Regulation of Extraadrenal Steroid

21-Hydroxylase ActivityINCREASEDCONVERSIONOF PLASMAPROGESTERONETO

DEOXYCORTICOSTERONEDURINGESTROGENTREATMENTOF WOMEN

PREGNANTWITH A DEADFETUS

PAUL C. MACDONALD,SUSANCUTRER, SUE C. MACDONALD,M. LINETTE CASEY,and C. RICHARDPARKER, JR., Cecil H. and Ida Green Center for ReproductiveBiology Sciences, Departments of Obstetrics-Gynecology and Biochemistry,The University of Texas, Southwestern Medical School, Dallas, Texas 75235

A B S T R A C T We measured deoxycorticosterone(DOC) and progesterone (P) in plasma of 47 womenpregnant with a dead fetus and sequentially through-out gestation in 35 women pregnant with a live fetus.When P levels in plasma were low, the plasma levelsof DOCin women pregnant with a dead fetus variedbut usually were similar to those in women pregnantwith a live fetus. However, when P levels were high,the levels of DOCin some women pregnant with adead fetus were considerably lower than those inwomenpregnant with a live fetus. To test whether thisfinding was due to loss of transfer of DOCfrom fetusto mother or else loss of extraadrenal steroid 21-hy-droxylase activity in the mother after death of thefetus, we conducted several studies. The levels of Pand DOCin plasma of one woman remained constantfrom 30 min after fetal death until delivery occurred13 h later. Estrogen treatment of four womenpregnantwith a dead fetus brought about an increase in plasmalevels of DOCin three of the women. In one womanthe ratio of plasma DOCto P was 0.015, a value similarto that found before fetal death, but was 0.003 afterfetal death but before estrogen treatment. In twowomen pregnant with a dead fetus the transfer con-stants of conversion of plasma P to DOCwere 0.011and 0.005 before, and 0.024 and 0.013, respectively,during estrogen treatment. In one woman pregnantwith a deformed fetus with adrenal agenesis, the met-abolic clearance rates of DOCbefore and during es-trogen treatment were similar, whereas the plasmaproduction rates of DOCwere 2.75 before and 4.31mg/24 h during estrogen treatment. Wesuggest that(a) the DOCin plasma of near-term pregnant womenarises in part by extraadrenal 21-hydroxylation of

Received for publication 17 July 1981 and in revised form25 September 1981.

plasma P and (b) estrogen stimulates steroid 21-hy-droxylase activity in extraadrenal tissues.

INTRODUCTIONDeoxycorticosterone (DOC)' and DOC-SO4 levels inplasma of pregnant women are extraordinarily highcompared with those found in men and nonpregnantwomen (1-8). Importantly, most of the DOCin plasmaof near-term pregnant women arises by a mecha-nism(s) other than adrenal secretion. This obtains be-cause dexamethasone treatment of near-term pregnantwomen does not cause a decrease in DOClevels inplasma (1, 3, 4, 6), and ACTH treatment of suchwomen does not cause an increase in the plasma con-centrations of DOC(3, 4, 6).

It has been suggested that DOCproduced in thefetus may be transferred to the mother because thelevels of DOCand DOC-SO4in umbilical cord plasma(1, 5, 8-10) are even greater than those in maternalplasma. Alternatively, we recently suggested that DOCin the maternal compartment arises, in part, by theextraadrenal 21-hydroxylation of plasma progesterone(P) (11). Plasma P is converted to DOC, but the trans-.fer constant of conversion of plasma P to DOC([P]BU-) varies widely among normal persons (11). In-terestingly, the kidney of the human adult and fetusmay be one site of extraadrenal synthesis of DOCbe-cause steroid 21-hydroxylase activity has been demon-strated in human adult (12) and fetal (13) kidneytissue.

1 Abbreviations used in this paper: DOC, deoxycorticoster-one; DOC-SO4, deoxycorticosterone sulfate; P, progesterone;[pJejc, transfer constant of conversion of plasma progesteroneto DOC; MCR, metabolic clearance rate; PR, production rate;tetrahydro-DOC, 3a,21-dihydroxy-513-pregnan-20-one; TLC,thin-layer chromatography; tetrahydro-DOC-diacetate, 5,B-pregnan-20-one-3a,21-diacetate.

J. Clin. Invest. The American Society for Clinical Investigation, Inc. * 0021-9738/82/00/0469/10 $1.00

Volume 69 February 1982 469-478469

In our investigation we sought initially to evaluatedirectly the fetal contribution of DOCto the maternalcompartment. Wedid so by measuring DOCand Pin plasma of women pregnant with a dead fetus. Wefound that in such women the plasma levels of DOCwere quite variable but in most instances were similarto those in women pregnant with a live fetus when Plevels in plasma of the women of the two groups werelow. However, if P levels were high, DOClevels inplasma of a number of women pregnant with a deadfetus were not nearly as high as those in women preg-nant with a live fetus with comparably high levels ofP. This finding could have been interpreted to meanthat the fetus does contribute DOCto the maternalcompartment, or else it could be interpreted to meanthat extraadrenal steroid 21-hydroxylase activity is in-creased by a hormonal event of pregnancy that is lostor diminished after fetal death. Based on the resultsof this study, we speculated that estrogen, the levelsof which ordinarily fall precipitously after fetal death,may stimulate the extraadrenal 21-hydroxylation ofP. In the present investigation this hypothesis wastested and evidence was obtained that estrogen, actingdirectly or indirectly, causes increased DOCformationfrom plasma P.

METHODS

Experimental design. