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

Evolution Of Feto-Placental Unit

Eytan R. Barnea, MD and Christine A. Brusato
SIEP, The Society for the Investigation of Early Pregnancy
Cherry Hill, NJ, USA

Textbook of Obstetrics and Gynecology
Editor, I. Munteanu

Introduction

Reproduction can be viewed as an odyssey. The embryo must struggle against staggering odds to survive. At the time of fertilization, the chances of a take home baby are rather small. This is mostly due to early embryonic demise that often occurs even prior to implantation. Following implantation, a delicate negotiation with the maternal organism has to take place to assure survival. Once embryo development has been achieved, the rate of pregnancy success is very high.

In the following discussion, it becomes readily apparent that pregnancy can not be both functionally or structurally viewed as a single entity, but as a chain of events where the successful completion of one in an almost perfect manner will lead to the next with an ultimate goal of the delivery of a healthy child. If a problem develops early during pregnancy, it may be clinically manifested differently later in gestation. For example, hyperglycemia early on may lead to a miscarriage or an increased rate of congenital anomalies. However, if the diabetes is not well controlled later in pregnancy macrosomia may result.

The goal of the present chapter is to provide a cross sectional as well as a longitudinal view of gestation using an integrative approach. For that purpose we will illustrate the different stages of gestation, which in our opinion are more numerous than traditionally described. The chapter will discuss the changing role of the embryo as it becomes a mature fetus and examine the trophoblast as it becomes a vascularized placenta. Finally, we will depict the intimate, although changing, relationship between the trophoblast and the embryo (creating the embryo-trophoblastic unit), and that between the fetus and placenta (creating the feto-placental unit) later in gestation.

The stages of gestation

Gestation can be divided into several stages. They include:

Fertilization: merging of the gametes
Early preimplantation embryo (until 8 cells): cellular division is symmetric and cells are toti-potential.
Late preimplantation embryo: cellular differentiation and embryo hatching.
Implantation: trophoblast invasion of the maternal decidua.
Embryogenesis: development of embryonic organs and their integration.
Transitions: the embryo becomes a fetus although it remains incapable of independent life.
The third trimester: the fetus is capable of independent life.

The following will delineate briefly the various features of the embryo and trophoblast throughout gestation.

From embryo to fetus

Early preimplantation embryo
Following fertilization, early preimplantation embryo development appears to be well ordered for the first couple of days as the zygote divides into two, four, and then eight cells. At this stage, if separated, each cell could form a full embryo. The embryo’s growth is initially autonomous, apparently independent of external maternal influences.

Late preimplantation embryo
In the late preimplantation embryonal stage, when the blastocyst develops, the toti-potentiality of these cells is lost as they are redirected towards specialized functions. The majority of the cells become the trophoblast, which surrounds the embryoblast in the blastocelic cavity. The embryoblast will develop into the post implantation embryo.

Implantation
During implantation, the role of the embryoblast is rather limited since, although it is located right behind the invading trophoblast, it is not in the immediate vicinity of the endometrial surface. The role and development of the embryoblast will probably be limited until implantation is secured.

Embryogenesis
Specific signals, most likely derived from the embryoblast, are needed for embryogenesis to begin. Since there is very limited room for mistakes, there are very specific requirements for the initiation of embryogenesis. If the embryo does not start to develop at the proper time, a blighted ovum will form and lead to spontaneous abortion. Any major structural damage or developmental defect will lead almost inevitably to lack of trophic support, therefore to miscarriage. During embryogenesis, the embryo’s function changes as strong metabolic activity develops. From the earliest stages of embryogenesis, both the morphology and functionality change very rapidly in a very ordinate manner. Proliferation, differentiation, cell migration, and programmed cell death are prominent within the embryo. Abnormal proliferation is controlled through the expression of local antiproliferative factors. In addition, there is a major push to develop various organs based on regional complexity principles. Once a cell is present in a given organ like the lung or liver, it is constrained by the surrounding cells to develop similarly in a given pathway. The major event in the development of the embryo is the formation of the circulatory system, which allows for blood to flow across the body and the brain. In practical terms, any major malformations almost inevitably will lead to pregnancy loss. There is a very well defined sequence of events that operate in the development process. For example, if the heart will not start pumping blood, the brain will not develop. Since embryogenesis is the most critical stage of gestation, its successful completion will lead to live birth in over 95% of cases.

