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

Summary

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.

Abstract

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.

Introduction

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,

Protocol

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.

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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