SIEP publications are protected by copyright and all rights are reserved.  SIEP publications may not be reproduced in any form or by any means without written permission from the copyright owner. This includes the posting of electronic files on the Internet, transferring electronic files to other persons, distributing printed output, and photocopying. Requests for authorization to make photocopies should be directed to:   SIEP, 1697 Lark Lane, Cherry Hill, NJ 08003-3157
Tel: (856) 429 2699, Fax:  (856) 429 7414, Email: barnea@earlypregnancy.org

---

EARLY PREGNANCY: Biology and Medicine Editor-in-Chief: Eytan R. Barnea MD, FACOG

July 2000
Volume IV, Number 3
ISSN: 1537-6583
Pages: 166-175

Eytan R.Barnea MD,* Young J. Choi PhD^, and Paul C. Leavis PhD^

*SIEP, The Society for the Investigation of Early Pregnancy, Cherry Hill, NJ
^ Boston Biomedical Research Institute, Boston

From Textbook of Obstetrics & Gynecology: Editor: I. Munteanu

Acknowledgements: The authors would like to thank Christine A. Brusato from Duke University, Durham, NC, for extensive editorial input.

Support for this project was provided, in part, by CONRAD [CIG-98-21] through a grant to BioIncept, Inc. from the CICCR Program of the Contraceptive Research and Development Program, Eastern Virginia Medical School.

Rationale

Mammalian reproduction represents the most complex form of parental gene interaction. This interaction requires a secure environment, namely the maternal organism, and the creation of specific safeguards to allow the fertilized egg there to survive. The issue of gamete preservation has been resolved by allowing for fertilization to occur close to the egg release site, within the ampullary portion of the fallopian tube, where the highly selected fertilizing sperm has to travel. This segregated area for fertilization, the tubal mucosa, apparently is located away from major sites of immune reaction, like those that are present in the vicinity of lymph nodes and systemic circulation. In addition, the zygote is surrounded by the zona pellucida and cumulus cells derived from the ovary that prevent direct contact between embryo and mother. The fallopian tube also will be the site of transit for the fertilized egg for the next few days while the embryonic mass continues to increase through cellular replication.

Unless the embryo continues to be recognized, it will be eliminated rather rapidly. It has been long debated how early the mother recognizes that the egg (of maternal origin), has been fertilized by the sperm (of paternal origin), creating a partial allograft. It can be argued that very early recognition is clearly advantageous since it allows for the development of a receptive endometrium where the conceptus can successfully implant. It also helps the embryo reach a critical size, which is required for sufficient surface to surface contact, expression of local recognition signals and adequate quantities of proteolytic enzymes to aid in the implantation process. However, if the maternal organism were to become aware of the presence of a partial allograft and not aid to accommodate it, then the mother would mount an immune reaction, likely destroying the fertilized egg.

In our view, this apparent conflict is resolved through embryo derived signaling that takes place prior to implantation. This signaling accomplishes two complementary goals: Protecting the embryo from maternal immune reaction while promoting the development a favorable nidation site.

Based on this rationale, it seems logical that the immune recognition of pregnancy would be a more generalized phenomenon than a local protection within the uterus. It could be hypothesized that the embryo derived signaling required for these two distinct tasks could be similar if not the same. Such a view will require confirmation by future studies. Since the embryo is very small, either the production of this signal and its secretion by the embryo is very high to reach the periphery shortly after fertilization, or alternately the embryo signal is amplified perhaps by the immune system.

