoprotein (AFP) which when bound to oestradiol- 17t3 (E-2) will in turn bind to E-2 receptors on maternal lymphocytes and mediate an immunosuppressive effect. It was considered likely that many different types of hormone receptors will be found distributed amongst lymphocyte populations in pregnancy and that hormone binding in lymphoid tissue may be an important facet of the maternal im- mune recognition mechanism. Recent studies on the immunosuppressive properties of progesterone were considered by Beer to support the conclusion that local suppression of the maternal cellular immune responses by high tissue concentrations of this hormone constitutes one important mechanism preventing rejection of the conceptus. The placenta may also be considered as an immunologic sponge and animal studies by T.G. Wegmann and his colleagues in Edmonton, Canada, have suggested that this mechanism is of prime importance in preventing access of cytotoxic antibodies to the feto-placental unit. Finally, Beer summarised experimental evidence for the generation of specific suppressor T lymphocytes in the draining lymph nodes of the pregnant uterus which are capable of modifying the maternal immune response to paternal antigens.

G. Voisin (Paris, France) was the second speaker in the session on feto-maternal im- munology and he dealt more specifically with the immune agents elaborated by the mother and able to prevent the induction and expression of the immune rejection of the alloantigen-bearing fetus. Specific anti-paternal antibodies can be demonstrated to be fixed on the placenta from which they can be eluted and found to be able to enhance the growth of paternal strain tumors grafted on maternal strain mice. Spleens of pregnant mice were found to contain cells able to accelerate the rejection of paternal strain tumors in maternal strain recipients. They also contained two populations of T suppressor lym- phocytes able to enhance the take and growth of relevant tumors and to inhibit relevant mixed lymphocyte reactions (MLR): the first population acting at the induction phase of the MLR, the second one at the proliferative phase. Both were genetically restricted for cell target co-recognition and for regulatory/reactive cell co-operation. Ia and Ly markers were also studied. Placental extracts injected mixed with H-2 antigens were able to induce suppressor ceils tested in mixed lymphocyte reaction and tumor allograft sys- tems. The phylogeny of this double rejection and facilitation reaction has been studied in a viviparous amphibian, Salamandra salamandra, where maternal spleen cells are able to kill in vitro cells isolated from their embryos. The serum from the same mother prevents this killing by two factors: one is specific IgM acting on the embryonic cells and the 'other

308

is non-specific a-2 protein acting on the mother's cells. These findings imply that from the beginning of viviparity the conceptus induces in the mother the formation of facili- tating agents preventing its own rejection.

W.R. JONES