placentation & trophoblast I and II Flashcards

1
Q

The placenta

A
  • The placenta is fetal in origin at term it weighs 500-1000g
  • Acts as the lungs, gut and kidneys of the fetus
  • Acts as an endocrine organ releasing hormones into the maternal circulation such as hCG and progesterone
    The placenta is a semi-allograft, the cells have genetic material from both the mother and the father
    In a human pregnancy, fetal cells are in direct contact with maternal blood this means it comes into contact with maternal immune system so requires mechanisms to evade the maternal immune system.
    However the fetal and maternal circulations do not mix
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2
Q

Placental development

A

· Blastocyst attaches and adheres to epithelial layer of uterine wall

· Trophoectoderm cells divide and migrate between epithelial cells through basement membrane and to uterine wall.

· Trophoectoderm proliferates and fuses to form a primitive syncytium (PS) beneath the implanted embryo.

· TC migrate or invade the decidua

· Lacunae (L) form by the action of proteases which later develop into the intervillous space.

· Behind the PS cytotrophoblasts proliferate and migrate through the syncytium into the uterine wall to form the anchoring villi.

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3
Q

implantation …

A

multistep process by which free floating blastocysts attaches to the endometrium , invades through epithelium and into the stroma beneath and begins to establish the placenta.

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4
Q

The placenta

A

As placenta develops , anchoring villi branch to form secondary and tertiary villi .
Blood vessels that supply foteus that supply are spiral arteries , which deliver maternal blood to the intervillious space . veins drain blood from intervillious space back into the maternal circualtion.
Highly branched with a large surface area for exchange.
Outer layer of fused cells- the syncytium
Underlying cytotrophoblast stem cells
Diffusion distance to vessels small
Growth is regulated by a number of factors including IGF I and II

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5
Q

Villious structure

A
  • highly branched vascular networks of arteries and veins that take blood to and from the developing fetus.
  • the vessels are very close to the surface which enables rapid exchange of material.
    -Hofbauer cells are macrophages and may be involved in immune protection of placenta and regulating the formation and branching of the vessels
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6
Q

Formation of the syncytium

A
  • combined action of hCG and syncytin 1/2 forms synctium
    1. hCG binds to the LH/CGR receptor
    2. this stimulates the production of cAMP
    3. cAMP will activate membrane protein called Scramblase and PKA
    4. Scramblase is responsible for the redistribution of phosphodyl serine from the inner surface of the plasma membrane to the outer surface of the membrane for apoptosis and fusion
    5. PKA phosphorylates Gilial Cell Missing homologue 1 (this is a transcription factor that will move to the nucleus and regulate the expression of syncytin 1/2 and hCG.
    6. Synytin 1/2 will move to the plasma membrane where it will induce self fusion and formation of primary syncytin and secondary
    7. Syncytium is continually shed in to the maternal circulation and cytotrophoblast has to fuse with syncytium to replace lost material.
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7
Q

What is the role of synctium?

A

help fusion of placental cells
made in the nucleus - Gililial cell missing homologue transcription factor involved in regulating expression of synctin 1/2 in the nucleus
synctium will move to the membrane where it is involved in fusion.

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8
Q

what protein do you use to stain syncytium and look at it under a microscope?

A
  • desmoplacin
  • multinucleated cells
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9
Q

What is the function of syncytium?

A

Syncytin-1 is a human endogenous retroviral envelope gene product that plays an important role in the formation and maintenance of normal syncytium throughout pregnancy.
Syncytium is constantly regenerated throughout gestation and slows down towards term( to allow placental rupture in pregnancy) – to do this, cytotrophoblasts have to fuse with the syncytium to replace lost material.
Syncytium is continually being shed in to the maternal circulation and is replaced by the underlying cytotrophoblasts.

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10
Q

What is the role of trophoblasts in placenta formation?

A

trophoblasts are important causing vascular smooth muscle loss in the placental arteries to remodel them from constricted high resitence low flow vessels to low resistance high flow vessels.

