Lehmann et al., Intraamniotic injection of DHEA-S in mid-pregnancy 255

J. Perinat. Med. in maternal plasma following intraamniotic injection of 4(1976)255 (3H)--sulfate in midpregnancy

W. D. Lehmann*, J. R. Strecker

Department of Gynecology and Obstetrics University of Ulm, Germany

This investigation was initiated to study the test was performed by precursor application to the kinetics of C19 and C18 in maternal plasma maternal compartment i.e. by intravenous injection following application of 3H-labelled · of DHEA-S into the maternal cubital vein. Since precursors to the fetal compartment. It was hoped the amniotic fluid has become more easily acces- to obtain Information about the quantity and sible due to better techniques the possibility of a quality of the fetoplacental -metabolism in new placenta function test using intraamniotic the direction of fetus to mother. DHEA-S injection was examined simultaneously. In the past years LAU RITZEN and coworkers [5,6, In the following investigations of 4 normal preg- 7] have developed a placental function test in nancies between the 18th to 20th week of ges- which dehydroepiandrosterone-sulfate s an estro- tation, we applied (3H)-labelled dehydroepi- gen-precursor was injected into the mother. There- -sulfate directly into the fetal com- after the increase of the estrogen-excretion in the partment i.e. intraamniotical. Subsequently these 24-h-urine or the estrogen level in maternal plasma pregnancies were legally terminated by injection of was determined s a parameter of placental meta- prostaglandine after the test-procedure. bolic activity [20]. Both urinary and plasma es- trogens rose markedly after application of the androgenic steroidprecursor s confirmed by l Materials and methods investigators[2, 21,19]. After locating the placenta by ultrasonic Vision in The DHEA-S induced increase in urinary or plasma 4 patients, ΙΟΟμΟί DHEA-7a-3H-sulfate (spez. estrogens provides Information about the reserve- activity 1000 mCi/mmol) + 5 mg DHEA in sterile capacity of the placenta. Using incubation studies 0.9% NaCl were injected transabdominally into with placental tissue we could demonstrate in the amniotic cavity. Ten milliliters of blood was vitro that the conversion of DHEA to estrogens by drawn from the cubital vein of the pregnant the placental aromatising enzyme-system is signif- women before injection and after 15, 30, 60,120, icantly reduced in pathological pregnancies (pre- 180 und 240 niinutes into heparinized tubes. After eclampsia, diabetes, postmaturity) s compared to centrifugation 5 ml of plasma was incubated with normal pregnancies [10, 12, 13]. These studies 5000 IU 0-glucuronidase sulfatase (BOEHRINGER) demonstrated that in complicated pregnancies the for 2 hrs. at pH 5.4 and 37°C in a shaking thermo- diminished capacity of the aromatizing enzyme- stat. Extraction of the total free metabolites was system can occur and may result in a reduced processed three times with ether/chloroform. estrogen production which is of great clinical importance. Untilnowthe dehydroepiandrosterone- * With support of DFG SFB 87, project C5.

J. Perinat. Med. 4(1976) 256 Lehmann et al., Intraamniotic injection of DHEA-S in mid-pregnancy

The extracts were evaporated to dryness and the From the C19-steroid-fr?ction, 16a-hydroxy- dry residues were redissolved in warm methanol DHEA was isolated with highest concentration in and pipetted into formamid-saturated paper for plasma. The recovery of unchanged (3H)-DHEA-S, chromatography. Separation of C18 and C19- the steroid that had been initially injected, was steroids was performed in formamid/monochlor- somewhat lower. benzene. The plasma-levels of A4- and The C18-steroids were rechromatographed in were almost equal. The plasma-levels formamid/chloroform, the C19-steroids in propyl- of the C19-steroids showed the same temporal englycol/cyclohexan (see KNÜPFEN [4] for further course äs those of the C18-steroids. The highest details). The resulting metabolites (Oej), values were detected at 180 minutes. 16a-hydroxyestrone (loa-OH-Oej) and - (OE2-17/3) äs well äs the , 4- androstenedione and testosterone were reduced or 3 Discussion oxidized, rechromatographed and characterized in The fetal adrenal cortex produces 75% of the total the following manner: DHEA-S which is then primarily C-l 6-hydroxylated *!. Oxidation by 3, 17/J-hydroxysteroid-reductase in the fetal liver. DHEA-S and 16a-OH-DHEA-S (EC 1.1.1.51) type I Sigma Chemicals, St. Louis, reach the placenta äs estrogen-precursors through USA. the umbilical arteries and are converted via 2. Reduction by sodiumboranate in methanol. androstenedione and 19-hydroxyandrostenedione to estrogens, mainly to [l, 14,17,18]. For Estriol (Oe3), DHEA and 16a-OH-DHEA were this conversion the placenta is provided with the partially acetylated and identified by recrystal- following enzymes arylsulfatase, 3j3-hydroxy- lization to constant specific activity. steroiddehydrogenase-A4-As-isomerase, 19-hy- After localization of the metabolites by parallel- droxylase and the aromatizing enzymesystem. The running, labelled test-chromatogramms, extraction additional activity of the placental microsomal from the paper was performed with methanol and enzyme 170-hydroxysteroiddehydrogenase causes radioactivity measured in a liquid-szintillation- formation of testosterone by reduction of andro- counter. The quantity of plasma-steroids was ex- stenedione and also of estradiol-17/3 from estrone pressed in nCi/100 ml plasma. [16, 8]. In our injection study we simulated physi- ological processes described above by applying the labelled and unlabelled DHEA directly to the feto- 2 Results placental unit by intraamniotical injection. The course of the plasma-levels for each steroid- The quantity of the exogenous hormones admin- fraction during the test-period is shown in Tab. L istered in our test is within the normal physiological The values for the 4 isolated C18-steroids over the ränge. The normal fetus produces about 70 mg test-period are additionally demonstrated in graphs DHEA-S in 24 hrs.; however, we injected 5 mg (see figures). From the C18-steroids, the two C16 unlabelled DHEA and measured its metabolites hydroxylated compounds, estriol and 16-hydroxy- over a 4 hr. period. Only 5 g of labelled DHEA estrone, have been isolated. Seventy-five percent was used to enable qualitative analysis. of all originating estrogens were hydroxylated at Previous investigators have shown that prior to the C16. Estradiol-17/3 and estrone were found in 20th week of gestation the fetus sw.allows signif- approximately the same quantity. In contrast the icant volumes of amniotic fluid. The high 16 - estriol level was 2 to 3 times higher than that of hydroxylaseactivity of the fetal liver [9, 11] causes the other estrogens. The peak plasma concentration C16-hydroxylation of 75% of all recovered met- of all C18-steroids appeared 180 minutes after abolites (Tab. I): The main 16-hydroxylated met- (3H)-DHEA-S injection with the exception of abolite is 16a-OH-DHEA and after placenta- estrone which reached its maximum concentration passage 16a-OH-estrone and estriol. 15 minutes slightly earlier, that is after 120 minutes. after intraamniotic injection of DHEA-S a signif-

J.Perinat. Med. 4(1976) Lehman n et aL, Intraamniolic injcction of DHBA-S in mid-prcgnancy 257

Tab. I. Level of ptaima eUrogcns (nCi/100 ml pla*ma) and of untijrogcnic precu/sors following intraamniotic injcction of 100 jjCi (JH)-dchydrocpiündroj>tcrone-sulfatc.

Time

(min) Oc3 Oer170 16a-OH-Oc, DHEA 16a-OH-DHEA A4-Androst. Testostcron Case l .

15 260 63 48 41 63 75 24 32 30 340 9l 100 60 78 102 48 52 60 490 130 190 70 97 120 65 67 120 520 150 200 85 100 125 79 98 180 710 262 240 100 82 140 85 100 240 640 220 203 90 60 79 43 91 Case 2

15 325 75 65 49 75 90 29 38 30 410 110 124 72 95 122 50 63 60 580 156 230 84 120 134 78 89 120 650 180 270 102 131 151 95 112 180 810 324 293 121 128 168 101 121 240 720 268 254 HO 75 96 52 99 Casc 3 15 340 100 90 51 90 121 34 44 30 430 110 130 71 102 142 52 63 60 600 170 200 85 112 154 69 74 120 675 200 250 90 140 160 85 90 180 870 305 170 105 120 187 97 80 240 750 270 100 80 82 136 66 56

Casc 4 15 240 72 81 68 75 101 28 37 30 325 89 122 79 98 112 38 49 60 456 120 154 85 110 134 49 62 120 528 172 163 108 130 158 59 78 180 690 218 134 145 122 177 85 72 240 581 154 103 110 91 127 44 41

icant quantity of labelled metabolites appears in several hours, since the elimination of steroids by the maternal circulation. As illustrated in the the kidney is rapid i.e. within 30 minutes. Twenty graphs, the plasmalevels of the metabolites in all 4 five percent of the swallowed DHEA is not C16- cases tested increased up to 3 hrs. after injection hydroxylated and therefore seems not to pass the and then decreased rather quickly. Our Inter- fetal liver and consequently to reach the placenta pretation of the results is, that the fetus continously without 16a-hydroxylation. Here it is converted swallows the amniotic fluid (approx. 300—500 ml to A4-androstenedionetestosterone and to estrone in the 20 th week fo gestation) containing the and estradiol-17/3 via the A4-metabolic pathway. dispersed radioactive steroid, metabolizes it and These fractions enter the maternal circulation and delivers it into the maternal circulation after were identified in plasma äs shown in table I. One placental passage. This consecutive release of steroid could assume that a small part of the intraamnioti- hormones by the placenta might explain the cally injected DHEA could be reabsorbed by the relatively high levels in maternal plasma over amnion and reaches the maternal circulation

J. Perinat. Med. 4(1976) 258 Lehmann et al., Intraamniotic injection of DHEA-S in mid-pregnancy

n Ci/100ml PLASMA n Ci/100ml PLASMA CASE 1 CASE 2

800-

Oe2-17

16a-OH-Oe1

1530 60 120 180 240 MINS. 240 MINS.

n Ci/100ml PLASMA nCi/100ml PLASMA 900-, CASE 3 90° CASE 4

600-

5500H

G§C

200- 200-

100- 100-

1530 60 120 180 240 MINS. 1530 60 120 180 240 MINS.

Fig. 1-4. Levels of the four main C18-steroids in maternal plasma of 4 patients following intraamniotic injection of ΙΟΟμ/Ci DHEA-7a-3H-sulfate in midpregnancy.

J. Perinat. Med. 4(1976) Lchmann et a L, Intiaamniotic injcction of DHEA-S in mid-prcgnancy 259 directly in this manner without 16a-hydroxylation. pregnancies with anencephalic fetuses. In these This assumption is supported by the recovery of cases the estriol excretion in 24-hr-urine was small amounts of unmetabolized labelled DHEA. significantly lower than in normal pregnancies. Our study shows that the application of labelled HAUSKNECHT and MANDELMAN [3] have found estrogen-precursors to the fetal compartment and an increase of estriol in 24-hr-urine samples after the following recovery of C19- and CJ8-steroids in intraamniotic application of unlabelled DHEA. maternal plasma give valuable Information about Other metabolites were not isolated. Similar thequantity of the fetoplacental steroid-metabolism investigations were carried out by MICH JE [15] in in midpregnancy.

Summary

In 4 patients with normal pregnancies between the 18th The following labelled metabolites were determincd and 20 th weck of gcstation (3H7a)-dehydroepiandro- quantitativeley: 3 sterone-sulfate (| H]-DHEA-S) was injected intraamnioti- Estriol (E3), estradiol-170 (E2-170), estrone (E^, 16 - cally. Maternal vcnous blood was drawn before and at hydioxy-estrone, (löa-OH-Ej), dehydroepiandrosterone rcgulai intcrvals for 240 minutes after DHEA-injection. (16a-OH-DHEA), A4-androstenedione (AD) and testo- Thereafter, legal abortion was performed by intra- sterone (T). amniotic instilJation of prostaglandine. The conjugated The maximal increase of all estrogen fractions in maternal steroids were hydrolyzed enzymatically and the total plasma occuried 120-180min after intraamniotic injection steroids were isolated and identified. of the precursor. The most prominent rise of the C18- steroids could be shown for estriol. 60-70% of all meta- bolites were C16-hydroxylated.

Keywords: Feto-maternal steroid metabolism, intraamniotic injection of dehydroepiandrosterone.

Zusammenfassung

Östrogenspiegel im mütterlichen Plasma nach intraamnialer (Oe2-170), Östron (Oe^, 16a-OH-Östron, DHEA, 16 - 3 Injektion von H-Dehydroepiandrosteron-Sulfat in der OH-DHea, A4-Androstendion und Testosteron. Der Mitte der Schwangerschaft. maximale Anstieg aller Östrogene erfolgte im Plasma (3H)-Dehydroepiandrosteron-Sulfat (DHEA^S) wurde bei 120-180 min nach Injektion des Vorläufers. Die Östriol- vier normalen schwangeren Frauen in der 18.-20. Ge- fraktion überwog bei weitem die der anderen C18-Steroide. stationswoche direkt intraamnial verabreicht. Die Schwan- 60-70% der Metabolite insgesamt sind an C16 hydroxy- gerschaften wurden später legal durch Prostaglandin- liert. Abschließend kann auf Grund unserer Ergebnisse Instillation unterbrochen. 15 bis 240 min nach der In- festgestellt werden, daß durch intraamniale Verabfolgung jektion wurde Blut aus der mütterlichen Kubitalvene ent- markierter Östrogenvorläufer und durch die Wiederfin- nommen. Im Plasma erfolgte die enzymatische Hydrolyse, dung der C19- und Cjg-Steroide im Plasma der Mutter die gesamten Steroide wurden isoliert und identifiziert. eine gute Auskunft über die Quantität und Qualität des Folgende radioaktive Metabolite wurden schließlich fetoplacentaren Steroidstoffwechsels erhalten werden quantitativ gemessen: Östriol (Oe3), Östradiol-17|3 kann.

Schlüsselwörter: Foeto-materneller Steroidstoffwechsel, intraamniale Injektion von Dehydroepiandrosteron.

Resume

Les oestrogenes dans le plasma maternel resultant d'une enzymatique s'ensuivit dans le plasma, tous les steroides injection intraamniotique de (3H)-deshydroepiandro- furent isoles et identifies. Enfin on a mesure quantitativ- steronesulfate en milieu de grossesse ement les metabolites radioactifs: Une injection intraamniotique, c.a.cL par le cöte foetal, de oestriol (E3), oestradiol-17/3 (E2-17/3), oestrone (Ei), (3H7a)-deshydro6piandrosteerone-sulfate ([3HJ-DHE A-S) 16a-hydroxy-oestrone, (löa-OH-Ej), deshydroepiandro- a ete faite chez 4 parturientes a la grossesse normale entre sterone (DHEA), 16a-hydroxy-d6shydroepiandrosterone, la l Seme et 20eme sernaine de gestation. Les grossesses 16a-OH-DHEA), A4-androstenedione (AD) et testosterone ont ete interrompues legalement par la suite par instillation (T). intraamniotique de prostaglandine. Du sang a ete preleve L'augmentation maximale de tous les oestrogenes survint dans la veine cubitale de la mere a intervalles reguliers dans le plasma 120-180 min apres l'injection du pre- avant et jusqu'a 240 min apres Finjection. L'hydrolyse curseur. La fraction d Oestriol depassa de beaucoup la

J. Perinat. Med. 4 (1976) 18 260 Lehmann et aL, Intraamniotic injection of DHEA-S in mid-pregnancy

hausse des äutres steroides Cig, 60-70% de tous les du metaboiisme steroide foeto-placentaire en administrant motabolites sont fiydroxyles a C^- E« conclusion on peut des precurseurs oestrogeniques marques de faQon physio- constater ä la sraite de rios resultats qu'fl est possible logique par le cote foetal et en retrouvant les steroi'des d'obtenir de bonnes indicaetions sur ia quantite et la qualite C}9 et Cjg dans le plasma de la mere.

Mots-cl&; Injection intraamniotique de deshydroopiandrosterone, metaboiisme steroide foeto-maternel

Bibliogiaphy

[i]BOLTE, E., N. WIQUIST, E. DICZFALUSY: Meta- [11] LEHMANN, W. D.: Untersuchungen über den bolism of dehydroepiandrosterone and dehydro- Östrogenstoffwechsel in der Leber. Fortschr. Geb. -sulphate by the human fetus at Gynäk. Vol. 46 (1972) 90 midpregnancy. Acta endocr. (Kbh.) 52 (1966) 583 [12] LEHMANN, w. D., GH. LAURITZEN: Aromati- [2JCRABBEN, VAN DER, H., K. KAMMACHER, sierung von (4-14C) Dehydroepiandrosteron zu H. SCHMIDT-ELMENDORFF, A. KUNKEL, Östron und Östradipl-170 durch Placenten von nor- E. KAISER: The early diagnosis of placental in- malen und pathologischen Schwangerschaften. Arch. suff iciency by a new dehy droepiandrosterone-sulphate Gynäk. 214 (1973) 436 loadtest compared to cardiotocography, placental [13] LEHMANN, W. D., GH. LAURITZEN, R. SCHUH- histology and other methods relating thereto. Acta MANN: In vitro conversion of [4-i4C] dehydroepi- N endocr. (Kbh.) Supp. 138 (l969) 244 androsteröne and androstendione to oestrogens by [3] HAUS KNECHT, R. U., N. MANDELMAN: The the microsomes of placentas from normal, toxaemic, metabolism of intraamniotically injected dehydro- diabetic and postmature pregnancies. Acta endocr. epiandrosteröne äs a placental function test. Am. J. 13(1973)771 Obstet, Gynec. 104 (1969) 443 [14JLEVITZ, M.: Conjugation and transfer of fetal- [4J KNÜPFEN, R.: Papierchromatographie phenolischer placental steroid hormones. J. clin. Endocr. 26 Steroide. Z. Vitamin-, Hormon-u. Fermentforsch. 12 (1966)773 (1962) 355 [15JMICHIE, E. A.: Oestrpgen levels in urine and [5] LAURITZEN, GH.: A clinical test for placental amniotic fluid in pregnancy with live anencephalic functional activity, using DHEA^ulfate and ACTH- foetus and the effect of intraamniotic injection of injections in pregnant women. Acta Endocr, (Kbh.) sodium dehydroepiandrosterone sulphate on these SuppL119(1967)88 levels. Acta endocr. (Kbh.) 51 (1966) 535 [6] LAURITZEN, CH.: Die hormoneile Diagnostik der [16JRYAN, K. J.: Biological aromatization of steroids. Placentainsuffizienz. In: SALING, E., K. A. HOFER: J. biol. Chem. 234 (1969) 268. Fortschritte der perinatalen Medizin- [17] SIMMER, H. H., W. E. EASTERLING, R. J. PlQN, 2. Deutscher Kongreß f. Perinatale Medizin, Berlin W. J. DIGNAM: Neutral C19-steroids and steroid 1969. Thieme, Stuttgart 1971 Sulfates in human pregnancy. Steroids 4 (1964) 125 [7] LAURITZEN, GH.: Beurteilung der Plazentain- [18JSIITERI, P. K., P. G. MACDONALD: Placental suffizienz mit Hilfe des Dehydroepiandrosteron- estrogen biosynthesis during human pregnancy. J. Sulfat-Tests. Geburtsh. u. Frauenheilk. 33 (1973) clin. Endocr. 26 (1966) 751 238 [19JSTEMBERA, Z. K., J. HERZMANN: Östriol-Unter- [8] LEHMANN, W. D., H. BREUER: Charakterisierung suchung nach DHEA-S als Test für placentare In- und Kinetik einer mikrosomalen 170-Hydroxysteroid- suffizienz.

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