Data generated by us and others supports a very early presence of a recognition signal(s), within hours after fertilization. However, nidation occurs about one week later. Thus, the separation between the time of pregnancy recognition (the term used here is general, implying any changes produced by the presence of a viable embryo) and the time of actual nidation would suggest that the immediate role of pregnancy recognition is to protect against systemic attack by the maternal organism. Consequently, pregnancy recognition is a widespread phenomena within the organism. The embryo creates its own local microenvironment through autocrine signals, seemingly independent of exogenous factors until the blastocyst stage. This is supported by in vitro evidence of the survival of cultured preimplantation embryos in rather simple media. It is unclear whether the preimplantation embryo is completely independent of its environment since it is always surrounded by an interactive environment. It appears that although in vitro development of preimplantation embryos can occur, it is slower than its in vivo counterpart. Evidence for autocrine control of preimplantation embryo development was demonstrated when increasing concentrations of secretory products in the embryo microenvironment led to higher embryonic survival and improved development.¹ Therefore, it is not surprising and is rather expected that pregnancies can also develop outside the uterus, albeit very rarely reaching term. Pregnancies have been described in the fallopian tube, ovary, in the abdominal cavity and in some cases elsewhere in the body in experimental systems. These observations prove that recognition of pregnancy and to a certain degree pregnancy development can occur in various sites within the organism. Our recent preliminary data would further support such a view.

With respect to the recognition phenomena, the immune system is the prime candidate to perform this important function. Indeed, the immune system is more ancient and was already functional long before mammalian reproduction evolved. Perhaps the earliest task of the developing embryo is to harness the immune system for its own advantage. If this is not accomplished rapidly, reproduction can not be successful.

It was earlier suggested that pregnancy is associated with immune suppression that allows the embryo to thrive and develop. Available data, however, does not support such a view. The immune system appears to be more modulated than actually suppressed. This issue was discussed in detail.² In order to exert an immune tolerance, a viable embryo must be present.

Embryo derived signaling appears shortly after fertilization. These signals are not only local but rather rapidly become generalized in the organism. The proof is that they can be detected in the maternal circulation already several days prior to implantation. With respect to the local signaling, cell surface ligands and adhesion molecules are likely to play a role during the stay of the embryo in the fallopian tube. The role of these cell surface recognition elements appear to be greatly enhanced during the implantation process. When the embryo reaches the endometrial cavity, which is a privileged site for implantation, several local factors come into play. This has been evidenced by in vitro systems of co-culture of endometria and blastocyst as well as in in vivo studies. The detailed description of such interaction and the mechanisms involved will be discussed in a later chapter. Among factors that appear to be involved in this implantation enhancing dialogue are the endometrium’s own secretory products and cell surface molecules as well as those of the embryo. These include, leukemia inhibitory factor (LIF), estradiol 17b, progesterone, hCG, Pregnancy-specific plasma protein C, histamine-releasing factors, prostaglandins, and inhibins.³ For example, LIF has been shown to promote blastocyst differentiation and hatching.⁴

In summary, two pathways of embryo derived maternal-recognition signals are present. The first is local, the cellular pathway which is related to cell surface receptors and compounds involved in recognition and adhesion, while the second system is humoral where compounds released from the embryo reach distant sites and lead to the systemic recognition of pregnancy. The second pathway may actually precede the first. These pathways resemble those that are involved in generalized immune response.

The current chapter describes our recent data as well as that of others on embryo derived signals that are involved in maternal recognition of pregnancy present prior to implantation.

Knowledge on early embryo development has been greatly enhanced for the past few years. However, the role of the embryo proper in the recognition process of pregnancy has not been well elucidated. There is an obvious need to identify specific embryo derived signals that are involved in immune modulation and which may help in initiating pregnancy recognition. Such a signal must be present only in viable pregnancy, secreted by the embryo prior to implantation and detected in the systemic circulation. However, only one of the currently investigated factors may fit this criterion. The following will review current knowledge on humoral signals that are present before implantation.

Early pregnancy factor (EPF)

The EPF phenomena was described in the peripheral sera of pregnant women over twenty years ago. However, in spite of very vigorous effort, it has remained rather elusive and the detection until present still relies on the rosette inhibition test (RIT), a semi quantitative assay which is complex to carry out and difficult to replicate.⁵ Also, it is by now clear that EPF is not specific to pregnancy. In addition to being expressed in the preimplantation embryo, in embryonal organs as well as in the placenta, it is also present in several non-pregnant tissues and regenerating organs as well as in cancer sera, several cell line culture media and even in yeast cultures.⁶

