RESUMEN
The placental microvasculature is a conduit for fetal blood allowing solute exchange between the mother and the fetus. Serial block-face scanning electron microscopy (SBF SEM) allows ultrastructure to be viewed in three dimensions and provides a new perspective on placental anatomy. This study used SBF SEM to study endothelial cells within the human placental microvasculature from uncomplicated pregnancies. Term human placental villi were aldehyde-fixed and processed for imaging by SBF SEM. Manual segmentation was carried out on a terminal villous capillary and an intermediate villous arteriole and venule. Twenty-seven SBF SEM stacks from terminal villi were analysed using stereological approaches to determine the volumes of microvascular components and the proportions of pericyte coverage. SBF SEM analysis of capillary endothelial cells revealed the presence of interendothelial protrusions (IEPs) originating from the donor cell at the endothelial junction and forming deep thin projections up to 7 µm into the adjacent endothelial cells. IEP density was estimated to be in the order of 35 million cm-3 placental tissue. Pericytes cover 15% of the fetal capillary surface area in terminal villi. In comparison, the cytotrophoblast covered 24% of the syncytiotrophoblast basal membrane. A trans-endothelial channel was observed in a region of the vasculo-syncytial capillary. Pericyte coverage was extensive in both arteriole and venule. Three-dimensional imaging of the placental microvasculature identified novel ultrastructural features and provided an insight into factors that may influence capillary permeability and placental function. We hypothesise that the IEPs may allow mechanosensing between adjacent endothelial cells to assist in the maintenance of vessel integrity. The numbers of endothelial junctions, the presence of trans-endothelial channels and the extent of pericyte coverage all provide an insight into the factors determining capillary permeability.
Asunto(s)
Vellosidades Coriónicas/ultraestructura , Microscopía Electrónica de Rastreo/métodos , Microvasos/ultraestructura , Placenta/ultraestructura , Células Endoteliales/ultraestructura , Femenino , Humanos , EmbarazoRESUMEN
The syncytiotrophoblast forms a continuous barrier between the maternal and fetal circulations. Here we present a serial block-face scanning electron microscopy (SBFSEM) study, based on a single image stack, showing pooling of fetal blood underneath a region of stretched syncytiotrophoblast that has become detached from the basement membrane. Erythrocytes are protruding from discrete holes in the syncytiotrophoblast suggesting that, under specific circumstances, the syncytiotrophoblast may be permeable to fetal cells. This observation represents a pathological process but it poses questions about the physical properties and permeability of the syncytiotrophoblast and may represent an early stage in the formation of fibrin deposits in areas of syncytial denudation. This study also illustrates how the 3D images generated by SBFSEM allow the interpretation of structures that could not be understood from a single histological section.
Asunto(s)
Eritrocitos/fisiología , Microscopía Electrónica de Rastreo/métodos , Placenta/fisiología , Femenino , Humanos , Placenta/ultraestructura , EmbarazoRESUMEN
INTRODUCTION: Extracellular vesicles are now believed to be important mediators of placental-maternal communication. However, little is known about the formation of extracellular vesicles by human placenta. This study uses nanoscale three-dimensional imaging to investigate how and where placental extracellular vesicles form. METHODS: Term and first trimester human placental villi were imaged by serial block face scanning electron microscopy. These images were analysed to quantify vesicle surface density. Segmentation was performed to reconstruct three-dimensional images of extracellular vesicles. Live imaging light microscopy of first trimester villous explants was performed. RESULTS: Vesicles were observed on the tips of placental microvilli in term and first trimester placenta. In term placenta these microvillous tip vesicles had a median size of 0.55 µm and their surface area density exceeded 22000 per mm2. Microvillous tip vesicle membranes had a lower electron density than the microvillous plasma membrane. Thirty seven percent of vesicles had a complex membrane structure including double membranes, internal vesicles and vesicle chains. Budding of smaller secondary vesicles from microvillous tip vesicle membranes was observed. Live imaging of a first trimester villus explant observed formation of vesicles which were larger but visually similar to the secondary vesicles observed by electron microscopy. DISCUSSION: These observations suggest that extracellular vesicles are forming on the tips of placental microvilli prior to release into maternal blood. However, it cannot be discounted that there are maternal extracellular vesicles that have bound to microvilli. In either case, the high surface area density of microvillous tip vesicles is consistent with an important role in placental-maternal signalling.
Asunto(s)
Vesículas Extracelulares , Placenta , Vellosidades Coriónicas , Femenino , Humanos , Microvellosidades , Placenta/metabolismo , Embarazo , Primer Trimestre del EmbarazoRESUMEN
The placental syncytiotrophoblast, a syncytium without cell-cell junctions, is the primary barrier between the mother and the fetus. Despite no apparent anatomical pathway for paracellular diffusion of solutes across the syncytiotrophoblast, size-dependent paracellular diffusion is observed. Here we report data demonstrating that the syncytiotrophoblast is punctuated by trans-syncytial nanopores (TSNs). These membrane-bound TSNs directly connect the maternal and fetal facing sides of the syncytiotrophoblast, providing a pathway for paracellular diffusion between the mother and fetus. Mathematical modeling of TSN permeability based on their 3D geometry suggests that 10-37 million TSNs per cm3 of placental tissue could explain experimentally observed placental paracellular diffusion. TSNs may mediate physiological hydrostatic and osmotic pressure homeostasis between the maternal and fetal circulations but also expose the fetus to pharmaceuticals, environmental pollutants, and nanoparticles.
RESUMEN
INTRODUCTION: Pericytes are a common feature in the placental microvasculature but their roles are not well understood. Pericytes may provide physical or endocrine support for endothelium and in some tissues mediate vasoconstriction. METHODS: This study uses serial block-face scanning electron microscopy (SBFSEM) to generate three-dimensional (3D) reconstructions of placental pericytes of the terminal villi and transmission electron microscopy (TEM) to study pericyte endothelial cell interactions. The proportion of endothelial cell junctions covered by pericytes was determined. RESULTS: The detailed 3D models of placental pericytes show pericyte structure at a new level of detail. Placental pericytes have many fingers extending from the cell body which can span multiple capillary branches. The proportion of endothelial cell-cell junctions covered by pericytes was significantly higher than pericyte coverage of capillary endothelium as a whole (endothelium: 14%, junctions: 43%, p < 0.0001). However, the proportion of endothelial cell-cell junctions covered by pericytes in regions adjacent to trophoblast was reduced compared to regions >3 µm away from trophoblast (27% vs 62% respectively, p < 0.001). No junctional complexes were observed connecting pericytes and endothelial cells but there were regions of cell membrane with features suggestive of intercellular adhesions. DISCUSSION: These data suggest that the localisation of pericytes on the villous capillary is not random but organised in relation to both endothelial junctions and the location of adjacent trophoblast. This further suggests that pericyte coverage may favour capillary permeability in regions that are most important for exchange, but limit capillary permeability in other regions.