Name: the 4-vessel sampling approach to integrative studies of human placental physiology in vivo
Uploaded: 2017-08-02T12:30:00.000+00:00
Duration: 12 min 17 s
Description: Watch this Scientific Journal Video about The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo at JoVE.com

First and foremost, we sincerely thank the mothers who participated in this project. Next, we acknowledge all the personnel that assisted and facilitated the sampling procedure, the Anesthesiologist, the nurse anesthesiologist and the surgical nurses. The project would not have been possible without funding from the South-Eastern Norway Regional Health Authority and Norwegian Advisory Unit on Women's Health, Oslo University and local funding provided by Oslo University Hospital.

The study was approved by the data protection officials at Oslo University Hospital and the Regional Committee for Medical and Health Research Ethics, Southern Norway 2419/2011. All participants signed a written informed consent at inclusion.
1. Preparations
NOTE: A timeline for the procedures is outlined in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55847/55847fig1.jpg" /> 
Figure 1: Flowchart Describing the Timing and the Personnel Involved in the 4-vessel Sampling Procedure. 
One color represents one person. Detailed description of the method is given in the protocol. <a href="http://cloudfront.jove.com/files/ftp_upload/55847/55847fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Staff
	<ol>
		<li>Make sure all required personnel are available: a highly skilled Fetal Medicine specialist conducting the ultrasound measurements, two obstetricians conducting the surgery, one of the obstetricians and two nurses collecting the samples, one assistant handling the blood gas analyses and one assistant handling the other samples consecutively and immediately following the collection. 
		NOTE: In cases of more advanced collection of placental tissue, an additional person is required.</li>
	</ol>
	</li>
	<li>Equipment
	<ol>
		<li>Prepare the equipment, 50 mL of ice cold 1 M phosphate buffered saline (PBS), 25 mL of cold RNA stabilizing solution and 5 x 0.5 mL of optimal cutting temperature compound (OCT). Label the vacutainers and tubes. See tentative list of equipment.</li>
	</ol>
	</li>
</ol>
2. Maternal Characteristics
<ol>
	<li>Record the maternal clinical and non-clinical characteristics at inclusion and repeat relevant questions and measurements, including weight, at the time of delivery. Record the duration of the fasting period prior to the cesarean section, and any hypotensive episodes occurring during the surgery. 
	Note: Include the minimal maternal clinical dataset reported in a recent publication from Global Pregnancy CoLaboratory (COLAB). This article also includes some very important aspects in choosing study populations and should be addressed while planning the study 18.</li>
	<li>Consider recording paternal characteristics, including ethnicity, age and body mass index (BMI).</li>
</ol>
3. Ultrasound
<ol>
	<li>Perform the Doppler ultrasound examination on the day of the delivery, with the women in a fasting state. Perform the examination during a period of fetal quiescence, with the woman in semi-supine position, tilted slightly laterally opposite to the region of interest in order to avoid compression of the aorta and vena cava. Monitor the output intensity by the mechanical and thermal indices on the display.</li>
	<li>Umbilical vein
	<ol>
		<li>Visualize the umbilical vein in a sagittal or oblique transection of the fetal abdomen. Measure the internal vessel diameter in the straight portion of the intra-abdominal umbilical vein, before any visible branches. Use regular B-mode and visualize the vessel in a perpendicular insonation angle for diameter measurements and keep several optimal frames for later measurements to minimize the effect of pulsatile diameter changes.
		<ol>
			<li>Repeat the measurements five to ten times 19.</li>
		</ol>
		</li>
		<li>At the same site, use Doppler ultrasound and adjust the probe to get an insonation angle as low as possible (always &#60;30&#176;) in order to measure the time-averaged maximum velocity (TAMX). Obtain the velocity over a period of 3 - 5 s (non-pulsating flow).</li>
	</ol>
	</li>
	<li>Uterine artery
	<ol>
		<li>Use Doppler ultrasound to visualize the uterine artery as it crosses the external iliac artery, immediately after it branches from the internal iliac artery. Adjust the probe at this site to get a low insonation angle (always &#60;30&#176;) and measure TAMX. Obtain the velocity as the mean velocity of three heart cycles.</li>
		<li>As it is unlikely to get a perpendicular angle at the same site as TAMX is measured, follow the vessel distally to get a correct angle for diameter measurements as close to the sites of diameter measurements as achievable. Exclude the diameter measurements if any visible vessels branch off before this site as evaluated by color Doppler ultrasound.
		<ol>
			<li>Use regular B-mode and visualize the vessel in a perpendicular insonation angle for diameter measurements and keep several optimal frames for later measurements to minimize the effect of pulsatile diameter changes.</li>
			<li>Repeat the measurements five to ten times 19.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Note the position of the placenta.</li>
</ol>
4. 4-vessel Blood Sampling
NOTE: The timeline for the procedures is outlined in Figure 1 and an overview of the samples is illustrated in Figure 2.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55847/55847fig2.jpg" /> 
Figure 2: Schematic Illustration of the Placental Vasculature and the Sampling Sites. 
In the 4-vessel sampling method blood samples are drawn from the uterine vein, the radial artery (as a proxy for the uterine artery) and the umbilical arteries and vein. Blood flow in the uterine artery and the umbilical vein is measured by ultrasound. Tissue samples from the placenta are collected. Illustration: &#216;ystein H. Horgmo, University of Oslo. <a href="http://cloudfront.jove.com/files/ftp_upload/55847/55847fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Safety procedures
	<ol>
		<li>Provide all personnel in the operation theater with gloves, surgical scrub suits, masks and headwear.</li>
		<li>Provide the surgeons and research personnel in contact with the operation field with surgical scrub suits, masks, headwear, gowns and double gloves. Glasses are optional.</li>
		<li>Provide personnel handling the blood samples with gloves.</li>
		<li>Provide personnel handling the placenta samples with gloves and surgical mask. Homogenization requires the use of hoods.</li>
	</ol>
	</li>
	<li>Preparation in the operation theater
	<ol>
		<li>Give a briefing and hand the equipment to all personnel that will assist the sampling before onset of surgery.</li>
		<li>Address the anesthesiologist and anesthesiology nurse who will assist with the necessary peripheral arterial and venous access, and ensure that no liquids are given intravenously before sampling.</li>
		<li>Give three syringes (10 mL) without needles to the person assisting with the antecubital vein sample and two syringes (one 20 mL and one 10 mL) and one blood gas syringe (with heparin) to the person assisting with the radial artery.</li>
		<li>Prepare two sterile syringes (20 mL), five sterile syringes (10 mL), three &#34;butterfly needles&#34; and two blood gas syringes for the operation field.</li>
	</ol>
	</li>
	<li>Access to blood vessels.
	<ol>
		<li>Follow standard procedure before the cesarean section to assure peripheral intravenous (iv) access. 
		NOTE: The antecubital vein is preferable because it is easier to draw samples from this site.</li>
		<li>Localize the radial artery at the wrist by ultrasound or by palpation. Following 0.5 mL of subcutaneous lidocaine analgesia, place an arterial line into the radial artery. Abandon the sampling from this site in case of three failed insertions, or if the woman experiences pain during the insertion. 
		NOTE: Perform the surgical procedure of cesarean section according to standard procedure. Only the adjustments needed for the sampling procedure are underlined below.</li>
	</ol>
	</li>
	<li>Maternal blood samples 
	NOTE: Obtain all three maternal blood samples (uterine vein, radial artery and antecubital vein) simultaneously before the uterine incision.
	<ol>
		<li>For the uterine vein, after opening the abdominal cavity, use a retractor to lift the abdominal wall and expose the main branches of the uterine veins on the anterolateral sides of the uterus. Obtain blood from uterine vein branches at the same side as the placenta whenever possible or use the most prominent vein plexus if the placenta is located in the uterine midline.
		<ol>
			<li>Insert a butterfly needle on a blood gas syringe in the uterine vein at an angle of approximately 30 degrees and collect blood through gentle aspiration to avoid hemolysis. While carefully securing the iv position of the butterfly needle, replace the filled blood gas syringe by a 20 mL and a 10 mL syringe consecutively. 
			NOTE: Optimal access is best ensured when standing on the contralateral side of the chosen uterine vein.</li>
		</ol>
		</li>
		<li>For the radial artery, aspirate from the intra-arterial line. Discard the first 5 mL, and then aspirate 3 mL in heparin syringe for blood gas analyses, followed by 3 mL in two syringes (20 + 10 mL).</li>