Transitions
Until now, the embryo has operated in a relatively low oxygen environment. This may have conferred a significant advantage since it protected the embryo from damage due to exposure to oxygen radicals. During the transitions period, in order to accommodate massive growth, the fetal environment becomes aerobic by the aid of the placental circulation. This allows the fetus, specifically the brain, to continue to grow and permits the completion of sexual differentiation. By the end of this period, the lung becomes potentially capable of extrauterine life. The delivery of a viable fetus becomes possible in the third trimester, the final stage of gestation.

From trophoblast to placenta

Late preimplantation embryo
The trophoblast develops in the late preimplantation period to serve as support and protection for the embryoblast as well as perform trophic functions. There is a rapid proliferation of the trophoblastic cells as compared to that of the embryo. This is necessary for implantation to be successful. Prior to implantation, the function of the trophoblast is mostly to protect the embryoblast, which is located in the blastocyst cavity. At this stage, the trophoblast is less likely to have a major metabolic role than to be related to the expression of growth factors and pregnancy recognition signals.

Implantation
At the time of implantation, cyto- and syncitio- trophoblastic cellular elements will have to project integrins and other recognition and adhesion molecules that will lead to attachment to the maternal decidua. The trophoblast will then strengthen the attachment to the maternal surface by burrowing into the decidua through the expression of proteases that break down the extra cellular matrix and establish links with the maternal circulation. Early in pregnancy, trophoblast invasiveness is a necessary pre-requisite for successful implantation. However, the invasive capacity of the trophoblast is largely lost after the first trimester. The trophoblast also has major role in protecting the embryo. Trophoblast cells reach the uterine maternal circulation and line the maternal blood vessel endothelium, creating a low pressure system. In addition, the trophoblast is involved in maintaining immune tolerance of pregnancy. This tolerance is likely to be exerted by the expression of modified HLA antigens (HLA G) that do not elicit an immune reaction by the mother, as well as by several other placental products. On the other hand, if the trophoblast does not function well it will lead to its own separation from the maternal decidua and consequently lead to the failure of the embryo. For example, if the aggression of the implanting trophoblast remained very high rather than fading after the first trimester (it actually possesses neoplastic-like features), then this invasiveness would ultimately damage the mother, defeating the purpose of support.

Embryogenesis
During embryogenesis, the trophoblast rapidly grows and serves as a peripheral brain for the embryo. Almost all of the factors present in the trophoblast that are likely to aid throughout pregnancy are similar to those present in the adult brain, including GnRH, hCG, steroids, neurotransmitters and growth factors. They support the embryo until its own brain develops and secretes similar factors. Through secretion of progesterone the trophoblast creates uterine quiescence. By secretion of hCG it supports the corpus luteum. Tolerance is aided by secretion of these factors and several immuno modulators. In addition, the trophoblast shields the embryo by creating a metabolic sink, activating and inactivating carcinogens and mutagens locally. It also protects the embryo through the creation of a very low oxygen environment, which guards against oxygen radical formation. The trophoblast creates a segregated area for the embryo, forming an environment where the passage of nutrients proceeds slowly through diffusion instead of through blood vessels. The trophoblast serves as a barrier, although incomplete, from infectious agents and it contains antiinfective agents such as interferons. Thus, when the embryo appears to be the most vulnerable to external adverse damage it is actually the best protected. Such an early protective ability of the trophoblast, and to the certain extent the embryo itself, leads to a relatively low vulnerability of the embryo to adverse environmental conditions and to a low rates of malformation. Those embryos that are seriously damaged are most commonly promptly rejected through separation of the trophoblast from the maternal decidua.

