The Birds and the Bees Talk: Why do we even care?
Why is it important to study placental development and embryo implantation? Pre-term deliveries account for 10 – 13% of all deliveries, and even though the survival rate with modern neonatal intensive care units is high, pediatric hospitals use a quarter of their spending on caring for these pre-term babies . Many of these births are due to placental pathologies . Examples of these placental pathologies include intrauterine growth restriction, which limits the amount of nutrients and oxygen obtained by the embryo, and preeclampsia, which causes an increase in maternal blood pressure and maternal seizures that place both the mother and baby at risk . By researching more into the process of placental development and embryo implantation, the rate of premature births may be reduced.
Importance of Placenta
Main functions of the placenta:
- To ensure adequate blood supply for the feto-placental unit in order to have exchange of nutrients, oxygen and wastes between the mother and the fetus 
- To protect the embryo and fetus from maternal immunologic attack 
- To anchor the conceptus, which includes both embryonic and extraembryonic structures of the zygote, to the uterine wall 
- To release hormones such as progesterone, estrogen and human chorionic gonadotropin (hCG) so pregnancy is maintained 
New functions of the placenta are still being discovered. In 2011, Bonnin et al. were able to support the placenta as the main source of the neurotransmitter serotonin (involved in regulating mood, appetite, sleep, and social behavior) for the fetal forebrain during early development. Tryptophan, a precursor of serotonin, supplied to the placenta through maternal blood, increases the levels of both placental and forebrain serotonin in mice between embryonic days 10.5 and 16.5. Read more about how Bonnin et al. were able to show this and how they eliminated other potential exogenous sources of serotonin for the fetal forebrain here: Placental Serotonin.
The following video provides illustrations of the structure of the placenta and discusses the importance of the placenta in fetal development:
The placentas of all placental (eutherian) mammals have common structures and functions. However, there are two distinct characteristics that form the different classifications of placental types.
Before the formation of the placenta, there are six layers of tissue separating maternal and fetal blood as shown in figure 1. These six layers consist of three layers from fetal extraembryonic membranes, which are all maintained in the mature placenta, and three layers on the maternal side, which vary in the layers that are retained in the mature placenta .
Primates including humans have discoid, hemochorial placentas. Discoid placentas are single placentas that are formed in a round shape. Hemochorial placentas do not retain any of the three maternal placental layers in the mature placenta, so the fetal chorionic epithelium is in direct contact with the maternal blood .
Embryo Implantation – Steps Involved
The uterine endometrium undergoes various morphological and physiological changes to accommodate the conceptus as demonstrated in figure 2.
Three general processes are needed for the embryo to degrade the endometrial extracellular matrix and properly implant into the uterine lining.
The three main processes are: attachment, apoptosis and invasion .
1. A floating blastocyst becomes attached to the uterine lining (endometrium)
- The blastocyst consists of two distinct cell types: an inner cell mass which develops into the fetus and the trophoblast which develops into the placenta and external membranes. For additional information on trophoblast development in primates, click here.
- The trophoblast starts to produce human chorionic gonadotropin hormone (hCG).
- The blastocyst is slowed down by molecules called mucins that extend from the endometrium, and the blastocyst is brought closer in contact with the endometrial cells .
2. Apoptosis of the maternal uterine epithelial cells
- The fetal/embryonic cells are always separated from maternal blood and tissues by two trophoblast layers: an inner mononuclear cytotrophoblast layer and an outer multinucleated syncytiotrophoblast layer. These layers protect the embryo and fetus from maternal immunologic attack .
- The syncytiotrophoblast layer produces lytic enzymes and secretes factors that induce the apoptosis of the endometrial epithelial cells . Apoptosis is essential to the proper implantation of the embryo.
- Syncytiotrophoblast cells phagocytize the apoptotic decidual cells of the endometrium and reabsorb the proteins, sugars and lipids present in those cells . Decidual cells are connective tissue cells in the endometrium.
3. Trophoblast cells begin to invade endometrium
- There are complex interactions between the trophoblast and the endometrium: trophoblasts have potent invasive capacity that is kept in check by the endometrium via locally secreted factors such as cytokines and protease inhibitors .
- hCG gradient is involved: lower concentrations of hCG is made in trophoblasts closer to the endometrium in order to allow them to differentiate into anchoring type cells that can invade the endometrium layer .
- The invading trophoblast begins to penetrate the maternal vessels and forms sinuses of maternal blood, which surround the growing trophoblasts. The maternal blood is supplied by the spiral arteries and drained by the uterine veins [3, 6].
- The chorionic villi, part of the border between maternal and fetal blood, develop .
- The fetal circulation develops and consists of capillary loops that terminate within the chorionic villi, which penetrates the maternal blood-filled sinuses .
Role of Frizzled Proteins
Secreted frizzled-related protein 4 (sFRP4) is known to competitively bind to Wnt signaling proteins and its receptors to inhibit the Wnt signaling pathway, which has a role in embryo implantation and placental development. The antagonistic role of sFRP4 in Wnt signaling is illustrated by figure 3. In addition, sFRP4 has been previously shown to increase the incidence of apoptosis, one of the three main processes needed for implantation .
