Disorders of early development tutorial Flashcards
LO
Notochord runs between ectoderm at top and endoderm at bottom and through mesoderm
- Embryo development: Summarise the key developmental events occurring in embryo in the first trimester.
- Pregnancy physiology: Summarise the key changes in maternal physiology across the course of pregnancy.
- Pregnancy disorders: Summarise the major pathology and pathophysiology of key disorders of pregnancy.
Early pregnancy loss is a frequent occurence in human populations, but quite how frequent is it, and what are the underlying caues?
What fraction of fertilised eggs are estimated to spontaneously miscarry in the early stages of pregnancy?
Range of estimates for loss before implantation/pregnancy detection:
- Probably around 40% but uncertain
- Estimate loss before implantation: 30-75%
- Before 6 weeks: 50%, 70%, 80%
- Before 8 weeks: 75%
- First trimester: 70%
- Total loss between fertilisation and birth: 46-90%
Human fecundity (monthly probability of conception) is around 15-25%, so very high estimates (>80%) of loss are unlikely.
High numbers are probably a little unfeasible as 15-25% of attempts to get pregnant are successful, then we can’t have 90% of all of these being lost.
Notes from him:
And there’s quite a range of estimates for pregnancy loss occurring prior to us being able to determine that the embryo has implanted or biochemical detection of a pregnancy by which we mean a positive test by a hCG pregnancy test. We think probably around 40 percent of pregnancies are lost prior to a detectable pregnancy. But the estimates range for somewhere between 30 percent and 75 percent. And more broadly, the total loss of pregnancy is between fertilisation and birth. So that’s across all three trimesters is estimated somewhere between 46 to 90 percent. So at least half, probably of all human pregnancies never make it to term. We’re not really sure whether these higher estimates of pregnancy loss are accurate, and indeed particularly estimates over 80 percent seem to be a little bit unlikely. And the reason for that is if we monitor the monthly probability of conception. So a young couple trying to conceive naturally will have a monthly probability of conception, a monthly chance of getting pregnant of roughly 15 to 25 percent. So one in eight to one in four. And obviously, if there’s a one in four chance of those getting to the point of a detectable pregnancy every month, in turn, that’s pretty difficult for then four fifths of all pregnancies to be lost. So there’s a bit of incompatibility between the monthly conception rate, which we can observe through pregnancy testing. And the estimates of overall loss of pregnancies. We can be quite a lot more accurate thinking about how many pregnancies are lost after they’ve been clinically determined. So, again, either detection of a hCG test or by embryonic ultrasound. And so we think probably around one in 10 of all clinically identified pregnancy to lost. And obviously, that’s on top of perhaps the 40 percent of early pregnancy losses prior to detection of a pregnancy. All of those clinically recognised pregnancies that are lost, around 80 percent of those losses occur in the first trimester. So early pregnancy loss really accounts for the vast majority of pregnancy loss across pregnancy. And it raises some interesting questions. The first of which is how might we be able to estimate with any degree of certainty how many embryos are lost? How many pregnancies are lost before they implant or before they’re clinically determined? And it also raises the question of whether in humans, what kind of natural background rate of embryo loss is how many embryos fail to implant, how many embryos that do implant fail to sustain the pregnancy. And those kind of questions are extremely difficult to ascertain, given that we’ve no way of monitoring them in real time.
What proportion of those are lost after a pregnancy has been biochemically confirmed?
- 10% of all clinically-recognised pregnancies lost
- 80% of those losses occur in the first trimester
Early pregnancy loss is a frequent occurence in human populations, but quite how frequent is it, and what are the underlying caues?
1.2 What is likely to be the major contributor to pregnancy loss before 12 weeks’ gestation?
- Maternal age, beyond mid 30s there is an exponential increase of having foetus with chromosomal abnormality
- Chromosomal defects eg aneuploidy is a major contributing factor
Notes from him:
So what are the major causes of early pregnancy loss?
