Placentation and the role of the trophoblast II Flashcards
Why is remodelling important in pregnancy?
- If the trophoblasts failed to invade or remodel the spiral arteries for any other reason (inadequate remodelling), this can lead to important obstetric complications, e.g.
1) Pre-eclampsia
2) Foetal growth restriction
3) Early pregnancy loss - These conditions often have an element which can be summarised as placental insufficiency.
Trophoblast invasion and remodelling the spiral arteries is important for pregnancy, but why is it important long-term?
- Major cause of maternal and fetal morbidity and mortality
- Diagnosed late in gestation with maternal hypertension
- However, the pathology is established in the first trimester
- Affects 2-5% of pregnancies
- Mother (within 10-15 yrs)
1) 4x hypertension
2) 2x ischemic heart disease and stroke - Children develop hypertension = 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
1) 3 million patients in UK
2) 15 million in the USA - The consequences of some form of placental insufficiency for both mother and child extends beyond the pregnancy itself. Mothers of a preeclamptic pregnancy are four times more likely to develop hypertension within 15 years of the pregnancy and twice as likely to suffer from ischaemic heart disease or stroke. Importantly, the child is also more likely to develop stroke. What initially appears to be a pregnancy related problem ends up being a lifelong problem for both the mother and the child. This has significant costs to the healthcare system.
What are the possible causes of poor placentation?
1) Poor trophoblasts invasion. There are a number of mechanisms that may be responsible for poor trophoblast invasion.
2) Failure to interact with the maternal spiral artery. The failure to interact with maternal spiral arteries in any context is something that could lead to the same outcome.
What are the difficulties in studying the mechanisms of human pregnancy in animal models?
1) Mouse
- Good for:
Trophoblast differentiation (Senner and Hemberger Placenta. 2010;31:944-950)
- Not good for:
Cell-cell interactions
Trophoblast invasion (shallow)
SA remodelling (different cells involved)
Three trophoblast cell layers vs one in humans
2) Rat
- Good for:
Maternal syndrome
(McCarthy et al. Placenta. 2011;32:413-419)
- These animal models do not seem to exhibit the same pregnancy complications that humans do.
- Great apes are probably the only exception to this.
Why are great apes the best models for human pregnancy?
- Great apes are probably the only exception to this.
- There is evidence to suggest that trophoblast invasion takes place to a similar extent and the remodelling of the spiral arteries follows a similar pattern to that seen in human pregnancies. This is illustrated by the slide on the right. The spiral arteries can be seen in white; the trophoblasts have been shown in red (using CK7). This indicates that there are trophoblasts present and the black indicates smooth muscle cell actin. The spiral artery in B1 has a discontinuous smooth muscle layer.
- There is recent evidence from this article that indicates a case of a lowland gorilla in Bristol Zoo being affected by preeclampsia and having to undergo an emergency caesarean section. This was assisted by an obstetrician from Bristol university.
- There are, however, ethical issues with performing studies on great apes.
How can pre-eclampsia be studied using human tissue?
- Human tissue is available only at two points normally; the first place is at term (when the baby is delivered) and the second is in the first trimester (following terminations of pregnancies).
- There are issues with using term placenta. Firstly, the organ is at the end of its functional life, so it has started to deteriorate as a tissue. It is terminally differentiated; the cells have undergone all of the changes that they are going to undergo and there is little more that can be achieved with them. They are, however, good for looking at transport studies. A lot of transport studies have been performed on term placentae. The responses of cells from different pregnancy disorders can be compared at term and some value can be obtained from those studies.
- First trimester placentae also has issues. They are of limited availability; not all jurisdictions allow work on first trimester terminations of pregnancies. In fact, not all places in the world allow terminations to take place at all. There are legal and ethical issues. One of the main issues, however, is that the pregnancy outcome is unknown.
- Breaking pre-eclampsia down, there are two phases = there is the maternal phase (endothelial dysfunction and hypertension) and the placental phase (placental insufficiency and poor spiral artery remodelling). The problem that researchers have when studying placental changes in pre-eclampsia is that diagnosis of the syndrome takes place relatively late in gestation (about 20 weeks) and comes at a point beyond which the initial changes may be taking place.
How have pregnancies been studied in the first trimester with spiral artery-related pathologies?
- The problem that researchers have when studying placental changes in pre-eclampsia is that diagnosis of the syndrome takes place relatively late in gestation and comes at a point beyond which the initial changes may be taking place. To overcome this, researchers at St. George’s have used an approach called uterine artery doppler ultrasound. This is a process that is used later on in pregnancy to diagnose preeclampsia. In the first trimester (9-14 weeks), it can be used to determine the uterine artery resistance.
- A pregnancy with a high uterine artery resistance has an increased chance of developing abnormally if the pregnancy continues. High RI group = decreased endovascular trophoblast invasion and artery plugging (Prefumo et al. 2004 Hum. Reprod. 19(1):206-209).
- If the uterine artery resistance lies within a normal range, then the pregnancy is most likely to develop without any complications. This assessment has been carried out on a number of ongoing pregnancies (pregnancies that reach term) and almost 10,000 pregnancies were followed. It was determined that only 5% of pregnancies with a normal uterine artery resistance developed an issue, whether it be pre-eclampsia, foetal growth restriction or resulted in stillbirth. Just under 3% of those pregnancies were at risk of developing pre-eclampsia. However, having a higher uterine artery resistance in the first trimester means an increased chance of developing pre-eclampsia, foetal growth restriction or a stillbirth. It is essentially a five-fold increase in pregnancies that have a high uterine artery resistance in the first trimester.
- However, not all high resistance pregnancies develop pregnancy complications.
- Normal RI (<95th percentile) = “Normal” remodelling
- High RI (>95th percentile) = “Abnormal” remodelling
- Normal UtA Doppler RI = 2.8% PE risk and 4.9% PE/FGR/stillbirth risk.
- High UtA Doppler RI = 15% PE risk and 24% PE/FGR/stillbirth risk (Leslie et al. 2015 Am. J. Path 185(10):2731-41)
Why don’t all high resistant pregnancies develop pregnancy complications?
1) High-RI EVT are less invasive
- Studies carried out at St. George’s looked at groups that are most and least likely to develop pre-eclampsia if the pregnancy reached term.
- The first question that was asked was is there was evidence of poor trophoblast invasion to go with migration in these pregnancies. One of the ways this can be assessed is to look for spiral artery plugging. In the first trimester, trophoblasts plug the spiral arteries and prevent maternal blood from entering the intervillous space. This is a normal phenomena, so the greater the degree of plugging, the more likely the pregnancy is to be normal. Taking material from a high resistance index and a normal resistance index to look for trophoblast plugging, a significant (but small) decrease in the number of spiral arteries that are plugged can be seen. This is just looking at the presence of the accumulation of trophoblasts within a particular spiral artery (seen in image). This was determined by screening a lot of sections of decidua. This suggests that the high resistance pregnancies are less invasive than the normal pregnancies.
- This study addressed whether this can be supported by direct assessment of the ability of extravillous trophoblasts to invade or migrate from the placental tissue. The explant cultures can be used to look at the extent of the outgrowth in both high and normal resistance index pregnancies. A significant reduction can be seen in the ability of these cells to migrate out. Relating this to the physiology, if a trophoblast is unable to migrate or invade into the decidua, it is less likely/will not be able to remodel the maternal spiral artery. This area can be measured quite simply.
2) High-RI EVT are more sensitive to apoptotic stimuli. One of the possibilities is that they fail to invade and remodel the spiral artery because they undergo apoptosis before they reach the vessel.
- Trophoblasts express
TNF, FasL or sFasL and TNF-related apoptosis-inducing ligand (TRAIL). Trophoblast also express the receptors
- Although placental cell apoptosis is a normal part of development and increases after the 40th week, it has also been shown that there is an exaggerated amount of apoptosis seen in placental tissues from preeclamptic and foetal growth restricted pregnancies.
- The opposite to that is that under normal circumstances, extravillous trophoblasts would appear to be resistant to apoptotic stimuli.
- Exaggerated in placental disease such as pre-eclampsia (PE) and fetal growth restriction (FGR)
- Are high resistance EVTs more sensitive to apoptotic stimuli?
Trophoblasts express both the ligand and the receptors for a number of apoptotic-inducing molecules, such as TNF-alpha, FasL and TRAIL.
- Can we see a difference between normal and the high risk pregnancies in the first trimester? To achieve this, extravillous trophoblasts (EVTs) are isolated and apoptosis is induced. The induction of apoptosis is followed by time-lapse microscopy. A particular advantage of this is that kinetics curves can be created of the induction of apoptosis. Apoptosis is a process by which, if intervened at a particularly crucial point, it can be stopped from continuing. A small time difference between a high and a low resistance pregnancy would have a significant effect on the extent of apoptosis in any particular tissue.
- The image on the left shows one of those kinetics curves. The red line is the high resistance and the blue line is the normal resistance pregnancies; there is a shift towards the right when moving from high resistance to normal resistance pregnancies,. This suggests that there is an increase in resistivity to apoptotic stimuli. To put it another way, the high resistance pregnancies are more sensitive to apoptotic stimuli than the normal resistance pregnancies. This particular experiment was performed using TNF-alpha.
- The third image shows a correlation between resistance and sensitivity to apoptosis when correlating the resistance measured in the first trimester before the termination takes place with the half time of the apoptotic curve.
What evidence suggests high-RI EVT are less invasive?
- High-RI EVT are less invasive
- Studies carried out at St. George’s looked at groups that are most and least likely to develop pre-eclampsia if the pregnancy reached term.
- The first question that was asked was is there was evidence of poor trophoblast invasion to go with migration in these pregnancies. One of the ways this can be assessed is to look for spiral artery plugging. In the first trimester, trophoblasts plug the spiral arteries and prevent maternal blood from entering the intervillous space. This is a normal phenomena, so the greater the degree of plugging, the more likely the pregnancy is to be normal. Taking material from a high resistance index and a normal resistance index to look for trophoblast plugging, a significant (but small) decrease in the number of spiral arteries that are plugged can be seen. This is just looking at the presence of the accumulation of trophoblasts within a particular spiral artery (seen in image). This was determined by screening a lot of sections of decidua. This suggests that the high resistance pregnancies are less invasive than the normal pregnancies.
- This study addressed whether this can be supported by direct assessment of the ability of extravillous trophoblasts to invade or migrate from the placental tissue. The explant cultures can be used to look at the extent of the outgrowth in both high and normal resistance index pregnancies. A significant reduction can be seen in the ability of these cells to migrate out. Relating this to the physiology, if a trophoblast is unable to migrate or invade into the decidua, it is less likely/will not be able to remodel the maternal spiral artery. This area can be measured quite simply.
How else might extravillous trophoblasts be affected in preeclamptic vs normal pregnancies?
What evidence suggests high-RI EVT are more sensitive to apoptotic stimuli?
- One of the possibilities is that they fail to invade and remodel the spiral artery because they undergo apoptosis before they reach the vessel.
- 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 disease such as pre-eclampsia (PE) and fetal growth restriction (FGR)
- EVT are resistant to apoptotic stimulation
- Trophoblasts express both the ligand and the receptors for a number of apoptotic-inducing molecules, such as TNF-alpha, FasL and TRAIL. - Although placental cell apoptosis is a normal part of development and increases after the 40th week, an exaggerated amount of apoptosis has been seen in placental tissues from of preeclamptic and foetal growth restricted pregnancies. Under normal circumstances, EVT would appear to be resistant to apoptotic stimuli.
- To identify whether a difference between normal and higher risk pregnancies can be seen in the first trimester, EVT can be isolated and apoptosis can be induced. The induction of apoptosis can be followed by time-lapse microscopy. An advantage of this is that kinetics curves can be created of the induction of apoptosis. Apoptosis is a process by which intervening at a particularly crucial point allows the process to be stopped. A small time difference between a low and high risk pregnancy would have a significant effect on the extent of apoptosis in any particular tissue. When moving from a high resistance to normal resistance pregnancy, there is a shift to the right on the kinetics curve. This suggests that there is an increase in resistivity to apoptotic stimuli. In other words, high resistance pregnancies are more sensitive to apoptotic stimuli than normal resistance pregnancies. This particular experiment was performed using TNF-alpha. Correlating the resistance that is measured in the first trimester before the termination takes place with the half time of the apoptotic curve shows a correlation between resistance and sensitivity to apoptosis.
What is a potential molecular explanation for the sensitisation of first trimester EVT to apoptosis?
- For a long time, NO has been implicated in pregnancy and a failure to synthesis NO has been proposed for pre-eclamptic pregnancies. The majority of these studies are looking at the maternal consequences of PE, rather than the placental aetiology of PE.
- This study looks for a link. L-NAME is an inhibitor of NO synthases. Looking at the induction of apoptosis, in the presence of L-NAME, some degree of apoptosis is seen. Looking at the normal inducers of apoptosis that are present within the placenta or decidua, i.e. Fas ligand, TNF-alpha and TRAIL, no significant apoptotic events can be seen taking place. However, combining the L-NAME (NO synthase inhibitor) with each of these stimulants, there is a significant increase in the levels of apoptosis occurring. These experiments have been carried out on a trophoblast cell line. When looking at whether a similar result occurs in first trimester EVT from two resistance groups (normal and high resistance). The hypothesis is that adding L-NAME will have no significant effect, whereas adding an NO donor may reduce the sensitivity of the cells to apoptosis. This is what happened. There was no change by inhibiting NO, but an increase in their resistance when providing an NO donor. This suggests that the NO donor is overcoming the factors that are inducing apoptosis. Performing the same experiment with EVT from a high resistance group, inhibiting NO reduces the T1/2 (increases the sensitivity). Providing more NO has only a marginal effect. This would suggest that in high resistance pregnancies, there may either be an abnormality in the ability to produce NO or to respond to it. This requires further investigation.
Summary.
- 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
