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Early pregnancy factor: its role in mammalian reproduction—research review
Vol. 35, No.4, April 1981 Printed in U.8A.
FERTILITY AND STERILITY Copyright c 1981 The American Fertility Society
EARLY PREGNANCY FACTOR: ITS ROLE IN MAMMALIAN REPRODUCTION-RESEARCH REVIEW
Y. CHENG SMART, M.Sc. * TIMOTHY K. ROBERTS, PH.D.t ROBERT L. CLANCY, B.Sc.(MED.), M.B., B.S., PH.D., F.R.A.C.P., F.R.A.C.P.(C) ALLAN W. CRIPPS, PH.D.:j:
Faculty of Medicine and Department of Biological Sciences, University of Newcastle, Newcastle, New South Wales 2308, and Hunter Immunology Unit, Royal Newcastle Hospital, Newcastle, New South Wales 2300, Australia
An immunosuppressive early pregnancy factor associated with mammalian reproduction is currently attracting considerable interest. Research into its detection, site of production, distribution, immunosuppressive property, characterization, and application is in progress in a number of laboratories. This review aims to crystallize the current research findings and to identify significant areas for further investigation and application. Fertil Steril35:397, 1981
Maternal nonrejection of the fetal allograft was initially attributed to inability of the uterus to mediate immunologic rejection, to the presence of a mechanical barrier, or to an absence of transplantation antigens in the fetoplacental unit. 1 However, recent studies have shown that graft rejection can occur within the endometrial wall,2 that no mechanical barrier exists at the deciduatrophoblast interface,3 and that transplantation antigens are evident in the blastocyst even prior to implantation. 4, 5 Maternal immune responses to fetus-related antigens occur6 but do not normally lead to embryo rejection. It is likely that the fundamental mechanism controlling this unique maternal immune response of pregnancy is one of suppression: suppression mediated by soluble serum factors,7 by locally released hormones,8 by placental antigens, or by factors released from immunologically competent cells. An exciting development is the detection of an immunosuppressive early pregnancy factor (EPF)9-12 which becomes evident in the serum
within hours of fertilization, that is, before implantation of the embryo. The possible role ofEPF in the immunologic protection of the early embryo and its practical role as an index of fertilization are the subject of this review. THE ASSAY SYSTEM
EPF is detected by using an in vitro rosette inhibition assay13 which measures the capacity of antilymphocyte serum (ALS) to inhibit rosette formation between T cells and heterologous erythrocytes. 14 The degree of inhibition by ALS is reflected in the level of the rosette inhibition titer (RIT), which is the reciprocal of that dilution of ALS resulting in the formation of 75% or fewer rosettes as compared with the internal spontaneous rosette formation control. The RIT value has been shown to reflect the in vivo immunosuppressive activity in patients receiving allografts. 15 Morton et al. 16 showed that the RIT of a particular ALS was dependent on the lymphocyte used. If lymphocytes from pregnant animals or normal lymphocytes incubated in serum from pregnant animals are used, the RIT is greater. The factor in pregnancy serum causing this enhancement of activity was termed early pregnancy factor (Fig. 1; Table 1). The sensitivity of the system
Received October 7, 1980; accepted November 20,1980. *Faculty of Medicine. University of Newcastle, Newcastle, New South Wales 2308, Australia. To whom reprint requests should be addressed. tDepartment of Biological Sciences, University of Newcastle. :j:Hunter Immunology Unit, Royal Newcastle Hospital.
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April 1981 In humans, EPF activity is evident in the maternal circulation within 48 hours after fertilization with a return toward normal in the third trimester of pregnancy 10, 20 (Fig. 2). Morton et al. 10 showed in two separate case studies that (1) spontaneous abortion was preceded by a return to normal RIT and (2) therapeutic abortion was followed by normal RIT within 4 weeks of surgery.
CHARACTERISTICS OF EPF
FIG. 1. Test for early pregnancy factor.
varies depending on the species studied. In mice, the RIT of lymphocytes obtained from pregnant and nonpregnant animals can differ by nine dilutions, whereas the human system is generally less sensitive, with a difference of only one and three dilutions. The mechanism of the rosette inhibition test system is unknown, although neither lymphocytotoxicity nor lymphoagglutinability is involvedp-19 ALS coating oflymphocytes has been suggested to provide the inhibition to rosette formation. 17 In view of its measure of the altered immunologic activity oflymphocytes, the test possibly depends on the blocking of lymphocytecombining sites by an immunosuppressive ALS, subsequently sterically hindering the binding of the heterologous red cells. EPF enhances this blocking effect of ALS, probably by saturating some binding sites on the lymphocytes. Further research to establish the mechanism of action of EPF on maternal T cells is essential. RELATIONSHIP TO PREGNANCY
The rosette inhibition assay consistently demonstrates a suppression of maternal lymphocyte activity by EPF during early pregnancy. Studies with mice 16 have shown a significant reduction of lymphocyte activity 6 hours after fertilization, coincidentally with formation of the pronuclei but before the first cell division. The titer values return to normal levels 4 to 6 days before parturition. EPF was detected in rats 24 hours after mating, with activity returning to normal 3 days before term.. 12 Sheep studies ll demonstrated EPF activity from 24 hours after mating, with serum titers correlating with subsequent pregnancy or return to estrus, indicating that EPF may provide a useful index of fertilization. Embryonic death was preceded by a decline in EPF activity, whereas surgical abortion led to a normalization of activity within 8 to 24 hours.ll
Physical Properties. Fractionation of mouse pregnancy serum through Ultrogel AcA34 gave two activity peaks of 180,000 and 40,000 molecular weight, respectively. The relative concentrations of the factors varied with duration of gestation. 21 The high molecular weight fraction appears soon after fertilization and its concentration decreases with progression of pregnancy. The low molecular weight factor appears coincidentally with implantation and its concentration increases with gestation. Human EPF is stable in storage at - 20° C for at least 19 weeks. 20 It is stable at 56° C but not at temperatures of 72° C or greater. 9 Its stability is also unaffected by low pH fractionation of pregnancy serum. 21 It is not species-specific, as it is detectable in sheep serum by mouse lymphocytes. 22 Comparison with Other Pregnancy-Associated Factors. Many pregnancy-associated serum factors, e.g., a-fetoprotein,23 pregnancy-zone protein,24 pregnancy-associated a-macroglobulin,25 human placental lactogen, 26 and human chorionic gonadotropin (hCG),27, 28 have been implicated in the suppression ofthe maternal immune response. They have been shown to inhibit mitogen and mixed lymphocyte cultures in vitro, and pregnancy-associated a-macroglobulin has also been . shown to reduce spontaneous E-rosette formation significantly. They appear in the maternal circulation at 6 to 9 weeks of gestation, with the exception of hCG, which is detected at implantation of the embryo. These factors increase in concentration with duration of pregnancy, reaching peak amounts in the last trimester. EPF, however, differs in both the time of appearance and TABLE 1. Rosetting Cells in Different Species Lymphocytes
Erythrocytes
Man Mouse Rat Sheep
Sheep Man Man Man
Vol. 35, No.4
EARLY PREGNANCY FACTOR
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FIG. 2. Rosette inhibition titers of pregnancy sera.
duration in the maternal serum, suggesting that it may indeed be a uniquely different pregnancyassociated substance. On the other hand, it is possible that the rosette inhibition system, given its high degree of sensitivity, may even respond positively to other potentially immunosuppressive pregnancy factors. Clarification must await establishment of the chemical identity of EPF. As hCG and hCG-like immunoreactive substances have been detected in preimplanting blastocysts,29, 30 attention has been focused on the relationship of hCG and EPF. In the RIT,10, 21 hCG obtained commercially (Sigma Chemical Co., St. Louis, Mo.) was shown to give increased rosette inhibition titers at concentrations of 1 pg/ml to 10 fJ.g/ml. Prior incubation of hCG solutions with anti-luteinizing hormone (Wellcome Reagents Ltd., Sydney, Australia) at 4° C for 18 hours resulted in normal titer values. Similar treatment of human pregnancy serum containing 85 and 325 nglml hCG with anti-luteinizing hormone, however, did not cause a reduction of rosette inhibition titer, presumably because EPF levels were significantly higher than hCG levels. lo Further studies are necessary to determine the dose response of purified hCG or synthetic hCG on the rosette inhibition system. Effective absorption of hCG should be performed on immunoadsorbent columns impregnated with anti-hCG. Immunosuppressive Property. The RIT demonstrates the suppression of lymphocyte activity by EPF in vitro. Its in vivo immunosuppressive characteristics are shown by indirect adoptive transfer 3l , 32 of contact sensitivity to trinitrochlorobenzene by lymphoid cells from sensitized mice. 22 Experimentation involved incubating lymph node cells from sensitized mice with serum or serum fractions containing sheep EPF, then injecting the cells into syngeneic mice. Complete suppression of delayed trinitrochlorobenzene sensitivity was observed in recipients when sensitized cells were incubated with 24-hour preg-
399
nancy serum or serum fractions. Incubation of cells with serum from nonpregnant sheep gave only partial suppression. Source. EPF is widely distributed in tissues of pregnant sheep, suggesting its nonspecific absorption by all tissues. 33 Experiments with mice during the first 48 hours after fertilization indicated involvement of the ovary, fertilized ovum, and the pituitary in the production ofEPF. 34 The pituitary component appears to be prolactin. 34 An immunosuppressive factor, EPF, was detected in human preimplantation embryo cultures35 (Fig. 3). Increased titer values were observed in 8 of9 cultures in which cleavage of the ovum from a 2- to > 16-cell stage was evident, representing in vitro embryo growth from 24 hours to 72 hours. Cultures in which no fertilization had occurred gave titer values within the normal range. The data suggest production of human EPF by the fertilized ovum within 24 hours after fertilization. It is not surpJ;ising that the mouse system differs from the human system, as mouse ovulation requires stimulation by prolactin. 36 RELEVANCE
Role in Pregnancy EPF is a pregnancy-associated substance demonstrated in the maternal circulation during early pregnancy. It appears capable of suppressing the
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400
April 1981
SMART ET AL.