		<li>For the antecubital vein, aspirate gently from the intravenous catheter. Discard the first 5 mL, and then aspirate 30 mL in three syringes (10 mL).</li>
		<li>Perform a final inspection of the sampling site on the uterine vein before starting to close the abdomen.</li>
	</ol>
	</li>
	<li>Fetal blood samples
	<ol>
		<li>When the child is born, immediately aspirate blood from the umbilical artery, without clamping the umbilical cord or delivering the placenta. Start with the syringe for blood gas analysis, and follow with three 10 mL syringes if possible.</li>
		<li>When the arterial samples are secured, clamp the cord and hand the child to the midwife before sampling from the umbilical vein (blood gas and 20 + 10 mL syringes). 
		NOTE: Obtain all umbilical samples within seconds of delivery and with the placenta in situ unless it has detached spontaneously.</li>
		<li>Follow the Norwegian recommendations on late cord clamping. In case of a distressed child, clamp and cut the cord immediately and the hand the child to the midwife and neonatologist.</li>
	</ol>
	</li>
	<li>Handling of blood samples
	<ol>
		<li>Put the blood gas syringes on ice while preparing the rest of the blood samples, and analyze them in a blood gas analyzer within 5 min.</li>
		<li>Transfer the blood samples immediately to vacutainers and place the plasma tubes on a rocker for 1 - 2 min before putting them on ice. Leave the serum tubes on the laboratory bench to settle for 30 minutes. 
		NOTE: This is a critical step in the procedure that needs extra attention because samples from all five sites have to be prepared simultaneously to ensure good quality.</li>
		<li>Centrifuge the plasma samples as soon as possible, and within 30 min, at 6 &#176;C, 2,500 x g for 20 min.</li>
		<li>After 30 min, centrifuge the serum samples at room temperature for 10 min at 2,500 x g.</li>
		<li>Aliquot the supernatants carefully to 2 mL cryo tubes, leaving 0.5 mL of the supernatant above the pellet to ensure platelet free plasma.</li>
		<li>Store the samples at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
5. Collection of Placental Tissue
<ol>
	<li>Place the placenta flat down on an ice chilled dissection tray as soon as possible after it has been delivered. Photographand measure the longest diameter and the diameter at 90 degrees. </li>
	<li>Weigh the placenta.</li>
	<li>Record the weight, the two diameters, any gross pathology, number of vessels in the cord and the time interval from delivery to when the placenta was placed on ice. 
	NOTE: Send the placenta to pathological examination if clinically indicated.</li>
	<li>Place the placenta with the maternal surface facing up and identify 4 - 5 sampling sites randomly located in each quadrant of the placenta, avoiding areas of frank pathology. Remove the decidua using scissors to cut away 3 - 5 mm from the maternal surface. Collect a 1 - 2 cm3 piece of villous tissue from each site.</li>
	<li>Wash the collected tissue gently in 50 mL of cold 1M PBS. Divide into several pieces from each sampling site and aliquot. 
	Note: The size of the placenta pieces will depend on the planned analyzes.</li>
	<li>Add aliquots of 0.1 - 0.5 cm3 tissue samples to 5 cryo tubes and snap freeze in liquid nitrogen.</li>
	<li>Add small pieces of 0.1 - 0.2 cm3 to the tube with 25 mL of RNA stabilization solution. Store at 4 &#176;C for 24 h, discard the RNA stabilization solution and replace it. Freeze.</li>
	<li>Add pieces of 0.5 cm3 to the 5 cryo tubes with 0.5 mL of OCT, top up with OCT, mix and freeze.</li>
	<li>Store the samples at -80 &#176;C until analysis. 
	NOTE: Burton et al. provides an excellent overview of practical aspects of placental sampling depending on the analyses planned. 20 Consider to prepare the remaining tissue for isolation of the microvillous and basal membranes, and to collect decidual tissue by vacuum suction technique. 21,22</li>
</ol>
6. Neonatal Characteristics
<ol>
	<li>Record the neonatal characteristics, including Apgar-score (1, 5 and 10 min), sex, weight, length, gestational age and admission to Newborn Intensive Care Unit (length and outcome of stay).</li>
	<li>Consider measuring neonatal body composition by anthropometric measurements, air-displacement plethysmograph or dual X-ray absorptiometry.23,24</li>
</ol>
7. Calculations
<ol>
	<li>Assume similar blood composition in the radial and uterine artery and calculate the uteroplacental arteriovenous concentration difference. 
	Uteroplacental arteriovenous concentration difference = CA - CV 
	Umbilical venous - arterial concentration difference = Cv - Ca 
	 