What is the importance of the Wnt pathway? The Wnt pathway involves the nuclear recruitment of beta-catenin, which activates the Wnt-dependent transcription factors. Wnt signaling is known to promote early trophoblast lineage development, blastocyst activation, implantation and chorion-allantois fusion . Wnt ligands have important roles in the cycling of endometrial cells and decidualisation, which is the postovulatory process of endometrial remodeling in preparation for pregnancy . The Wnt pathway also plays an important role in numerous other developmental processes such as hair follicle growth, limb development, mammalian sex determination, and body axis formation.
White et al. tested the expression of sFRP4 in both human and macaque monkey placentas at different gestational stages . sFRP4 is expressed predominantly in the villous syncytiotrophoblast and the invasive intermediate cytotrophoblast and in the amnion, which is a membranous sac that encloses the developing fetus. The results suggest that sFRP4 plays a role in placental development and implantation especially in trophoblast apoptosis and the development of the decidual fibrinoid zone. The fibrinoid zone provides the adhesiveness for the placenta to anchor to the uterine wall.
Macaque placental samples were taken and analyzed with semi-quantitative PCR (sQ-PCR). sQ-PCR involves reverse transcribing RNAs and adding primers for antisense sFRP4. A housekeeping gene standard (GAPDH) was also added, and both were run through electrophoresis. The results for sFRP4 were normalized with the GAPDH results. The electrophoresis results were then scanned and quantified by densitometry. As shown in figure 4, the expression of sFRP4 increased as gestation progressed especially after 50 days of gestation.
Immunohistochemistry and in-situ hybridization for sFRP4 were also performed on macaque placental samples. The results showed positive staining in the trophoblastic shell after 30 days of gestation; there was a high level of sFRP4 expression in the cytotrophoblasts. As shown in figure 5, the zone of apoptosis was found right beneath the sFRP4 staining of the intermediate trophoblast, indicating that sFRP4 may be a mediator for the zone of apoptosis. The zone of apoptosis was found by performing terminal deoxynucleotidyl transferase (TUNEL) assay, which detects the DNA fragmentation that results from apoptotic signaling cascades.
For the human placental samples, first trimester and term placentas were observed. The sFRP4 expression was shown in the first-trimester chorionic villi, confined mainly to the syncytiotrophoblast and not the cytotrophoblast as shown in figure 6. The infiltrating trophoblast cells stained positive for sFRP4 as expected because apoptosis is observed when the trophoblast invades the endometrium epithelial cells. When the proliferative regions of the intermediate trophoblast were tested for sFRP4 expression, the results showed that those regions do not produce sFRP4 because sFRP4 has an anti-proliferative role. In humans, there is a high level of expression of sFRP4 in the syncytiotrophoblast instead of the villous cytotrophoblast.
One criticism for this paper is that the experiment mainly examines the various areas sFRP4 is expressed in the endometrium-trophoblast interface and how the expression amount changes throughout gestation. However, it does not focus on the signaling pathway or the general mechanism that leads up to the phenotypic changes. Current findings only suggest that sFRP4 is involved in embryo implantation because of its role in apoptosis and its presence near the zone of apoptosis and the invading trophoblast cells. Future research into how sFRP4 brings about these changes would provide more validation for the role of sFRP4 in embryo implantation.
 Benirschke, Kurt and Peter Kaufmann. Pathology of the Human Placenta, 5th edition. New York: Springer-Verlag, 2006. Print.
 Bowen, Richard et al. “Placental Structure and Classification.” Hypertexts for Biomedical Sciences. 25 September 2011. Web. 23 April 2012. <http://www.vivo.colostate.edu/hbooks/pathphys/reprod/placenta/structure.html>
Bonnin, A., Goeden, N., Chen, K., Wilson, M.L., King, J., Shih, J.C., Blakely, R.D., Deneris, E.S., Levitt, P., 2011. A transient placental source of serotonin for the fetal forebrain. Nature 472, 347-350.
 Kliman, Harvey J. “From Trophoblast to Human Placenta.” Encyclopedia of Reproduction. Web. 16 March 2012. <http://med.yale.edu/obgyn/kliman/placenta/articles/EOR_Placenta/Trophtoplacenta.html>
 Maltepe, Emin et al. “The placenta: transcriptional, epigenetic, and physiological integration during development.” The Journal of Clinical Investigation 120.4 (2010): 1016 – 1025. Web. 9 March 2012.
 Sonderegger, Stefan et al. “Wnt Signaling in Implantation, Decidualisation, and Placental Differentiation -- Review.” Placenta 31.10 (2010): 839 – 847. Web. 25 April 2012.
 White, Lloyd et al. “Expression of secreted frizzled-related protein 4 in the primate placenta.” Reproductive BioMedicine Online 18.1 (2009): 104 – 110. Web. 9 March 2012.
Linked in webpage: http://en.wikipedia.org/wiki/Densitometry