130 Lemtis et al., Rapid procedure for testing the integrity of the maternal-fetal barrier

J. Perinat. Med.2 (1974) 130

A rapid procedure for testing the integrity of the maternal-fetal barrier in spontaneously delivered placentas

H. Lemtis, H. Kirchner

Department of Obstetrics and GynecologyKlinikum Steglitz of The Free University of Berlin(Director: Professor Dr. G. Hörmann)

Received February 27, 1974. Accepted April 25, 1974.

The study of the maternal-fetal passage of drugsand other alien substances, particularly those ofpotentially hazardous substances of the occupa-tional environment (with quantitative determin-ations in the blood of the fetal side of the placentalvascular System) is an important consideration ofperinatal medicine. Therefore, numerous authors(reviews by LEMTIS [18], GAGEL [5], and KIRCH-NER [10]) have attempted with their contributionsto solve the difficult problem of the physiologi-cally correct perfusion of the placenta. In spite ofremarkable advances in the field, the bilaterallong-term in vitro perfusion of the humanplacenta has not yet been accomplished satis-factorily. In part this is due to the failure ofsystematically studying the potential interferencesoccurring during perfusion with blood. On theother hand, investigators may have been impededin their study by the uncertainty äs to whether theavailable placenta was injured or intact. Overseveral years we have developed in collaborationwith GAGEL and following the experimentaldesigns of KRANTZ and coworkers [14, 15, 4, 8,9, 13] NESBITT et al. [21], äs well äs HAMRINet al. [7] a modern apparatus equipped for thebilateral long-term perfusion of spontaneouslydelivered human placentas [20], stressing thestudy of both the most favorable experimentalconditions äs well äs possible interfering factors(see also KIRCHNER and LEMTIS [11, 12], Duringthese experiments we designed a test whichenables us to determine before the Initiation of

Cürriculum vitae

HORST GÜNTHER LEMTISwas born in 1923. Hestudied Medicine and Scienceat the University of Hamburgand Kiel and obtained bisM. D. degree in 1954 atKieL Following bis residencyin Obstetrics and Gynecology attbe Universities of Marburgand Göttingen, he was ap-pointed to the senior staff oftbe Second Obstetric-Gyne-cology Service of the FreeUniversity, Berlin, in 1963.University lecturer 1967; Professor and Head of the Section öfExperimental Gynecology at the Department of Obstetrics andGynecology of the Klinikum Steglii^ of the Free University,Berlin since 1969.

a lengthy perfusion experiment within a fewminutes whether the maternal-fetal barrier ofthe experimental placenta is intact. j

1. Methode

We started with the basic assumption that with a idefective maternal-fetal barrier any dye present in 'the fetal vascular System should be transmitted lvery rapidly into the maternal placental circu- ;lation because of the pressure gradient from :80—90mm Hg in the fetal-placental to 10 to l20 mm Hg in the maternal intervillous capillary .}System [23, l, 3]. For this purpose, every ex-

J. Perinat. Med. 2 (1974)

Lcmtis et al., Rapid procedure for testing the integrity of thc maternal-fetal barrier 131

perimental placenta was placed in the "artificial"uterus of our experimental apparatus [20]. Thefetal vessels were filled with warm normal salinesolution and 2 ml aliquots of a 0.2% solution ofEvans Blue were injected at 2 minute intervalsinto the fetal placental circulation. The possibletransplacental passage of the dye was registeredphoto-electrically at the location of the maindrainage ("Collecting vein") of the maternalblood from the placenta in the artificial uterus.To this end our previously described experimentaldesign was augmented by a universal oximeter.The dye concentration of the circulating fluid wasmeasured with an ear electrode by the transmissionprinciple. The resulting dye dilution curve wasrecorded simultaneously with the perfusionpressure and the minute volume continuouslythroughout the entire experiment on a compen-sation linear recorder. The choice of varioussensitivities allowed the recording of evenminute changes in the variables. A continuouslyadjustable bridge circuit allowed the adjustmentof the units of measure of the apparatus to thecirculating fluid of the individual experiments.BLEYL [2] had demonstrated that the fetal-maternalbarrier is always penetrated after a substantialincrease of the peifusion pressure. This fact was

of great importance for our experiments. There-fore, it was attempted to determine the limit ofintravascular fetal placental pressure at whichfetal maternal passage of a dye occurs. Sinceaccording to the findings of GAUER [6] pressureand minute volume are directly dependent oneach other analogous to OHM'S law, we increasedthe intravascular pressure by increasing thevolume simultaneously with the EVANS Blueinjections at 2 minute intervals.

2. MaterialThe placentas used for establishing the techniquehad been delivered spontaneously from healthymothers after normal pregnancies and showed nogross injuries. Each placenta was prepared for theexperiment immediately after birth.

3. ResultsTab. I demonstrates the correlation between thepassage of dye from the fetal into the maternalplacental circulation and perfusion pressure andthus simultaneously from the volume. Damagedplacentas had a passage of EVANS Blue at theinitial pressure of 50 mm Hg. Thus the barrierbetween the two vascular Systems in these

Tab. I. Correlation between fetal-maternal passage of EVANS Blue and fetal-placental intravascular pressure. The increasein pressure was accomplished by increasing the minute volume. The passage of dye occurred at bold face values.

