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 ...

The overall goal of this procedure is to study human placental function in vivo using several methods, which together ensure the collection of comprehensive and integrative data of human fetal placental physiology. This method can help answer key questions about human placental physiology, such as the role of placenta in maternal-fetal interplay. The main advantage of this technique is that it makes it possible to test current concepts of placental transfer, uptake, and secretion in humans in vivo.Extrapolation of experimental studies to humans is hampered by the large differences between animals and humans in placental anatomy, duration of pregnancy, and pregnancy complications. Though this method can provide insight into placental metabolism and transfer of nutrients, it can also be applied to study placental release of compounds both in healthy and in complicated pregnancies, such as preeclampsia. This method requires a highly coordinated cooperation of several medical specialists, nurses, and researchers.The key to success is strict planning while all the time keeping the mother and her baby in focus. This chart illustrates the timeline and the personnel required to obtain the samples. Each individual assisting in the sampling is represented by a different color.This protocol consists of the combined use of three methods to study human placental physiology. First volume blood flow in the uterine artery and umbilical vein are performed by ultrasound. Second blood samples are collected from the incoming and outgoing vessels on the maternal and fetal sides of the human placenta during cesarean section.Third placenta tissue samples are collected for analysis. To begin, assist the woman into a semi-supine position, tilted slightly laterally opposite to the region of interest, in order to avoid compression of the aorta and vena cava. During a period of fetal quiescence, visualize the umbilical vein in a sagittal or oblique transection of the fetal abdomen and measure the internal vessel diameter in a straight portion of the intra-abdominal umbilical vein before any visible branches.Use regular B-mode and visual 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. Repeat the measurements five to 10 times. At the same site, adjust the probe and use Doppler ultrasound to get an insonation angle as low as possible in order to measure the time-averaged maximum velocity or TAMX.Obtain the velocity over a period of three to five seconds. Next, 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 and measure TAMX.Obtain the velocity as the mean velocity of three heart cycles. Follow the vessel distally to get a correct angle for diameter measurements as close to the sites of TAMX measurements as achievable. It is challenging to measure volume blood flow in the uterine artery because it is unlikely to get a perpendicular angle at the same site as TAMX is measured.Vessels that branch off between the two sites of measurements may result in exclusion of the diameter measurements. To carry out four-vessel blood sampling before the onset of surgery, prepare the solutions, tubes, and equipment. Then give a briefing and hand the equipment to all personnel that will assist with the sampling.Place an arterial line in the radial artery in addition to the standard peripheral intravenous access. The antecubital vein is preferable because it is easier to draw samples from this site. Next, 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.Identify a uterine vein branch on the same side as the placenta whenever possible or the most prominent vein plexus of the placenta is located in the uterine midline. When a suitable uterine vein has been identified, instruct the assisting personnel to draw blood samples from the radial artery and antecubital vein. Simultaneously, at a 30 degree angle, insert a butterfly needle on a blood gas syringe in the uterine vein and collect blood through gentle aspiration.While carefully securing the IV position of the butterfly needle, let an assistant replace the filled blood gas syringe with a 20 milliliter syringe. Immediately follow with a 10 milliliter syringe. From the radial artery, the antecubital vein, discard the first five milliliters, and then aspirate three milliliters into a heparin syringe for blood gas analyses.Then aspirate 30 milliliters from each site. When the child is born, begin with a syringe for blood gas analysis and immediately aspirate blood from the umbilical artery without clamping the umbilical cord or delivering the placenta. Follow with three 10 milliliter syringes if possible.When the arterial samples are secured, clamp and cut the cord before sampling from the umbilical vein, obtaining all umbilical samples with the placenta in situ. The four-vessel method requires coordinated blood sampling in addition to quick handling of samples from five different sites simultaneously. Pay attention to the umbilical artery, which is susceptible to contraction, hemolysis, and coagulation.Perform a final inspection of the sampling site on the uterine vein before starting to close the abdomen. Place all the blood gas syringes on ice and use a blood gas analyzer to analyze the samples within five minutes. Immediately transfer the blood samples to vacutainers and rock the plasma tubes for one to two minutes before putting them on ice.Centrifuge the plasma samples as soon as possible and within 30 minutes at 2, 500 times g and six degrees Celsius for 20 minutes. Then carefully aliquot the supernatants to two milliliter cryotubes. Analyze the blood samples for the desired substances and carry out calculations of placental transfer according to the text protocol.Place the placenta flat down on a ice-chilled dissection tray as soon as possible after it has been delivered. Record the weight of the tissue as well as the longest diameter and the diameter 90 degrees to the first measurement. Then record any gross pathology and the number of vessels in the cord.Place the placenta with the maternal surface facing up and identify four to five sampling sites randomly located in each quadrant of the placenta. Use scissors to remove the decidua, cutting away three to five millimeters from the maternal surface. Then collect a one to two centimeters cubed piece of villous tissue from each site.With 50 milliliters of cold, one molar PBS, gently wash the collected tissue. Then divide the tissue into several pieces from each sampling site and aliquot it. Add aliquots of 0.1 to 0.5 centimeters cubed tissue samples to five cryotubes and snap freeze the samples in liquid nitrogen.Add small pieces, 0.1 to 0.2 centimeters cubed in size, to previously prepared tubes containing 25 milliliters of RNA stabilization solution. For histological studies, add 0.5 centimeters cubed pieces to five cryotubes with 0.5 milliliters of OCT, top off with OCT, then mix the contents of the tubes. Freeze the samples at negative 80 degrees Celsius until further analysis.Blood samples from 209 mother-infant pairs have been successfully obtained using the four-vessel sampling method. Volume blood flow was measured in 128 of the samples. Complete four-vessel sampling and good quality flow measurements of both maternal and fetal vessels were obtained in 70 mother-fetus pairs.The four-vessel approach has been used to study placental transfer as exemplified by glucose, shown here. Significant arteriovenous glucose differences were found on both sides of the placenta, demonstrating an in vivo uteroplacental uptake of 0.29 millimoles per liter and a fetal uptake of 0.54 millimoles per liter. A median umbilical venous flow of 196.2 milliliters per minute was measured.Employing the Fick's Principle, a median fetal glucose uptake from the umbilical circulation of 0.10 millimoles per minute was calculated. Using the four-vessel method, the placental uptake of glutamic acid was studied, which is important as a fuel in metabolic pathways and for fetal-placental interconversion of amino acids. Placental uptake of glutamic acid from both the umbilical and the maternal circulation was observed.The in vivo release of progesterone by the placenta at term was also measured and a significant progesterone release into maternal circulation was found, demonstrating how the placental four-vessel sampling method can be used to detect substances released by the placenta. While attempting this procedure, it is important to underscore that volume blood flow measurements should be done by a fetal medicine specialist with experience in this type of sonography in order to obtain accurate measurements. Following this procedure, the blood samples can be analyzed to study placental transfer and uptake or release of the substance of interest, whether it is nutrients, metabolites, signaling molecules, exosomes, waste compounds, drugs, or toxins.Further, analysis of the placenta samples can answer additional questions concerning transfer regulation and placental metabolism. Altogether, this protocol demonstrates how coordinated collections of volume blood flow measurements, blood, and tissue samples can be achieved to gain an integrity in vivo model to study human placental physiology. After watching this video, an obstetrician should have a good understanding of how to safely collect arteriovenous blood samples on both sides of the human placenta during cesarean section.

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