	Where C is concentration with subscripts: A, the radial artery; V, the uterine vein; v the umbilical vein and a, the umbilical artery.</li>
	<li>Calculate the volume blood flow, mL/min (Q): 
	<img alt="Equation 1" src="/files/ftp_upload/55847/55847eq1.jpg" /> 
	Where D is the vessel diameter (cm), TAMX is time averaged maximum velocity and h is the coefficient for the spatial blood velocity profile. Use 0.5 as the coefficient for the umbilical vein and 0.6 for the uterine artery25,26.</li>
	<li>Calculate the placental uptake and release according to Fick&#39;s principle: 
	 
	Uteroplacental uptake = (CA - CV) x Qm 
	Fetal uptake = (Cv - Ca) x Qf 
	 
	Subscripts: m, maternal and f, fetal.</li>
</ol>

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C., Meschia, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+effect+on+fetal+glucose+utilization.">Insulin effect on fetal glucose utilization.</a> Pediatr Res. 12 (2), 90-92 (1978).</li><li>Bujold, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+supporting+that+the+excess+of+the+sVEGFR-1+concentration+in+maternal+plasma+in+preeclampsia+has+a+uterine+origin.">Evidence supporting that the excess of the sVEGFR-1 concentration in maternal plasma in preeclampsia has a uterine origin.</a> J Matern Fetal Neonatal Med. 18 (1), 9-16 (2005).</li><li>Jansson, T., Aye, I. L., Goberdhan, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+role+of+mTORC1+signaling+in+placental+nutrient-sensing.">The emerging role of mTORC1 signaling in placental nutrient-sensing.</a> Placenta. 33, 23-29 (2012).</li><li>Cetin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+transport+of+amino+acids+in+normal+and+growth-restricted+pregnancies.">Placental transport of amino acids in normal and growth-restricted pregnancies.</a> Eur J Obstet Gynecol Reprod Biol. 110, 50-54 (2003).</li></ol>

The authors have nothing to disclose.

The placenta 4-vessel sampling method is relevant for three main purposes. First, it can be used to study how specific substances are taken up by the placenta on the maternal side and possibly transferred to the umbilical circulation and the fetus, as demonstrated by our glucose and amino acid studies. Second, the method is highly relevant to study substances produced by the placenta and released to the maternal or the fetal circulations, as demonstrated by the progesterone results. Third, it may be useful to study how t...

