Studies of the isolated perfused human placenta

Studies of the isolated perfused human placenta I.

Methods and organ responses

R. JONATHAN GOERKE, M.D . CHARLES M. McKEAN, M . D. ALAN J . MARGOLIS, M.D. MARY BETH GLENDENING, PH.D. ERNEST W. PAGE, M.D. San Francisco, California

3.7 ± 0.9 ml. per kilogram per minute. They obscrvcd vasoconstrictIOn from increascd oxygcn concentration and vasodilatation in the prescnce of hypoxia. Chesley 6 perfused an individual cotyledon in a LindberghCarrel pump primarily for the purpose of detecting the elaboration of toxic or antidiurctic materials. Pincus,1 Romanoff,S Troen and Gordon,D and Trocn 10 have chiefly concerned themselves with the production of steroids, whereas, Krantz and PanosH have designed a double perfusion apparatus which is intended for thc study of placental tranSfcr. In all but the last instance, the perfusions have been nonpulsatilc with high pressures or low rates of flow, and with pcrfusion of the fetal circulation only. A suitable preparation can be used advantageously for the following purposes: (1) Products synthesized by the placenta can be isolated and identified, thus avoiding confusion about their sites of origin. It must be shown, howcver, that the quantities recovered in perfusates exceed those which can be extracted from the fresh organ. (2) During synthesis. products can be labeled with radioactive isotopes which are not safe to lise on the intact human subjcct. (3) The organ may be exposed to specific enzymatic poisons, to drugs, or to periods of severe hypoxia to assist in the elucidation of its metabolic activities. For these purposes,

P R () r: RES S in understanding the functional aspects of the human placenta is understandably slow becausc the organ is relatively inaccessible prior to dclivery. Many of the elegant techniques cvolvcd for studying the functions of thc kidncy, hcart, liver, or lungs arc not feasible or safe in the pregnant subject. There are certain advantagcs, therefore, in the study of the surviving human placenta in vivo, but there are also many limitations. A number of investigators have utilized the isolated perfused fetal circulation of the placenta for a variety of purposes. In many instances, the primary purpose has been to study the pharmacologic reactions of the fetal vascular bed or of the umbilical vessels. H Nyberg and 'Ves tin~ were the first to re]Jort the use of heparinized human blood, and they estimated the oxygen consumption of the intact, term placenta to he From the Department of ObJtetrics and Gynecology, Unil' ersity of California School of M edicine. Supported by Grant RG-4702 from the Nationallmtitute .\· 0/ Ilealth, United States Public II eIllth SeTl,ice, and by a grant from the A .(.\()cia tion for the Aid "f Crippled Children. 1''''Jcnterl at the T we nty-sel'ent h Annual Meeting of the Pacific Coa .' t Ob.\ Ietrical alld Gynecological Society, }'osemile National Park, California, Sept . 28Oct. 1, 1960.

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Studies of isolated perfused human placenta. I 1133

however, isolated pieces of placental tissue or specific extracts are frequently superior. (4) The metabolism of the intact organ can be readily studied. (5) Finally, it might be hoped that the isolated and doubly perfused organ might be utilized as a model for the study of the placental transfer of gases and foodstuffs, but over a period of 5 years we have not been able to achieve the proper conditions necessary to accomplish this goal. The difficulties have been twofold: (a) our inability to duplicate the in vivo circulation of the intervillous spaces without injury to the villi, and (b) increases of capillary permeability, which were probably due to insufficient amounts of specific serum globulins in our perfusion fluids. The latter difficulty could be obviated if one had an inexhaustible supply of intact human plasma. The perfusion apparatus

The primary design of the apparatus was based upon that described for the perfusion of rabbit livers by Young, Prudden, and Stirman. 12 When the placenta is being used for a study of its synthetic products, it is ~onvenient to combine the perfusates emergIng from the maternal surface and from the fetal circulation in a single "lung" for oxygenation. The circuit employed for this purpose is shown in Fig. 1. For transfer stUdies, a duplicate gassing chamber may readily be installed and the 2 circulations kept separate. The mechanical heart is a latex finger cot driven by an air piston which has an adjustable stroke volume and stroke rate. The unidirectional flow is maintained by a pair of glass ball valves. The systolic and diastolic pressures are recorded proximally to the point of entry through the 2 umbilical arteries. The bubble trap contains a Van Slyke thermometer. The flow meter is a sirnple chamber containing 2 electrodes so spaced that the distance between the points corresponds to an exact volume of 30 m!. When the flow is shunted through this meter by occluding the "vein" distal to the meter with a solenoid valve, the time for filling the chamber is automatically recorded. If

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..." .'.. .'· .'.'· Fig. 1. Schematic diagram of the circuit as arranged for combined fetal and maternal perfusates.

