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Perfusion of the human placenta in vitro
FETUS AND NEWBORN
Perfusion of the human placenta in vitro Study of the biosynthesis of estrogens
J. VARANGOT, M.D. L. CEDARD, PH.D. S. YANNOTTI, M.D. Paris, France
Material and methods
BEcAusE of its anatomical situationa single vascular pedicle that is easily accessible and an anatomically closed fetal circulatory system--the human placenta is an organ that is particularly suitable for perfusion. In France, as early as 1949, one of us together with Thomas 36 were the first to carry out perfusion of the placenta. Abroad, a number of authors have dealt with this problem, particularly, Pearlman, Pearlman, and Rakoff, 26 Levitz, Condon, and Dancis, 19 and Troen 3 H 4 who undertook perfusion of the placenta in vitro. In Sweden, Cassmer6 and Diczfaluzy and coworkers3· 22 • 23 were able to experiment on the placenta in vivo. Having taken up our previous experiments of 19598 - 11 we have at present performed iOi perfusions of the human placenta in vitro. Under these experimental conditions, we have been able to study the biosynthesis and metabolism of estrogens.
The perfusions of human placenta were carried out in vitro. Out of 10 l perfusions performed, 96 were effected on placentas at the time of delivery. The duration of the perfusion was 4 to 6 hours. Preparation of the placenta. In mo~t of the cases the placenta was derived from a natural confinement. Immediately after delivery it is collected aseptically and freed of its membranes. The umbilical blood vessels are dissected out over a length of 5 em. The two umbilical arteries were catheterized by means of perfusion needles with oliveshaped points fixed by a ligature and a Murphy artery clip (Fig. 1). The umbilical vein was then cut across and 10 mi. heparin was injected via the arteries. The perfusion equipment (Fig. 2). Briefly it can be described as 3 difl'erent circuits. The perfusion circuit. It is set in motion by a hand pump that ensures a pulsatile movement the pressure and speed of which may be adjusted. The perfusion liquid consists of a liter of Krebs-Ringer buffer solution at pH 7.5, to \vhich 1 Gm. of glucose, 0.25 mg. of papaverine, and antibiotics have been added. Thw..
From Clinique obstetricale et gynecologique-F aculte de M edecine Universite de Paris. With the support of the "Centre National de [a Recherche S cientifique ."
534
Volume 92
Perfusion of the human placenta
535
Number 4
Fig. 1. Catheterization of the two umbilical arteries by means of needles with olive-shaped points attached in position by Murphy artery clip. The perfusion liquid is seen flowing through the umbilical vein.
Fig. 2.
the fetal circulation is washed out. Very rapidly 200 mi. of preserved human blood is added. The perfusion outflow is from between 30 to 50 ml. per minute. The pressure varies from 20 to 30 mm. Hg. The oxygenation circuit. This is connected to the perfusion circuit at the exit of the umbilical vein. It consists of a mixture of 0 2 + C0 2 in proportions of 95 per cent and 5 per cent, respectively. The presence of C0 2 decreases vasoconstriction. So as to ensure satisfactory oxygenation the perfusion liquid is maintained in a thin layer by a rotating mixer, on its exit from the umbilical vein. The heating circuit. The temperature is kept constant by means of a thermostatic pump that maintains the heating circuit and placenta at 37° C. Analytical techniques. We determined estrone, estradiol, and estriol in both their free and combined forms. These determinations were performed by fluorometry following partition chromatography on a celite column according to the technique developed by one of us 7 which enables the fractional estimation of estrone. estradiol-17 ,8, and estriol. These determinations were carried out on 50 mi. of fluid sampled at regular intervals during the experiment. The quantities of estrogens were calculated by comparison with control curves and then related to the total volume of the perfusion liquid. Checking of the analytical techniques. We are able to check the precision, accuracy and specificity of our analytical technique as applied to the perfusion liquid. PRECISION. Measurement of the precision of the technique was calculated by Snedecor's method 31 in which S is determined from the difference between estimations carried out in double in a given series of analyses where d = the difference between the two results obtained in the doubles, and N = the number of analyses carried out in double. 1. For values between 0.5 p.g and 55 p.g (experiment without the addition of hor-
536
Varangot, Cedard, and Yannotti
mones): estrone ( 10 analyses) s = 0.28 /Lg; estradiol ( 10 analyses) s = 0.39 JLg; and estriol ( 11 analyses) s = 0.85 JLg. 2. For considerably higher values of between 100 and 1.400 vg (i.e., following the addition of hormones) the precision was obviously less: estrone ( 3 analyses) s = 4.4 vg; estradiol ( 4 analyses) s = 10.3 J-tg; and estriol (4 analyses) s = 11.3 vg. ACCURACY. The accuracy of these determinations was measured by making use of an internal standard, i.e., the recovery of pure hormones added to the sample. As in the case of the whole blood, recoveries were fairly low. Following the addition of pure hormones at the beginning of the analyses, the average percentage recovery is: for the estrone 50 ± 23 per cPnt ( 8 experiments); for the estradiol 49 ± 16 per cent (8 experiments); and for the estriol 69 ± 14 per cent ( 10 experiments) . SPECIFICITY. The substances measured showed the same chromatographic behavior as the pure estrogens. The maximum excitation and emission of fluorescence are identical, 455 and 480 to 490 miL for the pure hormones and the extracts, respectively. The extracts submitted to chromatography react with Brown's reagents (diluted S0 4 H~, quinol) and the absorption spectra of these reactions show the same maxima as those of the corresponding pure hormones. The estriol isolated from the perfusion liquid reacts with David's reagent* by producing a blue color of the same maximum wave length as the standard hormone. R. Ozon was kind enough to carry out the microsublimation of estrone and estriol on a glass slide according to Breuer and Kassau's technique'' in a pyrex cell (diameter 16 mm. and height 10 mm.) fitted on to a Reicher hot plate. In the case of estrone and estriol he was then able to observe the formation of typical microcrystals and was then able to determine the melting points that were found to be very close of the standard hormones. *Modification of Alexson and Diczfaluzy. 1
Am .
Estriol Temperature of mi crosublimation Melting point Estrone Temperature of microsublimation Melting point
Extract
.J.
June I'>. l'Jii:i Ob't. & Gynt·c.
Pure hormone
160° to 250° Ul0° to 240" 275° 280° Extract
Pure hormon<'
150° to 200° 150° to 200° 246° to 2+8° 2!6° to 2511'
W c were also able to carry out paper chromatography: Estrone and estradiol-17,8 in Kushinsky's system 17 2 benzene/! hcptanc;l.S mcthanol/1.5 water. The chromatographic analysis being carried out for 4 hours on Whatman paper No. 1 at room temperature. The rf's obtained were identical both for the pure hormones and the extracts, as well as positive Barton reactions ( rf estrone = l by definition; rf estradiol = 0.6 to 0. 7). The fraction corresponding to the estriol was submitted to chromatographic analysis in a Bush system: 2 benzene/1 methanol/1 water. Chromatography lasted 36 hours on Whatman paper No. 20. Here again we observed an identical rf to that of the pure hormone (the rf differs hy as little as 0.005) and a positive Barton reaction. This chromatographic process was either carried out directly or following elution of the starting point of a chromatogram in the system: 2 henzene/1 heptane/1.5 methanol/1.5 water. We studied the absorption spectra in ultraviolet light (UNICAM SP 500) of the three purified estrogenic fractions, after leaving them in the dark for 2 hours in the presence of concentrated sulfuric acid (micromethod according to Burnstein and Lcenhardt) and we observed in the case of these 3 fractions identical maximum wave lengths to these of the pure hormone despite several quantitative differences. Similarly, examination of the infra-red spectra shows the definite presence of estrone, estradiol, and estriol. However, the presence of some additional peaks and the weak amplitude of several typical maximums did not allow us to conclude as to the absolute pureness of our extracts, even after two successive partition chromatographs. Checking of the efficiency of perfusion.
Volum(' 92 1'\muher 4
Perfusion of the human placenta
Two essential conditions are required in order to effect an efficient perfusion: presence of a fetal circulatory system and survival of the perfused placenta. Presence of a fetal circulatory system is substantiated by several facts. From the mechanical point of view, at the very onset of perfusion the liquid escapes via the umbilical vein, sometimes in the form of a jet, whereas the arteries that are visible under the amnion become turgescent and pulsatile. In our opinion the rapidity of the venous return is not solely due to the presence of superficial arteriovenous anastomosis, which would reflect an incomplete perfusion. Indeed, by introducing stains into the perfusion liquid (e.g., Prussian blue, methyl green) the colored liquid is subsequently found in the histological examinations, in the blood vessels of the villous space. Nevertheless, perfusion is not entirely physiological. Indeed, considerable pressures of approximately 20 to 30 mrn. Hg are required to insure the venous return. These decrease during perfusion but are always definitely higher than the figures generally given. Furthermore, sometimes at the onset of perfusion slight ederna of the placenta may be observed. Finally, there is nearly always intercommunication between the fetal and maternal circuit during the perfusion
process; the liquid transudes via the maternal placental surface. But it is not known whether this, in fact, occurs in vivo. In any event these inconveniences are at present impossible to avoid. They have already been mentioned in a considerable number of publications on perfusion of the placenta. We only hope that they cause but slight disturbances in the endocrine activities of the placenta. From the chemical point of view, the concentration of estrogens increases in the perfusion liquid. Analyses carried out at the first and fourth hours demonstrate this. One hour following the beginning of perfusion we determined the estrogens. The average over-all value of 81 perfusions is assessed as: average over-all value, 45 p,g per liter (estrone, 28 per cent; estradiol, 8 per cent; and estriol, 64 per cent). There is prevalence of free estrogens over conjugated and of estriol over estrone as well as estradiol (Fig. 3). It will be seen that these proportions are in agreement with those found in the tissues of the placenta by various authors.l3, 14 • 24 • 29 But in the first hour we noticed considerable individual variations that bear no relation to the weight of the placenta. That is why during the first hour, in each experiment, we carried out a preliminary basic detern1ina-
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Fig. 3. Estrogens (expressed in ~£g) found in the perfusion liquid, one hour after the beginning of the experiment (average of 81 perfusions).
J
53B Vorangot, Cedard, and Yannotti
tion which is deducted from the final results. In six control experiments the determinations carried out at the fourth hour show an increase tljat is statistically significant as far as the estrogens are concerned, both for
10
Ill
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esh-ad1ol es~rone es~riol ' - - - . tolllbinf!d - - - - /
&nd
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bound.
Fig. 4. Placentas without addition of hormones (average of 6 experiments).
the free estrogens and the combined estriol. But the combined estrone and estradiol that only increases to a small extent are not found in the fresh placenta either (Fig. ! ) . Hence, it may be inferred that, during perfusion, the placenta liberates a quantity of estrogens that is higher than that destroyed within the system and that it contains at least 60 per rent of the free t'~ triol. This liberation, the progressive nature of which substantiates the actual extent of prrfusion, also, in our opinion, reflects the survival of the placenta. This survival is even more clearly demonstrated by the continuance of thr enzyme activities in the perfused placenta. lndced. the placenta is able to transform the estrogens supplied to it. In sc\·en experiments we added 20 mg. of rstrone and estradiol, in 5 cases, and 10 mg. of estrone and estradiol, in 2 cases, to the perfusion liquid following a preliminary sampling during the first hour. The determinations of the fourth hour show a considerable increase as far as the estradiol and particularly the estrone arc concerned whereas the estriol barely incrrases (Fig. 51. These results are identical whether estrone
514 --w
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F.ig. 5. Perfusions with addition of pure hormone; estradiol-17/3 ( 10 or 20 m~.).
Volume
9~
Number 4
or estradiol is added and this goes to preve the reversibility of the reaction: estrone ~ estradioL Identical results were obtained by Ryan and EngeF 7 with sections of placental tissue, by Levitz, Condon, and Dancis 20 in perfused cotyledons, and more recently by Troen on whole perfused placentas 33 " 34 and by Bolte and co-workers 3 during the perfusion of placenta in situ. This interconversion is effected by a placental 17 (J hydroxysteroid dehydrogenase. 18 • u, 37 - 39 The presence of an active enzyme thus testifies as to vitality of the placenta during these long-term perfusions. As a control experiment we effected two perfusions in the absence of a placenta. Under these conditions only a weak reduction of the estrone ( 1/10) occurred and no formation of estriol whatsoever. Results
The biosynthesis of estriol. In the light of our experiments it seems that the classical process of biosynthesis of estriol from estrone and estradiol would be quantitatively quite small. In 4 experiments we added 20 mg. of estrone or estradiol to the perfusion liquid. The quantity of estriol obtained is more than double that observed in the perfusions of the placenta without the addition of hormones. Statistical calculation shows that the difference between these two average mcreases (4 7.9 ± 14.5 fig and 19.9 ± 8.8 fig, respectively) is significant. In three experiments besides the estradiol we added 20,000 I.U. of chorionic gonadotropin. In this case the estriol is liberated in much larger quantities. The average increase in these three experiments was 96.5 ± 17.8 fig. Here again, this statistical calculation shows a marked significant difference as compared to the perfusions in which estradiol alone was added. The addition of chorionic gonadotropin (20,000 I.U.) without the addition of hormones causes no modifications of the liberated estrogens during the perfusions. Similar results were obtained by Troen 33
Perfusion of the human placenta
539
who, following the addition of chorionic gonadotropins and estradiol to the perfusion iiquid, found a peak of radioactivity and fluorescence after countercurrent distribution with a partition coefficient identical to the theoretical coefficient of the estriol. Hence, it is possible by the administration of estradiol alone or associated with chorionic gonadotropins to stimulate the secretion of estriol via the perfused placenta. This is the classical process in the human organism for the biosynthesis of estriol by hydroxylation at c16 of the estradiol. But, the quantity of estriol thus liberated is barely more than that observed in the fourth hour of a control perfusion with no additional hormones. And the percentage of estriol (maximum 5 per cent) is very different from the characteristic physiological percentage found in the urine at the end of gestation (95 per cent). In fact, in younger placentas, Bolte and co-workers 3 found no hydroxylation whatsoever at C 16 in placentas 17 to 20 weeks old, perfused in situ for several minutes with estradiol. During further perfusions in situ, the same authors observed no stimulating action of the chorionic gonadotropins. Hence, we then studied a second metabolic process which excludes the estradiol and in which the estriol is secreted directly from steroids previously hydroxylated at carbon-
16.z7 In four experiments the addition of 20 mg. of 16aOH estrone acetate causes 3 hours afterward an average increase of 1.400 fig of estrogens, collected in the perfusion liquid. Ninety-nine per cent of this increase consists of free estriol (Fig. 6). A more prolonged study with only 10 mg. of 16aOH estrone showed that this transformation is gradual and only reaches its maximum 4 hours after the addition of this substance. The same experiment carried out without the placenta does not liberate any estriol. In 4 experiments we introduced 10 mg. of 16aOH androsterone or 16aOH testosterone in the perfusion liquid together with 20.000 I.U. of chorionic gonadotropins. A considerable but very gradual increase of estriol
540 Varangot, Cedard, and Yonnotti
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is observed, up to 500 JJ.g 6 hours following addition of the hormones (Fig. 7\. The identical yield of these 2 substanc:es did not surprise us. Indeed, there is present in the placenta a 17 f3 hydroxysteroid dehydrogenase capable of transforming testosterone to .6. 4 androstene 3,17 dione as well as estradiol to estrone. 10 A very important factor for the success of these experiments is the necessity of the simultaneous addition of chorionic gonadotropins. Indeed, experiments carried out 'lNith simply the addition of neutral steroids hydroxylated at carbon-16 proved negative. The estriol having undergone but a very slight increase throughout the experiment, i.e., the 15 fJ.g usually found in the placenta following perfusion without the addition of hormones. Fig. 8 summarizes the results obtained during the different types of experiments. It shows the average increase of free estriol in the perfusion medium, this valnc
Fig. 6. Perfusion of placenta with addition of 16a hydroxy.estrone.
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Fig. 8. Increase in free estriol during different types of experiments. A, Control placenta without HP. B, +20 mg. of estrone or estradiol; C, +20 mg. of estrone or estradiol +20.000 I.V. HCG; D, +10 mg. of 16a0H testosterone or 16aOHA,, androstenedione; E, +10 mg. of 16aOH testosterone or 16aOHA, androstenedione +20.000 I.U. HCG: F, +20 mg. of 16<>0H estrone. ·
Volume 92 Number 4
Perfusion of the human placenta
being obtained by subtracting the quantities found at the end of the first hour in order to eliminate the variations clue to the content of the placenta itself. The problem remains of the origin of the steroids hyclroxylatecl at 16. I ncleecl, they are not normally synthetizecl in the placenta. Nevertheless, they have been identified.in the urine during pregnancy.15, 21 Neher and Stark 25 isolated 16aOH testosterone from the placenta m the proportion of 35 p.g per kilogram. Quite possibly the fetus 1s capable of effecting this hydroxylation at 16 and of providing the placenta with estrogen precursors. Zander'13 demonstrated that progesterone 4 CH when introduced into the umbilical vein of the human fetus is partially converted into 16a hydroxyprogesterone. Villee and associates 38 established the occurrence of a 16a hydroxylation by the fetal adrenal tissue in vitro as well as by the hyperplastic adrenal tissue·10 - 42 The biosynthesis of estrogens from neutral or substituted steroids. In a series of 35 perfusions we added 10 mg. or sometimes 20 mg. of steroids at 21, 18, and particularly 19 neutral or substituted carbon atoms, to the circulation liquid. Two or three experiments were undert;1ken for each steroid in
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order to eliminate vanatwns due to experimental h azards, and the results obtained were compared with those obtained by the use of radioactive steroids with preparations of placental microsomes + TNPH + 0 2 • The estrone and estradiol collected in the perfusion liquid were identified by means of infra-red spectrographic analysis and by microcrystallization of the estrone. The results of this experiment are to be found in Fig. 9. Several points appear to us to be significant : 1. As in the experiments carried out in vivo by Mikhail, Wiqvist, and Diczfaluzy 22 - 23 only very small amounts of conjugated estrogens were formed. 2. The aromatization of the neutral steroids by the perfused placenta results almost exclusively in the formation of estrone and estradiol, the estriol in all of these different cases only undergoes a very slight increase, of about the same order as that observed in the control experiments. 3. Under the experimental conditions we adopted, and as in the case of the microsome preparations, the maximum yield was obtained with 19aOH 6..1 androstene 3, 17 dione: 4.2 per cent of the latter being transformed into estrone and estradiol; the testosterone ( 17 f3 hydroxy androst 4 ene 3 one)
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542
Jum·
Varangot, Cedord, and Yonnotti
:\m .