immunologic competence of maternal lymphocytes both in vitro (by RIT) and in vivo (by adoptive transfer of contact sensitivity). Therefore, it is not unreasonable to implicate EPF in the active blocking of the maternal immune response toward the fetal allograft. Since only small variations are observed in the spontaneous rosetting capabilities of pregnancy and non pregnancy lymphocytes,1O, 16 it is reasonable to assume that pregnancy has little or no effect on the circulating T cell concentration, an observation supported by others. 37 Hence ALS suppression of lymphocyte activity is a functional, rather than a quantitative, effect. Of particular relevance is the production ofEPF by the fertilized ovum, which provides at a very early stage an immunosuppressive factor at the site of potential rejection. Many immunologically specific and nonspecific immunosuppressive factors have been described in pregnancy, many of which are concentrated at the maternal-fetal interface. However, other than EPF,20 none is produced in significant amounts at the implantation stage, providing for this factor possibly a unique role in preventing rejection of the oligocellular embryo. It is possible that many other factors combine to ensure fetal engraftment at different stages of development. Indeed, it must be remembered that the EPF assay system is nonspecific, and that different factors may give a positive result at different stages of pregnancy.21 The consistent reduction in EPF titer before parturition and spontaneous abortion supports the suggestion that reduced immunosuppression and immunologic rejection may contribute to abortion and parturition.26, 38
Application Since EPF has been validated as an indicator of fertilization and continued pregnancy,9-12,20 it has practical applications in investigation of the incidence of fertilization in infertile women, in elucidation ofthe mechanisms of intrauterine devices (IUDs), and in animal husbandry. It also may have a much broader role as a general immunosuppressant. Study ofFertilization in Women with a History of Infertility. Little is known about the occurrence of fertilization in infertile women, since available methods do not detect fertilization episodes but detect only the products of successful implantation. It is possible that fertilization occurs much more frequently than is indicated by confirmed pregnancies. With the establishment of
EPF as a fertilization index, such study is now possible and in progress in our laboratory in conjunction with standard hormone assays. Elucidation of the Mode of Action of IUDs. At present it is unclear whether IUDs act by preventing fertilization, by preventing implantation, or by initiating early abortion. Attempts to study the modes of action of IUDs have been confined to measurements of hCG in serum and urine ofIUD users during the luteal phase of the menstrual cycle. Conflicting evidence is reported relative to the presence or absence ofhCG-like material and the relative frequency of positive detection. 39-41 The presence ofhCG has been suggested as indicative of the presence of an implanted blastocyst. 42 Given the limitations of the technology used for hCG measurement and the lack of clinical symptoms of pregnancy in women, hCG or hCG-like substances detected at the lower limit of the system's sensitivity are unreliable. As EPF is an index of fertilization, we have used the rosette inhibition system to investigate the frequency of fertilization in IUD users over a number of menstrual cycles. Our data indicate the transient appearance of EPF in 6 of 22 cycles of IUD users, suggesting that fertilization had occurred but that the fertilized ovum probably failed to implant in the endometrium of the uterus. Thus the IUD is probably not an abortifacient, but rather it appears to act by interfering with the implantation process. 20 Role of EPF in Animal Husbandry. Until recently, failure of the animal to return to estrus was the earliest indication of successful mating. With the discovery of EPF, using the rosette inhibition assay, not only is the early detection of successful fertilization possible, but the progress of a continued pregnancy can be monitored. In the event of fetal distress or fetal death, therapeutic intervention or surgical abortion can be administered without time loss and unnecessary maternal stress. EPF may find applications in other areas of farm practices. For example, embryo transfers in animal breeding programs could be assisted by monitoring EPF during the pre- and post-transplantation processes. This would serve to provide breeders with better control and understanding of the operation, which, coupled with more refined techniques of transplantation, might lead to higher success rates. TECHNICAL CRITIQUE
The rosette inhibition assay, although sensitive and precise in demonstrating the presence of an
EARLY PREGNANCY FACTOR
Vol. 35, No.4
immunosuppressant in pregnancy sera, has many technical limitations. The parameters which require careful monitoring include activity of the different batches of ALS, activity and age of the guinea pig complement, source and age of the sheep red blood cells, and a reliable source of distilled water. It is essential that the assay be carried out by one person to minimize the inherent subjectivity of the system. Furthermore, it is important that the experimenter meticulously attend to details and precision of dilutions and screening for rosettes. Our main criticisms of the assay system are its lengthiness and its restrictiveness in the number of sera that can be processed per day. CONCLUSION
EPF is implicated in an immunosuppressive role with respect to the maternal tolerance of the antigenically alien fetus, especially during the preimplantation stage of pregnancy. After implantation of the blastocyst, a complex process involving many mechanisms may function to protect the fetus to term. EPF is established as an early index offertilization. As such, characterization and subsequent preparation of purified material may lead to the possible development of a contraceptive. EPF has made possible areas of research in pregnancy which, until recently, were difficult owing to a lack of suitable technology. These areas include IUD studies, study of fertility and infertility in women, and early detection of successful fertilization in farm breeding programs. However, technology remains a limiting factor in the routine clinical application of the assay. REFERENCES 1. Beer AE, Billingham RE: The Immunobiology of Mammalian Reproduction. Englewood Cliffs NJ, PrenticeHall Inc, 1976 2. Beer AE, Billingham RE: The embryo as a transplant. Sci Am 230:36,1974 3. Tekelioglu-Uysal M, Edwards RG, Kisnisci HA: Ultrastructural relationships between decidua, trophoblast and lymphocytes at the beginning of human pregnancy. J Reprod Fertil 42:431, 1975 4. Searle RF, Sellens MH, Elson J, Jenkinson EJ, Billington WD: Detection of alloantigens during preimplantation development and early trophoblast differentiation in the mouse by immunoperoxidase labeling. J Exp Med 143:348, 1976 5. Webb CG, Gall WE, Edelman GM: Synthesis and distribution of H-2 antigens in preimplantation mouse embryos. J Exp Med 146:923, 1977
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6. Youtananukorn V, Matangkasombut P: Human maternal cell mediated immune reaction to placental antigens. Clin Exp Immunol 11:549, 1972 7. Hellstrom KE, Hellstrom I, Brawn J: Abrogation of cellular immunity to antigenically foreign mouse embryonic cells by a serum factor. Nature 224:914, 1969 8. Lawrence R, Church JA, Richards W, Borzy M: Immunological mechanisms in the maintenance of pregnancy. Ann Allergy 44:166, 1980 9. Morton H, Hegh V, Clunie GJA: Studies of the rosette inhibition test in pregnant mice: evidence of immunosuppression? Proc R Soc Lond [Bioi] 193:413, 1976 10. Morton H, Rolfe B, Clunie GJA, Anderson MJ, MorrisonJ: An early pregnancy factor detected in human serum by the rosette inhibition test. Lancet 1:394, 1977 11. Morton H, Nancarrow CD, Scaramuzzi RJ, Evison BM, Clunie GJA: Detection of early pregnancy in sheep by the rosette inhibition test. J Reprod Fertil 56:75, 1979 12. Heywood LH, Goodall ET, Thorburn GD: Detection of early pregnancy in the rat using the rosette inhibition test. Presented at the Annual Meeting of the Australian Society for Reproductive Biology, Perth, 1979 13. Bach JF, Antoine B: In vitro detection of immunosuppressive activity of anti-lymphocyte sera. Nature 217:658, 1968 14. Bach JF, Dormont J, Dardenne M, BaIner H: In vitro rosette inhibition by antihuman antilymphocyte serum. Correlation with skin graft prolongation in subhuman primates. Transplantation 8:265, 1969 15. Munro A, Bewick M, Manuel L, Cameron JS, Ellis FG, Boulton-Jones M, Ogg S: Clinical evaluation of a rosette inhibition test in renal allotransplantation. Br Med J 3:271,1971 16. Morton H, Hegh V, Clunie GJA: Immunosuppression detected in pregnant mice by rosette inhibition test. Nature 249:459, 1974 17. Bach JF: Mechanism and significance of rosette inhibition by anti-lymphocyte serum. Symp Ser Immunobiol Standard 16:189, 1970 18. Brain P, Gordon J: Rosette formation by peripheral lymphocytes. II. Inhibition of the phenomenon. Clin Exp Immunol 8:441, 1971 19. Morton H, Hegh V, Clunie GJA: Production and assay of dog, pig and goat heterologous anti-rabbit lymphocyte serum. Clin Exp Immunol 13:595, 1973 20. Smart YC, Fraser IS, Roberts TK, Clancy RL, Cripps AW: Unpublished data 21. Clarke FM, Morton H, Clunie GJA: Detection and separation of two serum factors responsible for depression of lymphocyte activity in pregnancy. Clin Exp Immunol 32:318, 1978 22. Noonan FP, Halliday WJ, Morton H, Clunie GJA: Early pregnancy factor is immunosuppressive. Nature 278:649, 1979 23. Murgita RA, Tomasi TB: Suppression of the immune response by a-fetoprotein. II. The effect ofmouse a-fetoprotein on mixed lymphocyte reactivity and mitogen-induced lymphocyte transformation. J Exp Med 141:440, 1975 24. von Schoultz B, Stigbrand T: Purification of the "pregnancy-zone" protein. Acta Obstet Gynecol Scand 52:51,1973 25. Stimson WH: Studies on the immunosuppressive properties of a pregnancy-associated a-macroglobulin. Clin Exp Immunol 25:199, 1976
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26. Contractor SF, Davies H: Effect of human chorionic somatomammotrophin and human chorionic gonadotrophin on phytohaemagglutinin-induced lymphocyte transformation. Nature [New BioI] 243:284, 1973 27. Adcock EW III, Teasdale F, August CS, Cox S, Meschia G, Battaglia FC, Naughton MA: Human chorionic gonadotropin: its possible role in maternal lymphocyte suppression. Science 181:845, 1973 28. Beling CG, Weksler ME: Suppression of mixed lymphocyte reactivity by human chorionic gonadotrophin. Clin Exp ImmunoI18:537, 1974 29. Saxena BB, Hasan SH, Haour F, Schmidt-Gollwitzer M: Radioreceptor assay of human chorionic gonadotropin: detection of early pregnancy. Science 184:793, 1974 30. Haour F, Saxena BB: Detection of a gonadotrophin in rabbit blastocyst before implantation. Science 185:444, 1974 31. Moorhead JW: Soluble factors in tolerance and contact sensitivity to DNFB in mice. II. Genetic requirements for suppression of contact sensitivity by soluble suppressor factor. J Immunol 119:1773, 1977 32. Asherson GL, Zembala N: Anatomical location of cells which mediate contact sensitivity in the lymph nodes and bone marrow. Nature [New BioI] 244:176, 1973 33. Nancarrow CD, Wallace AL: Distribution of an early pregnancy factor in sheep. Presented at the Annual Meeting of the Australian Society for Reproductive Biology, Perth, 1979
April 1981 34. Morton H, Fitzgerald BE, Gidley-Baird AA, Cavanagh A, Clarke RM, Clunie GJA: Pituitary involvement in the production of early pregnancy factor. In Proceedings of the Sixth International Congress of Endocrinology, Melbourne, February 10-16, 1980. 35. Smart YC, Cripps AW, Clancy RL, Roberts TK, Lopata A, Shutt DA: Detection of an immunosuppressive factor in human preimplantation embryo culture. Med J Aust 1:78, 1981 36. Kwa HG, Verhofstad F: Prolactin levels in the plasma of female (C57BL x CBA!) F 1 mice. J EndocrinoI38:81, 1967 37. Baines MG, Pross HF, Millar KG: Lymphocyte populations in peripheral blood during normal human pregnancy. Clin Exp Immunol 28:453, 1977 38. Kerr MG: Immunological rejection as a cause of abortion. J Reprod Fertil [Suppl] 3:49, 1968 39. SaxenaBB, Landesman R: Does implantation occur in the presence of an IUD. Res Reprod 10:1, 1978 40. Beling CG, Cederquist LL, Fuchs F: Demonstration of gonadotropin during the second half ofthe cycle in women using intrauterine contraception. Am J Obstet Gynecol 125:855, 1976 41. Nilsson CG, Lahteenmaki P: Fertilisation in women with intrauterine devices. Lancet 2:1126, 1977 42. Landesman R, Coutinho EM, Saxena BB: Detection of human chorionic gonadotropin in blood of regularly bleeding women using copper intrauterine contraceptive devices. Fertil Steril 27:1062, 1976
FERTILITY AND STERILITY Copyright c 1981 The American Fertility Society
EARLY PREGNANCY FACTOR: ITS ROLE IN MAMMALIAN REPRODUCTION-RESEARCH REVIEW
Y. CHENG SMART, M.Sc. * TIMOTHY K. ROBERTS, PH.D.t ROBERT L. CLANCY, B.Sc.(MED.), M.B., B.S., PH.D., F.R.A.C.P., F.R.A.C.P.(C) ALLAN W. CRIPPS, PH.D.:j:
Faculty of Medicine and Department of Biological Sciences, University of Newcastle, Newcastle, New South Wales 2308, and Hunter Immunology Unit, Royal Newcastle Hospital, Newcastle, New South Wales 2300, Australia
An immunosuppressive early pregnancy factor associated with mammalian reproduction is currently attracting considerable interest. Research into its detection, site of production, distribution, immunosuppressive property, characterization, and application is in progress in a number of laboratories. This review aims to crystallize the current research findings and to identify significant areas for further investigation and application. Fertil Steril35:397, 1981
Maternal nonrejection of the fetal allograft was initially attributed to inability of the uterus to mediate immunologic rejection, to the presence of a mechanical barrier, or to an absence of transplantation antigens in the fetoplacental unit. 1 However, recent studies have shown that graft rejection can occur within the endometrial wall,2 that no mechanical barrier exists at the deciduatrophoblast interface,3 and that transplantation antigens are evident in the blastocyst even prior to implantation. 4, 5 Maternal immune responses to fetus-related antigens occur6 but do not normally lead to embryo rejection. It is likely that the fundamental mechanism controlling this unique maternal immune response of pregnancy is one of suppression: suppression mediated by soluble serum factors,7 by locally released hormones,8 by placental antigens, or by factors released from immunologically competent cells. An exciting development is the detection of an immunosuppressive early pregnancy factor (EPF)9-12 which becomes evident in the serum
within hours of fertilization, that is, before implantation of the embryo. The possible role ofEPF in the immunologic protection of the early embryo and its practical role as an index of fertilization are the subject of this review. THE ASSAY SYSTEM
EPF is detected by using an in vitro rosette inhibition assay13 which measures the capacity of antilymphocyte serum (ALS) to inhibit rosette formation between T cells and heterologous erythrocytes. 14 The degree of inhibition by ALS is reflected in the level of the rosette inhibition titer (RIT), which is the reciprocal of that dilution of ALS resulting in the formation of 75% or fewer rosettes as compared with the internal spontaneous rosette formation control. The RIT value has been shown to reflect the in vivo immunosuppressive activity in patients receiving allografts. 15 Morton et al. 16 showed that the RIT of a particular ALS was dependent on the lymphocyte used. If lymphocytes from pregnant animals or normal lymphocytes incubated in serum from pregnant animals are used, the RIT is greater. The factor in pregnancy serum causing this enhancement of activity was termed early pregnancy factor (Fig. 1; Table 1). The sensitivity of the system
Received October 7, 1980; accepted November 20,1980. *Faculty of Medicine. University of Newcastle, Newcastle, New South Wales 2308, Australia. To whom reprint requests should be addressed. tDepartment of Biological Sciences, University of Newcastle. :j:Hunter Immunology Unit, Royal Newcastle Hospital.
397
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April 1981 In humans, EPF activity is evident in the maternal circulation within 48 hours after fertilization with a return toward normal in the third trimester of pregnancy 10, 20 (Fig. 2). Morton et al. 10 showed in two separate case studies that (1) spontaneous abortion was preceded by a return to normal RIT and (2) therapeutic abortion was followed by normal RIT within 4 weeks of surgery.
CHARACTERISTICS OF EPF
FIG. 1. Test for early pregnancy factor.
varies depending on the species studied. In mice, the RIT of lymphocytes obtained from pregnant and nonpregnant animals can differ by nine dilutions, whereas the human system is generally less sensitive, with a difference of only one and three dilutions. The mechanism of the rosette inhibition test system is unknown, although neither lymphocytotoxicity nor lymphoagglutinability is involvedp-19 ALS coating oflymphocytes has been suggested to provide the inhibition to rosette formation. 17 In view of its measure of the altered immunologic activity oflymphocytes, the test possibly depends on the blocking of lymphocytecombining sites by an immunosuppressive ALS, subsequently sterically hindering the binding of the heterologous red cells. EPF enhances this blocking effect of ALS, probably by saturating some binding sites on the lymphocytes. Further research to establish the mechanism of action of EPF on maternal T cells is essential. RELATIONSHIP TO PREGNANCY
The rosette inhibition assay consistently demonstrates a suppression of maternal lymphocyte activity by EPF during early pregnancy. Studies with mice 16 have shown a significant reduction of lymphocyte activity 6 hours after fertilization, coincidentally with formation of the pronuclei but before the first cell division. The titer values return to normal levels 4 to 6 days before parturition. EPF was detected in rats 24 hours after mating, with activity returning to normal 3 days before term.. 12 Sheep studies ll demonstrated EPF activity from 24 hours after mating, with serum titers correlating with subsequent pregnancy or return to estrus, indicating that EPF may provide a useful index of fertilization. Embryonic death was preceded by a decline in EPF activity, whereas surgical abortion led to a normalization of activity within 8 to 24 hours.ll
Physical Properties. Fractionation of mouse pregnancy serum through Ultrogel AcA34 gave two activity peaks of 180,000 and 40,000 molecular weight, respectively. The relative concentrations of the factors varied with duration of gestation. 21 The high molecular weight fraction appears soon after fertilization and its concentration decreases with progression of pregnancy. The low molecular weight factor appears coincidentally with implantation and its concentration increases with gestation. Human EPF is stable in storage at - 20° C for at least 19 weeks. 20 It is stable at 56° C but not at temperatures of 72° C or greater. 9 Its stability is also unaffected by low pH fractionation of pregnancy serum. 21 It is not species-specific, as it is detectable in sheep serum by mouse lymphocytes. 22 Comparison with Other Pregnancy-Associated Factors. Many pregnancy-associated serum factors, e.g., a-fetoprotein,23 pregnancy-zone protein,24 pregnancy-associated a-macroglobulin,25 human placental lactogen, 26 and human chorionic gonadotropin (hCG),27, 28 have been implicated in the suppression ofthe maternal immune response. They have been shown to inhibit mitogen and mixed lymphocyte cultures in vitro, and pregnancy-associated a-macroglobulin has also been . shown to reduce spontaneous E-rosette formation significantly. They appear in the maternal circulation at 6 to 9 weeks of gestation, with the exception of hCG, which is detected at implantation of the embryo. These factors increase in concentration with duration of pregnancy, reaching peak amounts in the last trimester. EPF, however, differs in both the time of appearance and TABLE 1. Rosetting Cells in Different Species Lymphocytes
Erythrocytes
Man Mouse Rat Sheep
Sheep Man Man Man
Vol. 35, No.4
EARLY PREGNANCY FACTOR
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FIG. 2. Rosette inhibition titers of pregnancy sera.
