This work was supported by the New York State Office for People with Developmental Disabilities and Placental Analytics LLC, New Rochelle, NY.

This protocol follows the guidelines of New York State Institute for Basic Research in Developmental Disabilities human research ethics committee.
1. Villous Tree Dissection
<ol>
	<li>Rinse formalin fixed placenta tissue with phosphate buffed saline (PBS)14 to remove the formalin and place in a Petri dish on the stage of a dissection microscope. Use scalpel and fine forceps to tease the placenta tissue apart to locate villous trees (white threadlike branching structures). Dissect out pieces of villus tree (several mm long) and transfer the tissue to a micro-tube containing ~1 mL PBS.</li>
</ol>
2. Immunofluorescent Staining
<ol>
	<li>With a pipette, remove the PBS and replace with fresh PBS. Let stand for 10 min with occasional swirling. Repeat once more. 
	&#8203;NOTE: This step and all further steps are done at room temperature except the primary antibody incubation.</li>
	<li>Using a pipette, remove and replace the PBS with PBS +0.1% Triton-X100 + 2% goat serum (PBS-T-GS) to permeabilize the tissue and block non-specific binding of secondary antibodies. Incubate for 30 min.</li>
	<li>Replace the PBS-T-GS with PBS containing 2% goat serum, both mouse monoclonal anti-CK7 and rabbit anti-CK7.
	<ol>
		<li>Incubate tissue overnight at 4 &#176;C.</li>
		<li>Remove the remaining antibodies by washing with PBS-GS for 10 minutes with occasional swirling.</li>
		<li>Remove the PBS-GS and replace with PBS-GS containing both goat anti-mouse IgG coupled with a green emitting (520 nm) fluorophore and goat anti-rabbit IgG coupled an infrared emitting (652 nm) fluorophore.</li>
		<li>Remove the antibodies by repeating step 2.1.</li>
		<li>Store the immunolabeled tissue at room temperature in the dark.</li>
	</ol>
	</li>
</ol>
3. Clarification of the Tissue
<ol>
	<li>Wash the samples 3 times with PBS at 10 minute intervals.</li>
	<li>Dehydrate the tissue by removing the PBS with a pipette and replacing it with 50% ethanol (methanol may be used as well). Incubate for 10&#160;min. Replace with fresh 50% ethanol and incubate for 10&#160;min. Repeat this once more for a total of 3 incubations. Repeat incubations with 70%. Then replace with 100% ethanol and incubate for 15 minutes (alternatively methanol may be used).</li>
	<li>With a fine-tipped forceps, transfer tissue to a fresh micro-tube containing ~ 1 mL Solution-1 for 4 h.</li>
	<li>With fine-tipped forceps, transfer tissue to a fresh micro-tube containing Solution-2 for ~ 4 h. 
	NOTE: When stored in the dark, the immunofluorescence is stable at room temperature for at least 6 months.</li>
	<li>Do not mix with any aqueous solution, (e.g. mounting medium) as the clearing will lost.</li>
</ol>
4. Mounting for Microscopy
<ol>
	<li>Do not mount the tissue between a standard glass slide and a coverslip as the tissue is soft and easily deformed.</li>
	<li>Refer to Figure 5B and mount the tissue in a Sykes-Moore chamber as follows;
	<ol>
		<li>With fine-tipped forceps place a 25-mm round coverslip in the chamber base.</li>
		<li>With a fine-tipped forceps place the rubber gasket in the base on the coverslip.</li>
		<li>With a pipette place a drop (~40 &#181;L) of Solution-2 in the center of the coverslip.</li>
		<li>With the fine-tipped forceps transfer a piece tissue from Solution-2 to the drop in the center of the coverslip.</li>
		<li>Place a second coverslip on the top of the gasket.</li>
		<li>Thread the locking ring into the top of the base compressing the coverslips and gasket until firm. 
		NOTE: Be careful not to over-tighten or the coverslip will shatter.</li>
		<li>Insert a 22-gauge needle into a port on the base and through the gasket.</li>
		<li>Fill a 3-mL syringe with ~ 1.5 mL of Solution-2.</li>
		<li>Mount a 25-gauge needle on the syringe.</li>
		<li>Insert the syringe needle into the opposite port and through the gasket.</li>
		<li>Ensure that the air in the chamber is venting through the other 25-gauge needle, gently inject solution-2and fill the chamber.</li>
		<li>Remove the needle and syringe and place the chamber on a large glass slide on the confocal microscope stage.</li>
	</ol>
	</li>
	<li>Alternatively, cut custom wells out of PDMS silicone sheets.
	<ol>
		<li>With scissors cut a 25.4 &#215; 25.4 mm2 piece from the PDMS sheet.</li>
		<li>With a scalpel cut ~ 12.7 &#215; 12.7 mm2 well in the piece.</li>
		<li>Press the piece firmly onto a microscope slide.</li>
		<li>Fill the well to the top with Solution-2.</li>
		<li>Transfer the tissue piece from the micro-tube to the center of the well.</li>
		<li>With fine-tipped forceps place a coverslip on the top of the well such that the bottom of the coverslip is wetted with Solution-2.</li>
		<li>Mount the Sykes Moore Chamber assembly&#160;with the tissue on the confocal microscope stage.</li>
	</ol>
	</li>
</ol>
5. Confocal Microscopy
<ol>
	<li>Bring the tissue in the chamber into focus with a 10x objective.</li>
	<li>Move the microscope stage up and down through the focal plane to measure the distance between the top and bottom of the tissue.</li>
	<li>Move the stage in 2.5 &#181;m steps to collect a confocal image file containing a set of images through the tissue from top to bottom.</li>
</ol>
6. Reversing the Clearing
<ol>
	<li>Reverse the clearing by transferring the tissue to a micro-tube containing &#62; 20x the volume of 100% ethanol. At 1 h intervals replace with 70% ethanol, then 50% ethanol and finally PBS. Store at 4 &#176;C in the dark. 
	NOTE: Now process the tissue for standard paraffin embedding, sectioning and histological or immuno-histochemical staining.</li>
</ol>
7. Deconvolution
NOTE: For the following steps, software Commands, Tabs and drop-down menus are italicized.
<ol>
	<li>Start the deconvolution software and open the image file.</li>
	<li>Enter parameters listed in the &#39;Summary&#39; window.
	<ol>
		<li>For &#39;Spacings&#39; enter the voxel x, y, and z dimensions in &#181;m.</li>
		<li>For each color channel, select the fluorescent dye used for the immunostaining from the drop-down menu.</li>
		<li>For the &#39;Modality&#39;, select Laser Scanning Confocal from the drop-down menu.</li>
		<li>For the &#39;Objective Lens&#39;, select 10x from the drop-down menu.</li>
		<li>For the &#39;Immersion Medium&#39;, select Air from the drop-down menu.</li>
		<li>Click on the &#39;Apply&#39; button.</li>
	</ol>
	</li>
	<li>Click on the &#39;Deconvolution&#39; tab.</li>
	<li>Click on &#39;3D Deconvolution&#39; in the drop-down menu.</li>
	<li>In the &#39;3D -Deconvolution&#39; window insure that the settings are: 1) Adaptive PSF Blind, 2) Theoretical PSF, 3) Use (Default Adaptive PSF, 10 iterations, Medium noise), 4) Base Name.</li>
	<li>Click on the &#39;green Check button&#39; to start the program.</li>
	<li>When finished, repeat steps 7.3 -7.6 to restart the program. When finished restart it for third and last time.</li>
	<li>In the &#39;Data Manager&#39; window, click on the file prefixed with &#34;10-10-10-&#34;.</li>
	<li>Click on the &#39;File&#39; tab, then click on &#39;Save As&#39;.</li>
	<li>In the &#39;Save As&#39; window select &#39;8bit unsigned integer&#39; from the &#39;Data Type&#39; drop-down menu and click on Save.</li>
</ol>
8. Image Processing
<ol>
	<li>Start Fiji software.
	<ol>
		<li>Click on &#39;File&#39;, click on &#39;Open&#39; and select the Deconvolved image file and click on the &#39;Open&#39; button in the &#39;Open&#39; window.</li>
		<li>Click on &#39;Image/Color/Split channels&#39; and convert the three-color image file into three new files containing only one color. Save the red image file that contains the immunolabeled villous capillary image set. Delete the green and blue image files.</li>
		<li>Subtract the background fluorescence from the red image file with: &#39;Process/Subtract background&#39;.</li>
		<li>In the drop-down menu set Rolling ball radius to 10 pixels.</li>
		<li>Leave the 4 menu options unchecked and click &#39;OK&#39;.</li>
		<li>Click &#39;Yes&#39; on the Process Stack menu.</li>
		<li>Remove inherent tissue fluorescence (autofluorescence) by opening the &#39;Image/Adjust/Brightness-Contrast&#39; menu and adjusting the minimum, maximum, brightness and contrast settings. Be careful not to remove any of the capillary immunofluorescence.</li>
		<li>Minimize remaining irrelevant fluorescence by opening the &#39;Image/Adjust/Threshold&#39; menu and adjusting the sliders such that the capillary immunofluorescence is highlighted. Check the &#39;Dark background&#39; box and click on &#39;Apply&#39;. On the &#39;convert to binary drop-down&#39; menu check the &#39;Black background&#39; box and click on &#39;OK&#39;. Save the black and white (binary) image file for analyses below.</li>
	</ol>
	</li>
</ol>
9. Analysis
<ol>
	<li>Object Counting and Skeletonization.
	<ol>
		<li>Select the binary image file created in Step 8.1.8.</li>
		<li>Click on &#39;Analyze/3D OC Options&#39; and check only &#39;Volume&#39; and &#39;Store results &#8230;&#39; boxes in the drop-down menu. Insure that the &#39;Show numbers&#39; box is unchecked. Click on the &#39;OK&#39; button.</li>
		<li>Click on &#39;Analyze/3D Objects Counter&#39;. In the drop-down menu, set &#39;Size Filter: Min.&#39; to 5000. Insure that the &#39;Exclude objects on edges&#39; box is unchecked. Check that the &#39;Objects, Statistics&#39; and &#39;Summary&#39; boxes are checked. Click on the &#39;OK&#39; button. 
		NOTE: This may take several minutes depending on file size and computer configuration.</li>
		<li>Save the Statistics table and the Objects map file.</li>
		<li>Select the Objects map file. Click on &#39;Plugins/Skeleton/Skeletonize 2D-3D&#39; to skeletonize the capillary network in the binary file. Click on &#39;File/Save as&#39;. Select &#39;Tiff&#8230;&#39; in the drop-down menu. In the Save as TIFF window add &#34;Skel-&#34; to object map file name and click on the &#39;Save&#39; button.</li>
		<li>Click on &#39;Analyze/Skeleton/Analyze Skeleton 2D-3D&#39;. In the drop-down menu accept the default settings and click on the &#39;OK&#39; button. Save the Branch &#39;information&#39; table, the &#39;Results&#39; table, and two output files (Skel-Objects-labeled -skeletons and Tagged skeleton).</li>
	</ol>
	</li>
</ol>

<ol><li>Thornburg, K. L., Kolahi, K., Pierce, M., Valent, A., Drake, R., Louey, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biological+features+of+placental+programming%5BTitle%5D)+AND+%22Placenta%22%5BJournal%5D)">Biological features of placental programming.</a> Placenta. 48, Suppl 1 47-53 (2016).</li>
<li>Misra, D. P., Salafia, C. M., Charles, A. K., Miller, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Birth+weights+smaller+or+larger+than+the+placenta+predict+BMI+and+blood+pressure+at+age+7+years%5BTitle%5D)+AND+%22J+Dev+Orig+Health+Dis%22%5BJournal%5D)">Birth weights smaller or larger than the placenta predict BMI and blood pressure at age 7 years.</a> J Dev Orig Health Dis. 1 (2), 123-130 (2010).</li>
<li>Burton, G. J., Fowden, A. L., Thornburg, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Placental+Origins+of+Chronic+Disease%5BTitle%5D)+AND+%22Physiol+Rev%22%5BJournal%5D)">Placental Origins of Chronic Disease.</a> Physiol Rev. 96 (4), 1509-1565 (2016).</li>
<li>Srinivasan, A. P., Omprakash, B. O. P., Lavanya, K., Subbulakshmi Murugesan, P., Kandaswamy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+prospective+study+of+villous+capillary+lesions+in+complicated+pregnancies%5BTitle%5D)+AND+%22J+Pregnancy%22%5BJournal%5D)">A prospective study of villous capillary lesions in complicated pregnancies.</a> J Pregnancy. 2014, 193925(2014).</li>
<li>Jones, C. J. P., Desoye, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+possible+function+for+placental+pericytes%5BTitle%5D)+AND+%22Cells+Tissues+Organs%22%5BJournal%5D)">A new possible function for placental pericytes.</a> Cells Tissues Organs. 194 (1), 76-84 (2011).</li>
<li>Maly, A., Goshen, G., Sela, J., Pinelis, A., Stark, M., Maly, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histomorphometric+study+of+placental+villi+vascular+volume+in+toxemia+and+diabetes%5BTitle%5D)+AND+%22Hum+Pathol%22%5BJournal%5D)">Histomorphometric study of placental villi vascular volume in toxemia and diabetes.</a> Hum Pathol. 36 (10), 1074-1079 (2005).</li>
<li>Ellery, P. M., Cindrova-Davies, T., Jauniaux, E., Ferguson-Smith, A. C., Burton, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evidence+for+transcriptional+activity+in+the+syncytiotrophoblast+of+the+human+placenta%5BTitle%5D)+AND+%22Placenta%22%5BJournal%5D)">Evidence for transcriptional activity in the syncytiotrophoblast of the human placenta.</a> Placenta. 30 (4), 329-334 (2009).</li>
<li>Jirkovská, M., Kubínová, L., Janáček, J., Kaláb, J. <a target="_blank" href="https://www.ias-iss.org/ojs/IAS/article/view/820">3-D study of vessels in peripheral placental villi.</a> Image Anal Stereol. 26 (3), 165-168 (2011).</li>
<li>Pearce, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Image-Based+Modeling+of+Blood+Flow+and+Oxygen+Transfer+in+Feto-Placental+Capillaries%5BTitle%5D)+AND+%22PLOS+ONE%22%5BJournal%5D)">Image-Based Modeling of Blood Flow and Oxygen Transfer in Feto-Placental Capillaries.</a> PLOS ONE. 11 (10), 0165369(2016).</li>
<li>Plitman Mayo, R., Olsthoorn, J., Charnock-Jones, D. S., Burton, G. J., Oyen, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Computational+modeling+of+the+structure-function+relationship+in+human+placental+terminal+villi%5BTitle%5D)+AND+%22J+Biomech%22%5BJournal%5D)">Computational modeling of the structure-function relationship in human placental terminal villi.</a> J Biomech. 49 (16), 3780-3787 (2016).</li>
<li>Mayhew, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+stereological+perspective+on+placental+morphology+in+normal+and+complicated+pregnancies%5BTitle%5D)+AND+%22J+Anat%22%5BJournal%5D)">A stereological perspective on placental morphology in normal and complicated pregnancies.</a> J Anat. 215 (1), 77-90 (2009).</li>
<li>Teasdale, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gestational+changes+in+the+functional+structure+of+the+human+placenta+in+relation+to+fetal+growth%3A+a+morphometric+study%5BTitle%5D)+AND+%22Am+J+Obstet+Gynecol%22%5BJournal%5D)">Gestational changes in the functional structure of the human placenta in relation to fetal growth: a morphometric study.</a> Am J Obstet Gynecol. 137 (5), 560-568 (1980).</li>
<li>Richardson, D. S., Lichtman, J. W. <a target="_blank" href="http://www.cell.com/cell/abstract/S0092-8674(15)00837-5">Clarifying Tissue Clearing.</a> Cell. 162 (2), 246-257 (2015).</li>
<li>Dulbecco, R., Vogt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plaque+formation+and+isolation+of+pure+lines+with+poliomyelitis+viruses%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">Plaque formation and isolation of pure lines with poliomyelitis viruses.</a> J Exp Med. 99 (2), 167-182 (1954).</li>
<li>Gill, J. S., Salafia, C. M., Grebenkov, D., Vvedensky, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modeling+oxygen+transport+in+human+placental+terminal+villi%5BTitle%5D)+AND+%22J+Theor+Biol%22%5BJournal%5D)">Modeling oxygen transport in human placental terminal villi.</a> J Theor Biol. 291, 33-41 (2011).</li>
<li>Plitman Mayo, R., Charnock-Jones, D. S., Burton, G. J., Oyen, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three-dimensional+modeling+of+human+placental+terminal+villi%5BTitle%5D)+AND+%22Placenta%22%5BJournal%5D)">Three-dimensional modeling of human placental terminal villi.</a> Placenta. 43, 54-60 (2016).</li>
</ol>

The authors have nothing to disclose.

The Institutional Review Board approved the collection of placental villous tissues for formalin fixation from electively terminated pregnancies. Before the procedures were performed, a brief review of the medical record noted maternal age, parity, and confirmed the absence of any underlying medical (e.g., hypertension, diabetes, and lupus) or fetal (structural or chromosomal anomalies, abnormal growth) was performed. The data sheet and any collected specimens were labeled only with a Study ID. Thus, all the spe...

Our understanding of the developing placenta and its pathologies is, in large measure, limited to inferred spatial relationships between adjacent villi and contained capillaries derived from histological sections. In this study, we have addressed this issue by developing the means to generate three-dimensional (3D) renderings of human placental capillary networks that are suitable for analyses of capillary network features (e.g. branching, solidity). To do this we have combined immunofluorescent staining with two commercial tissue clearing products, Visikol-1 and Visikol-2 (referred to below as Solution-1 and Solution-2).
The human placenta is a vast complex of blood vessels located at the interface between the mother&#39;s intervillous blood and the developing fetus. Extending out from its insertion into the chorionic plate, the umbilical cord branches into a network of arteries and veins that ramify to cover the chorionic surface with an elaborate vascular network. Their ends then penetrate down into the interior or depth of the placental disk where they undergo several more branching generations and end in the terminal villi and their contained capillary networks, the site of the exchange of gases, nutrients, and metabolic waste between fetal and maternal blood.
Insults to the placental capillary network during development have lasting consequences for the health of the fetus, newborn infant and the emergent adult 1,2,3. In view of pregnancy-related pathologies such as miscarriage, intrauterine growth restriction, pre-eclampsia and maternal diabetes 4,5,6 there is a high value placed on developing methods of measurement and characterization of placental villous capillary networks. A major roadblock is that placental vascular networks encompass a broad range of scale. The surface vascular networks can be as great as 4-5 mm in diameter. The terminal villous capillaries are on the order of 10 -20 &#181;m in diameter; the placenta contains over 300 km of blood vessels 7. At present, there are few easy to use and rapid techniques that can capture these extremes of vessel scale. To date, only a small number of villi can be rendered by microscopy. For example, Jirkovska et al. focused on the placental villus at term, combining confocal microscopy with serial optical sections at 1 &#181;m intervals obtained from 120 &#181;m thick sections; no data on the number of samples studied nor statistics were provided 8. Capillary structures were identified, and contours of villi and capillaries were hand-drawn, with tracings exported for image analysis. While the authors discuss the implications of their findings for the &#34;growing villous vascular network&#34;, such conclusions are problematic when only &#34;term&#34; (36+ week gestational age) tissue is studied. Similarly, Mayo et al. and Pearce et al. relied on the tissue of the same age, for their simulations of blood flow and oxygen transfer, but their analyses were limited to only a few term, terminal villi 9,10. Stereology has also been applied to the study of the structure of villous vessels. But again, the focus has generally been on later pregnancies delivering liveborn infants with one or more pregnancy complications 11,12.
Until recently, confocal microscopy was limited to imaging to tissue depths of 100-200 &#181;m because of absorption of the excitation and emission of the fluorescence by the overlying tissue 13 . Though tissue clearing and 3D histology have been widely described in the literature and there are numerous methods for tissue clearing many are unsuitable for use with tissues in general, as they irreversibly damage cellular morphology through the hyperhydration of proteins or removal of lipids. Therefore, it is not possible to validate that these results are indicative of the tissue itself and not artifacts from processing whereas our tissue clearing process is a reversible technique that is able to validate against traditional histology. Tissue clearing generally involves one of three major approaches: 1) uniform matching of the refractive index (RI) of tissue components by submersion in RI matching solutions, which removes the cumulative light scattering caused from lensing due to continuous fluctuation of low RI (cytosol) and high RI (protein/lipids) constituents; 2) removing lipid components by way of embedding into hydrogel and utilizing electrophoresis/diffusion to remove lipid components; 3) expansion/denaturation of protein structure to allow increased penetration of solvents to encourage uniformity of RI 13. While these approaches can render tissues transparent and allow for 3D representations of biomarkers to be generated, these 3D representations are of questionable clinical value as it is challenging to determine if these images are indicative of tissue properties or of the tissue clearing process. On the other hand, since our tissue clearing is reversible standard histological and/or immunohistochemistry can be applied to the same tissue to evaluate whether the alterations are clinically significant.
This study presents an analysis of 47 distal villous samples obtained from a total of 23 clinically normal and electively terminated pregnancies between 9-23 completed weeks&#39; gestation and two full-term normal deliveries. Immunofluorescence labeling of trophoblast and endothelia has enabled a quantitative and automated analysis of changes in the villous vascular networks and their complexity.
With this protocol, we isolated and analyzed terminal villi and their capillary networks on a scale not possible previously. This approach, when applied to villous and capillary network development across gestation will identify those properties that are the foundation for the birth of a healthy child. When applied to studies of complicated pregnancies, it will also clarify when and how the placental pathologies modify the villous trees and the capillary networks they sheath, and how these impinge on fetal well-being.

<table><tbody><tr><td>10% Formaldehyde solution (w/v) in aqueous phosphate buffer</td><td>Macron Fine Chemicals</td><td>H-121-08</td><td>General fixation agent, ready to use formula, use caution as vapors are toxic</td></tr><tr><td>Scalpel blades</td><td>ThermoFisher Scientific</td><td>08-916-5B No. 11</td><td></td></tr><tr><td>Scalpel</td><td>ThermoFisher Scientific</td><td>08-913-5</td><td></td></tr><tr><td>Fine forceps</td><td>Electron Microscopy Services</td><td>78354-119</td><td></td></tr><tr><td>Micro Tube (1.7 mL)</td><td>PGC Scientifics</td><td>505-201</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma</td><td>D8537</td><td>PBS</td></tr><tr><td>Pipette</td><td>VWR</td><td>52947-948</td><td>disposable, 3ml transfer pipette</td></tr><tr><td>Triton X-100</td><td>Boehriner Mannheim</td><td>789 704</td><td>Dilute to 0.1% from stock</td></tr><tr><td>Goat Serum</td><td>Gibco</td><td>16210-064</td><td>Dilute to 2% in PBS solution</td></tr><tr><td>Mouse monoclonial anti-ck7 Keratin 7 Ab-2 (Clone OV-TL 12/30)</td><td>ThermoFisher Scientific</td><td>MS-1352-RQ</td><td></td></tr><tr><td>Rabbit Anti-CD31 antibody</td><td>Abcam</td><td>ab28364</td><td></td></tr><tr><td>green emitting (520 nm) fluorochrome&amp;nbsp;</td><td>Invitrogen</td><td>A11017</td><td>Alexa-Fluor 488</td></tr><tr><td>infrared emitting (652 nm) fluorochrome</td><td>Invitrogen</td><td>A21072</td><td>Alexa Fluor 633</td></tr><tr><td>Ethanol alcohol 200 proof</td><td>Pharmco-Aaper</td><td>111000200</td><td>Dilute down to lower concentrations using PBS as needed</td></tr><tr><td>Solution-1</td><td>Visikol Inc.</td><td></td><td>Visikol Histo-1</td></tr><tr><td>Solution-2</td><td>Visikol Inc.</td><td></td><td>Visikol Histo-2</td></tr><tr><td>Skyes-Moore chambers</td><td>BellCo Glass Inc.</td><td>P/N 1943-11111</td><td></td></tr><tr><td>25 gauge needle</td><td>ThermoFisher Scientific</td><td>14-826AA</td><td>BD Precision Glide Needes</td></tr><tr><td>3 mL syringe</td><td>ThermoFisher Scientific</td><td>14-823-40</td><td>BD disposable syringe</td></tr><tr><td>PDMS silicon sheets</td><td>McMaster-Carr</td><td>P/N 578T31</td><td></td></tr><tr><td>confocal microscope</td><td>Nikon Inc.</td><td></td><td>Nikon C1 Confocal Microscope</td></tr><tr><td>Deconvolution software</td><td>Media Cybernetics</td><td></td><td>AutoQuant X22</td></tr><tr><td>Fiji image processing software</td><td></td><td></td><td>free, Open source&amp;nbsp; software available at https://fiji.sc</td></tr><tr><td>Hematoxylin</td><td>Leica Biosystems</td><td>3801570</td><td>Component 1 of SelecTech H&amp;amp;E staining system</td></tr><tr><td>Alcoholic Eosin</td><td>Leica Biosystems</td><td>3801615</td><td>Component 2 of SelecTech H&amp;amp;E staining system</td></tr><tr><td>Blue Buffer</td><td>Leica Biosystems</td><td>3802918</td><td>Component 3 of SelecTech H&amp;amp;E staining system</td></tr><tr><td>Aqua Define MCX</td><td>Leica Biosystems</td><td>3803598</td><td>Component 4 of SelecTech H&amp;amp;E staining system</td></tr><tr><td>Immunohistochemistry detection system</td><td>ThermoFisher Scientific</td><td>TL-125-QHD</td><td>UltraVision Quanto Detection System HRP DAB</td></tr></tbody></table>

three-dimensional rendering and analysis of immunolabeled, clarified human placental villous vascular networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

The generated 3D renderings of the capillaries in clusters of terminal villi in human placenta from 8 weeks gestational age to term delivery were counted as individual clusters and skeletonized for network analysis. The functional units of the placenta (Figure 1A) are the villus trees that are an extension of surface vessels where they have penetrated the placenta parenchyma (Figure 1B, and enlar...

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Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

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Research

JoVE Journal

Developmental Biology

9.7K Views.  The New York State Office for People with Developmental Disabilities. Deze studie geeft een protocol voor het omkeerbare weefsel clearing, immunokleuring, 3D-rendering en analyse van vasculaire netwerken in menselijke placenta villi monsters over de volgorde van 1-2 mm,3.

Video: Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the maternal circulation. The maternal uterine vasculature needs to adapt to this temporary demand and the success of this arterial remodeling process has implications for fetal growth. Cells of the maternal immune system, especially natural killer (NK) cells, play a critical role in this process. Here we describe a method to assess the degree of remodeling of maternal spiral arteries during mouse pregnancy. Hematoxylin and eosin-stained tissue sections are scanned and the size of the vessels analysed. As a complementary validation method, we also present a qualitative assessment for the success of the remodeling process by immunohistochemical detection of smooth muscle actin (SMA), which normally disappears from within the arterial vascular media at mid-gestation. Together, these methods enable determination of an important parameter of the pregnancy phenotype. These results can be combined with other endpoints of mouse pregnancy to provide insight into the mechanisms underlying pregnancy-related complications.

This protocol describes a technique to assess changes in the maternal vasculature during pregnancy in mice. Using stereological methods, remodeling of the decidual spiral arteries is assessed quantitatively and the results confirmed qualitatively using immunohistochemistry.

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the ...

Accurate and Simple Evaluation of Vascular Anastomoses in Monochorionic Placenta using Colored Dye

The presence of placental vascular anastomoses is a conditio sine qua non for the development of twin-to-twin transfusion syndrome (TTTS) and twin anemia polycythemia sequence (TAPS)1,2. Injection studies of twin placentas have shown that such anastomoses are almost invariably present in monochorionic twins and extremely rare in dichorionic twins1. Three types of anastomoses have been documented: from artery to artery, from vein to vein and from artery to vein. Arterio-venous (AV) anastomoses are unidirectional and are referred to as "deep" anastomoses since they proceed through a shared placental cotyledon, whereas arterio-arterial (AA) and veno-venous (VV) anastomoses are bi-directional and are referred to as "superficial" since they lie on the chorionic plate. Both TTTS and TAPS are caused by net imbalance of blood flow between the twins due to AV anastomoses. Blood from one twin (the donor) is pumped through an artery into the shared placental cotyledon and then drained through a vein into the circulation of the other twin (the recipient). Unless blood is pumped back from the recipient to the donor through oppositely directed deep AV anastomoses or through superficial anastomoses, an imbalance of blood volumes occurs, gradually leading to the development of TTTS or TAPS. The presence of an AA anastomosis has been shown to protect against the development of TTTS and TAPS by compensating for the circulatory imbalance caused by the uni-directional AV anastomoses1,2. 
Injection of monochorionic placentas soon after birth is a useful mean to understand the etiology of various (hematological) complications in monochorionic twins and is a required test to reach the diagnosis of TAPS2. In addition, injection of TTTS placentas treated with fetoscopic laser surgery allows identification of possible residual anastomoses3-5. This additional information is of paramount importance for all perinatologists involved in the management and care of monochorionic twins with TTTS or TAPS. Several placental injection techniques are currently being used. We provide a simple protocol to accurately evaluate the presence of (residual) vascular anastomoses using colored dye injection.

Twin-to-twin transfusion syndrome and twin anemia polycythemia sequence are two potentially devastating problems in perinatal medicine. Both disorders occur only in monochorionic twins and result from unbalanced blood flow through placental vascular anastomoses. We provide a simple protocol to accurately evaluate the presence of vascular anastomoses using colored dye injection of placental vessels after birth.

The presence of placental vascular anastomoses is a conditio sine qua non for the development of twin-to-twin transfusion syndrome (TTTS) and twin anemia ...

Medicine

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent endothelial progenitors (EPs), which can differentiate into the cell types necessary to assemble mature, functional vasculature, have been derived from both embryonic and induced pluripotent stem cells. However, these cells have not been rigorously evaluated in three-dimensional environments, and a quantitative measure of their vasculogenic potential remains elusive. Here, the generation and isolation of iPSC-EPs via fluorescent-activated cell sorting are first outlined, followed by a description of the encapsulation and culture of iPSC-EPs in collagen hydrogels. This extracellular matrix (ECM)-mimicking microenvironment encourages a robust vasculogenic response; vascular networks form after a week of culture. The creation of a computational pipeline that utilizes open-source software to quantify this vasculogenic response is delineated. This pipeline is specifically designed to preserve the 3D architecture of the capillary plexus to robustly identify the number of branches, branching points, and the total network length with minimal user input.

Endothelial progenitors derived from induced pluripotent stem cells (iPSC-EPs) have the potential to revolutionize cardiovascular disease treatments and to enable the creation of more faithful cardiovascular disease models. Herein, the encapsulation of iPSC-EPs in three-dimensional (3D) collagen microenvironments and a quantitative analysis of these cells&#8217; vasculogenic potential are described.

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Immunology and Infection

Improving Ex Vivo Placental Explant Culture Using a Flow-Based Approach

Ex Vivo Placental Explant Flow Culture - Mimicking the Dynamic Conditions In Utero

The existing ex vivo placental explant culture models are primarily grounded in static culture systems using well plates. However, these models inadequately reflect the dynamic in utero setting, where the placenta encounters constant slight shear stress due to plasma or blood flow. To address this limitation, a flow culture system has been devised to bring ex vivo placental explant cultivation closer to the in utero flow conditions experienced within the maternal body. Within this approach, placental explants are cultivated in a sequence of five interconnected flow chambers. This setting&#160;maintains physiological oxygen concentrations and a consistent flow rate. The collected data reveals that under flow conditions, the preservation of tissue morphology exhibits notable enhancement compared to conventional static methods. This innovative technique introduces a straightforward means of ex vivo placental explant culture, offering a more faithful representation of the dynamic in vivo environment. Moreover, this study introduces new&#160;possibilities for investigating the functional dynamics of the feto-maternal interface. By embracing feasible dynamic methodologies, a deeper comprehension of placental biology is facilitated, underscoring its relevance for maternal-fetal health.

Author Spotlight: Advancing Research Through Single Cell Sequencing and Spatial Histology in Placental Tissues 

Here is a protocol for culturing placental explants under constant flow conditions. This approach enhances traditional static villous culture systems by enabling the replication of dynamic physiological environments.

The existing ex vivo placental explant culture models are primarily grounded in static culture systems using well plates. However, these models ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Biology

Retinal Pericytes Visualization Using Tissue-Clearing and Fluorescent Labeling

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining 

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal vasculature. To provide a detailed view of the morphological characteristics of pericytes, this study described a new approach combining the retro-orbital injection of a fluorescent agent, immunofluorescent-staining, and tissue-clearing treatment. Firstly, the fluorescent tomato lectin was injected into the retro-orbital sinus of the live mouse to label the retinal vasculature. After 5 min, the mouse was sacrificed, and its intact retina was carefully removed from the retinal cup and immunofluorescently stained with platelet-derived growth factor receptor &#946; to reveal the pericytes. Additionally, the stained retina was treated with tissue-clearing reagent and whole mounted on the microscope slide. Through these approaches, the retinal vasculature and pericytes were clearly observed in the transparent retina. Under a confocal microscope, we obtained more opportunities to take high-resolution images for further reconstructing and analyzing the morphological characteristics of pericytes along the retinal vascular tree in a three-dimensional view. Methodologically, this protocol offers an effective approach for visualizing pericytes within the retinal vasculature, providing valuable insights into their role under both physiological and pathological conditions.

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining

The pericytes in retinal vasculature were examined by immunofluorescent staining with platelet-derived growth factor receptor &#946; after retro-orbital injection of fluorescent tomato lectin. The labeled retina was further treated with the tissue-clearing method and whole mounted for visualizing the three-dimensional views of pericytes surrounding retinal vasculature under a confocal microscope.

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal ...

Biochemistry

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore largely based on animal studies, despite the high diversity among species in placental anatomy, hemodynamics and duration of the pregnancy. The vast majority of human placenta studies are ex vivo perfusion studies or in vitro trophoblast studies. Although in vitro studies and animal models are essential, extrapolation of the results from such studies to the human placenta in vivo is uncertain. We aimed to study human placenta physiology in vivo at term, and present a detailed protocol of the method. Exploiting the intraabdominal access to the uterine vein just before the uterine incision during planned cesarean section, we collect blood samples from the incoming and outgoing vessels on the maternal and fetal sides of the placenta. When combining concentration measurements from blood samples with volume blood flow measurements, we are able to quantify placental and fetal uptake and release of any compound. Furthermore, placental tissue samples from the same mother-fetus pairs can provide measurements of transporter density and activity and other aspects of placental functions in vivo. Through this integrative use of the 4-vessel sampling method we are able to test some of the current concepts of placental nutrient transfer and metabolism in vivo, both in normal and pathological pregnancies. Furthermore, this method enables the identification of substances secreted by the placenta to the maternal circulation, which could be an important contribution to the search for biomarkers of placenta dysfunction.

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

We present a detailed method to study human placental physiology in vivo at term. The method combines blood sampling from the incoming and outgoing vessels on the maternal and fetal sides of the placenta with ultrasound measurements of volume blood flow and placental tissue sampling.

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

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