Well, we think predominantly the major driver is likely to be aneuploidy to chromosomal errors occurring within the embryo. And indeed, if we look at early pregnancies, if we look at the embryonic material from early pregnancies that we can detect, at least half of them display some kind of chromosomal error, which certainly supports aneuploidy being a major causative factor in early pregnancy loss. If we look at the graph on the right here, this is the incidence of trisomy. So this is the percent, the fraction of clinically recognised pregnancies where there are three or more copies whether three copies of a particular chromosome, which is a aneuploidy error. And you can see very strikingly that there is essentially an exponential increase in the frequency of aneuploidy. Then the percentage of pregnancies that have a chromosomal disorder. As we proceed up through maternal age and then particularly in the late 30s upwards, that number increases very sharply. And if we were to look at other areas of aneuploidy, they follow a very similar trajectory with maternal age.
How does maternal age affect the chance of miscarriage?
- Risk is lowest between 25-29 (10% risk of it happening)
- Increases with maternal age (53% at >45)
- Degeneration of cohesin proteins which hold together chromosomes. Breakdown of that support structure with age causes uneven splitting of chromosomes.
- Above 45 risk gets very high ie above 50%
Which leads us neatly onto the next question, which is what’s the impact of maternal age on the risk of miscarriage or pregnancy loss?
This is work from a group in Norway monitoring the risk of miscarriage, the risk of pregnancy loss across increasing maternal age. And you can see quite strikingly that the lowest risk is women in their late 20s and early 30s when there’s a risk of roughly one in 10 of losing the pregnancy. But as we proceed up into the 40s, that becomes a very, very significant risk of early pregnancy loss, perhaps upwards of 50 percent. You’ll note also that there’s actually a slight increase in women who are under 21.
So the risk of pregnancy loss in women under 20 appears to be elevated relative to women in their 20s and early 30s as well. And we don’t really understand what the driver of this is. So why is this happening?
Well, we think the major driver of age related increases in miscarriage and age related increases in aneuploidy pregnancies is to do with a loss of cohesion between homologous chromosomes in the oocyte so early on in oocyte formation. The pattern or copy of each chromosome and the maternal copy. So the copy that was inherited from the father and the copy that was inherited from the mother. And this is prior to fertilisation. Remember, the paternal and the maternal copies pair up and there’s an exchange of genetic material in a process called recombination. These homologous chromosomes are held together by these little black lines here, which denote proteins called cohesins, and they essentially act as a ring which holds the two chromatids that make up each chromosome together. If we go through a normal process of meiosis in the first division, the two homologues get pulled apart by the spindle. So the spindle would bind here and here. The pattern or copy gets pulled this way. The maternal this way. And so they separate into the two daughter cells. And then the second meiotic division, the two chromatids, the two individual sticks here get pulled apart, creating four haploid gametes However, as age progresses, these cohesin proteins don’t get replaced. So. As the woman ages and the eggs are ageing in her ovary, we see a progressive decline in the amount of cohesin proteins holding together these homologous chromosomes.
And so what happens is eventually cohesion can break down. We can get a loss of cohesion between the chromatids. And one of these chromatics can essentially drift off. And when the spindle comes in to grab them, to separate them, it can’t find one of the chromatids because it’s drifted off limits, partner, and therefore it gets separated at cell division randomly and we end up with aneuploidy.
We don’t understand why cohesin isn’t replaced over time. But obviously, a woman’s eggs are all synthesised in the foetal ovary. So all the eggs are formed in the foetal ovary and then menopause happens around the age of 50. So eggs can be hanging around in the ovary for up to 50 years with this progressive loss of cohesion with age. And it seems to become particularly striking once we’re kind of into the late 30s and particularly into the 40s.
Why might this be the case, and what are the molecular mechanisms that underpin this?
- Miscarriage risk mirror increase in aneuploidy risk
- Primary driver of age-related miscarriage is aneuploidy
- Degeneration of cohesin proteins which hold together chromosomes. Breakdown of that support structure with age causes uneven splitting of chromosomes.
- Under normal circumstances in first mitotic division the homolgous chromosomes are pulled apart in anaphase and cohesin breaks
- Cohesions deplete with age and are not replaced.
- Oocytes formed when woman was foetus so these eggs are very old and have depleted cohesin. If homologous chromosomes start to drift the spindle may not collect all of them and we get drifting and consequent aneuploidy.