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11
Q

Trophoblast differentiation and function

A

Extravillous trophoblast differentiate into endovascular and intristial trophoblasts.
Endovascular and interstitial trophoblast work together to ultimately remodel the maternal spiral arteries.
-in the initial stages of pregnancy the endovascular trrophoblast forms a trophoblast plug which prevents the maternal blood from entering the intervillus space.
-interstitial extravillous trophoblast (EVT) invades into the decidua and migrates towards spiral arteries.

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12
Q

What happens when the trophoblast moves into the decidua (extravillious cytotrophoblast)

A

as cells move into the decidua they undergo epithelial mesoncymal transition during this process the cells lose their polarity and lose adherens and as this progresses they express different cell surface markers.

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13
Q

What regulates growth of the trophoblast?

A

Growth of the trophoblast column regulated by factors such as IGF1
produced by the underlying mesenchymal cells

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14
Q

What factors stimulates proliferation and inhibit differentiation?

A

HIF-1α and Stox1

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15
Q

Oxygen tension and gestational age

A

trophoblast plug reduces level of oxygen
the villus tissue is exposed to in early gestation.

Up until 12th week the uterine spiral arteries are plugged with trophoblasts

Placental development therefore occurs under relative hypoxia 2-3% O2

While the spiral arteries are plugged nutrition is histiotrophic nutrients being secreted by the glandular cells

Following dissolution of the trophoblast plug the placenta switches to haemotrophophic nutrition

Oxygen concentration:
- 8-10 weeks gestation 2-3%
- 12-13 weeks gestation 7-8%

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16
Q

Why is it important to have low oxygen in early stages of pregnancy but not good to have prolonged low oxygen?

A
  • Prolonged low oxygen leads to placental pathologies
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17
Q

Limitation of using animal model to study human placental development.

A

Animal models
There are significant differences in the placental development between mammals
Human studies are limited for ethical reasons

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18
Q

The similarities and difference in mice compared to humans.

A
  • Trophoblasts invade the decidua and maternal arterial wall and come in to direct contact with maternal blood (similar)
  • However there is no deep interstitial invasion of the decidua
  • Mice do not exhibit the same obstetric complications as humans
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19
Q

Why are Apes a better animal model than mice?(bc more simialrities)

A
  • Trophoblasts invade the decidua and maternal arterial wall and
    come in to direct contact with maternal blood
  • Deep interstitial invasion of the decidua does occur
  • Some evidence that they do exhibit the same obstetric
    complications as humans
  • Ethically unacceptable to experiment on these animals (limitation)
    more expensive and need licesnsing
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20
Q

If animal models arent appropriate what can we use to study placental development?

A

human tissue

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21
Q

What are two types of human tissue used?

A
  1. Trophoblast cell lines
  2. Tissue transfected with oncogenes
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22
Q

Problem with using human tissue?

A

Human tissue can only be obtained either in the first trimester from miscarriages or at term whereas pre eclampsia manifests in week 20 after first trimester , after birth you can observe physiological changes live while they happen

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23
Q

human tissue : trophoblast lines used derived from …

A

JEG3, Jar and BeWo
=> grow well
=> have lost some characteristics

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24
Q

Human tissue : examples of cells developed following transfection with oncogenes …

A

such as t- and T-antigen of SV40 or more
recently hTERT
- grow well
- have lost some characteristics but this can be manipulated by altering how they are cultured

25
Q

In vitro methods used to study human placental development

A

Human embryonic stem cell-derived trophoblast cells (hESCs)

26
Q

In vitro methods used to study human placental
development

A

Human trophoblast stem cells (hTSCs) derived from the trophectoderm and first trimester placentae

27
Q

What determines the phenotype of all cells grown in vitro?

A
  • culture conditions
28
Q

Culture formats

A
  1. simple mono layer cultures
  2. Simple co-cultures
  3. Addition of extracellular matrix
  4. effect of flow
  5. 3D environment
  6. organoid cultures - cells are isolated but tissue is reconstructed
29
Q

What regulates trophoblast invasion?

A
  • using Ex Vivo and in-vitro methods we have been able to identify different factors hCG, EGF, IGF-1/2 and a number of pathways can be activated.
30
Q

What factors influence trophoblast invasion?

A
  1. Growth factors and cytokine regulate trophoblast invasion
    - HGF
    - IGF-1
    - Prolactin
  2. Matrix proteases
    - MMP-2, 9, 10, 12

These factors negatively regulate trophoblast invasion
3. Tissue inhibitors matrix
metalloproteinases
4. Inhibitory factors
- TNF
- TGFβ
- IGFBP-1

=> upsetting the balance between growth factors and inhibitory factors leads to pregnancy complications

31
Q

Trophoblast and cancer cells have common mechanisms in invasion but the difference is …

A

trophoblast invasion is tightly regulated , positive and negative feedback

32
Q

A major source of factors that regulate trophoblast invasion are….

A

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 a number factors which has two roles:
1. prepare spiral artery for invading trophoblast
2. to release chemokine which will attract invading trophoblast

33
Q

Why is placental development so important?

A
  • 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
34
Q

Overview of extravillous trophoblast function

A
  • early in pregnancy cells of the
    placenta (trophoblasts) invade the
    decidua
  • they plug the maternal spiral arteries
  • they interact with and replace the
    cells of the vessel wall
  • ultimately this will result in increased
    blood flow to the developing baby
35
Q

Spiral artery is remodelled from ….to ….

A

Low-flow, high-resistance
vessel (200 mcirometers) > High-flow, low-resistance remodelled vessel (2mm diameter)

36
Q

What is the physiological reason of the interaction between endovascualr and interstitial trophoblast?

A
  • spiral arteries under normal condition respond to vasoconstrictors and dilators by removing the ability to constrict the fetus more blood flows into the intervillus space enabling nutrients exchange
37
Q

two phases of spiral artery remodelling:

A
  • Trophoblast independent
    – Immune cell
    – Pregnancy hormones
  • Trophoblast dependent
    => We know this by studying ectopic pregnancies
38
Q

Unmodelled decidual spiral artery -> remodelling decidual spiral artery

A

unmodelled decidual spiral cells phenotype:
contractile smooth muscle cells and endothelial cells
remodelled cells = loss of SMC and endothelial cells and replaced by fetal cell invading trophoblast, ie. extrovillus trophoblast (EVT)

some of the influences that cause loss of SMC when remodelling are:
- EVT
-decidual NK
-stromal cells
-decidual Macrophages

39
Q

some of the influences that cause loss of SMC when remodelling are:

A

Hypothesis = Extravillous trophoblasts in uterine spiral arteries bring about changes leading
to the loss of vascular cells - crucial for the vascular remodelling

Mechanism of vascular cell loss
- Migration
- De-differentiation
- Loss of adhesion (Anoikis)
- Vascular cell apoptosis

40
Q

Apoptosis

A
  1. programmed cell death
    2.cell death without the inflammatory response
  2. characterised by distinct biochemical changes
    Cell shrinkage
    – Chromatin condensation
    – DNA fragmentation
    – Membrane blebs and blisters
  3. 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
  4. Induced by cellular stress such as nutrient deprivation, hypoxia and viral infection
  5. Mediated by a family of enzymes called caspases. Although caspase independent apoptosis does occur
41
Q

Can apoptotic changes be detected in endothelial and vascular smooth muscle cells on co-culture with trophoblasts?

A

yes
there is significantly more apoptosis in the presence of trophoblast - secrete factors such as caspases which induce apoptosis of vascular cells - involved in spiral artery remodelling
end stage of apoptosis is PARP cleavage - in trophoblast there is higher levels of PARP (suggesting more apoptosis)
To confirm morphological changes was due to apoptosis , inhibitor of caspases (zVAD) was used, which showed lower levels of apoptosis and less morphological changes.