According to the National Institutes of Health, USA, the placenta is the least understood organ in the human body 1,2,3. It is difficult to access and study the human placenta in vivo without imposing unethical risks on the ongoing pregnancy. Studies of placental function in the human are therefore largely based on in vitro and ex vivo models. The majority of previous in vivo studies of placental transport and metabolism have been performed in animals 4,5,6. However, as placental structure and functions vary considerably between species, extrapolation of results from animals to humans must be done with caution. Only a few smaller human in vivo studies have investigated placental and fetal uptake and transport under normal physiological conditions, and none have explored the integrated transfer of several compounds 7,8,9,10,11,12,13. These fundamental studies illustrate that in vivo studies of the human placenta are feasible, and that they may serve several purposes. First, current concepts of placental functions mainly derived from in vitro, ex vivo and animal studies may be tested in a human setting and thus provide novel and more specific insight into the human placenta. Second, properties of the dysfunctional placenta associated with aberrant fetal growth, preeclampsia, maternal diabetes, metabolic syndrome and other maternal metabolic disturbances may be better characterized. Third, human in vivo studies provide an opportunity to develop diagnostic and predictive tools of placental function.
On this background we aimed to establish a comprehensive collection of physiological data to investigate human placental function in vivo. During a planned cesarean section, we exploit the intraabdominal access to the uterine vein to collect blood samples from the incoming and outgoing vessels on the maternal and fetal sides of the placenta (the 4-vessel sampling method). These samples are used to calculate the paired arteriovenous concentration differences of nutrients and other substances 14. In addition, we measure volume blood flow on both sides of the placenta by ultrasound. Consequently, placental and fetal uptake of any compound may be quantified. Further, it is possible to determine substances released by the placenta to the maternal and fetal circulations 15,16,17. When combined with clinical parameters of mother and child, and analyses of placental and other relevant tissues, this method has the exciting potential to integrate many aspects of placental functions in vivo in the same mother-fetus pairs.

<table><tbody><tr><td>&lt;strong&gt;Maternal body composition&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Impedance scale</td><td>Tanita</td><td></td><td>or similar</td></tr><tr><td>&lt;strong&gt;Ultrasound measurements&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sequoia 512 ultrasound machine</td><td>Acuson</td><td></td><td>equipped with a curved transducer with colour and pulsewave Doppler (frequency bandwidth 2 - 6 MHz)</td></tr><tr><td>&lt;strong&gt;Blood samples&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Arerial cannula</td><td>BD Medical</td><td>682245</td><td>or similar</td></tr><tr><td>20 cc Eccentric Luer Tip Syringe without Needle</td><td>Termo</td><td>SS-20ES</td><td>or similar. 3 needed.</td></tr><tr><td>10 cc Eccentric Luer Tip Syringe without Needle</td><td>Termo</td><td>SS-10ES</td><td>or similar. 9 needed.</td></tr><tr><td>5 cc 6% Luer Syringe without Needle</td><td>Termo</td><td>SS-05S1</td><td>or similar. 2 needed.</td></tr><tr><td>Arterial blood gas syringe&amp;nbsp;</td><td>Radiometer Medical</td><td></td><td>or similar. 4 needed.</td></tr><tr><td>Sterile winged needle connected to flexible tubing, 21 gauge</td><td>Greiner Bio-One</td><td>450081</td><td>(intended for single use).3 needed.</td></tr><tr><td>Vacutainer tube 6 mL EDTA&amp;nbsp;</td><td>Greiner Bio-One</td><td>456043</td><td>or similar. Label with sample site. 10 needed.</td></tr><tr><td>Vacutainer tube 5 mL LH Lithium Heparin Separator</td><td>Greiner Bio-One</td><td>456305</td><td>or similar. Label with sample site. 5 needed.</td></tr><tr><td>Vacutainer tube 6 mL Serum Clot Activator&amp;nbsp;</td><td>Greiner Bio-One</td><td>456089</td><td>or similar. Label with sample site. 5 needed.</td></tr><tr><td>Vacutainer tube 3 mL&amp;nbsp; 9NC Coagulation sodium citrate 3.2%</td><td>Greiner Bio-One</td><td>454334</td><td>or similar. Label with sample site. 5 needed.</td></tr><tr><td>Cryogenic vials, 2.0 mL</td><td>Corning</td><td>430488</td><td>or similar. Label with sample site, serum/type of plasma and ID. 90 needed.</td></tr><tr><td>Marked trays to transport the syringes</td><td></td><td></td><td>to transport the blood samples in the operation theatre</td></tr><tr><td>Rocker</td><td></td><td></td><td>for gentle mixing of the samples</td></tr><tr><td>Ice</td><td></td><td></td><td>in styrofoam box</td></tr><tr><td>Liquid nitrogen</td><td></td><td></td><td>in appropriate container</td></tr><tr><td>&lt;strong&gt;Placenta samples&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Metal tray</td><td></td><td></td><td></td></tr><tr><td>Ice</td><td></td><td></td><td>in styrofoam box</td></tr><tr><td>Calibrated scale</td><td></td><td></td><td></td></tr><tr><td>Metal ruler</td><td></td><td></td><td></td></tr><tr><td>1 M Phosphate buffered saline</td><td>Sigma</td><td>D1408</td><td>or similar. Dilute 10 M to&amp;nbsp; 1 M before use</td></tr><tr><td>RNA stabilization solution</td><td>Sigma</td><td>R0901-500ML&amp;nbsp;</td><td>or similar</td></tr><tr><td>Optimal Cutting Temperature (O.C.T.) compound</td><td>vwr</td><td>361603E</td><td>or similar</td></tr><tr><td>Cryogenic vials, 2.0 mL</td><td>Corning</td><td>430488</td><td>or similar. Label with sample site. content and ID. 10 needed.</td></tr><tr><td>Centrifuge tubes, conical bottom 50 mL</td><td>Greiner Bio-One</td><td>227,285</td><td>or similar. Label with &quot;RNA later&quot;, sample site and ID. 2 needed.</td></tr><tr><td>Liquid nitrogen</td><td></td><td></td><td>in appropriate container</td></tr><tr><td>&lt;strong&gt;Fetal body composition&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Calibrated scale</td><td></td><td></td><td></td></tr><tr><td>Measuring tape</td><td></td><td></td><td></td></tr></tbody></table>