Part 2 - Recurrent Miscarriage and Recurrent Implantation Failure
Recurrent miscarriage and recurrent or repeated implantation failures are distressing disorders characterised by repeated loss or failure of pregnancy. Using the resources you have available, consider the questions below:
What are recurrent miscarriage and recurrent implantation failure and what is the key difference between them?
Recurrent miscarriage: loss of three or more consecutive pregnancies, affects 1% of couples trying to conceive
Recurrent implantation failure: refers to cases in which women have had three failed in vitro fertilisation (IVF) attempts with good-quality embryos
Difference: Failure to implant or sustain pregnancy by natural conception (recurrent miscarriage) versus failure of transferred embryo to implant/sustain pregnancy (RIF)
But the approaches that one might take to diagnosing these conditions or investigating these conditions would be similar:
So the key difference between recurrent miscarriage and recurrent implantation failure is recurrent miscarriage arises really from natural conception, whereas recurrent implantation failure is the failure of a transferred embryo during an IVF cycle to implant or be sustained as a pregnancy. But the approaches that one might take to diagnosing these conditions or investigating these conditions would be similar.
So in both instances, we’d be checking the uterine anatomical defects or perhaps the presence of fibroids or polyps. So these are growths within the endometrium or the myometrium that might disrupt implantation. We do know that some auto immune conditions, particularly anti phospholipid antibodies, have the capacity to attack the developing embryo and therefore compromise implantation. And there is increasing interest in whether perhaps fragmentation of the sperm DNA so damaged DNA being delivered by the sperm into the oversight at fertilisation might also contribute to early pregnancy loss. And so there is some interest in perhaps following up to determine whether the DNA integrity of the paternal sperm is as good as it could be.
What potential common/overlapping causes would you explore in the first instance for patients experiencing these conditions?
- Check for uterine anatomical defects or presence of fibroids/polyps that might disrupt implantation
- Determine the presence of auto-immune antibodies within circulation (anti-nuclear or anti- phospholipid antibodies)
- Test for paternal DNA sperm integrity or fragmentation
What signalling pathway(s) might underpin recurrent miscarriage or RIF?
- LIF: leukaemia inhibitory factor (cytokine)
- It was found that there was normal embryo development but failed implantation in LIF-deficient mouse models.
- LIF promotes the decidualisation of human endometrial stromal cells in cultures
- Reduced LIF in the uterine secretions of subfertile women would cause recurrent miscarriage/implantation failure
Notes from him:
We thought a little bit about the signalling pathways that might underpin recurrent miscarriage or recurrent implantation failure. And really we identified perhaps the leukaemia inhibitory factor pathway as being a potential regulator of this process. So this is hypothetical, but it does seem like a good candidate. If we make genetically engineered mouse models that lack LIF, we see that the embryos of those mice develop normally. And actually, if you take them out of that mouse and put them into a mouse, which has normal levels of LIF those embryos will implant and develop. Okay. But although the embryos develop okay within the reproductive tracts of the LIF deficient mice, they can’t implant. So we see implantation failure in the absence of life in mouse models. If we take human endometrial stromal cells out of the endometrium and put them in culture, then treat them with LIF, we can see that LIF contributes to the decidualization of those cells. So although progesterone is the primary driver of decidualization, it appears that LIF can contribute to that as well. So you can imagine that the situation where disrupted lif signalling might lead to compromised decidualization. And that compromises visualisation that impairs implantation and subsequent embryo development. And indeed, if we examine the uterine secretions of women who have some fertility, we can see that the level of LIF in those uterine secretions is decreased. So this is perhaps some circumstantial evidence that there may be some disruption in the embryo to maternal signalling through the LIF signalling pathway. In some situations of poor implantation or implantation failure.
What is endometrial scratching and how might it help a patient experiencing RIF?
- This is the use of pipette or hysteroscope to damage endometrial mucosa before embryo transfer in IVF.
- The mechanism is unknown but thought to stimulate immune cell infiltration and wound healing cytokine production.
- Evidence in support of is variable but there is possible benefit in selected groups (e.g. RIF).
- Large NZ study suggests no benefit in any group.
Notes from him:
We then touched on this idea of endometrium scratching. So what is it? And how does it work?