42
Q

Interaction between trophoblasts and vascular smooth muscle cells

A

Primary first trimester EVT co-cultured with human aortic vascular smooth muscle cells
Experiment showed smooth muscle ells secrete a factor that attracts trophoblasts this was tested based on the direction, speed and persistence of trophoblast.

43
Q

Trophoblast use Fas/FasL to induce apoptosis in EC and VSMC

A

NOK2 is neutralizing antibody that binds to FasL and prevent it from working.
With NOK2 there is a significant decrease in trophoblasts caused apoptosis in both EC and VSMC.

44
Q

Trophoblast-induced apoptosis in an in vivo co-
culture model

A

Red-Horse et al. 2006. Cytotrophoblast induction of arterial apoptosis and
lymphangiogenesis in an in vivo model of human placentation.
=> Transplanted placental villi into the mammary fat pads of Scid mice for 3 weeks.
=> CK+ve TC invaded and interacted with vessels.
=>Induction of vascular cell apoptosis in vivo and in vitro co-cultures

45
Q

Hypothesis:

A

Extravillous trophoblasts in uterine spiral arteries bring about changes leading to the loss of vascular cells - crucial for the vascular remodelling

46
Q

What are phenotypic modulation of undifferentiated SMC?

A

VSMC have a plastic phenotype and can move from a contractile phenotype to a de-differentiated non-plastic phenotype in pathological conditions.

This de-differentiation is stimulated by factors, some include TGF-b and PDGF-BB.

Changes in phenotype are characterized by loss of contractile proteins e.g Calponin

In vivo proof of de-differentiation:

in non-remodelled spiral arteries there is a tight ring of smooth muscle cells. In remodeled spiral arteries the smooth muscle is less tightly packed and evidence of less well stained cells that have moved away from the vessel wall so evidence in vivo for muscle de-differentiation.

In vitro proof:

If there is trophoblast conditioned media where media is grown with trophoblasts and contains factors secreted by them, that is stimulating smooth muscle cells, ther eis dose dependent drop in expression of SMC suggesting there is de-differentiationt aking place – more motile. Looking at motility of SMC there is a dose-dependent rise.

47
Q

What are modulations of differentiated and defifferentiated SMC?

A
48
Q

Trophoblast dependent remodelling

A

To examine the effects of endovascular trophoblast on these vascular spheroids, we first simply conducted a gene expression study. Conditioned media from an extravillous trophoblast cell line, SGHPL-4 (also grown in 3-D) was added to our vascular spheroids. Changes in gene expression were examined by Illumina bead-chip microarray, and genes which were significantly altered by the trophoblast conditioned media were examined.

49
Q

Working model

A
  • Communication between trophoblasts and vascular wall. Trophoblast stimulates VSMC to release factors which recruit more trophoblasts.
  • Trophoblasts induce VSMC apoptosis
  • Endovascular trophoblasts interact with Vascular endothelial cells
  • Interstitial trophoblasts produce factors which interact with VSMCs
  • Trophoblast conditioned media stimulates factors e.g PDGF which is important for regulating de-differentiation of VSMCs and stimulates metalloproteinases to breakdown elastin in the wall and produce elastin derived peptides which are chemoattractants for trophoblasts.
50
Q

Why is placentation and trophoblast studies important?

A

Failed/inadequate remodelling leads to common pregnancy complications such as pre-eclampsia, fetal growth restriction and early pregnancy loss.

® Major cause of maternal and fetal morbidity and mortality

® Diagnosed late in gestation with maternal hypertension

® Pathology established in the first trimester

® Affects 2-5% of pregnancies

® Mother (within 10-15 yrs)

o 4x hypertension

o 2x ischemic heart disease and stroke

® Children develop hypertension

o 2x likely to have a stroke

® PE is associated with FGR and prematurity

® Low birth weight is linked to diseases such as type 2 diabetes

® High cost to health system

o 3 million patients in UK

o 15 million in the USA

51
Q

Possible causes of poor placental perfusion

A

Poor trophoblasts invasion
Failure to interact with the maternal spiral artery

52
Q

Difficulties in studying the mechanisms important in human pregnancy.

