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The influence of trophoblast plugs on spiral artery hemodynamics
Abstracts / Placenta 45 (2016) 63e133
The villous trees of human placentas delineate the feto-maternal border and are complex 3D structures. Common 2D histological sections cannot provide access to key aspects of a 3D villous tree and have specific problems in recognizing and recording structural parameters like branching points or branching angles. Thus, analysis of the architecture of the villous tree should include true 3D analysis at microscopic scale. We used the novel 3D analysis and algorithms provided by the new software PlacentaLucida. PlacentaLucida is a computer-assisted camera lucida software which allows optimal and straightforward access to 3D analysis of villous trees at microscopic scale. It enables e.g. to analyze branching nodes and their topology (e.g., branching hierarchy, branching angles, diameters and lengths of branches) observer independent and efficiently in whole-mount preparations of isolated villous trees using high-end light microscopy. To use the software properly, tissue pretreatment has to be adapted and optimized for computer-assisted 3D analysis. Important steps include isolation of villous trees, fixation and subsequent staining and immunohistochemistry of isolated villous trees. Various options of data analysis will be demonstrated. This study focuses on the feasibility to generate 3D data of villous trees and will guide through the process steps of 3D analysis with PlacentaLucida. P2.6 GENERATION AND COMPARATIVE ANALYSIS OF CHORIOCARCINOMA TUMOR ORGANOIDS USING A NEW HANGING-DROP CULTURE METHOD Timothy Lyden, Stephanie Strohbeen. University of Wisconsin-River Falls, River Falls, WI, USA Using hanging-drop 3D culture techniques, we produced a staged series of artificial tumor tissues from the choriocarcinoma cells lines; BeWo, JEG-3 and JAR. Each of these cell lines has a distinct and characteristic set of properties and display varying degrees of malignancy. Interestingly, these three tumor cell-lines also display very different responses to hanging-drop culture environments which are designed to generate spherical organoids. The major difference observed in this study between these cell-lines and contrasting with previous hanging-drop work done in our lab using breast cancer cell lines, was that BeWo and JEG-3 did actually form clearly rounded spheroids by 72-96 hours in culture and then retained this shape as they grew larger going out to 120 hours. In contrast, JAR cells form very small spheroids early in culture which immediately coalesced into branching columns of cells. These then expanded out to form large, relatively flat “snowflake-like” structures by 72 hours. These geometric structures continued to grow through 120 hours of culture, but never formed truly rounded structures. Spheroid labeling studies are ongoing and western blot analysis will be performed to examine the pattern of cell adhesion molecules expressed by each type of 3D structure/cell-line as well as markers of both epithelial/mesenchymal transitions (EMT/MET) and stem cell population status. It is also expected that structural differences observed in these early studies will be reflected in differential adhesion and potential invasive behaviors of the 3D organoids. An expansion of these studies will therefore use 3D artificial tissue constructs of stromal and endometrial origin as target tissues for choriocarcinoma spheroids or “snowflakes” from each of these trophoblast cell-lines. It is our working hypothesis is that choriocarcinoma spheroids are working models of macrometastasis and trophoblast invasion. P2.7 THE INFLUENCE OF TROPHOBLAST PLUGS ON SPIRAL ARTERY HEMODYNAMICS Rojan Saghian 1, Joanna James 2, Merryn Tawhai 1, Alys Rachel Clark 1. 1 Auckland Bioengineering Institute, University of Auckland, Auckland, New Zealand; 2 Department of Obstetrics & Gynaecology, University of Auckland, Auckland, New Zealand
105
Background: Until 12 weeks of gestation trophoblast plugs (TPs) are observed in uterine spiral arteries (SAs). TPs appear to restrict maternal plasma flow to the intervillous space (IVS) maintaining a physiologically hypoxic environment. TPs are also theorised to lower wall shear stress (WSS) within the SA. WSS <1-2 dyne/cm2 can promote trophoblast migration and endothelial cell apoptosis, allowing remodelling of SAs to accommodate increasing uterine blood flows. Loose trophoblast plugging is associated with complicated pregnancies, but the impact of TPs on SA haemodynamics is not well understood. Methods: A computational model of a plugged SA, including upstream arteriovenous shunts, was used to predict blood flow and WSS in and around the TP. The model was parameterized to Doppler ultrasonography in spiral and radial arteries distal to the TP and in the IVS1 as well as measurements of uterine vessel dimensions and TP porosity (ratio of empty space to cell volume) from histology. Results: Our model predicts an IVS blood flow rate of 9.4ml/min with no TP compared with <0.4ml/min over a physiological range of TP porosities (consistent with ultrasound rates of 0.15-0.34ml/min1). Predicted WSS at the site of trophoblast migration (upstream from the SA) increases exponentially with TP porosity, so small increases in porosity (looser plugs) result in rapid increases in WSS. WSS >2 dyne/cm2 is predicted with TP porosity >0.5, within the range seen histologically (0.250.6). Conclusions: Our model supports evidence that TPs can increase SA resistance to flow sufficiently to provide the low flow and shear environment theorised in early pregnancy. Maternal blood is predicted to preferentially flow through arteriovenous shunt pathways. A high sensitivity of both IVS blood flow and SA WSS to plug porosity suggests loose plugging in complicated pregnancies may be sufficient to provide conditions unfavourable to SA remodelling. 1 Merce, 1996, J Ultrasound Med 15:135-142. P2.8 VILLUS PACKING DENSITY AND LACUNARITY: MARKERS OF PLACENTAL EFFICIENCY? Ruchit Shah 1, Carolyn Salafia 1, 2, Theresa Girardi 1, George Merz 2. 1 Placental Analytics, New Rochelle, NY, USA; 2 Institute for Basic Research, Staten Island, NY, USA Introduction: The interplay between the maternal intervillus flow and the placental villi and their capillary beds plays a crucial role in the maternofetal oxygen exchange. A quantitative analysis of the 2D distribution of villus packing density (and, by extension, the intervillus geometry that impacts maternal intervillus flow) may provide a microscopic measure of placental function and efficiency that can be reliably obtained using routine histology stains (hematoxylin and eosin, H&E). Here we investigated the relationships between placental efficiency (b,log(placental weight)/log(birthweight)), and villus packing density and lacunarity or ‘gappiness’ of villus distribution from placental micrographs. Methods: 88 term (37-41 weeks) cases were selected from a large cohort (Pregnancy Infection and Nutrition Study, University of North Carolina) to include a wide range of birth weights. 264 high-resolution H&E images (3 images from each placenta, Fig. 1) were segmented to extract all villus pixels (Fig. 2). Villus packing density and lacunarity were computed for each image using Matlab. The average and range of these measures were compared to their bs. Results: Range of lacunarity and villus packing density were significantly correlated with b (r¼0.23, 0.31, respectively), while average value was not. Discussion: Packing density and lacunarity are quantitative measures of the extent and nature of villus distribution. The significant relationship between b and intra-placental variation in villus packing density and lacunarity highlights the importance of studying the morphology and spatial distribution of villi. We encourage the use of simple quantitative measures to provide a window into the complex functioning of placenta.
The villous trees of human placentas delineate the feto-maternal border and are complex 3D structures. Common 2D histological sections cannot provide access to key aspects of a 3D villous tree and have specific problems in recognizing and recording structural parameters like branching points or branching angles. Thus, analysis of the architecture of the villous tree should include true 3D analysis at microscopic scale. We used the novel 3D analysis and algorithms provided by the new software PlacentaLucida. PlacentaLucida is a computer-assisted camera lucida software which allows optimal and straightforward access to 3D analysis of villous trees at microscopic scale. It enables e.g. to analyze branching nodes and their topology (e.g., branching hierarchy, branching angles, diameters and lengths of branches) observer independent and efficiently in whole-mount preparations of isolated villous trees using high-end light microscopy. To use the software properly, tissue pretreatment has to be adapted and optimized for computer-assisted 3D analysis. Important steps include isolation of villous trees, fixation and subsequent staining and immunohistochemistry of isolated villous trees. Various options of data analysis will be demonstrated. This study focuses on the feasibility to generate 3D data of villous trees and will guide through the process steps of 3D analysis with PlacentaLucida. P2.6 GENERATION AND COMPARATIVE ANALYSIS OF CHORIOCARCINOMA TUMOR ORGANOIDS USING A NEW HANGING-DROP CULTURE METHOD Timothy Lyden, Stephanie Strohbeen. University of Wisconsin-River Falls, River Falls, WI, USA Using hanging-drop 3D culture techniques, we produced a staged series of artificial tumor tissues from the choriocarcinoma cells lines; BeWo, JEG-3 and JAR. Each of these cell lines has a distinct and characteristic set of properties and display varying degrees of malignancy. Interestingly, these three tumor cell-lines also display very different responses to hanging-drop culture environments which are designed to generate spherical organoids. The major difference observed in this study between these cell-lines and contrasting with previous hanging-drop work done in our lab using breast cancer cell lines, was that BeWo and JEG-3 did actually form clearly rounded spheroids by 72-96 hours in culture and then retained this shape as they grew larger going out to 120 hours. In contrast, JAR cells form very small spheroids early in culture which immediately coalesced into branching columns of cells. These then expanded out to form large, relatively flat “snowflake-like” structures by 72 hours. These geometric structures continued to grow through 120 hours of culture, but never formed truly rounded structures. Spheroid labeling studies are ongoing and western blot analysis will be performed to examine the pattern of cell adhesion molecules expressed by each type of 3D structure/cell-line as well as markers of both epithelial/mesenchymal transitions (EMT/MET) and stem cell population status. It is also expected that structural differences observed in these early studies will be reflected in differential adhesion and potential invasive behaviors of the 3D organoids. An expansion of these studies will therefore use 3D artificial tissue constructs of stromal and endometrial origin as target tissues for choriocarcinoma spheroids or “snowflakes” from each of these trophoblast cell-lines. It is our working hypothesis is that choriocarcinoma spheroids are working models of macrometastasis and trophoblast invasion. P2.7 THE INFLUENCE OF TROPHOBLAST PLUGS ON SPIRAL ARTERY HEMODYNAMICS Rojan Saghian 1, Joanna James 2, Merryn Tawhai 1, Alys Rachel Clark 1. 1 Auckland Bioengineering Institute, University of Auckland, Auckland, New Zealand; 2 Department of Obstetrics & Gynaecology, University of Auckland, Auckland, New Zealand
105
Background: Until 12 weeks of gestation trophoblast plugs (TPs) are observed in uterine spiral arteries (SAs). TPs appear to restrict maternal plasma flow to the intervillous space (IVS) maintaining a physiologically hypoxic environment. TPs are also theorised to lower wall shear stress (WSS) within the SA. WSS <1-2 dyne/cm2 can promote trophoblast migration and endothelial cell apoptosis, allowing remodelling of SAs to accommodate increasing uterine blood flows. Loose trophoblast plugging is associated with complicated pregnancies, but the impact of TPs on SA haemodynamics is not well understood. Methods: A computational model of a plugged SA, including upstream arteriovenous shunts, was used to predict blood flow and WSS in and around the TP. The model was parameterized to Doppler ultrasonography in spiral and radial arteries distal to the TP and in the IVS1 as well as measurements of uterine vessel dimensions and TP porosity (ratio of empty space to cell volume) from histology. Results: Our model predicts an IVS blood flow rate of 9.4ml/min with no TP compared with <0.4ml/min over a physiological range of TP porosities (consistent with ultrasound rates of 0.15-0.34ml/min1). Predicted WSS at the site of trophoblast migration (upstream from the SA) increases exponentially with TP porosity, so small increases in porosity (looser plugs) result in rapid increases in WSS. WSS >2 dyne/cm2 is predicted with TP porosity >0.5, within the range seen histologically (0.250.6). Conclusions: Our model supports evidence that TPs can increase SA resistance to flow sufficiently to provide the low flow and shear environment theorised in early pregnancy. Maternal blood is predicted to preferentially flow through arteriovenous shunt pathways. A high sensitivity of both IVS blood flow and SA WSS to plug porosity suggests loose plugging in complicated pregnancies may be sufficient to provide conditions unfavourable to SA remodelling. 1 Merce, 1996, J Ultrasound Med 15:135-142. P2.8 VILLUS PACKING DENSITY AND LACUNARITY: MARKERS OF PLACENTAL EFFICIENCY? Ruchit Shah 1, Carolyn Salafia 1, 2, Theresa Girardi 1, George Merz 2. 1 Placental Analytics, New Rochelle, NY, USA; 2 Institute for Basic Research, Staten Island, NY, USA Introduction: The interplay between the maternal intervillus flow and the placental villi and their capillary beds plays a crucial role in the maternofetal oxygen exchange. A quantitative analysis of the 2D distribution of villus packing density (and, by extension, the intervillus geometry that impacts maternal intervillus flow) may provide a microscopic measure of placental function and efficiency that can be reliably obtained using routine histology stains (hematoxylin and eosin, H&E). Here we investigated the relationships between placental efficiency (b,log(placental weight)/log(birthweight)), and villus packing density and lacunarity or ‘gappiness’ of villus distribution from placental micrographs. Methods: 88 term (37-41 weeks) cases were selected from a large cohort (Pregnancy Infection and Nutrition Study, University of North Carolina) to include a wide range of birth weights. 264 high-resolution H&E images (3 images from each placenta, Fig. 1) were segmented to extract all villus pixels (Fig. 2). Villus packing density and lacunarity were computed for each image using Matlab. The average and range of these measures were compared to their bs. Results: Range of lacunarity and villus packing density were significantly correlated with b (r¼0.23, 0.31, respectively), while average value was not. Discussion: Packing density and lacunarity are quantitative measures of the extent and nature of villus distribution. The significant relationship between b and intra-placental variation in villus packing density and lacunarity highlights the importance of studying the morphology and spatial distribution of villi. We encourage the use of simple quantitative measures to provide a window into the complex functioning of placenta.