Individual scratching is increasingly common in IVF units. It’s offered as a treatment. And really, it’s the use of a pipette or a hytsteroscope to damage the endometrial mucosa. So we’re damaging the lining of the endometrium here before we put back an embryo through IVF. Really, the thinking behind this is by inflicting a wound on the endometrium lining. This stimulates immune cell infiltration and perhaps the promotion of a wound healing response in the endometrium might be might result in cytokine productions or a growth factor environment which is more conducive to the embryo implanting. Initially, there was some support, there was some evidence to support the use of endometrial scratching, particularly in highly selected populations of patients. So, for example, patients who were experiencing recurrent implantation failure, if you just used endometrial scratching in that population of patients, it did indicate perhaps that there was some level of benefit and improved implantation and and successful pregnancy in those patients.
However, a large study from New Zealand a couple of years ago now has suggested that actually there is no benefit in any group. So even when you so highly select your patients for endometrial scratching, it doesn’t really appear to be doing anything compared to scratching in terms of promoting implantation or getting more life births out of the end of the procedure.
Part 3
Ectopic pregnancies arise from the implantation of the embryo at a site other than the uterine endoetrium. 98% of these implanatation events occur in the fallopian tube. Around 1-1.5% of pregnancies are ectopic, and treatment ranges from chemotherapy (methotrexate) to surgery to remove the affected tube. Rupture of an ectopic pregnancy can lead to severe internal bleeding.
Histologically, how is the fallopian tube adapted to support transit of the fertilised embryo to the uterus?
- Smooth muscle - contractions drive the embryo along the fallopian tube
- Epithelium - coated in cilia (microvilli) promote fluid movement
There have been cases where ectopic pregnancies have developed where the fertilised egg moved up into the peritoneum but this leads to placenta attaching to the peritoneum and is impossible to remove without severe fatal bleeding.
Notes from him:
We looked at the development of the fallopian tube and thought about some signalling pathways and external disruptions of implantation, particularly thinking about ectopic pregnancy. So if we think about the fallopian tube, we can see here, it’s really geared to delivering the embryo to the uterus because it’s coated here in this smooth muscle, which is going to drive contractions, which will push the embryo along the fallopian tube from the ovary down to the uterus. And importantly, the epithelium of the fallopian tube is covered in cilia - microvilli and the beating of these microvilli promotes a fluid flow along the tract of the fallopian tube. Which again, will also assist in moving the embryo along the fallopian tube down to the uterus. So we can imagine that anything that perhaps perturbs the function of the smooth muscle or disrupts the ability of the cilia to move the fluid along the tube might contribute to the embryo getting trapped in the fallopian tube. And if it gets trapped in the fallopian tube, it has the potential to implant and to cause an ectopic pregnancy.
How might this help explain how cigarette smoking increases the risk of ectopic pregnancy?
- Cotinine is an alkaloid found in tobacco and is also the predominant metabolite of nicotine. Cotinine is a component of cigarette smoke and has been shown to regulate the expression of PROKR1
- This is a regulator of fallopian tube smooth muscle contractility
- Cotinine increases expression of pro-apopotic proteins the fallopian tube explants
- Tobacco smoke also likely to inhibit ciliary function, reducing transit fo the embryo through the fallopian tube
- Epidemiological studies: OR: 1.7-4 for tubal pregnancy in smokers
- Smoking is a major risk factor for EP. FTs from women with EP exhibit altered prokineticin receptor-1 (PROKR1) expression, the receptor for prokineticins (PROK). PROK1 is angiogenic, regulates SMC, and is involved in intrauterine implantation. We hypothesized that smoking predisposes women to EP by altering tubal PROKR1 expression.
Notes from him:
So we then thought about, well, what can smoking do to the fallopian tube?
We know that maternal smoking, maternal cigarette smoking. So tobacco use during pregnancy is a risk factor for ectopic pregnancy. And some studies and these are mostly laboratory studies where bits of the fallopian tube have been taken out. They’ve been put in tissue culture and treated with various chemicals that are present in cigarette smoke. An example of that is cotinine. So cotinine is a component of cigarette smoke. And if we treat explant - bits of fallopian tubes - in a culture dish with cotinine, we see disruption of the expression of this. PROKR1, the Prokineticin Receptor 1. We know PROKR1 is important in regulating fallopian tube, smooth fallopian tubes, smooth muscle contractility. So there’s evidence here that a component of cigarette smoke, which does get into the systemic circulation cotinine has the capacity to disrupt the expression of a particular gene, which in turn is important for regulating fallopian tube smooth muscle contractility. So we can imagine potentially that the cotinine in cigarette smoke might be inhibiting the action of the smooth muscle, and that might be contributing to a failure to transmit the embryo along the fallopian tube. And therefore, ectopic pregnancy. Cottoning also seems to increase the expression of pro- apoptotic proteins in fallopian tube plants.
