Module 2 Essay Plans Flashcards
Describe the development of the feto-maternal interface between day 5 post-fertilisation and the end of the second trimester. Describe what goes wrong with this in pre-eclampsia and how this leads to the characteristic features of the condition. Summarise developments in, and challenges of, treatment.
Between day 6-7 post-fertilisation, the blastocyst implants within the wall of the uterus. The trophectoderm cells carry out a very controlled invasion of the decidua, moving between epithelial cells rather than destroying them. Cytotrophoblasts and syncytiotrophoblasts layers have become distinct by day 9, and by day 13 the primary villi have appeared. Shortly afterwards, cytotrophoblasts invade the syncytium, forming secondary villi with syncytiotrophoblasts and cytotrophoblast layers, and a mesenchymal core. Around this time, maternal sinusoids open allowing blood to flow into the trophoblastic lacunae. Meanwhile cytotrophoblasts penetrate the syncytium and anchor the placenta in the decidua.
By day 21 the tertiary villi are developing i.e. blood vessels are appearing in their cores. By day 28 the embryo lies fully within the maternal tissue, but contact between the syncytium and the maternal capillary blood is starting to be lost as cytotrophoblasts proliferate to form the cytotrophoblast shell and separate villi from intervillous spaces. There is no further contact with maternal blood till week 10-12, and during that time the oxygen tension of the fetal tissue is low (~3%). During the fourth week, endometrial glands undergo hypertrophy underneath the cytotrophoblast shell, and secrete nutrients to diffuse across the cytotrophoblasts to the fetus (histotrophic nutrition).
At week 8, cytotrophoblasts form plugs in the ends of the spiral arteries to prevent flow of maternal blood into the intervillous space. Cytotrophoblasts also migrate along the artery endothelium and replace endothelial and smooth muscle cells. This serves to further anchor the placenta, and to remodel the spiral arteries making them wider, and removing their capacity to constrict. This cytotrophoblast invasion continues till it reaches the inner 3rd of the myometrium (~16 weeks post-fertilisation). Between weeks 10-12 the cytotrophoblast plugs are removed and the placenta is exposed to full maternal blood flow. This is the cause of the relatively high rate of pregnancy loss at this time, as the placenta may be dislodged by the exposure to high pressure blood.
Pre-eclampsia is a relatively common complication of pregnancy, occurring in 1-5% of pregnancies, and accounting for 20-80% of mortality in pregnancy (depending on location worldwide). Pre-eclampsia is characterised by abnormal systemic vascular responses to placental implantation, and is defined by new onset hypertension (>140/90) with proteinuria >0.5g/day on two separate occasions. Pre-eclampsia presents in the second trimester or later (almost never before 20 weeks gestation), and the later it presents, the better the outcome for mother and child.
The pathophysiology of pre-eclampsia is not totally understood, but is believed to originate with insufficient remodelling of the spiral arteries. This results in narrow, high pressure vessels, which promotes platelet clotting. Platelets release inflammatory mediators which cause vasoconstriction which, in combination with the occlusive clot, leads to poor flow through spiral arteries. This leads to a decrease in oxygen tension within the placenta, which rises back to normal levels once the clot is thrombolysed via normal pathways. The reoxygenation produces oxygen free radicals which damage the syncytium, releasing syncytiotrophoblast microparticles (STBMs) into the maternal circulation. STBMs damage the maternal endothelial cells, which causes proteinuria, and hypercoagulability and hypertension (due to endothelial dysfunction and decreased prostacyclin production). STBMs also lead to maternal leucocyte activation causing further vascular damage. The cycle of clotting and thrombolysis repeats, producing multiple batches of free radicals.
Pre-eclampsia may lead to multiple maternal complications including oedema, eclampsia (seizures due to cerebral oedema.) cerebral haemorrhage, acute kidney injury, and HELLP syndrome, and increases the risk of cardiovascular disease in later life due to cardiac remodelling in response to chronic hypertension.
Currently the only solution to pre-eclampsia is to remove the placenta, either through birth or termination. However, treatment with anti-platelets has been shown to decrease incidence of pre-eclampsia and complications from it. Increased platelet volume has been observed in patients who later develop pre-eclampsia – this was presumed to be due to release of larger, immature platelets to compensate for depletion in the clotting-thrombolysis cycles in spiral arteries.