Major uncertainties persist with the precise identity of EPF. Some authors believe that it is thioredoxin which is a 10 kDa heat shock protein or chaperonin 10,⁷ a 12 kDa protein.⁸ However, other investigators suggest that the EPF phenomenon is not due to a single factor alone since it behaves anomalously and may exert different effects. EPF activity actually was found when the pregnant sera were fractionated at the <1- >500 kDa region.⁹ Several compounds have been identified as being active in the assay including arachidonic acid and several leukotrienes. More recent work strongly points to the fact that thioredoxin has only a permissive role in the EPF phenomena. Its activity, as shown by the RIT, is enhanced by the addition of platelet activating factor (PAF) to spleen cells.¹⁰ In addition, pregnant sera, unlike non-pregnant sera, induces EPF activity, suggesting that thioredoxin is part of the components that are required for the expression of EPF.⁹

According to recent data, although EPF antibodies did not affect mouse blastocyst development, they did perturb trophoblast outgrowth. This data implies that the effect of EPF on the embryo is indirect and may act through an autocrine loop.⁸ Also, mice immunized with anti EPF antibodies led to a decrease in implantation rates but no noted interference with the number of corpora lutea.¹¹ This suggests a direct effect on the embryo or on the implantation site itself. Evidence for binding of EPF to specific sites on the blastocyst surface was shown as well. Localization studies have shown that EPF antibodies are located mostly in the trophoectoderm with very few binding to the inner cell mass (the site for cells that later lead to embryo development). Therefore, EPF could be viewed as both an immunosuppressive agent and a growth factor.⁸

Clinical studies have shown that EPF can be detected shortly after fertilization in the maternal system and its presence is related to the presence of a viable embryo. EPF persists in the sera until two thirds of pregnancy in many mammals. Its presence in sera varies in different species where alternate forms of EPF related molecules can be detected. Overall, these EPF molecules have been shown to suppress both T and B cell proliferation by binding to specific receptors expressed by lymphocytes after mitogenic stimulation.¹²

The presence of EPF-like activity in the embryo has been correlated with successful pregnancy in patients undergoing In Vitro Fertilization (IVF). However, preimplantation embryos secrete several factors that make identification of EPF elusive. Antibodies raised against EPF activity in culture media have shown that EPF induced immunosuppression can be abolished. These monoclonal antibodies revealed that EPF is present in the placenta. However, this phenomenon was not specific for pregnancy since the antibodies were expressed in choriocarcinoma cancer cell lines as well.¹³ The generation of these monoclonal antibodies has also led to identification of several immunosupressive factors (24-37 kDa range) which so far remain unidentified.¹⁴ EPF may interact with follicle stimulating hormone and estradiol to aid in follicular development as well as early embryonic cell division following IVF.

In conclusion, EPF is involved in immune response to the presence of pregnancy, or more generally to the presence of proliferating entities in the organism. However, since EPF is not embryo specific it has a limited role in clinical assessment of embryo viability prior to implantation.

Platelet activating factor (PAF)

These are a class of low molecular weight acetylated glycero phospholipids that can be detected in the media surrounding the embryo shortly after fertilization. It has been suggested that PAF is responsible for the transient thrombocytopenia that is present shortly after fertilization in mice and human.¹⁵ In the pregnant subject PAF is found in follicular fluid, endometrium, and amniotic fluid.¹⁶ PAF has an important function in the non-pregnant subject with respect to cell to cell interaction, changes in vascular permeability, activation of inflammatory processes and immunoregulation of graft rejection.¹⁷ PAF is expressed by white cells, platelets, and endothelial cells.¹⁸ The PAF effect and release varies across the organism. Current knowledge on PAF with respect to early embryonal events suggests that it is detected in the culture media of the fertilized eggs within eight hours after fertilization.¹

PAF may be important for pregnancy maintenance at least in the early stages. It appears that embryos of high quality secrete greater amounts of PAF than those of poor quality. The PAF secreted may aid in tubal transport of the embryo. The highest expression of PAF by the embryo may be in the two-cell stage and decline thereafter.^19,20,21 Addition of PAF to embryonic cultures had variable results. In some cases it had no effect while in others it affected mouse embryonal viability and reduced blastocyst volume.

PAF related system expression by the embryo is complex. The expression of the gene for the intracellular form of the PAF:acetylhydrolase enzyme receptor/ transducer for PAF, a G protein linked receptor, was recently examined. Genes of maternal origin were present in the oocyte and the zygote and subsequently degraded. At the four cell stage expression of the embryonal genome begins, creating higher levels of PAF related complex than present in the oocyte. In contrast, PAF:acetylhydrolase receptor transcripts were not identified in embryos following IVF. This suggests that culture conditions modify expression of the receptor.²² Experimental data suggested that PAF antibodies injected into mice prior to mating decreased the number of implanted embryos,²³ but this was not confirmed by other studies.²⁴

PAF is likely to have a role in early development. However, PAF is not a specific embryo derived marker since it is obiquitous in the organism. Therefore, the maternal source of PAF would mask the signal that would be coming from embryonic origin.^25,26

Interleukin signalling process in the embryo

The expression of interleukins (IL) in the embryo was recently examined. It was found that IL-B1 was expressed after the four-cell stage of development. In contrast, IL-A1 was not expressed in the preimplantation embryos. It is unlikely that IL-1 had a direct effect on the embryo since IL-1 receptor mRNA was not expressed in the preimplantation embryo. In contrast, IL-1 could have a direct effect on the uterus since there the receptor was expressed in all developmental stages.²⁷ The question remains whether IL signaling is involved in communications between mother and embryo prior to implantation and whether the fallopian tube contains IL receptors that could be helpful in amplifying the signal prior to implantation.

The role of insulin and insulin-like growth factors (IGFs) in embryo development have been examined in several studies. It was found that both the receptor and the ligands are expressed in early stages.²⁸ Transcripts were present in human oocytes and preimplantation embryos for insulin receptor and insulin-like growth factor receptors 1 and 2 (IGF1R and IGF2R) as well as for the ligand IGF2.²⁹ Another study has shown that insulin acts in mice to increase the total blastocyst mass while insulin-like growth factor 1,2 (IGF1 and IGF2) act only on the inner cell mass proliferation.³⁰ When these growth factors were added to the culture media, agent specific changes in the expression of several proteins were documented by a 2D dimensional gel.³¹ It remains unclear whether these factors only exert a local autocrine effect or if they also have a role in interacting with the maternal organism at the initiation of pregnancy.³² In both human and mouse, IGF2 is the only ligand in this family that has been shown to have an autocrine effect on preimplantation development. Insulin and IGF1, produced by the mouse maternal tissues in the oviduct and uterus, have a paracrine effect on the preimplantation embryo. A study suggests that similar relationships exist in humans and that preimplantation development may be regulated by IGFs from both embryonic (IGF2) and maternal (insulin and IGF1) sources.²⁹ More recently, the expression of IGF binding proteins has been documented in the preimplantation embryo. Such proteins are likely to modulate local effects of these growth factors.³³ Gene expression for TGF-alpha and its action as an autocrine factor has also recently been shown in the blastocyst.³⁴

From the previous description of signals present prior to implantation it appears that they are not specific to pregnancy, although they have a role in early development. As for early pregnancy recognition, factor(s) that would exert such a pregnancy specific signal for the embryo can not be found elsewhere, since pregnancy-like conditions are unique. We believe that our recent data would support the view that preimplantation factor (PIF) may have such a role.

Preimplantation factor (PIF)

Our laboratory has been involved for the past several years in the study of preimplantation factor (PIF). Initially we found in clinical samples that PIF, as recognized by the increased autorosette formation between lymphocytes and platelets in the presence of sera and CD2 antibody, was highly accurate in documenting pregnancy.³⁵ Through repeated testing on blind samples in several independent laboratories, the phenomenon of increased rosette formation was found only in the presence of pregnant samples.³⁶ This opened the possibility of examining the sources and the roles of these putative agent(s) in modulating the immune system. As reported recently, PIF is embryo-derived factor(s) that modulates the cellular immune system. PIF is already present in human and mouse embryo cultures at the two cell stage,³⁷ is detected in the maternal circulation four days after embryo transfer and 5-6 days after intrauterine insemination, and it persists in pregnant sera until term.³⁶