So, again, if we take these fallopian tubes out, we put them in culture and we treat them with cotinine, we see an increase in apoptosis, particularly in the epithelial cells. And again, if we think about those cilia, if we’re getting death of the epithelium, we’re likely to be reducing the number of silly cells and therefore, perhaps we might be damaging transit along the fallopian tube.
We know that tobacco smoke itself is likely to inhibit ciliary functions, and that might have an impact on reducing embryo transit along the tube. And indeed, epidemiological studies have shown that the odds ratio for tubal pregnancies, ectopic pregnancy is in smokers is about one point seven to four. So the strong epidemiologic epidemiological evidence that smoking through pregnancy increases the risk of ectopic pregnancy.
At the molecular level, how might cannabis use elevate the risk of tubal pregnancy?
- Fallopian tube expressed CB1 and CB2 cannabinoid receptor. So if pregnant women takes cannabis it will trigger these receptors and cause downstream signalling.
- CB1 reduced in ectopic pregnancy patients and CB1 KO in mice causes embryo retention in the fallopian tubes.
- Tetrahydrocannabinol (THC) is the main psychoactive component of cannabis, can get through to the fallopian tubes and affect there
- Levels of endocannabinoids elevated in ectopic pregnancy fallopian tubes
- Components such as THC in cannabis may act directly on the fallopian tube to perturb transit or alter the balance of endocannabinoids in the fallopian tube leading to a disrupted embryo environment
Notes from him:
And the last thing we looked at in the tutorial was the impact of cannabis use on ectopic pregnancy and on the fallopian tube, the fallopian tube expresses the cannabinoid receptors, CB1 and CB2. And you can see in this lower panel here, I think this is CBD2. But this brown staining is the epithelium here of the fallopian tube is staining for CB2 receptor. And you can see it’s expressed predominantly in the epithelium. And I think this is CB1, a slightly lower level again in the in the epithelial tissues. And these are the two controls here. We know from studies in genetically engineered mice that if we knock out, if we inactivate the CB1 gene and so we get no production of the CB1 receptor in the fallopian tube epithelium. What happens is embryos collect in the fallopian tubes of those mice. So there is an essential role for CB1 in ensuring that the embryos can transit the fallopian tube in mice. So that’s pretty good evidence that cannabinoid signalling because one of the cannabinoid receptors here is involved. This is pretty good evidence that cannabinoid signalling is clearly important in embryo transit and therefore dysregulation of it perhaps might be involved in ectopic pregnancy.
If we think about what’s going on in humans, if we just think about women who’ve had ectopic pregnancy and so these are not cannabis users needed, just women who’ve had ectopic pregnancy, we can see that the levels of endocannabinoids. So cannabinoids produced by the fallopian tube appear to be elevated. So if we take biopsies of fallopian tubes from women who’ve had an ectopic pregnancy and look at the level of the cannabinoids in those tubes, they are elevated in the tubes of women who’ve had ectopic pregnancies compared to women who have not had ectopic pregnancies. And so there’s a kind of theory now emerging about the idea of this endocannabinoid tone. So the idea of this is that perhaps there is a natural balance of endocannabinoid production, endocannabinoid metabolism and endocannabinoid signalling through the CB1and CB2 receptors within the fallopian tube.
Under normal conditions, which is important for the embryo being able to transmit along the fallopian tube normally and perhaps in situations of cannabis use where we’re introducing additional cannabinoids such as THC, the addition of those extra cannabinoids or the introduction of those extra cannabinoids might then in turn disrupt this endocannabinoid tone. And that, in turn, may lead to a disrupted tuval environment and perhaps a failure of the embryo to be transmitted along the tube, appropriately leading or predisposing to ectopic pregnancy.