A

Animals don’t exhibit the same pregnancy complications that humans do.
Best models for pregnancy are the great apes.
Mice are good for:
- Trophoblast differentiation

Mice are not good for:
- Cell-cell interactions
- Trophoblast invasion (shallow)
- SA remodelling (different cells involved)
- 3 trophoblast cell layers vs one in humans

Rats are good for:
- Maternal syndrome

53
Q

Difficulties in Studying pre-eclampsia human pregnancy

A

Humans
Studies on term placentae
– Organ at the end of its functional life
– Terminally differentiated
– Good for transport studies

First trimester most appropriate
– Limited availability
– Pregnancy outcome not known
– Legal and ethical issues Placental

The manifestation of preclampsia symptoms is at 20 weeks , whereas etiology of spiral artery remodeling happens earlier so it is harder to diagnose and detect earlier on

54
Q

What is the role of trophoblasts in placenta formation?

A

trophoblasts are important causing vascular smooth muscle loss in the placental arteries to remodel them from constricted high resitence low flow vessels to low resistance high flow vessels.

55
Q

First trimester studies of pregnancies with spiral artey related pathologies

A

In first trimester, can determine spiral artery resistance. In a pregnancy with higher uterine artery resistance, the embryo has an increased chance of developing abnormally if the pregnancy continues.

Of a normal uterine artery resistance then pregnancy is likely to not have complications.

FGR = fetal growth restriction

56
Q

High RI is a problem because…

A

High RI group – decreased endovascular trophoblast invasion and artery plugging.
Not all high resistant pregnancies develop pregnancy complications.

High-RI EVT are less invasive:
- Is there poor trophoblast migration/invasion in these pregnancies? – look for spiral artery plugging
- The more trophoblast plugging, less likely to have complications

Experiment shows there is a significant reduction in the ability of trophoblasts to migrate out of decidua with high-RI. if trophoblast can’t migrate out of the decidua then it is less likely to remodel maternal spiral artery.

57
Q

High RI EVT are more sensitive to apoptic stimuli

A

Trophoblasts express:
- TNF, FasL or sFasL and TNF-related apoptosis-inducing ligand (TRAIL)
- Trophoblast also express the receptors
-Normal part of placental development increases after week 40
-Exaggerated in placental diseases such as pre-eclampsia (PE) and fetal growth restriction (FGR)
-Normally EVT are resistant to apoptotic stimulation

A small time difference between high and low-risk pregnancy would have a significant effect on the extent of apoptosis in a particular tissue.

The kinetic curve shows a shift towards the right from high à normal RI pregnancy showing an increase in resistance to apoptotic stimuli= in high resistance index pregnancy there is less resistance to apoptosis.
Correlation between resistance and sensitivity to apoptosis on the right graph.

58
Q

Sensitisation of first trimester EVT by inhibition of NO synthesis

A

Failure to synthesise NO in pregnancies occur in pre-eclampsia patients.
NO involved in maintaining normal preganancy.

L-NAME is an inhibitor of NO synthases and in the presence of L-NAME there is some degree of apoptosis.

In the presence of apoptosis inducers such as Fas, TNF and TRAIL there isn’t little bit apoptosis taking place.

If you add NO inhibitor to apoptosis inducers then significant increase in apoptosis.

In high risk group adding L-NAME has no significant effect but by adding NO donor may reduce the sensitivity of these cells to apoptosis. NO donor overcomes pathways that induce apoptosis. Pregnancies with high risk may have an abnormality to produce NO or to respond to it.

59
Q

summary

A
  • The symptoms of pre-eclampsia appear late in gestation yet the aetiology is believed to be in the first trimester
  • It is presently not possible to identify in the first trimester those pregnancies that will develop pre-eclampsia
  • It is possible to identify those at increased risk
  • Cells isolated from high-risk patients are different from those low or normal risk