Placentano.

123456

7

89

10

11121314

Pressureincrease(mm Hg)

"aj[jgΛo>·ωI6

M-CO

%

i

3

50

303025203530

25

403030

30252025

60

354530254035

35

454035

35303030

70

405535354540

50

654540

40404540

80

456050457065

55

705060

80505550

90

707560509080

60

857080

95607065

100

751208580

12595

80

1007590

10080

10070

120

9512590

115130100

95

11590

100

11510010595

140

120140105130145115

110

130110125

140110120105

160

150145135150160120

125

145135140

155120135130

Remarks

placentadefectiveplacenta

defective

placentadefective

J. Perinat. Mcd. 2 (1974)

132 Lemtis et al., Rapid procedure for tcsting the integrity of the maternal-fetal barrier

placentas was defect. In intact placentas thepassage of the dye occurred only after a pressurerise to 100—160 mm Hg (see Tab. II). Figs. land 2 demonstrate the difference between aplacenta with intact and one with defectivematernal-fetal barrier.

Tab. II. Pressure and minute volumes at which fetal-maternal dye passage occurred in intact placentas. S (x) =sum of all measurements; = arithmetic mean.

S(x)X

median80% ränge

Pressure(mm Hg)

1300118.2120

100—140

Minute volumeml/min

1165105.9105

80—120

Fig. 1. Intact placenta. Dye dilution curve of EVANS Blue(below) which has been injected at two-minute intervalsinto the fetal placental vascular System simultaneouslyincreasing the intravascular pressure. Above: the recordingof intravascular pressure, middle: registration of minutevolume.

Fig. 2. Placenta with defective maternal-fetal barrier. Dyedilution curve following injection of EVANS Blue into thefetal placental vascular System recorded photo-electricallya t the "main vein" of the maternal placental circulation.

4. Discussion• ;

In the great majority of the placentas with intactmaternal-fetal barrier which we have examined,the passage of Evans Blue from the fetal intothe maternal circulation occurred only withunphysiologic high intravascular pressuresat a mean of 120 mm Hg (see Tabs. I and II).Since both vascular Systems were closed, theinjected dye remained constantly in the fetalplacental circulation with pressures under 100 mmHg. Because the oximeter was not recalibrated to2ero following the injections of EVANS Blue at2 minute intervals, the curve must demonstrate astep-wise rise (Fig. 1).Completely different results were found inplacentas with a defective maternal-fetal barrier.Passage of dye from the fetal into the maternalcirculation occurred already at the lowestinitial pressures at the beginning of theexperiments. The graphic depiction of the ex-tinction curve shows a typical dye dilution curve(Fig. 2). Because the curves of an intact and adefective placenta are distinctly different fromeach other and the brief experimeiit can becarried out very quickly, it is ideally suited fortesting the maternal-fetal barrier for its integrity.With the aid of this test we were able to demon-strate [19] that contrary to some assumptionsonly 30% of the spontaneously delivered.placentashave a defective maternal-fetal barrier.As demonstrated in Tabs. I and II the pressurelimit at which even placentas with intact maternal-fetal barrier will always have a dye passage fromthe fetal into the maternal placental circulationranges from 100—160 mm Hg. Possibly eachplacenta has its own threshold regarding this"opening pressure". Apparently, in each placentastomata in the area of the maternal-fetal barrierare opened when the individual "maximalpressure" is reached. These gaps in the capillärywalls in the fetal-placental vacular System havebeen demonstrated by PANIGEL [22] after theexistence of stomata had been suspected by ussince the experiments of BLEYL [2]. This authorhad utilized the wash-out technique describedby us in 1952 [16] and 1955 [17] and had suc-cessfully accomplished a complete exsanguinationof placentas if following the introduction of glass

J. Perinat. Med. 2 (1974)

Lerntis et al., Rapid procedure for testing the integrity of the maternal-fetal barrier 133

cannulas into the umbilical arteries and closure of abnormally high, intravascular pressure, all com-the umbilical vein, the placentas were perfused ponents of blood may pass through certain gapswith pressures of over 100 mm Hg. Thus he in the capillary walls of the fetal vascular Systemhad unknowingly proven that with sufficient, i. e. which are not normally open.

Summary

In the course of several years we developed in collaborationwith GAGEL based on three American models a modernapparatus for the bilateral long-term perfusion ofnormally born human placentas, stressing the study offavorable experimental conditions äs well äs that of possibleinterfering factors. Because the bilateral long-term per-fusion of placentas with blood is costly, it should beascertained that a planned experiment is worthwhile (i. e.that the experimental placenta is intact). Thus far no practi-cal experimental design had been described. The authorshave now developed a test allowing to determine within

a few minutes before the Initiation of a lengthy ex-periment whether the maternal-fetal barrier is defective.To this end the fetal vascular System of the placentaplaced in the "artificial" uterus is filled with warm normalsaline solution to which Evans Blue is added. With asmall photo-electric attachment at the "collecting vein"of the maternal circulation similarly filled with normalsaline it can be determined immediately whether theplacenta in question is damaged. Normally the maternal-fetal barrier is not passed by EVANS Blue. The test detectedeven minute defects.