Aspirin was shown to be effective in reducing severe outcomes of pre-eclampsia, especially when given in the first trimester. This implies that the damage leading to pre-eclampsia begins before the classic presentation time of >20 weeks. One trial used maternal vitamin supplements to try and mediate the oxidative damage of oxygen free radicals to prevent endothelial damage in pre-eclampsia. The trial was halted when it became apparent that the incidence of low birth weight had been raised. This led to speculation that the hypertension in pre-eclampsia is beneficial to the foetus, as it results in increased blood flow through core blood vessels, including the placenta.
Describe the investigations you would carry out in a couple with recurrent miscarriage and discuss treatments. Describe the management of miscarriage
Miscarriage is defined as loss of a intrauterine pregnancy before 24 weeks gestation. Recurrent miscarriage is defined as at least 3 miscarriages in a row, and occurs in 1-2% of fertile women.
Thrombophillic: Antiphospholipid syndrome is the most important and treatable cause of RM. Antibodies bind to platelets and endothelial cells, activating them and triggering clotting and infarction in placental vessels. Investigations are ELISA for anti-cardiolipin antibodies, tests for anti-B2 glycoprotein antibodies, and dilute Russel’s viper venom time test with platelet neutralisation test for lupus anticoagulant. Treatment with aspirin and unfractionated heparin has been shown to significantly reduce RM rates (54%). RM patients should also be screened for factor V leiden, protein C&S deficiency, and antithrombin and prothrombin mutations.
Karyotypic disorders
Parents can be carriers of balanced translocations which lead to unbalanced translocations in the foetus, leading to miscarriage. Screening and IVF with PGD can be done to only select euploid, normal embryos, but has been shown to not affect healthy birth rate. Aneuploidy is also more common in older women, due to prolonged meiotic arrest of their oocytes, but this may be addressed with IVF with PGS, or egg donation.
Uterine malformation
There is a wide variety of possible uterine malformations that can increase RM rates. These are often investigated initially using ultrasound, but the gold standard is using hysteroscopy, which also allows the malformation to be fixed as it is examined. Laparascopy will sometimes be necessary (usually in the case of didelphys - two uteri). Successful pregnancy rates after surgery are between 65-85%, but around 60% of women will have a successful pregnancy without surgery, so management depends on the specific malformation.
Metabolic and Endocrine disease
PCOS (40% in RM women), thyroid disease, and insulin resistance are all associated with RM. TFTs, thyroid antibody tests, Hba1c, ovarian USS, and gonadotrophins could all be done to investigate. Control of thyroid disease and diabetes improves live birth rate, as does metformin in PCOS. hCG polymorphisms may cause RM, as they result in insufficient production of hCG from the conceptus to stimulate sufficient progesterone release from the corpus luteum. hCG production may also be decreased to to aneuploidy or immune/ thrombophillic disturbance. hCG supplementation does not improve outcomes, but progesterone supplementation in women with RM does reduce spontaneous miscarriage rate.
Dysregulation of implantation:
Decidual tissue will usually only allow embryos of sufficient quality to implant. In some women this selectivity fails, leading to implantation of aneuploid embryos which are later miscarried. In a normal patient these embryos would never implant and would never develop into a clinical pregnancy. Furthermore, the time period for implantation is usually strictly regulated, and implantation outside this window leads to higher rates of miscarriage. If the selectivity of the decidua fails, and allows implantation outside the normal window, this may also lead to recurrent miscarriage.
Sperm fragmentation
Sperm samples from RM men have revealed higher incidence of sperm fragmentation. There are multiple tests to assess sperm, but the alkaline COMET assay results in better prediction for male infertility. Antioxidants may provide some benefit, if not sperm donation should be considered.
Management of miscarriage
Diagnosed via ultrasound when gestation sac >25mm with no evidence of yolk sac
Expectant: wait to pass products of conception at home. First line treatment for 7-14 days, safe but unpleasant.
Medical: vaginal misoprostol to induce passage, 1% chance of bleeding requiring medical attention
Surgical: Aspiration of products under local or evacuation under general. Can leave adhesions and carries normal risks of anaesthetic
IVF has a low success rate in women over 40, what are the reasons behind this? Explain ways in which we can ameliorate the effects of advanced maternal age on IVF success.
Discuss the factors affecting success rate of IVF and the risks of IVF
The main factor in decreased IVF success in women over 40 is the increase in aneuploidy. Oocytes that have been held in prolonged meiotic arrest are more vulnerable to aneuploidy, which decreases the likelihood of a viable pregnancy. This increased incidence of aneuploid oocytes is thought to be due to long-term oxidative stress, decreased microcirculation around the dominant ovarian follicle, and exposure to environmental toxins leading to degeneration of cohesins.