Transitions
The transitions stage is where hCG levels plateau, the trophobast acquires stromal cells, and blood vessels start to form and become functional. At that time, expansion of the amniotic cavity will allow for fetal growth to proceed optimally. The transition from embryo to fetus requires significant resources from the mother, including the transfer of nutrients and oxygen. In addition, the transfer of CO2 and refuse products from the fetus must proceed efficiently. Thus, the placenta by now provides nutrients through the umbilical cord and a secure and comfortable environment in the amniotic fluid for the developing fetus. At the transitions stage, the placenta is an almost complete barrier, allowing the passage of only small molecules and preventing direct contact between maternal and fetal circulation. Consequently, the passage of infectious agents to the fetus is rather limited. The placenta also serves as a metabolic engine where the secretion of hPL, placental growth hormone, and several growth factors may have important trophic roles on the fetus both directly and indirectly. A complex interaction between fetal and placental steroidogenesis begins to evolve, creating the feto-placental unit.

The third trimester
In the third trimester the placenta begins to age, its function gradually becoming less efficient as it partly undergoes calcification and fibrosis. When term is reached, the placenta has a significant contributing role in the delivery of the infant, including a shift in local steroid milieu to favor estrogen over progesterone production through the effect of oxytocin and other factors.

The embryo-trophoblastic unit

Embryogenesis is an extremely complex coordination of processes that rapidly leads to embryo formation. Despite an expeditious rate of cell proliferation in the early trimester that could easily facilitate mutations as well as tumor development, embryonic malignancy is very rare and pregnancy loss in this period is quite low. The rate of early trimester loss is only approximately 15 percent, suggesting the presence of some controlling mechanism that checks and balances the system, allowing normal cells to proliferate and differentiate into specific organs while regulating or eliminating abnormal cells. Evidence indicates that the embryo controls itself as well as the trophoblast. While the trophoblast has some autonomy over its own proliferation and differentiation, it must be kept in check so it will not develop overly aggressive features resembling neoplasia. If the trophoblast develops abnormally and embryo derived controls fail, instead of acting as support, the trophoblast will interfere with embryo development and lead to its demise.

Embryo-trophoblastic dependence
A viable trophoblast is necessary for embryo development. Studies indicate that the proximity of the trophoblast to the embryo may contribute to allowing for the trophoblast’s supportive role on the embryo.¹ Data suggest that the embryo harnesses trophoblastic resources for its own advantage, regulating trophoblast function according to its needs while promoting its own development. One of the most compelling indications that the embryo does regulate trophoblast function is an experiment where the embryo was removed surgically from mice at 10 days of gestation.² After removal of the embryo, the trophoblast in culture became spontaneously cancerous (choriocarcinoma). These results were confirmed in a later study.³ A study on primates revealed that a trophoblast remaining in situ does not appear to undergo malignant transformation.⁴ However, in humans during a molar pregnancy associated with an embryo lead to lower hCG levels and to a less aggressive behaviour of the molar gestation than if the embryo is absent. These experiments suggest that the embryo may have a mitigating effect of trophoblast aggressiveness even when trophoblast invasiveness is prominent.

Several facts suggest that the critical period for embryo-trophoblast interdependence is during embryogenesis and shortly thereafter.⁵ A study has demonstrated that trophoblast cell cultures are impaired in tissues obtained from pregnancies where the embryo is not viable. In addition, trophoblast cultures grow poorly following recent death of the embryo. Altered dynamics of hCG secretion by the trophoblast has been demonstrated in pregnancies with abnormal embryonal development. For example, ectopic pregnancies with a viable embryo have higher levels of hCG than anembryonic pregnancies.⁶ The decreased levels of hGC in anembryonic pregnancies is due to downregulation of the hCGa and hCBb genes in the trophoblast as well as decreased placentation.⁷ In cases of multiple embryo reduction, hCG levels drop within a short time although the trophoblast should not be directly affected. In pregnancies with chromosomal anomalies, hCG and estradiol levels are lower and the hCG levels do not appear to plateau as would be expected at 9-10 weeks in normal pregnancy.⁸ Instead, hCG levels in the second trimester of chromosomally abnormal pregnancies are higher or lower, depending on the type of aberration. Based on these changes, hCG-b together with alphafeto-protein and unconjugated estriol has become an important marker in screening for chromosomal anomalies.⁹ Data suggests that embryo derived developmental proteins (DPs) are responsible for the embryo’s effect on trophoblastic hCG secretion.

Developmental proteins
Evidence indicates that the embryo contains regulatory compounds that help its own development and are also involved in controlling trophoblast function. Developmental proteins modulate trophoblast hormone secretion, controlling secretion of hCG and progesterone by placental explants in a dose-, time-, and gestational age dependant manner.¹⁰ DPs were found to be of low molecular weight and secreted by embryo tissue explants.^10,11 Embryonic spinal cord extracts exert inhibitory as well as stimulatory effects on hCG secretion which is evidenced by separating the extracts into different molecular weights. Thus, the embryo contains both hCG inhibitors and stimulators which contribute to early pregnancy development.

Evidence for the ability of the embryo to control neoplastic like features of the trophoblast was provided when addition of DPs reduced the ability of mouse embryos to implant on an extracellular matrix feeding layer.

The embryo, the trophoblast, and cancer
When cells from a normal embryo’s neural crest were transplanted into adult mouse testes, they led to the development of teratocarcinoma.¹² The transformation of the cells was due to the imposition of an abnormal environment. However, when teratocarcinoma cells were injected into mouse blastocysts, this led to the development of normal mouse offspring that were mosaic, containing cells derived from both the original blastocyst and from the tumor.¹³ This suggests that the embryo is capable of transforming cancerous cells into normally differentiated cells, enabling them to incorporate into the fetus. Pregnancy has also been shown to protect against the transplantation of lymphoma cells in rats. In addition, the injection of pregnant serum into non-pregnant rats provided some cancer protection in non-pregnant rats.¹⁴

Data has shown that the hCGb subunit and whole hCG secreted by the trophoblast exert an inhibitory effect on Kaposi’s sarcoma cell line proliferation and prevent the development of tumors in nude mice. In some mice, becoming pregnant provided protection, while in others it caused the tumor to regress. However, inoculation of the tumor with hCG during the first ten days of gestation led to complete protection against Kaposi’s sarcoma development and with later inoculation only small tumors developed as compared to that in controls.¹⁵ Since pregnant mice do not express a chorionic gonadotrophin (CG) gene, the anti-tumor effect seen must be exerted by other factors. This shows that early pregnancy is unique in its methods of cell proliferation control.

Based on the previous discussion, it seems likely that DPs might also control cancer cell proliferation. Evidence of this has been reported recently.¹⁶ Two partially purified DPs suppress the proliferation of several disparate human cancer lines. The effect was exerted in low concentrations while having no effect on normal cells. DPs expression is not limited to humans. Although DPs are of neural origin, they affect epithelial and mesenchymal proliferation, indicating that in addition to their local effect, they may have a multi-targeted role during embryonal development. For example, kidney morphogenesis is dependent on the expression of nerve growth factor (GF) receptors.¹⁷ The gestational age-dependent expression of DPs might reflect a transition from cell proliferation and differentiation to organ development. The identity and the mechanisms of DPs are currently under investigation. It has been speculated that the factors may limit GF action, or possibly bind to specific sites that the embryonal cells may share with neoplastic cells.¹⁸

In summary
This discussion of the embryo-trophoblastic unit has shown that while the trophoblast has certain neoplastic features, it is different from cancer cells in that it loses most of its neoplastic properties as it develops. Data suggests that the embryo provides at least part of this control of the trophoblast. The data also show that embryo-derived compounds are capable of modulating trophoblast hormones as well as cancer cell proliferation, perhaps because of the similarities that exist between cancer and trophoblast cells. Further studies in the identity and mechanism of these embryo-derived compounds may lead to important implications for pregnancy and the control of proliferation in general.

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