duration in the maternal serum, suggesting that it may indeed be a uniquely different pregnancyassociated substance. On the other hand, it is possible that the rosette inhibition system, given its high degree of sensitivity, may even respond positively to other potentially immunosuppressive pregnancy factors. Clarification must await establishment of the chemical identity of EPF. As hCG and hCG-like immunoreactive substances have been detected in preimplanting blastocysts,29, 30 attention has been focused on the relationship of hCG and EPF. In the RIT,10, 21 hCG obtained commercially (Sigma Chemical Co., St. Louis, Mo.) was shown to give increased rosette inhibition titers at concentrations of 1 pg/ml to 10 fJ.g/ml. Prior incubation of hCG solutions with anti-luteinizing hormone (Wellcome Reagents Ltd., Sydney, Australia) at 4° C for 18 hours resulted in normal titer values. Similar treatment of human pregnancy serum containing 85 and 325 nglml hCG with anti-luteinizing hormone, however, did not cause a reduction of rosette inhibition titer, presumably because EPF levels were significantly higher than hCG levels. lo Further studies are necessary to determine the dose response of purified hCG or synthetic hCG on the rosette inhibition system. Effective absorption of hCG should be performed on immunoadsorbent columns impregnated with anti-hCG. Immunosuppressive Property. The RIT demonstrates the suppression of lymphocyte activity by EPF in vitro. Its in vivo immunosuppressive characteristics are shown by indirect adoptive transfer 3l , 32 of contact sensitivity to trinitrochlorobenzene by lymphoid cells from sensitized mice. 22 Experimentation involved incubating lymph node cells from sensitized mice with serum or serum fractions containing sheep EPF, then injecting the cells into syngeneic mice. Complete suppression of delayed trinitrochlorobenzene sensitivity was observed in recipients when sensitized cells were incubated with 24-hour preg-
399
nancy serum or serum fractions. Incubation of cells with serum from nonpregnant sheep gave only partial suppression. Source. EPF is widely distributed in tissues of pregnant sheep, suggesting its nonspecific absorption by all tissues. 33 Experiments with mice during the first 48 hours after fertilization indicated involvement of the ovary, fertilized ovum, and the pituitary in the production ofEPF. 34 The pituitary component appears to be prolactin. 34 An immunosuppressive factor, EPF, was detected in human preimplantation embryo cultures35 (Fig. 3). Increased titer values were observed in 8 of9 cultures in which cleavage of the ovum from a 2- to > 16-cell stage was evident, representing in vitro embryo growth from 24 hours to 72 hours. Cultures in which no fertilization had occurred gave titer values within the normal range. The data suggest production of human EPF by the fertilized ovum within 24 hours after fertilization. It is not surpJ;ising that the mouse system differs from the human system, as mouse ovulation requires stimulation by prolactin. 36 RELEVANCE
Role in Pregnancy EPF is a pregnancy-associated substance demonstrated in the maternal circulation during early pregnancy. It appears capable of suppressing the
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4 2
o
a
e
Sample Size FIG. 3. Human EPF activity in culture media in vitro. a, Controls; b, insemination media; c, day 2 fertilized cultures (2 to 8 cells); d, day 3 fertilized cultures (2 to > 16 cells); e, day 2 unfertilized cultures.
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immunologic competence of maternal lymphocytes both in vitro (by RIT) and in vivo (by adoptive transfer of contact sensitivity). Therefore, it is not unreasonable to implicate EPF in the active blocking of the maternal immune response toward the fetal allograft. Since only small variations are observed in the spontaneous rosetting capabilities of pregnancy and non pregnancy lymphocytes,1O, 16 it is reasonable to assume that pregnancy has little or no effect on the circulating T cell concentration, an observation supported by others. 37 Hence ALS suppression of lymphocyte activity is a functional, rather than a quantitative, effect. Of particular relevance is the production ofEPF by the fertilized ovum, which provides at a very early stage an immunosuppressive factor at the site of potential rejection. Many immunologically specific and nonspecific immunosuppressive factors have been described in pregnancy, many of which are concentrated at the maternal-fetal interface. However, other than EPF,20 none is produced in significant amounts at the implantation stage, providing for this factor possibly a unique role in preventing rejection of the oligocellular embryo. It is possible that many other factors combine to ensure fetal engraftment at different stages of development. Indeed, it must be remembered that the EPF assay system is nonspecific, and that different factors may give a positive result at different stages of pregnancy.21 The consistent reduction in EPF titer before parturition and spontaneous abortion supports the suggestion that reduced immunosuppression and immunologic rejection may contribute to abortion and parturition.26, 38
Application Since EPF has been validated as an indicator of fertilization and continued pregnancy,9-12,20 it has practical applications in investigation of the incidence of fertilization in infertile women, in elucidation ofthe mechanisms of intrauterine devices (IUDs), and in animal husbandry. It also may have a much broader role as a general immunosuppressant. Study ofFertilization in Women with a History of Infertility. Little is known about the occurrence of fertilization in infertile women, since available methods do not detect fertilization episodes but detect only the products of successful implantation. It is possible that fertilization occurs much more frequently than is indicated by confirmed pregnancies. With the establishment of
EPF as a fertilization index, such study is now possible and in progress in our laboratory in conjunction with standard hormone assays. Elucidation of the Mode of Action of IUDs. At present it is unclear whether IUDs act by preventing fertilization, by preventing implantation, or by initiating early abortion. Attempts to study the modes of action of IUDs have been confined to measurements of hCG in serum and urine ofIUD users during the luteal phase of the menstrual cycle. Conflicting evidence is reported relative to the presence or absence ofhCG-like material and the relative frequency of positive detection. 39-41 The presence ofhCG has been suggested as indicative of the presence of an implanted blastocyst. 42 Given the limitations of the technology used for hCG measurement and the lack of clinical symptoms of pregnancy in women, hCG or hCG-like substances detected at the lower limit of the system's sensitivity are unreliable. As EPF is an index of fertilization, we have used the rosette inhibition system to investigate the frequency of fertilization in IUD users over a number of menstrual cycles. Our data indicate the transient appearance of EPF in 6 of 22 cycles of IUD users, suggesting that fertilization had occurred but that the fertilized ovum probably failed to implant in the endometrium of the uterus. Thus the IUD is probably not an abortifacient, but rather it appears to act by interfering with the implantation process. 20 Role of EPF in Animal Husbandry. Until recently, failure of the animal to return to estrus was the earliest indication of successful mating. With the discovery of EPF, using the rosette inhibition assay, not only is the early detection of successful fertilization possible, but the progress of a continued pregnancy can be monitored. In the event of fetal distress or fetal death, therapeutic intervention or surgical abortion can be administered without time loss and unnecessary maternal stress. EPF may find applications in other areas of farm practices. For example, embryo transfers in animal breeding programs could be assisted by monitoring EPF during the pre- and post-transplantation processes. This would serve to provide breeders with better control and understanding of the operation, which, coupled with more refined techniques of transplantation, might lead to higher success rates. TECHNICAL CRITIQUE
The rosette inhibition assay, although sensitive and precise in demonstrating the presence of an
EARLY PREGNANCY FACTOR
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immunosuppressant in pregnancy sera, has many technical limitations. The parameters which require careful monitoring include activity of the different batches of ALS, activity and age of the guinea pig complement, source and age of the sheep red blood cells, and a reliable source of distilled water. It is essential that the assay be carried out by one person to minimize the inherent subjectivity of the system. Furthermore, it is important that the experimenter meticulously attend to details and precision of dilutions and screening for rosettes. Our main criticisms of the assay system are its lengthiness and its restrictiveness in the number of sera that can be processed per day. CONCLUSION
EPF is implicated in an immunosuppressive role with respect to the maternal tolerance of the antigenically alien fetus, especially during the preimplantation stage of pregnancy. After implantation of the blastocyst, a complex process involving many mechanisms may function to protect the fetus to term. EPF is established as an early index offertilization. As such, characterization and subsequent preparation of purified material may lead to the possible development of a contraceptive. EPF has made possible areas of research in pregnancy which, until recently, were difficult owing to a lack of suitable technology. These areas include IUD studies, study of fertility and infertility in women, and early detection of successful fertilization in farm breeding programs. However, technology remains a limiting factor in the routine clinical application of the assay. REFERENCES 1. Beer AE, Billingham RE: The Immunobiology of Mammalian Reproduction. Englewood Cliffs NJ, PrenticeHall Inc, 1976 2. Beer AE, Billingham RE: The embryo as a transplant. Sci Am 230:36,1974 3. Tekelioglu-Uysal M, Edwards RG, Kisnisci HA: Ultrastructural relationships between decidua, trophoblast and lymphocytes at the beginning of human pregnancy. J Reprod Fertil 42:431, 1975 4. Searle RF, Sellens MH, Elson J, Jenkinson EJ, Billington WD: Detection of alloantigens during preimplantation development and early trophoblast differentiation in the mouse by immunoperoxidase labeling. J Exp Med 143:348, 1976 5. Webb CG, Gall WE, Edelman GM: Synthesis and distribution of H-2 antigens in preimplantation mouse embryos. J Exp Med 146:923, 1977
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6. Youtananukorn V, Matangkasombut P: Human maternal cell mediated immune reaction to placental antigens. Clin Exp Immunol 11:549, 1972 7. Hellstrom KE, Hellstrom I, Brawn J: Abrogation of cellular immunity to antigenically foreign mouse embryonic cells by a serum factor. Nature 224:914, 1969 8. Lawrence R, Church JA, Richards W, Borzy M: Immunological mechanisms in the maintenance of pregnancy. Ann Allergy 44:166, 1980 9. Morton H, Hegh V, Clunie GJA: Studies of the rosette inhibition test in pregnant mice: evidence of immunosuppression? Proc R Soc Lond [Bioi] 193:413, 1976 10. Morton H, Rolfe B, Clunie GJA, Anderson MJ, MorrisonJ: An early pregnancy factor detected in human serum by the rosette inhibition test. Lancet 1:394, 1977 11. Morton H, Nancarrow CD, Scaramuzzi RJ, Evison BM, Clunie GJA: Detection of early pregnancy in sheep by the rosette inhibition test. J Reprod Fertil 56:75, 1979 12. Heywood LH, Goodall ET, Thorburn GD: Detection of early pregnancy in the rat using the rosette inhibition test. Presented at the Annual Meeting of the Australian Society for Reproductive Biology, Perth, 1979 13. Bach JF, Antoine B: In vitro detection of immunosuppressive activity of anti-lymphocyte sera. Nature 217:658, 1968 14. Bach JF, Dormont J, Dardenne M, BaIner H: In vitro rosette inhibition by antihuman antilymphocyte serum. Correlation with skin graft prolongation in subhuman primates. Transplantation 8:265, 1969 15. Munro A, Bewick M, Manuel L, Cameron JS, Ellis FG, Boulton-Jones M, Ogg S: Clinical evaluation of a rosette inhibition test in renal allotransplantation. Br Med J 3:271,1971 16. Morton H, Hegh V, Clunie GJA: Immunosuppression detected in pregnant mice by rosette inhibition test. Nature 249:459, 1974 17. Bach JF: Mechanism and significance of rosette inhibition by anti-lymphocyte serum. Symp Ser Immunobiol Standard 16:189, 1970 18. Brain P, Gordon J: Rosette formation by peripheral lymphocytes. II. Inhibition of the phenomenon. Clin Exp Immunol 8:441, 1971 19. Morton H, Hegh V, Clunie GJA: Production and assay of dog, pig and goat heterologous anti-rabbit lymphocyte serum. Clin Exp Immunol 13:595, 1973 20. Smart YC, Fraser IS, Roberts TK, Clancy RL, Cripps AW: Unpublished data 21. Clarke FM, Morton H, Clunie GJA: Detection and separation of two serum factors responsible for depression of lymphocyte activity in pregnancy. Clin Exp Immunol 32:318, 1978 22. Noonan FP, Halliday WJ, Morton H, Clunie GJA: Early pregnancy factor is immunosuppressive. Nature 278:649, 1979 23. Murgita RA, Tomasi TB: Suppression of the immune response by a-fetoprotein. II. The effect ofmouse a-fetoprotein on mixed lymphocyte reactivity and mitogen-induced lymphocyte transformation. J Exp Med 141:440, 1975 24. von Schoultz B, Stigbrand T: Purification of the "pregnancy-zone" protein. Acta Obstet Gynecol Scand 52:51,1973 25. Stimson WH: Studies on the immunosuppressive properties of a pregnancy-associated a-macroglobulin. Clin Exp Immunol 25:199, 1976
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26. Contractor SF, Davies H: Effect of human chorionic somatomammotrophin and human chorionic gonadotrophin on phytohaemagglutinin-induced lymphocyte transformation. Nature [New BioI] 243:284, 1973 27. Adcock EW III, Teasdale F, August CS, Cox S, Meschia G, Battaglia FC, Naughton MA: Human chorionic gonadotropin: its possible role in maternal lymphocyte suppression. Science 181:845, 1973 28. Beling CG, Weksler ME: Suppression of mixed lymphocyte reactivity by human chorionic gonadotrophin. Clin Exp ImmunoI18:537, 1974 29. Saxena BB, Hasan SH, Haour F, Schmidt-Gollwitzer M: Radioreceptor assay of human chorionic gonadotropin: detection of early pregnancy. Science 184:793, 1974 30. Haour F, Saxena BB: Detection of a gonadotrophin in rabbit blastocyst before implantation. Science 185:444, 1974 31. Moorhead JW: Soluble factors in tolerance and contact sensitivity to DNFB in mice. II. Genetic requirements for suppression of contact sensitivity by soluble suppressor factor. J Immunol 119:1773, 1977 32. Asherson GL, Zembala N: Anatomical location of cells which mediate contact sensitivity in the lymph nodes and bone marrow. Nature [New BioI] 244:176, 1973 33. Nancarrow CD, Wallace AL: Distribution of an early pregnancy factor in sheep. Presented at the Annual Meeting of the Australian Society for Reproductive Biology, Perth, 1979
April 1981 34. Morton H, Fitzgerald BE, Gidley-Baird AA, Cavanagh A, Clarke RM, Clunie GJA: Pituitary involvement in the production of early pregnancy factor. In Proceedings of the Sixth International Congress of Endocrinology, Melbourne, February 10-16, 1980. 35. Smart YC, Cripps AW, Clancy RL, Roberts TK, Lopata A, Shutt DA: Detection of an immunosuppressive factor in human preimplantation embryo culture. Med J Aust 1:78, 1981 36. Kwa HG, Verhofstad F: Prolactin levels in the plasma of female (C57BL x CBA!) F 1 mice. J EndocrinoI38:81, 1967 37. Baines MG, Pross HF, Millar KG: Lymphocyte populations in peripheral blood during normal human pregnancy. Clin Exp Immunol 28:453, 1977 38. Kerr MG: Immunological rejection as a cause of abortion. J Reprod Fertil [Suppl] 3:49, 1968 39. SaxenaBB, Landesman R: Does implantation occur in the presence of an IUD. Res Reprod 10:1, 1978 40. Beling CG, Cederquist LL, Fuchs F: Demonstration of gonadotropin during the second half ofthe cycle in women using intrauterine contraception. Am J Obstet Gynecol 125:855, 1976 41. Nilsson CG, Lahteenmaki P: Fertilisation in women with intrauterine devices. Lancet 2:1126, 1977 42. Landesman R, Coutinho EM, Saxena BB: Detection of human chorionic gonadotropin in blood of regularly bleeding women using copper intrauterine contraceptive devices. Fertil Steril 27:1062, 1976