and the .6. 4 androstene 3,17 dione are also transformed into estrone and estradiol to quite a considerable extent, with a similar yield for both these hormones ( 3.0 and 3.3 per cent, respectively). The uehydroepiandrosterone ( 3[3 hydroxyandrost 5 ene 17 one) as opposed to what occurs in the case of the placental microsomes seems to be a more efficient precursor of estrogens than the .6. 4 , 3 ketones, indeed the average percentage recovery is 4 per cent following addition of 10 mg. of this steroid. The presence of the sulfate of dehydroepiandrosterone in abundance in the maternal and fetal blood, and the presence of a sulfatase in the placenta capable of rapidly liberating free dehydroepiandrosterone from its ether sulfate suggest that DHA is the principal precursor of placental estrogens. In 1963, Baulieu and Dray" on the one hand, and Siiteri and MacDonald" 0 on the other, as well as Bolt{- and co-workers 3 demonstrated by means of experiments carrie-d out in vivo that DHA and its sulfate, act as precursors to the major part of the synthesized estrogens during pregnancy, hydrolysis by the placental sulfatase preceding aroma tiza tion. On the other hand, the introduction of an additional double bond ( 1,4 androstadiPne. 3, 17 dione or .6. 1,-1- androstadiene 3 one 17 (3-ol) or the preliminary demethyla-
1:•. 1'16.\
Obxt. & Gyn
tion at C 19 (19 nortestosterone, 19 nor~. androstene 3,17 dione) do not in any way facilitate transformation into estrogem. The average yields being 1,+ to 1,5 per cent, n·spectively, for the ~,J"OR deriYatives and O.i per cent for the~ 1,4 diem•. The presence of axial substituents on carbon-11 upset the aromatization of the A nucleus, as was the case in the preparations of placental microsomes, and similarly the 17a OH~~ androstene 3 one is converted, to a very small extent, into estrone and estradiol raverage yield 0.1 per cent). Finally the 17a OH progesterone. just like progesterone, shows practically no transformation to estrogen under our experimental conditions, as in the case of the placental nucrosomes. 4. We should like to point out that thr placental 17 {3-ol dehydrogenase maintains a ~tate of equilibrium between the estrone and estradiol in a ratio of 3 to 1, the presence in the steroid precursor of a ketone group at C 1,, or of a 17 {3 hydroxyl st·ems to Ita 1·e no influence whatsoever. Having been unable for material reasom to utilize radioactive substances, which allow to be used like tracers, we intended to l'erify that quantities of steroids employed by us usually 10 mg.) are not abnormally high and correspond to the catalytic possibilities of plact'ntal microsomes. 1
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Fig. 10. lJ)crease in estrone and estracliol in perfusion fluid, with growing quantities of dehydroepiandrosterone added.
Volume 9:.! ~mnbt>r
Perfusion of the human placenta
4
543
Table I. Increase of estrone and estradiol free and conjugated 3 hours after addition of
cl9
steroids
----
+ 20.000 I.U. HCG
Without HCG
--
Precursor added
20 mg. ~. androstene-3, 17dione 20 mg. clio!
~,
androstene-3, 17-
I 0 mg. 19 OH 3,17 dione Ill mg. DHEA
~.
androstene-
I
No. of ex periments
%
Precursor added (mg.)
18.1
0.09
10
15 ..)
0.07
10
548.6
5.4
287.0
2.8
Value obtained
I
p.g
As shown by Fig. 10, we could observe in a range of experiments leaded with growing quantities of dehydroepiandrosterone (from :1 to 50 mg.) that the increase of estrone and estradiol measured 4 hours after addition of DHEA, grown according to the quantity of substract added in an absolutely typical manner of enzymatic reactions. The product of the enzymatic reactions raises in fact according to the concentration of substrate, first in a linear way, then more slowly until a saturation landing, which for our experiments of perfusion seems to be near 500 /hg of estrogens. We see therefore that for quantities of 10 or 20 mg. of neutral steroids usually utilized, we are far from this landing of saturation and thus in good experimental conditions.
The problem of chorionic gonadotropins. We observed that for the hydroxylation of the estrone at C 16 as well as for the aromatization of neutral or hydroxylated steroids at C ,r, to estrogens, the addition of chorionic gonadotropins improved the yield, whereas they did not increase the aromatization of the 19 OH androstene dione (Table I) . This gives rise to two problems: l. It is difficult to explain why gonadotropins should be supplied via the perfusion liquid whereas the placenta is reputed to produce large quantities. Several hypotheses have been put forward: (a) According to
I
No. of ex periments
Mean valua obtained p.g
I
%
323.8
3.2
2
94.3 ( 92.8 to 95.8)
0.9
10
4
424.1 ( 103.7 to 725.7)
4.2
10
2
403.3 (339.0 to 467.1)
4.0
Troen* the placenta is incapable of elaborating gonadotropic hormones in vitro. We were able to establish, together with Vassy, that the quantity of chorionic gonadotropins contained in the perfusion liquid was identical both at the beginning and end of the experiment (approximately 10.000 I.U.). (b) It is also possible that the placentas that have reached time of delivery, which was the case for most of our experiments, are generally senescent and elaborate only very small quantities of gonadotropic hormones. (c) Finally one could point out the fact that gonadotropic hormones are elaborated on the maternal surface of the placenta which, to a certain extent, is not included in the perfusion of the fetal circuit which we carry out. For this reason we devised a system of perfusion with a double fetal and maternal circulation, and we intend to analyze the chorionic gonadotropins in both these circulations. Already, it should be emphasized that perfusion of the maternal surface, particularly of the intervillus chamber, is technically extremely difficult to achieve. 2. It would be interesting to establish with precision the exact part played by the chorionic gonadotropins in the metabolism of estrogens. In our opinion the chorionic gonadotropin appear to act directly in the placenta on the hydroxylation process and in this way activate aromatization. *Personal communication.
544 Varangot, Cedard, and Yannotti
;\m .
Following the advice of Breuer we undertook the kinetic study of the aromatization process by adding successively, 10 mg. of steroid and 20,000 I.U. of chorionic gonadotropin at an hour interval, and subsequently took 5 or 6 successive samples in each experiment. (a) In the absence of chorionic gonadotropins the ,;1 4 androstenedione is progressively transformed into estradiol and particularly into estrone essentially in its free form. (b) The addition of HCG produces a considerable increase in yield with a veritable angulation of the curve. In both cases the maximum values were obtained in 3 hours (227 {J-g, 465 p.g). (c) With 19 OH L1 4 androstenedione the maximum estrone and estradiol concentration ( 435 fLg) was obtained much more rapidly in one hour. But above all the addition of HCG does not in any way modify the yield, which if anything is lower (Fig. 11). (d) This effect on hydroxylation is produced by means of TPNH, and this led us to envisage like a possible pathway the basic reactions Chorionic gonadotropin
~ TPNH + 0 2
t A4 androstenedione --:. 19 OH A, androstenedione
t hydroxylase
1
desmolase
estrone
suggested by Breuer: 5 This schema is actually an hypothesis that we have to verify by addition of TPNH generators into perfusions fluid. (e) This action of the chorionic gonadotropins is moreover analogous to the action of ACTH on the steroidogenesis of the adrenal cortex, and of LH in corpus luteurn preparations. At least it deserves the credit of attempting to explain the purpose of chorionic gonadotropins which up until the present was unknown other than for the biological diagnosis of pregnancy. Comment
.J,
June 1.-), l:HJ~J Obst. & l;\'lli't.
centas during 4 to 5 hours, sufficient time to allow a dynamic study of estrogen metabolism. Progressive formation of estrogens and especially of free estriol vvas observed_~ and exogenous estrogens can also be transformed. We could also make conspicuous the ability of the placenta to aromatize androgens in estrogens, and study the respectiH· output of different substances. We could at last verify that the biosynthesis of estriol. on one hand, and of estrone and estradiol on the other hand follow two distinct metabolic channels and that the chorionic gonadotropins seem to have a promoting action on the hydroxylation process and b\ this way on the estrogen aromatization. The part played by the fetus in the metabolism of placental hormones thus appears to be of considerable importance, because a previous hydroxylation of neutral steroids seems to be necessary for biosynthesis of estriol, in notable amount. The presence of a 16 hydroxylase has been demonstrated in the adrenals and liver of the fetus. Cassmern inferred from his experimf'nts on the perfusion of young placenta in 't\"(J that the part played by the fetus was Lo maintain an adequate placental circulation. When the pulsatile outflow maintained by the pump ceases, the percentage of estrogens decreases. Many authors observed a decrease that affected particularly the estriol in cases of fetal death. Hence, it is quite possible to belieye that the excretion of estriol during pregnancy reflects the efficient functioning of the fetoplacental system. The fetus being responsible for hydroxylation at C 1 u and the sulfaconjugation which facilitates urinary excretion, and the placenta for effecting the intermecliarr synthesis of the steroids. The decrease in estriol is therefore of considerable interest from the practical point of view durin~r observation of endamtered v pregnancies. The recent investigations·' carried out un0
\tVe have been able to maintain in a satisfactory condition of survival 101 term pia-
:
Volume 92 Number 4
Perfusion of the human placenta
~9 es1-rli''e
t..S\1adool
\ +2oooo1.u
545
~cu-J
500
\00
200
lnHl!l2.nc.e of HC(;. on
aromatiz.a~ion
o~ 6'J a.ndro~.tenediooe J
and '\9 hydroxy_
b. 1t androstenedionL.
Fig. 11. Influence of HCG on aromatization of t., androstenedione and 19 hydroxy-~, androstenedione.
der conditions very different from our own (perfusion of the placenta in situ, injections of dehydroepiandrosterone or radioactive dehydroepiandrosterone disulfate, in vivo, in the fetal and maternal compartments), produced results that agree with ours. Like ourselves, they also found in the placenta a 17 (3-ol dehydrogenase, extremely high capacity of aromatization, and a hydroxylation power of almost nil. We have however noticed a slight hydroxylation of estradiol in estriol chiefly after addition of chorionic gonadotropins, that Bolte and co-workers 3 did not find again in their experimental conditions. We can think the difference is due to the fact that these authors have utilized early placentas of 17 to 20 weeks, while we have employed term placentas which get perhaps different enzymatic abilities. In order to eliminate an error clue to the transformation of endogenous precursors we are actually studying the metabolism of estradiol 17 (3 6-7 H 3 in term placentas perfused in vitro. On the other hand, in the fetus, considerable sulfurylation occurred and slight hydroxylation at 16 together with probable formation of 16aOH estrone which subsequently becomes reduced by the placenta to estriol (a reaction we managed to produce in vitro). According to Diczfaluzy, the estriol thus formed would pass partly into the maternal
organism, and partly into the fetal organism, where it would be sulfurylated before returning into the placenta following hydrolysis. They also believe that starting with dehydroepiandrosterone sulfate the fetus is able to form a neutral intermediary at c19 hydroxylated at 16a before its subsequent aromatization (we demonstrated in vitro, the formation of estriol in appreciable quantities from these metabolites). The formation of these steroids from sources other than dehydroepiandrosterone for the moment cannot be excluded, but does not seem to be of any quantitative importance. The parts played by the fetus and the placenta, respectively, in the biosynthesis of the steroid nucleus still remain to be known and particularly of progesterone, and dehydroepianclrosterone from cholesterol or acetate from the food supply. Conclusion
Agreeing with the other experiments, our in vitro results confirm that full-term placentas possess 17 (3-ol dehydrogenase, extremely high capacity of aromatization, and very poor hydroxylation power in C 16 • This lack explains that the excretion of estriol during pregnancy reflects the efficient functioning of the fetoplacental unit.
J11nf' }.1 ~~~#u
546 Varongot, Cedard, and Yonnotti :\!!!.
Summary
We have been able to maintain in a satisfactory condition of survival 101 term placentas for 4 to 5 hours-sufficient time to allow a dynamic study of estrogen metabolism. Progressive formation of estrogens and especially of free estriol was observed, and exogenous estrogens can also be transformed. We could also appraise the ability of the placenta to aromatize androgens in estroO'Pns. :1nrl rPsnPrtivP fl1Jtnut n----J -·---- stuclv ------; thf' ---- ---r----·----r--- nf -- clif---
ferent substances. We could verify that the biosynthesis of estriol, on one hand, and of estriol and estradiol, on the other hand, follow two distinct metabolic pathways and that the chorionic gonadotropins seem to have a promoting action on the hydroxylation process and, by this, on the estrogen aromatization. Agn·eing with in uiuo cxperi-
REFERENCES
1. Axelson, E., and Diczfaluzy, E.: Rec. Trav. Chim.- Pays-Bas. 80: 971, 1961. 2. Baulieu, E. E., and Dray, F.: ]. Clin. Endocrinol. 23: 1298, 1963. 3. Bolte, E., Mancuso, S., Eriksson, G., Wiqvist, N., and Diczfaluzy, E.: Acta endocrinol. 45: 535, 1964. 4. Breuer, H., and Kassau, E.: Acta endocrin. Suppl. 51: 1113, 1960. 5. Breuer, H.: Internationales symposion der gynakoloschen endokrinologie Berlin, May, 1963. 6. Cassmer, 0.: Acta endocrinol. Suppl. 45. 1959. 7. Cedard, L.: Sem. Hop. path. et bioi. 8: 901, 1960. 8. Cedard, L., Varangot, J., and Yannotti, S.: Compt, rend. Acad. Sci. 254: 1870, 1962. 9. Cedard, L., Varangot, ]., and Yannotti, S.: Compt. rend. Acad. Sci. 254: 3896, 1962. 10. Cedard, L., Varangot, ]., and Yannotti, S.: Compt. rend. Acad. Sci. 257: 2725, 1963. I I. Cedard, L., Varangot, J., and Yannotti, S.: Compt. rend. Soc. Bioi. 155: 458, 1961. 12. Coyle, M. G.: J. Endocrinol. 25: VIII, 1962. 13. Diczfaluzy, E., and Lindkvist, P.: Acta endocrinol. 22: 203, 1956. 14. Diczfaluzy, E., and Halla, M.: Acta endocrinol. 27: 303, 1958. 15. Fotherby, K., Colas, A., Atherden, S. M., and 1-vfar.ria:n, G. E.: Biochen1. j. 66: 664l 1957.
J. Ohst. 8.:
f;yt!t•;"
menls, our in r·itro results , on firm that {uf/-
lcrrn placenta.r j>ossc.\s 17{3 OH dchydu•<;cnase. extrf'rnrly high capacitr of aromati-:cation and an hydroxylation powo iu ( ., of almost nil. The authors are much indeb1ed to Prnlt'ssor agregc R. Weiman, Dr. Vassy, ;mel R. Ozon for their cooperation in some special assays, tn Or. E. Diczfalm.y and to Professor Drcu
16. Jarabak, J., Adams, ]. A., Williams-Ashman. H. G., and Talalay, P.: ]. Biol. Chern. 237: 3+5, 1962. 17. Kushinsky, S., Demetriou, J. A., Wu, J., and Nasutavicus, W.: ]. Clin. Endocrinol. 20: 719, 1960. 18. Langer, L. ]., and En~,SeL L. L.: J. Bioi. Chern. 223: 583, 1958. 19. Levitz, M., Condon, G. P., and Dancis . .). : Fed. Proc. 14: 245, 1955. 20. Levitz, M., Condon, G. P., and Danris, J.: Endocrinology 58: 3 76, 1956. 21. Marrian, G. F.: Nature 154: 19. 19H. 22. Mikhail, G., Wiqvist, N., and Diczfaluzy. E.: Acta endocrinol. 42: 519, 196:1. 2:.1. Mikhail. G .. Wiqvist, N., and Diczfaluzy. E .. Acta endocrinol. 43: 21 :l, 196:1. 2+. Mitchell, F. L., ;:md Davi<'s, R. E.: Biochern. J 56: 690, 1954. ~5. Neher, R., and Stark, ( ;,: Experientia 17: 510, 1961. ~6. PeZ~rlman, W. H., Pearlman, M. R. J, and Rakoff, A. E.: AM. ]. OnsT. & GY:-.JEC. 6G: :no, 1953. 27. Ryan, K. J, and Engel, L. L.: Endocrinolos;v 52: "287, 1953. . 28. Ryan, K. ].: ]. Bioi. Chern. 234: 2006, 1959. 29. Schmidt-Elmendorff, H. W.: Acta endocrinoL 38: 5~7. 1961. :lO. Siiteri, P. K., and MacDonald, P. C.: Stt"roids 2: 7I:cl, 1963. 31. Snedecor~ G. \V.: Biometrics 8: 85, i952. 32. Talalay, P., and Williams-Ashman, H. (; · Proc. Nat. Acad. Sc. 44: 1S, 1958.
Volume 92
Perfusion of the human placenta
547
Number 4
3:1. Troen, P.: In Recent Progress in the Endocrinology of Reproduction, New York, 1959, ~A.. cademic Press, Inc. 34. Troen, P.: J. Clin. Endocrinol. 21: 895, 1961. 35. Varangot, J., Cedard, L., and Yannotti, S.: Excerpta Medica International Congres Series No. 51, Abstract No. 396, Milan, 1962. :16. Varangot, J., and Thomas, J. A,: Compt. rend. Acad. Sci. 228: 132, 1949. :n. Villee, C. A.: J. Biol. Chern. 205: 113, 1953. ~l!l. Villee, D. B., Engel, L. L., Loring, J. M., and Villee, C. A.: Endocrinology 69: 354, 1961.
39. Villee, c. A.: AM. J. 0BST. & GY~EG. 84: 1684, 1962. n:rv..n.l;nn l<'nrr~l ...__, .... .... .... , ~A".' .&..0.1.15"-"'' L. T........ , 40. ''111e~o,. .................... , D. R Villee, C. A., Raker, J.: J. Clin. Endocrinol. 22: 726, 1962. 41. Ward, P. J., and Grant, J. K.: Biochern. J. 84: 117P, 1962. 42. Weliky, I., and Engel, I. L.: J. Bioi. Chern. 238: 1302, 1964. 43. Zander, J., Thijssen, J., and Munsterrnann, A.M.: J. Clin. Endocrinol. 22: 861, 1962. ~-,
89 rue D' Ass as Paris VI', France
~~._,
~~
Perfusion of the human placenta in vitro Study of the biosynthesis of estrogens
J. VARANGOT, M.D. L. CEDARD, PH.D. S. YANNOTTI, M.D. Paris, France
Material and methods
BEcAusE of its anatomical situationa single vascular pedicle that is easily accessible and an anatomically closed fetal circulatory system--the human placenta is an organ that is particularly suitable for perfusion. In France, as early as 1949, one of us together with Thomas 36 were the first to carry out perfusion of the placenta. Abroad, a number of authors have dealt with this problem, particularly, Pearlman, Pearlman, and Rakoff, 26 Levitz, Condon, and Dancis, 19 and Troen 3 H 4 who undertook perfusion of the placenta in vitro. In Sweden, Cassmer6 and Diczfaluzy and coworkers3· 22 • 23 were able to experiment on the placenta in vivo. Having taken up our previous experiments of 19598 - 11 we have at present performed iOi perfusions of the human placenta in vitro. Under these experimental conditions, we have been able to study the biosynthesis and metabolism of estrogens.
The perfusions of human placenta were carried out in vitro. Out of 10 l perfusions performed, 96 were effected on placentas at the time of delivery. The duration of the perfusion was 4 to 6 hours. Preparation of the placenta. In mo~t of the cases the placenta was derived from a natural confinement. Immediately after delivery it is collected aseptically and freed of its membranes. The umbilical blood vessels are dissected out over a length of 5 em. The two umbilical arteries were catheterized by means of perfusion needles with oliveshaped points fixed by a ligature and a Murphy artery clip (Fig. 1). The umbilical vein was then cut across and 10 mi. heparin was injected via the arteries. The perfusion equipment (Fig. 2). Briefly it can be described as 3 difl'erent circuits. The perfusion circuit. It is set in motion by a hand pump that ensures a pulsatile movement the pressure and speed of which may be adjusted. The perfusion liquid consists of a liter of Krebs-Ringer buffer solution at pH 7.5, to \vhich 1 Gm. of glucose, 0.25 mg. of papaverine, and antibiotics have been added. Thw..
From Clinique obstetricale et gynecologique-F aculte de M edecine Universite de Paris. With the support of the "Centre National de [a Recherche S cientifique ."
534
Volume 92
Perfusion of the human placenta
535
Number 4
Fig. 1. Catheterization of the two umbilical arteries by means of needles with olive-shaped points attached in position by Murphy artery clip. The perfusion liquid is seen flowing through the umbilical vein.
Fig. 2.
the fetal circulation is washed out. Very rapidly 200 mi. of preserved human blood is added. The perfusion outflow is from between 30 to 50 ml. per minute. The pressure varies from 20 to 30 mm. Hg. The oxygenation circuit. This is connected to the perfusion circuit at the exit of the umbilical vein. It consists of a mixture of 0 2 + C0 2 in proportions of 95 per cent and 5 per cent, respectively. The presence of C0 2 decreases vasoconstriction. So as to ensure satisfactory oxygenation the perfusion liquid is maintained in a thin layer by a rotating mixer, on its exit from the umbilical vein. The heating circuit. The temperature is kept constant by means of a thermostatic pump that maintains the heating circuit and placenta at 37° C. Analytical techniques. We determined estrone, estradiol, and estriol in both their free and combined forms. These determinations were performed by fluorometry following partition chromatography on a celite column according to the technique developed by one of us 7 which enables the fractional estimation of estrone. estradiol-17 ,8, and estriol. These determinations were carried out on 50 mi. of fluid sampled at regular intervals during the experiment. The quantities of estrogens were calculated by comparison with control curves and then related to the total volume of the perfusion liquid. Checking of the analytical techniques. We are able to check the precision, accuracy and specificity of our analytical technique as applied to the perfusion liquid. PRECISION. Measurement of the precision of the technique was calculated by Snedecor's method 31 in which S is determined from the difference between estimations carried out in double in a given series of analyses where d = the difference between the two results obtained in the doubles, and N = the number of analyses carried out in double. 1. For values between 0.5 p.g and 55 p.g (experiment without the addition of hor-
536
Varangot, Cedard, and Yannotti
mones): estrone ( 10 analyses) s = 0.28 /Lg; estradiol ( 10 analyses) s = 0.39 JLg; and estriol ( 11 analyses) s = 0.85 JLg. 2. For considerably higher values of between 100 and 1.400 vg (i.e., following the addition of hormones) the precision was obviously less: estrone ( 3 analyses) s = 4.4 vg; estradiol ( 4 analyses) s = 10.3 J-tg; and estriol (4 analyses) s = 11.3 vg. ACCURACY. The accuracy of these determinations was measured by making use of an internal standard, i.e., the recovery of pure hormones added to the sample. As in the case of the whole blood, recoveries were fairly low. Following the addition of pure hormones at the beginning of the analyses, the average percentage recovery is: for the estrone 50 ± 23 per cPnt ( 8 experiments); for the estradiol 49 ± 16 per cent (8 experiments); and for the estriol 69 ± 14 per cent ( 10 experiments) . SPECIFICITY. The substances measured showed the same chromatographic behavior as the pure estrogens. The maximum excitation and emission of fluorescence are identical, 455 and 480 to 490 miL for the pure hormones and the extracts, respectively. The extracts submitted to chromatography react with Brown's reagents (diluted S0 4 H~, quinol) and the absorption spectra of these reactions show the same maxima as those of the corresponding pure hormones. The estriol isolated from the perfusion liquid reacts with David's reagent* by producing a blue color of the same maximum wave length as the standard hormone. R. Ozon was kind enough to carry out the microsublimation of estrone and estriol on a glass slide according to Breuer and Kassau's technique'' in a pyrex cell (diameter 16 mm. and height 10 mm.) fitted on to a Reicher hot plate. In the case of estrone and estriol he was then able to observe the formation of typical microcrystals and was then able to determine the melting points that were found to be very close of the standard hormones. *Modification of Alexson and Diczfaluzy. 1
Am .
Estriol Temperature of mi crosublimation Melting point Estrone Temperature of microsublimation Melting point
Extract
.J.
June I'>. l'Jii:i Ob't. & Gynt·c.
Pure hormone
160° to 250° Ul0° to 240" 275° 280° Extract
Pure hormon<'
150° to 200° 150° to 200° 246° to 2+8° 2!6° to 2511'
W c were also able to carry out paper chromatography: Estrone and estradiol-17,8 in Kushinsky's system 17 2 benzene/! hcptanc;l.S mcthanol/1.5 water. The chromatographic analysis being carried out for 4 hours on Whatman paper No. 1 at room temperature. The rf's obtained were identical both for the pure hormones and the extracts, as well as positive Barton reactions ( rf estrone = l by definition; rf estradiol = 0.6 to 0. 7). The fraction corresponding to the estriol was submitted to chromatographic analysis in a Bush system: 2 benzene/1 methanol/1 water. Chromatography lasted 36 hours on Whatman paper No. 20. Here again we observed an identical rf to that of the pure hormone (the rf differs hy as little as 0.005) and a positive Barton reaction. This chromatographic process was either carried out directly or following elution of the starting point of a chromatogram in the system: 2 henzene/1 heptane/1.5 methanol/1.5 water. We studied the absorption spectra in ultraviolet light (UNICAM SP 500) of the three purified estrogenic fractions, after leaving them in the dark for 2 hours in the presence of concentrated sulfuric acid (micromethod according to Burnstein and Lcenhardt) and we observed in the case of these 3 fractions identical maximum wave lengths to these of the pure hormone despite several quantitative differences. Similarly, examination of the infra-red spectra shows the definite presence of estrone, estradiol, and estriol. However, the presence of some additional peaks and the weak amplitude of several typical maximums did not allow us to conclude as to the absolute pureness of our extracts, even after two successive partition chromatographs. Checking of the efficiency of perfusion.
Volum(' 92 1'\muher 4
Perfusion of the human placenta
Two essential conditions are required in order to effect an efficient perfusion: presence of a fetal circulatory system and survival of the perfused placenta. Presence of a fetal circulatory system is substantiated by several facts. From the mechanical point of view, at the very onset of perfusion the liquid escapes via the umbilical vein, sometimes in the form of a jet, whereas the arteries that are visible under the amnion become turgescent and pulsatile. In our opinion the rapidity of the venous return is not solely due to the presence of superficial arteriovenous anastomosis, which would reflect an incomplete perfusion. Indeed, by introducing stains into the perfusion liquid (e.g., Prussian blue, methyl green) the colored liquid is subsequently found in the histological examinations, in the blood vessels of the villous space. Nevertheless, perfusion is not entirely physiological. Indeed, considerable pressures of approximately 20 to 30 mrn. Hg are required to insure the venous return. These decrease during perfusion but are always definitely higher than the figures generally given. Furthermore, sometimes at the onset of perfusion slight ederna of the placenta may be observed. Finally, there is nearly always intercommunication between the fetal and maternal circuit during the perfusion
process; the liquid transudes via the maternal placental surface. But it is not known whether this, in fact, occurs in vivo. In any event these inconveniences are at present impossible to avoid. They have already been mentioned in a considerable number of publications on perfusion of the placenta. We only hope that they cause but slight disturbances in the endocrine activities of the placenta. From the chemical point of view, the concentration of estrogens increases in the perfusion liquid. Analyses carried out at the first and fourth hours demonstrate this. One hour following the beginning of perfusion we determined the estrogens. The average over-all value of 81 perfusions is assessed as: average over-all value, 45 p,g per liter (estrone, 28 per cent; estradiol, 8 per cent; and estriol, 64 per cent). There is prevalence of free estrogens over conjugated and of estriol over estrone as well as estradiol (Fig. 3). It will be seen that these proportions are in agreement with those found in the tissues of the placenta by various authors.l3, 14 • 24 • 29 But in the first hour we noticed considerable individual variations that bear no relation to the weight of the placenta. That is why during the first hour, in each experiment, we carried out a preliminary basic detern1ina-
~4,5
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Fig. 3. Estrogens (expressed in ~£g) found in the perfusion liquid, one hour after the beginning of the experiment (average of 81 perfusions).
J
53B Vorangot, Cedard, and Yannotti
tion which is deducted from the final results. In six control experiments the determinations carried out at the fourth hour show an increase tljat is statistically significant as far as the estrogens are concerned, both for
10
Ill
esh·adiol e&~rvne es~riol \......--- fr~~ ____....,
esh-ad1ol es~rone es~riol ' - - - . tolllbinf!d - - - - /
&nd
pro~in
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Fig. 4. Placentas without addition of hormones (average of 6 experiments).
the free estrogens and the combined estriol. But the combined estrone and estradiol that only increases to a small extent are not found in the fresh placenta either (Fig. ! ) . Hence, it may be inferred that, during perfusion, the placenta liberates a quantity of estrogens that is higher than that destroyed within the system and that it contains at least 60 per rent of the free t'~ triol. This liberation, the progressive nature of which substantiates the actual extent of prrfusion, also, in our opinion, reflects the survival of the placenta. This survival is even more clearly demonstrated by the continuance of thr enzyme activities in the perfused placenta. lndced. the placenta is able to transform the estrogens supplied to it. In sc\·en experiments we added 20 mg. of rstrone and estradiol, in 5 cases, and 10 mg. of estrone and estradiol, in 2 cases, to the perfusion liquid following a preliminary sampling during the first hour. The determinations of the fourth hour show a considerable increase as far as the estradiol and particularly the estrone arc concerned whereas the estriol barely incrrases (Fig. 51. These results are identical whether estrone
514 --w
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Volume
9~
Number 4
or estradiol is added and this goes to preve the reversibility of the reaction: estrone ~ estradioL Identical results were obtained by Ryan and EngeF 7 with sections of placental tissue, by Levitz, Condon, and Dancis 20 in perfused cotyledons, and more recently by Troen on whole perfused placentas 33 " 34 and by Bolte and co-workers 3 during the perfusion of placenta in situ. This interconversion is effected by a placental 17 (J hydroxysteroid dehydrogenase. 18 • u, 37 - 39 The presence of an active enzyme thus testifies as to vitality of the placenta during these long-term perfusions. As a control experiment we effected two perfusions in the absence of a placenta. Under these conditions only a weak reduction of the estrone ( 1/10) occurred and no formation of estriol whatsoever. Results
The biosynthesis of estriol. In the light of our experiments it seems that the classical process of biosynthesis of estriol from estrone and estradiol would be quantitatively quite small. In 4 experiments we added 20 mg. of estrone or estradiol to the perfusion liquid. The quantity of estriol obtained is more than double that observed in the perfusions of the placenta without the addition of hormones. Statistical calculation shows that the difference between these two average mcreases (4 7.9 ± 14.5 fig and 19.9 ± 8.8 fig, respectively) is significant. In three experiments besides the estradiol we added 20,000 I.U. of chorionic gonadotropin. In this case the estriol is liberated in much larger quantities. The average increase in these three experiments was 96.5 ± 17.8 fig. Here again, this statistical calculation shows a marked significant difference as compared to the perfusions in which estradiol alone was added. The addition of chorionic gonadotropin (20,000 I.U.) without the addition of hormones causes no modifications of the liberated estrogens during the perfusions. Similar results were obtained by Troen 33
Perfusion of the human placenta
539
who, following the addition of chorionic gonadotropins and estradiol to the perfusion iiquid, found a peak of radioactivity and fluorescence after countercurrent distribution with a partition coefficient identical to the theoretical coefficient of the estriol. Hence, it is possible by the administration of estradiol alone or associated with chorionic gonadotropins to stimulate the secretion of estriol via the perfused placenta. This is the classical process in the human organism for the biosynthesis of estriol by hydroxylation at c16 of the estradiol. But, the quantity of estriol thus liberated is barely more than that observed in the fourth hour of a control perfusion with no additional hormones. And the percentage of estriol (maximum 5 per cent) is very different from the characteristic physiological percentage found in the urine at the end of gestation (95 per cent). In fact, in younger placentas, Bolte and co-workers 3 found no hydroxylation whatsoever at C 16 in placentas 17 to 20 weeks old, perfused in situ for several minutes with estradiol. During further perfusions in situ, the same authors observed no stimulating action of the chorionic gonadotropins. Hence, we then studied a second metabolic process which excludes the estradiol and in which the estriol is secreted directly from steroids previously hydroxylated at carbon-
16.z7 In four experiments the addition of 20 mg. of 16aOH estrone acetate causes 3 hours afterward an average increase of 1.400 fig of estrogens, collected in the perfusion liquid. Ninety-nine per cent of this increase consists of free estriol (Fig. 6). A more prolonged study with only 10 mg. of 16aOH estrone showed that this transformation is gradual and only reaches its maximum 4 hours after the addition of this substance. The same experiment carried out without the placenta does not liberate any estriol. In 4 experiments we introduced 10 mg. of 16aOH androsterone or 16aOH testosterone in the perfusion liquid together with 20.000 I.U. of chorionic gonadotropins. A considerable but very gradual increase of estriol
540 Varangot, Cedard, and Yonnotti
-\w.
1000
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800
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___;
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is observed, up to 500 JJ.g 6 hours following addition of the hormones (Fig. 7\. The identical yield of these 2 substanc:es did not surprise us. Indeed, there is present in the placenta a 17 f3 hydroxysteroid dehydrogenase capable of transforming testosterone to .6. 4 androstene 3,17 dione as well as estradiol to estrone. 10 A very important factor for the success of these experiments is the necessity of the simultaneous addition of chorionic gonadotropins. Indeed, experiments carried out 'lNith simply the addition of neutral steroids hydroxylated at carbon-16 proved negative. The estriol having undergone but a very slight increase throughout the experiment, i.e., the 15 fJ.g usually found in the placenta following perfusion without the addition of hormones. Fig. 8 summarizes the results obtained during the different types of experiments. It shows the average increase of free estriol in the perfusion medium, this valnc
Fig. 6. Perfusion of placenta with addition of 16a hydroxy.estrone.
·n 'h
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'
'
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Fig. 7. Perfusion of placenta with addition of 10 mg. 16a hydroxyA, androstenedione.
,
,
Fig. 8. Increase in free estriol during different types of experiments. A, Control placenta without HP. B, +20 mg. of estrone or estradiol; C, +20 mg. of estrone or estradiol +20.000 I.V. HCG; D, +10 mg. of 16a0H testosterone or 16aOHA,, androstenedione; E, +10 mg. of 16aOH testosterone or 16aOHA, androstenedione +20.000 I.U. HCG: F, +20 mg. of 16<>0H estrone. ·
Volume 92 Number 4
Perfusion of the human placenta
being obtained by subtracting the quantities found at the end of the first hour in order to eliminate the variations clue to the content of the placenta itself. The problem remains of the origin of the steroids hyclroxylatecl at 16. I ncleecl, they are not normally synthetizecl in the placenta. Nevertheless, they have been identified.in the urine during pregnancy.15, 21 Neher and Stark 25 isolated 16aOH testosterone from the placenta m the proportion of 35 p.g per kilogram. Quite possibly the fetus 1s capable of effecting this hydroxylation at 16 and of providing the placenta with estrogen precursors. Zander'13 demonstrated that progesterone 4 CH when introduced into the umbilical vein of the human fetus is partially converted into 16a hydroxyprogesterone. Villee and associates 38 established the occurrence of a 16a hydroxylation by the fetal adrenal tissue in vitro as well as by the hyperplastic adrenal tissue·10 - 42 The biosynthesis of estrogens from neutral or substituted steroids. In a series of 35 perfusions we added 10 mg. or sometimes 20 mg. of steroids at 21, 18, and particularly 19 neutral or substituted carbon atoms, to the circulation liquid. Two or three experiments were undert;1ken for each steroid in
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order to eliminate vanatwns due to experimental h azards, and the results obtained were compared with those obtained by the use of radioactive steroids with preparations of placental microsomes + TNPH + 0 2 • The estrone and estradiol collected in the perfusion liquid were identified by means of infra-red spectrographic analysis and by microcrystallization of the estrone. The results of this experiment are to be found in Fig. 9. Several points appear to us to be significant : 1. As in the experiments carried out in vivo by Mikhail, Wiqvist, and Diczfaluzy 22 - 23 only very small amounts of conjugated estrogens were formed. 2. The aromatization of the neutral steroids by the perfused placenta results almost exclusively in the formation of estrone and estradiol, the estriol in all of these different cases only undergoes a very slight increase, of about the same order as that observed in the control experiments. 3. Under the experimental conditions we adopted, and as in the case of the microsome preparations, the maximum yield was obtained with 19aOH 6..1 androstene 3, 17 dione: 4.2 per cent of the latter being transformed into estrone and estradiol; the testosterone ( 17 f3 hydroxy androst 4 ene 3 one)
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Fig. 9. Average values of free and combined estrone and estradiol following the add ition of neutral or substituted steroids at C1s and c, (after deduction of the values observed after one hour of perfusion without hormone) .
542
Jum·
Varangot, Cedord, and Yonnotti
:\m .
and the .6. 4 androstene 3,17 dione are also transformed into estrone and estradiol to quite a considerable extent, with a similar yield for both these hormones ( 3.0 and 3.3 per cent, respectively). The uehydroepiandrosterone ( 3[3 hydroxyandrost 5 ene 17 one) as opposed to what occurs in the case of the placental microsomes seems to be a more efficient precursor of estrogens than the .6. 4 , 3 ketones, indeed the average percentage recovery is 4 per cent following addition of 10 mg. of this steroid. The presence of the sulfate of dehydroepiandrosterone in abundance in the maternal and fetal blood, and the presence of a sulfatase in the placenta capable of rapidly liberating free dehydroepiandrosterone from its ether sulfate suggest that DHA is the principal precursor of placental estrogens. In 1963, Baulieu and Dray" on the one hand, and Siiteri and MacDonald" 0 on the other, as well as Bolt{- and co-workers 3 demonstrated by means of experiments carrie-d out in vivo that DHA and its sulfate, act as precursors to the major part of the synthesized estrogens during pregnancy, hydrolysis by the placental sulfatase preceding aroma tiza tion. On the other hand, the introduction of an additional double bond ( 1,4 androstadiPne. 3, 17 dione or .6. 1,-1- androstadiene 3 one 17 (3-ol) or the preliminary demethyla-
1:•. 1'16.\
Obxt. & Gyn
tion at C 19 (19 nortestosterone, 19 nor~. androstene 3,17 dione) do not in any way facilitate transformation into estrogem. The average yields being 1,+ to 1,5 per cent, n·spectively, for the ~,J"OR deriYatives and O.i per cent for the~ 1,4 diem•. The presence of axial substituents on carbon-11 upset the aromatization of the A nucleus, as was the case in the preparations of placental microsomes, and similarly the 17a OH~~ androstene 3 one is converted, to a very small extent, into estrone and estradiol raverage yield 0.1 per cent). Finally the 17a OH progesterone. just like progesterone, shows practically no transformation to estrogen under our experimental conditions, as in the case of the placental nucrosomes. 4. We should like to point out that thr placental 17 {3-ol dehydrogenase maintains a ~tate of equilibrium between the estrone and estradiol in a ratio of 3 to 1, the presence in the steroid precursor of a ketone group at C 1,, or of a 17 {3 hydroxyl st·ems to Ita 1·e no influence whatsoever. Having been unable for material reasom to utilize radioactive substances, which allow to be used like tracers, we intended to l'erify that quantities of steroids employed by us usually 10 mg.) are not abnormally high and correspond to the catalytic possibilities of plact'ntal microsomes. 1
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Fig. 10. lJ)crease in estrone and estracliol in perfusion fluid, with growing quantities of dehydroepiandrosterone added.
Volume 9:.! ~mnbt>r
Perfusion of the human placenta
4
543
Table I. Increase of estrone and estradiol free and conjugated 3 hours after addition of
cl9
steroids
----
+ 20.000 I.U. HCG
Without HCG
--
Precursor added
20 mg. ~. androstene-3, 17dione 20 mg. clio!
~,
androstene-3, 17-
I 0 mg. 19 OH 3,17 dione Ill mg. DHEA
~.
androstene-
I
No. of ex periments
%
Precursor added (mg.)
18.1
0.09
10
15 ..)
0.07
10
548.6
5.4
287.0
2.8
Value obtained
I
p.g
As shown by Fig. 10, we could observe in a range of experiments leaded with growing quantities of dehydroepiandrosterone (from :1 to 50 mg.) that the increase of estrone and estradiol measured 4 hours after addition of DHEA, grown according to the quantity of substract added in an absolutely typical manner of enzymatic reactions. The product of the enzymatic reactions raises in fact according to the concentration of substrate, first in a linear way, then more slowly until a saturation landing, which for our experiments of perfusion seems to be near 500 /hg of estrogens. We see therefore that for quantities of 10 or 20 mg. of neutral steroids usually utilized, we are far from this landing of saturation and thus in good experimental conditions.
The problem of chorionic gonadotropins. We observed that for the hydroxylation of the estrone at C 16 as well as for the aromatization of neutral or hydroxylated steroids at C ,r, to estrogens, the addition of chorionic gonadotropins improved the yield, whereas they did not increase the aromatization of the 19 OH androstene dione (Table I) . This gives rise to two problems: l. It is difficult to explain why gonadotropins should be supplied via the perfusion liquid whereas the placenta is reputed to produce large quantities. Several hypotheses have been put forward: (a) According to
I
No. of ex periments
Mean valua obtained p.g
I
%
323.8
3.2
2
94.3 ( 92.8 to 95.8)
0.9
10
4
424.1 ( 103.7 to 725.7)
4.2
10
2
403.3 (339.0 to 467.1)
4.0
Troen* the placenta is incapable of elaborating gonadotropic hormones in vitro. We were able to establish, together with Vassy, that the quantity of chorionic gonadotropins contained in the perfusion liquid was identical both at the beginning and end of the experiment (approximately 10.000 I.U.). (b) It is also possible that the placentas that have reached time of delivery, which was the case for most of our experiments, are generally senescent and elaborate only very small quantities of gonadotropic hormones. (c) Finally one could point out the fact that gonadotropic hormones are elaborated on the maternal surface of the placenta which, to a certain extent, is not included in the perfusion of the fetal circuit which we carry out. For this reason we devised a system of perfusion with a double fetal and maternal circulation, and we intend to analyze the chorionic gonadotropins in both these circulations. Already, it should be emphasized that perfusion of the maternal surface, particularly of the intervillus chamber, is technically extremely difficult to achieve. 2. It would be interesting to establish with precision the exact part played by the chorionic gonadotropins in the metabolism of estrogens. In our opinion the chorionic gonadotropin appear to act directly in the placenta on the hydroxylation process and in this way activate aromatization. *Personal communication.
544 Varangot, Cedard, and Yannotti
;\m .
Following the advice of Breuer we undertook the kinetic study of the aromatization process by adding successively, 10 mg. of steroid and 20,000 I.U. of chorionic gonadotropin at an hour interval, and subsequently took 5 or 6 successive samples in each experiment. (a) In the absence of chorionic gonadotropins the ,;1 4 androstenedione is progressively transformed into estradiol and particularly into estrone essentially in its free form. (b) The addition of HCG produces a considerable increase in yield with a veritable angulation of the curve. In both cases the maximum values were obtained in 3 hours (227 {J-g, 465 p.g). (c) With 19 OH L1 4 androstenedione the maximum estrone and estradiol concentration ( 435 fLg) was obtained much more rapidly in one hour. But above all the addition of HCG does not in any way modify the yield, which if anything is lower (Fig. 11). (d) This effect on hydroxylation is produced by means of TPNH, and this led us to envisage like a possible pathway the basic reactions Chorionic gonadotropin
~ TPNH + 0 2
t A4 androstenedione --:. 19 OH A, androstenedione
t hydroxylase
1
desmolase
estrone
suggested by Breuer: 5 This schema is actually an hypothesis that we have to verify by addition of TPNH generators into perfusions fluid. (e) This action of the chorionic gonadotropins is moreover analogous to the action of ACTH on the steroidogenesis of the adrenal cortex, and of LH in corpus luteurn preparations. At least it deserves the credit of attempting to explain the purpose of chorionic gonadotropins which up until the present was unknown other than for the biological diagnosis of pregnancy. Comment
.J,
June 1.-), l:HJ~J Obst. & l;\'lli't.
centas during 4 to 5 hours, sufficient time to allow a dynamic study of estrogen metabolism. Progressive formation of estrogens and especially of free estriol vvas observed_~ and exogenous estrogens can also be transformed. We could also make conspicuous the ability of the placenta to aromatize androgens in estrogens, and study the respectiH· output of different substances. We could at last verify that the biosynthesis of estriol. on one hand, and of estrone and estradiol on the other hand follow two distinct metabolic channels and that the chorionic gonadotropins seem to have a promoting action on the hydroxylation process and b\ this way on the estrogen aromatization. The part played by the fetus in the metabolism of placental hormones thus appears to be of considerable importance, because a previous hydroxylation of neutral steroids seems to be necessary for biosynthesis of estriol, in notable amount. The presence of a 16 hydroxylase has been demonstrated in the adrenals and liver of the fetus. Cassmern inferred from his experimf'nts on the perfusion of young placenta in 't\"(J that the part played by the fetus was Lo maintain an adequate placental circulation. When the pulsatile outflow maintained by the pump ceases, the percentage of estrogens decreases. Many authors observed a decrease that affected particularly the estriol in cases of fetal death. Hence, it is quite possible to belieye that the excretion of estriol during pregnancy reflects the efficient functioning of the fetoplacental system. The fetus being responsible for hydroxylation at C 1 u and the sulfaconjugation which facilitates urinary excretion, and the placenta for effecting the intermecliarr synthesis of the steroids. The decrease in estriol is therefore of considerable interest from the practical point of view durin~r observation of endamtered v pregnancies. The recent investigations·' carried out un0
\tVe have been able to maintain in a satisfactory condition of survival 101 term pia-
:
Volume 92 Number 4
Perfusion of the human placenta
~9 es1-rli''e
t..S\1adool
\ +2oooo1.u
545
~cu-J
500
\00
200
lnHl!l2.nc.e of HC(;. on
aromatiz.a~ion
o~ 6'J a.ndro~.tenediooe J
and '\9 hydroxy_
b. 1t androstenedionL.
Fig. 11. Influence of HCG on aromatization of t., androstenedione and 19 hydroxy-~, androstenedione.
der conditions very different from our own (perfusion of the placenta in situ, injections of dehydroepiandrosterone or radioactive dehydroepiandrosterone disulfate, in vivo, in the fetal and maternal compartments), produced results that agree with ours. Like ourselves, they also found in the placenta a 17 (3-ol dehydrogenase, extremely high capacity of aromatization, and a hydroxylation power of almost nil. We have however noticed a slight hydroxylation of estradiol in estriol chiefly after addition of chorionic gonadotropins, that Bolte and co-workers 3 did not find again in their experimental conditions. We can think the difference is due to the fact that these authors have utilized early placentas of 17 to 20 weeks, while we have employed term placentas which get perhaps different enzymatic abilities. In order to eliminate an error clue to the transformation of endogenous precursors we are actually studying the metabolism of estradiol 17 (3 6-7 H 3 in term placentas perfused in vitro. On the other hand, in the fetus, considerable sulfurylation occurred and slight hydroxylation at 16 together with probable formation of 16aOH estrone which subsequently becomes reduced by the placenta to estriol (a reaction we managed to produce in vitro). According to Diczfaluzy, the estriol thus formed would pass partly into the maternal
organism, and partly into the fetal organism, where it would be sulfurylated before returning into the placenta following hydrolysis. They also believe that starting with dehydroepiandrosterone sulfate the fetus is able to form a neutral intermediary at c19 hydroxylated at 16a before its subsequent aromatization (we demonstrated in vitro, the formation of estriol in appreciable quantities from these metabolites). The formation of these steroids from sources other than dehydroepiandrosterone for the moment cannot be excluded, but does not seem to be of any quantitative importance. The parts played by the fetus and the placenta, respectively, in the biosynthesis of the steroid nucleus still remain to be known and particularly of progesterone, and dehydroepianclrosterone from cholesterol or acetate from the food supply. Conclusion
Agreeing with the other experiments, our in vitro results confirm that full-term placentas possess 17 (3-ol dehydrogenase, extremely high capacity of aromatization, and very poor hydroxylation power in C 16 • This lack explains that the excretion of estriol during pregnancy reflects the efficient functioning of the fetoplacental unit.
J11nf' }.1 ~~~#u
546 Varongot, Cedard, and Yonnotti :\!!!.
Summary
We have been able to maintain in a satisfactory condition of survival 101 term placentas for 4 to 5 hours-sufficient time to allow a dynamic study of estrogen metabolism. Progressive formation of estrogens and especially of free estriol was observed, and exogenous estrogens can also be transformed. We could also appraise the ability of the placenta to aromatize androgens in estroO'Pns. :1nrl rPsnPrtivP fl1Jtnut n----J -·---- stuclv ------; thf' ---- ---r----·----r--- nf -- clif---
ferent substances. We could verify that the biosynthesis of estriol, on one hand, and of estriol and estradiol, on the other hand, follow two distinct metabolic pathways and that the chorionic gonadotropins seem to have a promoting action on the hydroxylation process and, by this, on the estrogen aromatization. Agn·eing with in uiuo cxperi-
REFERENCES
1. Axelson, E., and Diczfaluzy, E.: Rec. Trav. Chim.- Pays-Bas. 80: 971, 1961. 2. Baulieu, E. E., and Dray, F.: ]. Clin. Endocrinol. 23: 1298, 1963. 3. Bolte, E., Mancuso, S., Eriksson, G., Wiqvist, N., and Diczfaluzy, E.: Acta endocrinol. 45: 535, 1964. 4. Breuer, H., and Kassau, E.: Acta endocrin. Suppl. 51: 1113, 1960. 5. Breuer, H.: Internationales symposion der gynakoloschen endokrinologie Berlin, May, 1963. 6. Cassmer, 0.: Acta endocrinol. Suppl. 45. 1959. 7. Cedard, L.: Sem. Hop. path. et bioi. 8: 901, 1960. 8. Cedard, L., Varangot, J., and Yannotti, S.: Compt, rend. Acad. Sci. 254: 1870, 1962. 9. Cedard, L., Varangot, ]., and Yannotti, S.: Compt. rend. Acad. Sci. 254: 3896, 1962. 10. Cedard, L., Varangot, ]., and Yannotti, S.: Compt. rend. Acad. Sci. 257: 2725, 1963. I I. Cedard, L., Varangot, J., and Yannotti, S.: Compt. rend. Soc. Bioi. 155: 458, 1961. 12. Coyle, M. G.: J. Endocrinol. 25: VIII, 1962. 13. Diczfaluzy, E., and Lindkvist, P.: Acta endocrinol. 22: 203, 1956. 14. Diczfaluzy, E., and Halla, M.: Acta endocrinol. 27: 303, 1958. 15. Fotherby, K., Colas, A., Atherden, S. M., and 1-vfar.ria:n, G. E.: Biochen1. j. 66: 664l 1957.
J. Ohst. 8.:
f;yt!t•;"
menls, our in r·itro results , on firm that {uf/-
lcrrn placenta.r j>ossc.\s 17{3 OH dchydu•<;cnase. extrf'rnrly high capacitr of aromati-:cation and an hydroxylation powo iu ( ., of almost nil. The authors are much indeb1ed to Prnlt'ssor agregc R. Weiman, Dr. Vassy, ;mel R. Ozon for their cooperation in some special assays, tn Or. E. Diczfalm.y and to Professor Drcu
16. Jarabak, J., Adams, ]. A., Williams-Ashman. H. G., and Talalay, P.: ]. Biol. Chern. 237: 3+5, 1962. 17. Kushinsky, S., Demetriou, J. A., Wu, J., and Nasutavicus, W.: ]. Clin. Endocrinol. 20: 719, 1960. 18. Langer, L. ]., and En~,SeL L. L.: J. Bioi. Chern. 223: 583, 1958. 19. Levitz, M., Condon, G. P., and Dancis . .). : Fed. Proc. 14: 245, 1955. 20. Levitz, M., Condon, G. P., and Danris, J.: Endocrinology 58: 3 76, 1956. 21. Marrian, G. F.: Nature 154: 19. 19H. 22. Mikhail, G., Wiqvist, N., and Diczfaluzy. E.: Acta endocrinol. 42: 519, 196:1. 2:.1. Mikhail. G .. Wiqvist, N., and Diczfaluzy. E .. Acta endocrinol. 43: 21 :l, 196:1. 2+. Mitchell, F. L., ;:md Davi<'s, R. E.: Biochern. J 56: 690, 1954. ~5. Neher, R., and Stark, ( ;,: Experientia 17: 510, 1961. ~6. PeZ~rlman, W. H., Pearlman, M. R. J, and Rakoff, A. E.: AM. ]. OnsT. & GY:-.JEC. 6G: :no, 1953. 27. Ryan, K. J, and Engel, L. L.: Endocrinolos;v 52: "287, 1953. . 28. Ryan, K. ].: ]. Bioi. Chern. 234: 2006, 1959. 29. Schmidt-Elmendorff, H. W.: Acta endocrinoL 38: 5~7. 1961. :lO. Siiteri, P. K., and MacDonald, P. C.: Stt"roids 2: 7I:cl, 1963. 31. Snedecor~ G. \V.: Biometrics 8: 85, i952. 32. Talalay, P., and Williams-Ashman, H. (; · Proc. Nat. Acad. Sc. 44: 1S, 1958.
Volume 92
Perfusion of the human placenta
547
Number 4
3:1. Troen, P.: In Recent Progress in the Endocrinology of Reproduction, New York, 1959, ~A.. cademic Press, Inc. 34. Troen, P.: J. Clin. Endocrinol. 21: 895, 1961. 35. Varangot, J., Cedard, L., and Yannotti, S.: Excerpta Medica International Congres Series No. 51, Abstract No. 396, Milan, 1962. :16. Varangot, J., and Thomas, J. A,: Compt. rend. Acad. Sci. 228: 132, 1949. :n. Villee, C. A.: J. Biol. Chern. 205: 113, 1953. ~l!l. Villee, D. B., Engel, L. L., Loring, J. M., and Villee, C. A.: Endocrinology 69: 354, 1961.
39. Villee, c. A.: AM. J. 0BST. & GY~EG. 84: 1684, 1962. n:rv..n.l;nn l<'nrr~l ...__, .... .... .... , ~A".' .&..0.1.15"-"'' L. T........ , 40. ''111e~o,. .................... , D. R Villee, C. A., Raker, J.: J. Clin. Endocrinol. 22: 726, 1962. 41. Ward, P. J., and Grant, J. K.: Biochern. J. 84: 117P, 1962. 42. Weliky, I., and Engel, I. L.: J. Bioi. Chern. 238: 1302, 1964. 43. Zander, J., Thijssen, J., and Munsterrnann, A.M.: J. Clin. Endocrinol. 22: 861, 1962. ~-,
89 rue D' Ass as Paris VI', France
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