Placentation & Trophoblast I Flashcards
What is the placenta?
Is an organ unique to pregnancy
Forms the interface between the mother and the fetus.
The placenta is fetal in origin at term it weighs 500-1000g
Acts as the lungs, gut and kidneys of the fetus
It also acts as an endocrine organ releasing hormones into the maternal circulation such as hCG and progesterone
The placenta is a semi-allograft, what does this mean?
the cells have genetic material from both the mother and the father
Foetal and Matenal blood
In a human pregnancy, fetal cells are in direct contact with maternal blood
As it is in contact with maternal blood this requires mechanisms to evade the maternal immune system
However although the fetal cells and maternal cells are in direct contact, the fetal and maternal circulations do not mix
How does the placenta develop?
Implantation is the multistep process by which the free-floating blastocyst attaches to the endometrium, invades through the epithelium and into the stroma beneath and begins to establish the placenta.
The blastocyst attaches to and adheres to the epithelial layer of the uterine wall.
The Trophoectoderm cells will proliferate and migrate through the epithelial cells through the basement membrane and into the underlying uterine wall.
Later the Trophoectoderm cells fuses to form a primitive syncytium (PS) (synctiotrophoblasts) beneath the implanted embryo.
The Trophoectoderm cells will continue to migrate or invade into the decidua.
They will form an increasing primitive syncytium (PS), and within the syncytium will form holes (Lacunae).
Lacunae (L) form by the action of proteases which later develop into the intervillous space
Behind the primitive syncytium, Cytotrophoblasts will proliferate and migrate through the syncytium and into the uterine wall to form the anchoring villi (dark blue).
Placental formation at later stage in gestation
Can see the decidua basalis underneath the placenta (light blue).
Can see the anchoring villi
As the placenta develops the villi start to branch and form both secondary and tertiary villi.
Within these villi are the blood vessels the supply the placenta, from the foetus. They are responsible for exchanging the nutrients and respiratory gases that come from the mother and are delivered back from the foetus.
Can see the presence of spiral arteries that deliver maternal blood to the intervillous space.
The are also veins that will drain the blood from the intervillous space back into the maternal circulation.
The villi are highly branched with a large surface area for exchange of nuitrents and respiratory gasses.
The Outer layer of the villi is formed of fused cells- the syncytium
Underlying this are the cytotrophoblast stem cells
Diffusion distance to vessels small
Growth is regulated by a number of factors including IGF I and II
Define the villous structure and cross section appearance:
Within each of the villi are a highly branched vascular network of arteries and veins which takes blood to and from the developing foetus.
Cross section:
On the outside there is a fused layer of cells called the syncytium
Underneath are the cytotrophoblast stem cells
The vessels are very close the surface of the villi, this is important for the delivery and exchange of nutrients.
Within the villi there are macrophages known as hofbauer cells. Their function may be involved in immune protection of the placenta and regulating the formation and branching of the vessels.
Formation of the syncytium
It is formed by the combined action of hCG and an endogenous retroviral protein called Syncytin-1/2.
hCG binds to the LH/CRG receptor
This will stimulate the production of cAMP
cAMP will then activate a membrane enzyme called Scramblase.
Scramblase is responsible for the redistribution of phosphatidylserine from the inner surface of the plasma membrane, to the outer surface of the plasma membrane.
cAMP will also increase the activity of PKA
PKA will phosphorylate the protein GCM1 (Glial Cells Missing Homolog 1), which is a transcription factor
GCM1 will then move to the nucleus and regulate the expression of Syncytin-1/2
Syncytin 1 and 2 will then be transported to the plasma membrane where it will induce cell fusion and the formation of the syncytium.
The syncytium is being regenerated constantly throughout gestation, although it does slow down.
In order to do this the underlying cytotrophoblasts have to fuse with the syncytium, thereby increasing/replacing lost material.
Villous cytotrophoblast proliferation decreases with gestation by term the syncytium close to placental vessels
Syncytium is continually been shed in to the maternal circulation and is replaced by the underlying cytotrophoblasts
Using the stain desmoplacin you can see that the membranes between cells are lost and multinucleated cells are formed.
Trophoblast differentiation and function
Have mentioned the villous pathway of the cytotrophoblast stem cells, will now focus on the Extravilous pathway:
These cells are derived from the cytotrophoblast cell columns and shell. They form two different subtypes:
The endovascular Extravilous trophoblast
The interstitial Extravilous trophoblast
These two cells types work together to remodel the maternal spiral arteries.
BUT in the initial stages of pregnancy, the endovascular extravilous trophoblasts form the trophoblast plug. This prevents maternal blood from entering into the intervillous space.
The interstitial extravilous trophoblasts invade into the decidua and migrate towards the maternal spiral arteries.
Extravillous cytotrophoblast invasion
As the cells from the column move into the decidua, they undergo epithelial mesenchymal transition.
During this process cells lose the polarity they have. They loose adherence. They become more motile and invasive in nature.
As this progresses they will also start to express different cell surface markers.
Initially they express: α6β4, αVβ6 and E cadherin
As the cells migrate away from the column they express: αVβ3, α1β1, VE-cadherin, VCAM-1, and PECAM-1
Oxygen tension and gestational age
The main purpose of the trophoblast plug is that it reduces the amount of oxygen that the villous tissue is exposed to in early gestation.
Up until the 12th week of gestation, the uterine spiral arteries are plugged with trophoblasts.
Placental development therefore occurs under relative hypoxia 2-3% O2
While the spiral arteries are plugged, the nutrition that is supplied to the growing foetus is through the process of histiotrophic nutrients being secreted by the glandular cells
Following the dissolution of the trophoblast plug, the placenta switches to a normal haemotrophophic nutrition. This occurs around the 12 week.
Low oxygen early in pregnancy is important normal pregnancy progression
Prolonged low oxygen leads to placental pathologies
Methods used to study human placental development
Human studies are limited for ethical reasons
Animal models:
BUT There are significant differences in the placental development between mammals:
The mouse is not always a suitable model:
Trophoblasts invade the decidua and maternal arterial wall and come in to direct contact with maternal blood. SMILARITY
However there is no deep interstitial invasion of the decidua. The extent of interstitial invasion is significantly less than in humans.
Mice do not exhibit the same obstetric complications as humans. DIFFERENCE
The best model of human pregnancy is the great apes:
Trophoblasts invade the decidua and maternal arterial wall and come in to direct contact with maternal blood (maternal spiral artery) SMILARITY
Deep interstitial invasion of the decidua does occur. SMILARITY
Some evidence that they do exhibit the same obstetric complications as humans. SMILARITY
BUT it is ethically unacceptable to experiment on these animals.
In vitro methods used to study human placental development
Human tissue can only be obtained either in the first trimester from Termination of pregnancy or at term upon delivery.
Tissues can either be used whole or cells can be isolated from them.
Also available from cell lines:
Trophoblast cell lines derived from choriocarcinomas JEG3, Jar and BeWo
grow well in culture
but have lost some characteristics
Developed cell lines following transfection with oncogenes such as t- and T-antigen of SV40 or more recently hTERT
grow well
but have lost some characteristics but this can depend on how they are cultured
An approach that showed initial promise: use of Human embryonic stem cell-derived trophoblast cells (hESCs)
BUT characterisation has proved problematic
It cannot be used in a number of regions due to legal considerations.
The most recent development showing initial promise: Use of Human trophoblast stem cells (hTSCs) derived from the trophectoderm and first trimester placentae
express characteristics of first trimester trophoblasts
can be induced to differentiate along either syncytial or extravillous lineages
BUT can be difficult to prepare and grow
Not allowed in some regions.
For ALL models: The phenotype of all cells grown in vitro will be dependent the
culture conditions used. (eg differences between monolayer and 3D cultures)
Culture formats
Simple mono layer culture
Simple co-culture models
Mixing two cell types together and growing in a monolayer
Or separate them using multi cell inserts – so factors produced by each cell type can freely diffuse between the two.
Addition of extracellular matrix
Communication can take place between cells in a more direct fashion.
Effect of flow
Grow cells and look at the effects of flow
3D environment
Spheroid model or a perfusion model of dissected arteries.
Organoid cultures
More recent approach where cells are isolated but then the tissue is reconstructed.
What regulates trophoblast invasion- how can these models be used?
Ex vivo model using human placental tissue.
The anchoring villous can be identified and dissected, then placed on an extracellular matrix and the migration of cells from the villous tissue can be monitored by time-lapse microscopy. These processes can be manipulated by using both pharmacological can molecular techniques.
An alternative approach:
In vitro model using a multicell insert.
Have an extracellular matrix, on top of that are seeded cells: Extravillous trophoblast.
A stimulus can then be put into the lower chamber and if this stimulates the trophoblast, it may stimulate the trophoblast to migrate through the matrix.
Using a combination of approaches, we have identified a number of factors that regulate trophoblast invasion:
hCG
EGF
HGF
IGF-I/II
What factors influence trophoblast invasion?
Trophoblast and cancer cells have mechanisms of invasion in common
However trophoblast invasion is tightly regulated unlike metastasis. There are both positive and negative regulators of trophoblast invasion.
Positive regulators of trophoblast invasion:
Growth factors and cytokines
HGF
IGF-1
Prolactin
Matrix proteases
MMP-2, 9, 10, 12 – that will digest the extracellular matrix
Negative regulators of trophoblast invasion:
Tissue inhibitors of matrix metalloproteinases
Inhibitory cytokines and growth factors:
TNF
TGFβ
IGFBP-1
Upsetting the balance between stimulatory and inhibitory factors can lead to pregnancy complications
Major source of factors that regulate trophoblast invasion are the maternal immune cells
Immune cells in decidua basalis
70% uterine natural killer cells (uNK cells)
20% are macrophages
They are recruited following implantation
They localise to maternal spiral arteries
They precede the invasion trophoblasts
They secrete factors that regulate trophoblast invasion that have two roles:
1) to prepare the spiral artery for the invading trophoblast
2) to release chemokines that will attract the invading trophoblast.
Failure of placental development
Failure of the placental to develop normally can lead to common pregnancy disorders such as early pregnancy loss, pre-eclampsia and fetal growth restriction.
Failure to thrive in utero has lifelong consequences including increased risk of developing hypotension and diabetes
Overview of extravillous trophoblast function
Early in pregnancy cells of the placenta (extravillous trophoblasts) invade the decidua
They migrate from the anchoring villi into the decidua
They will form plugs that block the maternal spiral arteries
These two functions lead to two different phenotypes:
Interstitial extravillous trophoblasts
Endovascular extravillous trophoblasts
The invading interstitial extravillous trophoblasts migrate towards the spiral artery and will interact with the vascular smooth muscle cells, line the vessels, and replace the cells of the vessel wall
The endovascular extravillous trophoblasts will initially plug the spiral arteries and will then migrate down the lumen of the vessels interacting with the endothelial cells, ultimately will lead to their loss
The physiological reason for this interaction is that the spiral arteries under normal circumstances will respond to vasoconstrictors and dilators, these arteries will be under control of the maternal needs. By removing the ability of these vessels to contract, the foetus removes the ability of the mother to regulate the flow of blood into the intervillous space.
Ultimately this will result in increased blood flow to the developing baby
Spiral artery remodelling
In a non pregnant state they have a diameter of 200um. They consist of an endothelial cell lumen, surrounded by both connective tissue (elastin and/or collagen) and then surrounded by smooth muscle cells.
Following the remodelling process, interaction with trophoblasts and the stimulation by pregnancy related hormones:
The diameter of the vessel increases significantly (2mm diameter).
This results in a change of system from a low flow high-resistance vessel -> High flow, low resistance vessel.
There are two phases of spiral artery remodelling:
Trophoblast independent phase
Maternal Immune cells (specifically the uterine natural killer cells (UNK))
Pregnancy hormones (Such as progesterone)
Trophoblast dependent phase
We know about the trophoblast independent phase by studying ectopic pregnancies, as when the blastocyst implants in a place other than the uterine wall, it still has spiral artery remodelling.
How are the vascular cells lost from spiral arteries?
Mechanisms from the contractile phenotype of smooth muscle cells to a vessel that is highly modified with loss of smooth muscle cells and endothelial cells. These are replaced by invading foetal cells (invading Extravilous trophoblasts).
Some of the influences are:
dNK cells
dMacro (decidual macrophages)
Stromal cells (within the decidua)
EVT (Extravilous trophoblasts)
The extravillous trophoblasts will replace both the endothelial and smooth muscle cells within the wall of the spiral artery.
Mechanisms that could be involved:
Migration – cells could induce vascular cells to migrate away from the vessel
De-differentiation – the trophoblasts could induce de-differentiation of both the smooth muscle and endothelial cells.
Loss of adhesion (Anoikis) – Anoikis is a form of programmed cell death.
Vascular cell apoptosis
The hypothesis is that:
Extravillous trophoblasts in uterine spiral arteries bring about changes leading to the loss of vascular cells - crucial for the vascular remodelling
Mechanisms of vascular cell loss
Vascular cell apoptosis
Migration
Dedifferentiation
Loss of adhesion
What is apoptosis?
Programmed cell death
Cell death without the inflammatory response
Apoptosis is characterised by distinct morphological changes, what are they?
Characterised by distinct morphological changes:
Cell shrinkage
Chromatin condensation
DNA fragmentation
Apoptosis is characterised by distinct biochemical changes
Cleavage of lamins and actin filaments in the cytoskeleton
The breakdown of chromatin in the nucleus leading to nuclear condensation
Translocation of phosphatidylserine to outer membrane
Cleavage of key enzymes such as poly ADP ribose phosphate (PARP) involved in DNA repair
What is apoptosis induced and mediated by?
Induced by cellular stress such as nutrient deprivation, hypoxia and viral infection
Mediated by a family of enzymes called caspases. Although caspase independent apoptosis does occur