the pressure on the arterial side should fall below 20 mm. Hg or rise above 200 mm. Hg (indicating some mechanical failure), an electronic device shuts off the pump. The venous return goes into a plastic chamber through which the gas mixture flows. The 95 per cent oxygen and 5 per cent carbon dioxide mixture is prewarmed and enters the "lung" through fritted glass funnels. It is necessary to utilize a small quantity of Dow anti foam at the top of the chamber to prevent foaming. Although the antifoam, a strong surface active agent, is sparingly soluble, it may well affect membrane permeability. T-tubes are inserted at various points so that samples may be withdrawn or glucose may be infused continuously without opening the door of the surrounding box. The entire circuit is contained in an airconditioned cabinet maintained at 37° C. and 90 per cent humidity by means of a 4

PLACENTA -62. (720 6M5) RATE.~/.l tsM/KG/HR.

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motor-driven fan, heater clement, and humidifier. A sliding Plexiglas door permits frequent observation or access.

Preparation of the placenta Placentas delivered vaginally were found to be heavily contaminated with bacteria and frequently lacerated ; so studies were limited to those placentas delivered at the time of elective cesarean sections. With the usc of an aseptic technique, the placenta was rinsed with warm saline solution, membranes were trimmed, and the umbilical cord was cut 1 inch from its insertion. The 2 umbilical arteries were mechanically dilated with tapered glass rods and plastic intravenous tubing was introduced into each artery and threaded past the cord to the fetal surface. Avoidance of the thick-walled umbilical arteries is absolutely essential for the achievement of high flow rates. A cannula was inserted into the vein which was then tied in place. The fetal circulation was then washed free of blood with warm Krebs-Ringer bicarbonate solution containing heparin, using a gravity flow with a pressure of 60 mm. Hg. The maternal surface was then placed upon a perforated plastic plate in a sterile basin. The basin was perforated with tubes on either side so that perfusion fluid could flow continuously over the decidual plate. On several occasions, multiple tubes were placed through the decidual plate into the intervillous space but in our hands this resulted

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in injury to the fetal capillaries. Every effort was made to begin the actual perfusion within 30 minutes after delivery.

Perfusion fluids The earliest perfusions were attempted with defibrinated, heparinized fetal cord blood. In each instance, obstruction of the fetal capillaries occurred within an hour. The obstruction may have been caused by cell sludging and appeared to be in the postcapillary limb, inasmuch as the rate of transfer of water from the fetal to the maternal circulation increased markedly. The difficulty was not due to ABO incompatibility or to gross clotting, but might have resulted from residual fibrinogen. The preliminary report by Krantz and Panosl l indicates that success with whole blood may be achieved by pretreatment of the blood with fibrinolysin. In any event, calculations revealed that red blood cells or hemoglobin were not

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Studies of isolated perfused human placenta. I 1135

essential for adequate oxygenation. The extrapolation of oxygen utilization data from Warburg experiments indicates an oxygen requirement of 3.5 m\. per minute per kilogram, which is very close to the direct observations made by Nyberg and Westin. s The solubility of oxygen in Ringer's solution at 37 0 C. is 23.8 ml. per liter, so that a flow rate of 74 m!. per minute would supply an adequate quantity for a 500 gram organ. Flow rates were usually several times this minimal requirement. The perfusion fluid which was finally . adopted had the following constitution: Each liter contained 80 m!. of the commercial tissue culture medium (Mixture 199) * described by Morgan, Morton, and Parker,I3 but did not contain bicarbonate; 50 Gm. of human albumin (which contained 1.2 per cent sodium chloride by weight)t; 0.8 Gm. of additional glucose; 75 mg. each of penicillin and streptomycin; and su pplementary electrolytes sufficient to bring the final concentrations to the following composition (in milliequivalents per liter) : Na 152, l( 5.9, Ca 5.9, Mg 2.3, Cl 103, and total P0 4 1.4. The human albumin is needed for the maintenance of oncotic pressure and for the transport of steroids. Despite the antibiotics, colonies of aerogenes bacteria could be cultured from the medium in many instances after 8 to 12 hours.

Metabolic factors One of the parameters of viability was the rate of glucose utilization. For any given Organ, this was remarkably constant, and Showed no signs of decreasing for at least 12 hours, which was the usual limit of perfUsion time. Fig. 2 illustrates the rate of ?isappearance of glucose from the perfusate In a typical experiment. The shaded area of the curve is due to the movement of glUcose into the intracellular compartment of the placenta which, in this instance,

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*Obtained from Microbiological Associates, Inc., 4846 ctlaesda Ave ., Washington 14, D . C. tOonated by CUller Laboratodes, Berkeley, California.

amounted to 56 per cent of the organ weight. The average rate of glucose utilization is 1 Gm. per Kg, wet weight per hour. Troen and Gordon u showed that citrate utilization is about one tenth of this value. The metabolism of the placenta results in the production of organic acids. This, or possibly the release of intracellular hydrogen ions, causes a steady fall in the pH of the perfusate. This is accompanied by a nearly parallel rise in the potassium ion concentration of the perfusing medium (or fall in the pK+) as illustrated in Fig. 3. During this experiment, . no adjustments were made, but it is ordinarily necessary to adjust the pH hourly by the addition of sodium bicarbonate. When bilirubin was introduced into the fetal circuit, there was a slow but definite transfer to the maternal circuit in every instance. The absolute rates varied from one organ to another and are probably meaningless in terms of the in vivo situation. The important observation was the complete absence of any conjugation with glucuronic acid (i.e., formation of the soluble or direct bilirubin ) . Neither the fetus nor the placenta appears to possess this capability.

Response of the fetal circulation to variations of oxygen and selected drugs The typical response of the fetal vascular bed to hypoxia or to high oxygen is shown in Fig. 4. In this instance the placenta weighed 488 grams, the perfusion medium contained 0.2 per cent fetal serum in place of the human albumin, and the stroke rate was 120 per minute throughout. The apparatus is so designed that vasoconstriction produces both a rise in the systolic and diastolic pressures and a decrease in the flow rate. It can be seen that hypoxia results in vasodilatation and high oxygen produces transient vasoconstriction. There is indirect evidence that the fetal vascular bed of the placenta behaves in a similar fashion in utero. After studying the effects of oxygen administered to pregnant women at term, PrystowskyH concludes: "In the normal parturients who received oxygen

1136 Goerke et 01.

there was a rise in p02 on the maternal and fetal sides, but the latter to a lesser degree. In other words, what the fetus apparently does is decrease the size of the capillary bed, so that he gets what he wants at the pressures he desires. These findings are not at all surprising, for one would expect that the fetus in utero would have some means of expanding and contracting his exposed capillary bee\." The fetal vessels respond to minute doses of histamine or serotonin by vigorous contraction, but we were unable to confirm earlier reports about a response to epinephrine and norepinephrine . The response to the injection of 1 mg. of histamine into the fetal circuit is illustrated in Fig. 5 . In this experiment, the organ weight was 848 grams, the volume of perfusion fluid on the fetal side was 750 mI., the pH was 7.8, and the pulse rate was constant at 120 per minute. On occasions, the placenta would lose its responsiveness to serotonin or histamine while maintaining its normal glucose utilization.

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The problem of viability or functional integrity, therefore, is complex and can be answered only with respect to each individual function in question. Summary

Methods of perfusing the isolated, surviving human placenta under nearly physiologic circumstances are presented. During periods up to 12 hours, the rate of glucose utilization is about 1 Gm. per kilogram per hour. The production of organic acids causes . a steady rise in hydrogen ion concentration with a parallel rise in potassium concentration. No conjugation of bilirubin with glucuronic acid could be observed. Hypoxia results in vasodilata tion with increased rates of flow through the fetal vascular bed, whereas high oxygen pressures cause the reverse. It is believed that similar respoI}ses occur in utero. The fetal circulation is not responsive to epinephrine or norepinephrine but responds with vigorous vasoconstriction to serotonin and histamine.

REFERENCES

1. Kiistner, H., and Siedentopf, H.: Arch. Gynlik. 138: 131, 1929. 2. Veda, K.: Jap. J. Obst. & Gynec. 14: 225, 1931. 3. von Euler, V. S.: J. Physiol. 93: 129, 1938. 4. Eliasson, R., and Astrom, A.: Acta pharmacol. 11: 254, 1955. 5. Nyberg, R., and Westin, B.: Acta physiol. scandinav. 39: 216, 1957. 6. Chesley, L. C.: AM . J. OaST. & GVNEC. 58: 159, 1949. 7. Pincus, G.: In VilIee, C. A., editor: Transactions of the third conference on gestation, New York, 1957, Josiah Macy, Jr., Foundation, p. 91. 8. Romanoff, E. B.: In Lloyd, C. W., editor: Recent progress in the endocrinology of re-

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production, New York, 1959, Academic Press, Inc., p. 283. Trocn, P., and Gordon, E. E.: J. Clin. Invest. 37: 1516, 1958. Troen, P.: In Lloyd, C. W., editor : Recent progress in the endocrinology of reproduction, New York, 1959, Academic Press, Inc., p. 299. Krantz, K. E., and Panos, T . C.: A . M. A. J . Dis. Child. 98: 674, 1959. Young, M. K., Jr., Prudden, J. F., and Stirman, J. A.: J. Lab. & Clin. Med. 46: 155, 1955. Morgan, J. F., Morton, H. J., and Parker, R. C .: Proc. Soc. Exper. BioI. & Med. 73: 1, 1950. Prystowsky, H.: AM. J. OaST. & GVNEC. 78: 483, 1959.