ヒト胎盤生理学の統合研究への4容器サンプリングアプローチ

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.