Keywords: Diaplacental passage, dye passage (transplacental), maternal-fetal barrier, penetration, permeability, placentalpassage, placental perfusion, rapid placental test.

ZusammenfassungEin Schnelltest zur Prüfung der Mutter-Kind-Schrankespontan geborener Plazenten auf Unversehrtheit.Im Verlaufe mehrerer Jahre wurde gemeinsam mit GAGELin Anlehnung an 3 amerikanische Modelle eine mit mo-dernsten Geräten ausgestattete Anlage zur beidseitigenDauerperfusion geborener menschlicher Plazentenentwickelt, wobei auf das Studium der günstigsten Ver-suchsbedingungen sowie der möglichen Störfaktoren be-sonderer Wert gelegt wurde. — Da die doppelseitigeDauerperfusion von Plazenten mit Blut sehr kostpielig ist,muß gesichert sein, daß ein geplantes Experiment auchlohnt, d. h. daß der zu untersuchende Mutterkuchenintakt ist. Hierzu gab es bisher noch keine in der Praxisbewährte Versuchsanordnung. Die Autoren entwickeltenjetzt einen Test, der es gestattet, vor Beginn eines großen

Schlüsselwörter: Diaplazentare Passage, FarbstofFpassage (transplazentare), Mutter-Kind-Schranke, Penetration, Perme-abilität, Plazentapassage, Plazentaperfusion, Plazenta-Schndltest.

Experiments innerhalb weniger Minuten festzustellen,ob die Mutter-Kind-Schranke defekt ist. Dazu wirdder fetale Gefäßapparat der in den „künstlichen" Uteruseingebrachten Plazenta lediglich mit temperierter phy-siologischer Kochsalzlösung aufgefüllt, der EvansBlue zugesetzt wird. Mittels einer kleinen photoelek-trischen Vorrichtung läßt sich dann an der „Sammelvene"des ebenfalls mit Kochsalzlösung beschickten mütter-lichen Kreislaufs der Plazenta sofort feststellen, ob derFarbstoff aus den feto-plazentaren Strombahnen über-getreten ist, d. h. ob der betreffende Mutterkuchen ver-letzt ist. Normalerweise wird die Mutter-Kind-Schrankevom EVANS Blue nicht passiert. Mit dem Test werdenselbst kleinste Defekte erfaßt.

R<§sumeUn test rapide pour Pexamen de l^tat de la barrierem&re-foetus dans les placentas humains nes spontan£-ment.Apr£s plusieurs annees d'etudes realisees avec le concoursde GAGEL et s'appuyant sur trois modeles americains, lesauteurs du present article ont mis au point un systfcmedoto d'appareils ultra-modernes pour perfusionscontinu£es ambilatorales de placentas humains nes. Unsoin particulier a ete porte sur Petude des conditionsoptimales d'experimentation et des facteurs de troubleseventuels. La perfusion placentaire continue ambilateraleavec du sang etant tres coüteuse, il faut lui garantir le

maximum de chance de succes, c. a. d. s'assurer au prealableque le placenta a examiner est intact. Or, il n'existait aucuntest sur jusqu'a ce que les auteurs de cet article aient mis aupoint une mothode qui permet de detecter en quelquesminutes avant l'intervention une d£fection eventuellede la barriere m&e-foetus. Pour cela, le Systeme vasculairefoetal du placenta place dans Puterus «artificiel» est remplid'une solution saline physiologique tempe~ree, ad-ditionne'e de Bleu d'Evans. Au moyen d'un petitdispositif photo-e"lectrique fixe sur la «veine principale»de la circulation maternelle du placenta remplie aussi desolution saline physiologique, il est alors possible d'ob-

J. Pcrinat. Mcd. 2 (1974)

134 Lemtis et al., Rapid procedure for testing the integrity of the maternal-fetal barrier

Server immediatement si le colorant a traverse" la barriereplacentaire entre les circulations maternelle et foetale,c. a. d. si le placenta maternel concerne comporte deslesions. Normalement en effet, le Bleu d'EvANS ne franchit

pas cette barriere entre la mere et le foetus. Le test permetd'enregistrer meme les plus petites lesions de la paroi descapillaires du foetus.

Mots-cles: Barriere mere-foetus, passage de colorant (transplacentaire), passage diaplacentaire, passage placentaire,penetration, perfusion placentaire, permeabilite, test placentaire rapide.

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This work is dedicated toProfessor HÖRMANN on the occasionof his sixtieth birthday.

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Prof. Dr. med. H. LemtisFrauenklinik im Klinikum Steglitzder Freien Universität BerlinHindenburgdamm 30D-1000 Berlin 45/Germany

J. Pcrinat. Med. 2 (1974)