Placentation and trophoblast 1

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 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 requires mechanisms to evade the maternal immune system However the fetal and maternal circulations do not mix

Trophoectoderm proliferates and fuses to form a primitive syncytium (PS) beneath the implanted embryo TC migrate or invade into the decidua Lacunae (L) form by the action of proteases which later develop into the intervillous space Cytotrophoblasts proliferate and migrate through the syncytium to form the anchoring villi

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

2 circles, syncytium on the outside cytotrophoblast stem cells on the inside placental vessels like circles on the inside Hofbauer cells are small circles around the placental vessels the cytoplasm empty space are the ‘placental stromal cells’

A positive feedback loop governs GCM1 activity, hCGβ expression, and placental cell differentiation. The binding of hCG to LH/CGR elevates cAMP which activates PKA. PKA phosphorylates GCM1 on Ser269 and Ser275. This phosphorylation of GCM-1 results in hCG and syncytin 1/2 expression. Syncytin-1 promotes placental cell fusion and differentiation. 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. 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 (GCM1 - Glial Cells Missing Homolog 1)

Growth of the trophoblast column Regulated by factors such as IGF1 produced by the underlying mesenchymal cells HIF-1α and Stox1 stimulate proliferation and inhibit differentiation

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 Low oxygen early in pregnancy is important normal pregnancy progression Prolonged low oxygen leads to placental pathologies

1)Trophoblasts invade the decidua and maternal arterial wall and come in to direct contact with maternal blood However there is no deep interstitial invasion of the decidua Mice do not exhibit the same obstetric complications as humans 2)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

Human tissue can only be obtained either in the first trimester from TOPs or at term Trophoblast cell lines derived from choriocarcinomas JEG3, Jar and BeWo grow well have lost some characteristics Developed following transfection with oncogenes such as t- and T-antigen of SV40 or more recently hTERT grow well have lost some characteristics but this can depend on how they are cultured Human embryonic stem cell-derived trophoblast cells (hESCs) characterisation has proved problematic 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 difficult to prepare and grow

Simple mono layer cultures simple co-cultures addition of extracellular matrix effect of flow 3D environment organoid cultures

Trophoblast and cancer cells have mechanisms of invasion in common However trophoblast invasion is tightly regulated. 1)Growth factors and cytokine HGF IGF-1 Prolactin 2)Matrix proteases MMP-2, 9, 10, 12 3)Tissue inhibitors matrix metalloproteinases 4)Inhibitory factors TNF TGFβ IGFBP-1 Upsetting the balance between stimulatory and inhibitory factors can lead to pregnancy complications

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? 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

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

Two phases Trophoblast independent Immune cell Pregnancy hormones Trophoblast dependent We know this by studying ectopic pregnancies

Migration De-differentiation Loss of adhesion (Anoikis) Vascular cell apoptosis Extravillous trophoblasts in uterine spiral arteries bring about changes leading to the loss of vascular cells - crucial for the vascular remodelling

Programmed cell death Cell death without the inflammatory response Characterised by distinct morphological Cell shrinkage Chromatin condensation DNA fragmentation Membrane blebs and blisters 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 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 Trophoblast use Fas/FasL to induce apoptosis in EC and VSMC

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

Dedifferentiated SMC Migratory Proliferative Produce extracellular matrix components phenotypic modulation: TGF B (transforming growth factor-B) Differentiated SMC Non-migratory Low growth rate Primary function is to carry out contraction To go back to dedifferentiated form- need Platelet derived growth factor (PDGF-BB)

Trophoblast conditioned media generated Control or conditioned media added to vascular spheroids for 24 hours RNA extracted from vascular spheroids Gene expression altered by trophoblast examined by Illumina Bead-chip array

PDGF, KL4, CXCL 10, IL-6 These are genes involved in VSMC differentiation MMP10- Matrix degradation and possibly elastin derived peptides which stimulate trophoblast invasion IL-8- Stimulates trophoblast invasion. IL-11, regulats VSMC phenotype CCL20- chemokine Gene ontologies Blood vessel morphogenesis Inflammatory response Angiogenesis Blood vessel development Vasculature development Response to stress