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 method is applicable in clinical practice and we have successfully obtained blood samples from 209 mother/infant-pairs. In 128 of these we also achieved to measure volume blood flow. Complete 4-vessel sampling and good quality flow measurements of both maternal and fetal vessels were obtained in 70 mother-fetus pairs (Figure 3). In addition, we have so far collected blood and placenta samples from 30 preeclamptic patients. We have previo...

Watch this Scientific Journal Video about The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo at JoVE.com

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 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 ...

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Research

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Medicine

10.5K Views.  Oslo University Hospital. 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.

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

Guide Wire Assisted Catheterization and Colored Dye Injection for Vascular Mapping of Monochorionic Twin Placentas

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC twin pregnancies develop complications arising from the shared placenta and associated vascular connections1. Severe twin-to-twin syndrome (TTTS) is reported to account for approximately 20% of these complications2,3. Inter-twin vascular connections occur in almost all MC placentas and are related to the prognosis and outcome of these high-risk twin pregnancies. The number, size and type of connections have been implicated in the development of TTTS and other MC twin conditions. Three types of inter-twin vascular connections occur: 1) artery to vein connections (AVs) in which a branch artery carrying deoxygenated blood from one twin courses along the fetal surface of the placenta and dives into a placental cotyledon. Blood flows via a deep intraparenchymal capillary network into a draining vein that emerges at the fetal surface of the placenta and brings oxygenated blood toward the other twin. There is unidirectional flow from the twin supplying the afferent artery toward the twin receiving the efferent vein; 2) artery to artery connections (AAs) in which a branch artery from each twin meets directly on the superficial placental surface resulting in a vessel with pulsatile bidirectional flow, and 3) vein to vein connections (VVs) in which a branch vein from each twin meets directly on the superficial placental surface allowing low pressure bidirectional flow. In utero obstetric sonography with targeted Doppler interrogation has been used to identify the presence of AV and AA connections4. Prenatally detected AAs that have been confirmed by postnatal placental injection studies have been shown to be associated with an improved prognosis for both twins5. Furthermore, fetoscopic laser ablation of inter-twin vascular connections on the fetal surface of the shared placenta is now the preferred treatment for early, severe TTTS. 
Postnatal placental injection studies provide a valuable method to confirm the accuracy of prenatal Doppler ultrasound findings and the efficacy of fetal laser therapy6. Using colored dyes separately hand-injected into the arterial and venous circulations of each twin, the technique highlights and delineates AVs, AAs, and VVs. This definitive demonstration of MC placental vascular anatomy may then be correlated with Doppler ultrasound findings and neonatal outcome to enhance our understanding of the pathophysiology of MC twinning and its sequelae. Here we demonstrate our placental injection technique.