Furthermore the number of antral follicles declines with age, which negatively impacts fertility.
Outcomes can be improved by transferring more embryos into the mother, thereby increasing the chance that at least one will implant. This also increases the chance of multiple pregnancy, which carries a greater risk of complications
Egg or embryo cryopreservation at a young age allows the egg or embryo to be thawed when a woman’s fertility has declined
Egg donation from a younger person improves the chance of success, though is sometimes unpopular as the resulting child would not be genetically related to the mother.
Pre-implantation genetic screening can be used to identify aneuploid embryos so only euploid embryos are implanted. Morphology of the cells can also be examined, but this does not always predict accurately which embryos will develop successfully.
Factors influencing IVF success chance include age, ovarian reserve (AMH, antral follicle count, or or FSH early follicular measurement) BMI, caffeine, alcohol, smoking, drugs, obstetric history (underlying conditions, previous births, previous miscarriages or failure to conceive).
The risks of |IVF include OHSS (especially if a GnRH agonist is used to down-regulate the pituitary), ectopic pregnancy, multiple pregnancy (with associated risks of TTTS, cord entanglement, IUGR, congenital malformation, preterm death), and drug reactions
Describe the epigenetic regulation of chromatin in embryonic stem cells (ESCs)
Chromatin can be modelled into different states via epigenetic modification, which affects accessibility of gene sequences to transcription machinery. Euchromatin refers to accessible chromatin, and is characterised by H3K9 and 14 acetylation, and H3K4 methylation. Heterochromatin refers to inaccessible, tightly wound chromatin, and is characterised by H3K9 and 14 de-acetylation, and H3K27 methylation.
Chromatin in ESCs must be kept accessible in order to make the cells pluripotent, and not commit them to any one lineage. Specialised epigenetic regulation of ESC DNA allows expression of the crucial components for pluripotency: Oct4, Sox2, and Nanog. However, chromatin must also be regulated so that transcription of all genes does not occur.
A defining characteristic of embryonic stem cells is the presence of bivalent methylation markings in promoters of key developmental genes – i.e. the presence of both activating and repressing marks. This serves to maintain the cell’s pluripotency, and to prevent unregulated transcription of all genes. Polycomb repressive complexes (PRCs) aid this process: PRC 1 prevents RNA polymerase II from elongating DNA after it has been assembled by activating methylation marks, while PRC 2 makes repressive gene marks (e.g. methylation of H3K27).
As cells differentiate, they acquire more repressive epigenetic modifications, and their chromatin becomes less accessible to transcription machinery, consolidating their differentiated identity.
Define DOhAD
Discuss an example of how maternal lifestyle and diet affects the fetus
a. Dutch hunger winter
b. Monozygotic twins
c. Agouti mouse/ BPA
DOhAD: how environmental factors acting during the development of an embryo/ child alter the individual’s gene expression
a) The Dutch Hunger Winter refers to the winter of 1944-45 when food to the west of the Netherlands was restricted by the Nazis. As a result there was a cohort of children exposed to famine in utero (mothers having 400-800 calories per day), but were then well nourished throughout childhood. These children has a higher risk of CVS disease (early gestation starvation), and obesity/ insulin resistance (prenatal starvation). The neonates had high head: birth weight ratio, suggesting brain sparing. This phenomenon supports the thrifty gene hypothesis - that exposure to starvation in utero results in adaptations that prepare the foetus to live under the conditions it is exposed to in utero, but will become detrimental under more abundant conditions. The idea of predictive adaptive responses built on the thrifty gene hypothesis to try and explain the ongoing high levels of metabolic disease in society by proposing exhibited continued plasticity, i.e. people were vulnerable to epigenetic changes throughout life, not just in utero.
b) A study examined normal metaphase chromosomes of monozygotic twins. 3-year-old twins had virtually identical chromosomes, but 50 year-old twins had very different patterns of methylation. This supports the idea that methylation is due to environment
c) The Agouti gene is normally expressed in the mouse hair growth cycle, but when hypomethylated becomes continuously active and ectopically expressed, and leads to a yellow, obese phenotype. Agouti is theorised to have target sites in adipocytes and the hypothalamus, which lead to its metabolic effects. Pregnant Agouti mice were exposed to BPA, which increased the number of offspring with yellow coats. BPA was found to demethylate the Agouti gene in offspring of pregnant mice, resulting in over-activation and continuous expression - causing the yellow-coat phenotype. A follow-up study deomnstrated this effect could be countered with nutritional supplementation of the mothers
Immunology of pregnancy
Immune modulation during pregnancy is critical as the foetus represents a large amount of foreign genetic material which must be allowed to grow within the mother for 9 months.
