Lehmann et al., Intraamniotic injection of DHEA-S in mid-pregnancy 255 J. Perinat. Med. Estrogens in maternal plasma following intraamniotic injection of 4(1976)255 (3H)-dehydroepiandrosterone-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 steroids in maternal plasma maternal compartment i.e. by intravenous injection following application of 3H-labelled estrogen· 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 steroid-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- androsterone-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-androstenedione and The C18-steroids were rechromatographed in testosterone 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 estrone (Oej), values were detected at 180 minutes. 16a-hydroxyestrone (loa-OH-Oej) and estradiol- (OE2-17/3) äs well äs the androgens, 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 estriol [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.