Name: the 4-vessel sampling approach to integrative studies of human placental physiology in vivo
Uploaded: 2017-08-02T12:30:00
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" /> 
<stro...

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+distribution+of+arachidonic+acid+in+plasma+and+tissues+of+patients+near+term+undergoing+elective+or+emergency+Caesarean+section.">The distribution of arachidonic acid in plasma and tissues of patients near term undergoing elective or emergency Caesarean section.</a> Br J Obstet Gynaecol. 85 (2), 119-123 (1978).</li><li>Haberey, P. P., Schaefer, A., Nisand, I., Dellenbach, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fate+and+importance+of+fetal+lactate+in+the+human+placenta+-a+new+hypothesis.">The fate and importance of fetal lactate in the human placenta -a new hypothesis.</a> J Perinat Med. 10 (2), 127-129 (1982).</li><li>Prendergast, C. 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M., Henriksen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+glucose+transfer:+a+human+in+vivo+study.">Placental glucose transfer: a human in vivo study.</a> PLoS One. 10 (2), 0117084 (2015).</li><li>Holme, A. M., Roland, M. C., Henriksen, T., Michelsen, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+uteroplacental+release+of+placental+growth+factor+and+soluble+Fms-like+tyrosine+kinase-1+in+normal+and+preeclamptic+pregnancies.">In vivo uteroplacental release of placental growth factor and soluble Fms-like tyrosine kinase-1 in normal and preeclamptic pregnancies.</a> Am J Obstet Gynecol. 215 (6), 781-782 (2016).</li><li>Paasche Roland, M. 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S., 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=Uteroplacental+unit+as+a+source+of+elevated+circulating+prorenin+levels+in+normal+pregnancy.">Uteroplacental unit as a source of elevated circulating prorenin levels in normal pregnancy.</a> Am J Obstet Gynecol. 155 (6), 1223-1226 (1986).</li><li>Myatt, L., 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=Strategy+for+standardization+of+preeclampsia+research+study+design.">Strategy for standardization of preeclampsia research study design.</a> Hypertension. 63 (6), 1293-1301 (2014).</li><li>Kiserud, T., Rasmussen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+repeat+measurements+affect+the+mean+diameter+of+the+umbilical+vein+and+the+ductus+venosus.">How repeat measurements affect the mean diameter of the umbilical vein and the ductus venosus.</a> Ultrasound Obstet Gynecol. 11 (6), 419-425 (1998).</li><li>Burton, G. J., 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=Optimising+sample+collection+for+placental+research.">Optimising sample collection for placental research.</a> Placenta. 35 (1), 9-22 (2014).</li><li>Illsley, N. P., Wang, Z. Q., Gray, A., Sellers, M. C., Jacobs, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+preparation+of+paired,+syncytial,+microvillous+and+basal+membranes+from+human+placenta.">Simultaneous preparation of paired, syncytial, microvillous and basal membranes from human placenta.</a> Biochim Biophys Acta. 1029 (2), 218-226 (1990).</li><li>Staff, A. C., Ranheim, T., Khoury, J., Henriksen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+contents+of+phospholipids,+cholesterol,+and+lipid+peroxides+in+decidua+basalis+in+women+with+preeclampsia.">Increased contents of phospholipids, cholesterol, and lipid peroxides in decidua basalis in women with preeclampsia.</a> Am J Obstet Gynecol. 180 (3), 587-592 (1999).</li><li>Catalano, P. M., Thomas, A. J., Avallone, D. A., Amini, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anthropometric+estimation+of+neonatal+body+composition.">Anthropometric estimation of neonatal body composition.</a> Am J Obstet Gynecol. 173 (4), 1176-1181 (1995).</li><li>Ellis, K. J., 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=Body-composition+assessment+in+infancy:+air-displacement+plethysmography+compared+with+a+reference+4-compartment+model.">Body-composition assessment in infancy: air-displacement plethysmography compared with a reference 4-compartment model.</a> Am J Clin Nutr. 85 (1), 90-95 (2007).</li><li>Haugen, G., Kiserud, T., Godfrey, K., Crozier, S., Hanson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Portal+and+umbilical+venous+blood+supply+to+the+liver+in+the+human+fetus+near+term.">Portal and umbilical venous blood supply to the liver in the human fetus near term.</a> Ultrasound Obstet Gynecol. 24 (6), 599-605 (2004).</li><li>Acharya, G., 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=Experimental+validation+of+uterine+artery+volume+blood+flow+measurement+by+Doppler+ultrasonography+in+pregnant+sheep.">Experimental validation of uterine artery volume blood flow measurement by Doppler ultrasonography in pregnant sheep.</a> Ultrasound Obstet Gynecol. 29 (4), 401-406 (2007).</li><li>Wu, X., 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=Glutamate-glutamine+cycle+and+exchange+in+the+placenta-fetus+unit+during+late+pregnancy.">Glutamate-glutamine cycle and exchange in the placenta-fetus unit during late pregnancy.