When pregnancy is failing, PIF disappears rapidly from the circulation, preceding the decline in hCG, the standard marker, by a number of weeks. These observations were confirmed by retrospective as well as prospective studies.³⁸

Actually, the presence of PIF in the circulation after four days predicted a high percent >70 % take home babies, while absence of PIF at the same time led to only a 3% rate of viable pregnancy.³⁹ This suggests that the presence of PIF is likely to be a recognition signal and perhaps in addition is involved in maintenance of pregnancy. The quest for identifying the specific factors that are responsible for the PIF phenomena is ongoing. According to our recent data, PIF activity in the purified mouse culture media is due to a small peptide of 1300 daltons. Further studies using pregnant pig sera showed that PIF activity was also due to small peptides less than 1000 daltons.² This raises the possibility that the PIF that is seen in the periphery may actually be a breakdown product of embryo derived PIF. The effort to finally identify PIF is ongoing, but sequencing is hampered by the very low concentration of the active compounds. It remains an open question whether the PIF activity that is seen in the periphery actually reflects the PIF that is secreted by the embryo proper. Only peptide sequencing and characterization would be able to answer this question. Since the egg is the largest cell in the body it is not unlikely that it may have the possibility of secreting sufficient quantities of very potent agents to modulate the immune system shortly after fertilization.

It was further investigated whether there are any analogies between the PIF, EPF and PAF described above. Our data suggest that PIF is a separate phenomena which is likely to be due to novel compounds.⁴⁰

Another point of importance is that PIF so far has been found in preliminary studies in five different mammalian species including human, mouse, pig, horse and more recently cow. This would imply that PIF is involved in immune modulation in several. A further support for this view is the finding that PIF is not only present in sera, but it is detectable in pregnant cows milk. This data, which requires further confirmation, would suggest that the presence of PIF reflects a generalized phenomena of pregnancy recognition in a rather segregated area in the mammary gland. Milk is produced by active transport of nutrients from the periphery. Such an observation would help to support the hypothesis that PIF is involved in generalized immune recognition and that pregnancy may proceed in several locations in the body since the viable embryo can create an immunologically favorable environment for itself. Obviously this does not negate the value of the uterus where the additional support systems of a highly favorable site of implantation and perhaps further immune privilege are conferred.

In conclusion, the present chapter described the logic and hypothesis of systemic over initial local pregnancy recognition that takes place prior to implantation in mammals. It described various early pregnancy phenomena like EPF, PAF, ILs and insulin that have a role in the success of pregnancy however are not unique for pregnancy. In our view, it is very unlikely that a combination of several factors that are not embryo specific acting in concert would be so well tuned that it would create such a unique environment as that seen in early pregnancy.

Finally, we described progress made in characterization and identification of a unique embryo derived factor, PIF, that it is found only in pregnancy due to very potent small size peptides. We have further described that PIF could be used for pregnancy identification and monitoring in human as well as in several mammalian species. Its widespread clinical application is contingent upon development of a widely usable assay such an ELISA.

For personal use. Only reproduce with permission from SIEP.

1. O’Neill, C. (1998). Autocrine mediators are required to act on the embryo by the 2-cell stage to promote normal development and survival of mouse preimplantation embryos in vitro. Biol. Reprod., 58, 1303-1309.
   
2. Barnea, E.R., Simon, J.H., Levine, S.P., Coulam, C.B., Taliadouros, G.S. and Leavis, P.C. (1998). Progress in characterization of preimplantation factor (PIF) in embryo cultures and in vivo. Am. J. Reprod. Immunol. (in press).
   
3. Edwards, R.G. and Brody, S. (1995). Principles and practice of assisted human reproduction. W.B. Saunders, Philadelphia.
   
4. Tsai, H.D., Chang, C.C., Hsieh, Y.Y., Lo, H.Y., Hsu, L.W. and Chang, S.C. (1999). Recombinant human leukemia inhibitory factor enhances the development of preimplantation mouse embryo in vitro. Fertil. Steril., 71, 722-725.
   
5. Morton, H., Rolfe, B.E., Clunie, G.J.A., Anderson, M.J. and Morrison, J. (1977). An early pregnancy factor detected in human serum by the rosette inhibition test. Lancet., 1, 394-397.
   
6. Clarke, F.M. (1997). Does "EPF" have an identity? J. Assist. Reprod. Genet., 14, 489-491.
   
7. Cavanagh, A.C. and Morton, H. (1994). The purification of early pregnancy factor to homogeneity from human platelets and identification as chaperonoin 10. Eur. J. Biochem., 222, 551-560.
   
8. Cavanagh, A.C. (1997). An update on the identity of early pregnancy factor and its role in early pregnancy. J. Assist. Reprod. Genet., 14, 492-494.
   
9. Di Trapani, G., Orozco, C., Cock, I. and Clarke, F. (1997). A re-examination of the association of ‘early pregnancy factor’ activity with fractions of heterogeneous molecular weight distribution in pregnancy sera. Early Preg.: Biol. & Med., 3, 312-323.
   
10. Cavanagh, A.C., Tolfe, B.E., Athanasas-Platsis, S., Quinn, K.A. and Morton, H. (1991). Relationship between early pregnancy factor, mouse embyro-conditioned medium and platelet activating factor. J. Reprod. Fert., 93, 355-365.
    
11. Athanasas-Platsis, S., Morton, H., Dunglison, G.F. and Kaye, P.L. (1991). Antibodies to early pregnancy factor retard embryonic development in mice in vivo. J. Reprod. Fert., 92, 443-451.
    
12. Lash, G.E. and Legge, M. (1997). Early pregnancy factor: an unresolved molecule. J. Assist. Reprod. Genet., 14, 39-43.
    
13. Bose, R. (1997). An update on the identity of early pregnancy factor and its role in early pregnancy. J. Assist. Reprod. Genet., 14, 497-499.
    
14. Bose, R. and Lacson, A.G., (1995). Embryo-associated immunosuppressor factor is produced at the maternal-fetal interface in human pregnancy. Am. J. Reprod. Immunol., 33, 373-380.
    
15. O’Neill, C. (1985). Thrombocytopenia is an initial maternal response to fertilization in mice. J. Reprod. Fertil., 73, 559-566.
    
16. Critser, E. and English, J. (1992). The role of platelet-activating factor in reproduction. In Immunological obstetrics, (ed. C.B. Coulam, W.P. Faulk, and J.A. McIntyre), pp. 202-215. W.W. Norton, New York.
    
17. Fuerstein, G. and Siren, A. (1988). Platelet activating factor and shock. Progress Biochem. Pharmacol., 22, 181-199.
    
18. Braquet, P., Touqui, I., Shen, T. and Vargaftig, B. (1987). Perspectives in platelet-activating factor research. Pharmacol. Rev., 39, 97-115.
    
19. Collier, M., O’Neill, C., Ammit, A. and Saunders, D. (1988). Biochemical and pharmacological characterization of human embryo-derived platelet activating factor. Hum. Reprod., 3, 993-998.
    
20. Velasquez, L., Aguilera, J. and Crozatto, H. (1995). Possible role of platelet-activating factor in embryonic signaling during oviductal transport in the hamster. Biol. Reprod., 52, 1302-1307.
    
21. Elias, K., Das, A., Pardue, D., Coulam, C.B., Critser, J. and Critser, E. (1989). Alteration in platelet count during early pregnancy in the mouse. Am. J. Reprod. Immunol., 21, 82-86.
    
22. Stojanov, T. and O’Neill, C. (1999). Ontogeny of expression of a receptor for platelet-activating factor in mouse preimplantation embryos and the effects of fertilization and culture in vitro on its expression. Biol. Reprod., 60, 674-682.
    
23. Spinks, N.R., Ryan, J.P., O’Neill, C. (1990). Antagonists of embryo-derived platelet activating factor act by inhibiting the ability of the mouse embryo to implant. J. Reprod. Fertil., 88, 241-248.
    
24. Milligan, S.R. and Finn, C.A. (1990). Failure of platelet-activating factor (PAF-acether) to induce decidualisation in mice and failure of antagonists of PAF to inhibit implantation. J. Reprod. Fertil., 88, 105-112.
    
25. Abisogun, A., Braquet, T. and Tsafriri, A. (1989). The involvement of platelet activating factor in ovulation. Science, 234, 381-383.
    
26. O’Neill, C., Ryan, J., Collier, M., Saunders, D., Ammit, A.J. and Pike, I. (1992). Outcome of pegnancies resulting from a trial of supplementing human IVF culture media with platelet activating factor. Reprod. Fertil. Dev., 4, 109-112.
    
27. Takacs, P. and Kauma, S. (1996). The expression of interleukin-1 alpha, interleukin-1 beta and interleukin-1 receptor type I mRNA during preimplantation mouse development. J. Reprod Immunol, 32, 27-35.
    
28. Doherty, A.S., Temeles, G.T. and Schultz, R.M. (1994). Temporal pattern of IGF-I expression during mouse preimplantation embryogenesis. Mol. Reprod. Dev., 57, 469-79.
    
29. Lighten, A.D., Hardy, K., Winston, R.M. and Moore, G.E. (1997). Expression of mRNA for the insulin-like growth factors and their receptors in human preimplantation embryos. Mol. Reprod. Dev., 47, 134-139.
    
30. Smith, R.M., Garside, W.T., Aghayan, M., Shi, C.-Z., Shah, N., Jarret, L. and Heyner, S. (1993). Mouse preimplantation embryos exhibit receptor-mediated binding and transcytosis of maternal insulin-like growth factor I. Biol. Reprod., 49, 1-12.
    
31. Shi, C.-Z., Collins, H.W., Garside, W.T., Buettger, C.W., Matschinsky, F.M. and Heyner, S. (1994). Insulin family growth factors have specific effects on protein synthesis in preimplantation mouse embryos. Mol. Reprod. Develop., 37, 398-406.
    
32. Heyner, S. (1997). Growth factors in preimplantation development: role of insulin and insulin-like growth factors. Early Preg.: Biol. & Med., 3, 153-163.
    
33. Liu, H.C., He, Z.Y., Tang, Y.X., Mele, C.A., Veeck, L.L., Davis, O. and Rosenwaks, Z. (1997). Simultaneous detection of multiple gene expression in mouse and human individual preimplantation embryos. Fertil. Steril., 67, 733-741.
    
34. Onohara, Y., Harada, T., Tanikawa, M., Iwabe, T., Yoshioka, H., Taniguchi, F., Mitsunari, M., Tsudo, T. and Terakawa, N. (1998). Autocrine effects of transforming growth factor-alpha on the development of preimplantation mouse embryos. J. Assist. Reprod. Genet., 15, 395-402.
    
35. Barnea, E.R., Lahijani, K.I., Roussev, R., Barnea, J.D. and Coulam, C.B. (1994). Use of lymphocyte platelet binding assay for detecting a preimplantation factor: A quantitative assay. Am. J. Reprod. Immunol., 32, 133-138.
    
36. Roussev, R.G., Barnea, E.R., Thomason, E.J. and Coulam, C.B. (1995). A novel bioassay for detection of preimplantation factor (PIF). Am. J. Reprod. Immunol., 33, 68-73.
    
37. Roussev, R.G., Coulam, C.B. and Barnea, E.R. (1996). Development and validation of an assay for measuring preimplantation factor (PIF) of embryonal origin. Am. J. Reprod. Immunol., 35, 281-287.
    
38. Coulam, C.B., Roussev, R.G., Thomasson, E.J. and Barnea, E.R. (1995). Preimplantation factor (PIF) predicts subsequent pregnancy loss. Am. J. Reprod. Immunol., 34, 88-92.
    
39. Barnea, E.R. and Coulam, C.B. (1997). Embryonic signals. In Embryonic Medicine and Therapy, (ed. E. Jauniaux, E.R. Barnea, and R.G. Edwards), pp. 63-75. Oxford University Press, Oxford.
    
40. Roussev, R.G., Coulam, C.B., Kaider, B.D., Yarkoni, M., Leavis, P.C. and Barnea, E.R. (1996). Embryonic origin of preimplantation factor (PIF): biological activity and partial characterization. Mol. Human Reprod., 2, 883-887.