There are several maternal mechanisms to induce tolerance including: presence of uterine natural killer cells (uNK); Th1-Th2 switch; and limited access for T-cells and antibodies to the uterus due to the placenta.
uNK cells are a unique Nk cell variant found mostly in the uterus, particularly early in pregnancy. They are thought to have a role in vascular remodelling, and are involved in immune tolerance of the foetus, as they express inhibitory MHC Ia and b receptors that prevent them from attacking cytotrophoblasts.
There is a switch from Th1 to Th2 mediated response in pregnant women. As a result, maternal Th1-mediated autoimmune conditions will often improve in pregnancy, whereas Th2-mediated conditions will worsen. This switch results in an overall reduction in inflammatory processes, which aids immune tolerance of the foetus. This is also the reason pregnant women are more susceptible to viral infections, which are dealt with by a Th1-mediated response, and why maternal Th1-mediated autoimmune conditions (e.g. rheumatoid arthritis) tend to improve in pregnancy, but Th2-mediated conditions (e.g. SLE) tend to worsen.
There are also fetal mechanisms to enable immune tolerance, mainly through HLA allele expression, and FasL expression. Trophoblasts express the inhibitory HLA E and G variants, and do not express A, B, or C, which inhibits immune rejection. Trophoblasts also express Fas ligand, allowing them to trigger apoptosis in any maternal lymphocytes that invade.
The placental sink has been speculated as an explanation for the prevention of maternal antibodies that would otherwise cross the placenta and damage the foetus. There is evidence for placental production of asymmetric (one glycosylated arm) IgE which is thought to attach to the Fc portions of maternal antibodies and t-cells, thereby preventing foetal attack.
Describe the characteristics of IPSCs and their uses in regenerative medicine
Characteristics:
iPSC are pluripotent cells re-programmed from somatic cells and form ESC-like colonies, express ESC genetic markers, and have similar epigenetic signatures to ESCs. They also form teratomas and will form chimeras when injected into a blastocyst. iPSCs have similar differentiation potency to ESCs and can be amplified significantly in vitro.
Pros:
There are no associated ethical issues regarding obtaining embryos
They can be patient-matched to overcome immune rejection
They can be used to produce any required cell type
They can be amplified greatly in vivo
Cons:
They are inefficient
It may be tricky to culture the exact cell type needed
Reprogramming does not totally erase epigenetic marks, so they may still have some differentiation preference
There is a risk of tumourigenesis due to residual undifferentiated cells
Uses:
Modelling of disease through producing diseased cells and tissue, or using the cells to induce gene KOs in mice
Testing of drug toxicity on models
Cell therapy/ regenerative medicine
e.g. cardiac repair where they are a good option because they can differentiate into authentic mature cardiomyocytes (as opposed to other stem cell options like bone-marrow derived stem cells). However the cardiomyocytes produced are immature, and there may be mixed phenotypes which contribute to arrhythmia, and the process of reprogramming of somatic cells may cause genetic/ epigenetic changes in the cells. Overall iPSCs have been shown to be effective in improving ejection fraction in heart failure
Describe three reprogramming technologies, and discuss the factors affecting reprogramming
Somatic cell nuclear transfer:
This is how Dolly the sheep was cloned. An oocyte has its nucleus removed, then a whole somatic cell is inserted into the oocyte. The new cell is activated with an electric shock and cultured. The cells can then be used for reproductive cloning (e.g. Dolly) or therapeutic cloning (cells are taken and differentiated to somatic cells). Somatic cell nuclear transfer could allow creation of tissue that could be transplanted without being rejected, and could produce all cell types. However there are ethical issues RE where to get the oocytes. Furthermore the process is currently inefficient (Dolly took 400 nucleated oocytes)
Fusion with embryonic stem cells:
Somatic and embryonic cells are fused to create a pluripotent tetraploid cell. This cell is unstable and used for research, but not regenerative medicine
Reprogramming with defined factors:
Yamanaka in 2006 identified 24 highly and specifically expressed factors in ESCs and removed one at a time, whilst monitoring ESCs. Oct4 and Sox2 were found to be essential - they form a complex which binds to the Nanog promoter and are sufficient to activate reprogramming of somatic to pluripotent cells
Factors:
Degree of differentiation - the more differentiated a cell, the less efficient reprogramming will be
Cell age - the older the cell, the less efficient the reprogramming
Technical issues e.g. transduction efficiency, mechanical factors