</a> Amino Acids. 47 (1), 45-53 (2015).</li><li>Tuckey, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progesterone+synthesis+by+the+human+placenta.">Progesterone synthesis by the human placenta.</a> Placenta. 26 (4), 273-281 (2005).</li><li>Simmons, M. A., Meschia, G., Makowski, E. L., Battaglia, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+metabolic+response+to+maternal+starvation.">Fetal metabolic response to maternal starvation.</a> Pediatr Res. 8 (10), 830-836 (1974).</li><li>Simmons, M. A., Jones, M. D., Battaglia, F. 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. 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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 border="0" cellpadding="0" cellspacing="0" width="1323">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:557px;">Maternal body composition</td>
			<td style="width:173px;"></td>
			<td style="width:107px;"></td>
			<td style="width:487px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Impedance scale</td>
			<td>Tanita</td>
			<td></td>
			<td>or similar</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultrasound measurements&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="20">
			<td height="20" style="height:20px;">Blood samples</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Arerial cannula</td>
			<td>BD Medical</td>
			<td>682245</td>
			<td>or similar</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">20cc Eccentric Luer Tip Syringe without Needle</td>
			<td>Termo</td>
			<td>SS-20ES</td>
			<td>or similar. 3 needed.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10cc Eccentric Luer Tip Syringe without Needle</td>
			<td>Termo</td>
			<td>SS-10ES</td>
			<td>or similar. 9 needed.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5cc 6% Luer Syringe without Needle</td>
			<td>Termo</td>
			<td>SS-05S1</td>
			<td>or similar. 2 needed.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Arterial blood gas syringe&#160;</td>
			<td>Radiometer Medical</td>
			<td></td>
			<td>or similar. 4 needed.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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 height="20">
			<td height="20" style="height:20px;">Vacutainer tube 6 mL EDTA&#160;</td>
			<td>Greiner Bio-One</td>
			<td>456043</td>
			<td>or similar. Label with sample site. 10 needed.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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 height="20">
			<td height="20" style="height:20px;">Vacutainer tube 6 mL Serum Clot Activator&#160;</td>
			<td>Greiner Bio-One</td>
			<td>456089</td>
			<td>or similar. Label with sample site. 5 needed.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:557px;">Vacutainer tube 3 ml&#160; 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 height="20">
			<td height="20" style="height:20px;">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 height="20">
			<td height="20" style="height:20px;">Marked trays to transport the syringes</td>
			<td></td>
			<td></td>
			<td>to transport the blood samples in the operation theatre</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rocker</td>
			<td></td>
			<td></td>
			<td>for gentle mixing of the samples</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ice</td>
			<td></td>
			<td></td>
			<td>in styrofoam box</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liquid nitrogen</td>
			<td></td>
			<td></td>
			<td>in appropriate container</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Placenta samples</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Metal tray</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ice</td>
			<td></td>
			<td></td>
			<td>in styrofoam box</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calibrated scale</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Metal ruler</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 M Phosphate buffered saline</td>
			<td>Sigma</td>
			<td>D1408</td>
			<td>or similar. Dilute 10 M to&#160; 1M before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA stabilization solution</td>
			<td>Sigma</td>
			<td>R0901-500ML&#160;</td>
			<td>or similar</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Optimal Cutting Temperature (O.C.T.) compound</td>
			<td>vwr</td>
			<td>361603E</td>
			<td>or similar</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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 height="20">
			<td height="20" style="height:20px;">Centrifuge tubes, conical bottom 50 mL</td>
			<td>Greiner Bio-One</td>
			<td>227,285</td>
			<td>or similar. Label with &#34;RNA later&#34;, sample site and ID. 2 needed.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liquid nitrogen</td>
			<td></td>
			<td></td>
			<td>in appropriate container</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal body composition</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calibrated scale</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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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