Vascular mapping of monochorionic (MC) twin placentas after birth provides a means for detailed demonstration of vascular connections between the twins&#x2019; circulations. 
Imbalance of these connections is thought to play a pivotal role in the development of complications of MC twinning including twin-to-twin transfusion syndrome.

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC ...

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 ...

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Biology

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Bioengineering

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer. One of the most useful techniques in determining the cellular roles of droplets has been proteomic identification of bound proteins, which can be isolated along with the droplets. Here, two methods are described to isolate lipid droplets and their bound proteins from two wide-ranging eukaryotes: fission yeast and human placental villous cells. Although both techniques have differences, the main method - density gradient centrifugation -&#160;is shared by both preparations. This shows the wide applicability of the presented droplet isolation techniques.

In the first protocol, yeast cells are converted into spheroplasts by enzymatic digestion of their cell walls. The resulting spheroplasts are then gently lysed in a loose-fitting homogenizer. Ficoll is added to the lysate to provide a density gradient, and the mixture is centrifuged three times. After the first spin, the lipid droplets are localized to the white-colored floating layer of the centrifuge tubes along with the endoplasmic reticulum (ER), the plasma membrane, and vacuoles. Two subsequent spins are used to remove these other three organelles. The result is a layer that has only droplets and bound proteins.

In the second protocol, placental villous cells are isolated from human term placentas by enzymatic digestion with trypsin and DNase I. The cells are homogenized in a loose-fitting homogenizer. Low-speed and medium-speed centrifugation steps are used to remove unbroken cells, cellular debris, nuclei, and mitochondria. Sucrose is added to the homogenate to provide a density gradient and the mixture is centrifuged to separate the lipid droplets from the other cellular fractions.

The purity of the lipid droplets in both protocols is confirmed by Western Blot analysis. The droplet fractions from both preps are suitable for subsequent proteomic and lipidomic analysis.

Two techniques for isolating cellular lipid droplets from 1) yeast cells and 2) human placentas are presented. The centerpiece of both procedures is density gradient centrifugation, where the resulting floating layer containing the droplets can be readily visualized by eye, extracted, and quantified by Western Blot analysis for purity.

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of ...

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.

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 ...

Developmental Biology

Ex Vivo Perfusion of the Rodent Placenta

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An assay is described here to perfuse an isolated rat placenta and evaluate the maternal-to-fetal translocation of xenobiotics ex vivo. In addition, the evaluation of physiological processes such as fluid flow to the fetus and placental metabolism may be conducted with this methodology. This technique is suitable for evaluating maternal-to-fetal kinetics of pharmaceutical candidates or environmental contaminants. In contrast to current alternative approaches, this methodology allows the evaluation of the isolated maternal-fetal vasculature, with the systemic neural or immune involvement removed, allowing any observed changes in physiological function to be attributable to local factors within the isolated tissue.

Here is presented a protocol of ex vivo maternal-fetal vascular perfusion to enable the administration of a test article into maternal vasculature and to evaluate placental transfer of xenobiotic particles or pharmacological agents in addition to alterations in placental physiology.

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An ...

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 ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

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

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Guide Wire Assisted Catheterization and Colored Dye Injection for Vascular Mapping of Monochorionic Twin Placentas

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

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

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

Ex Vivo Perfusion of the Rodent Placenta

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

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

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods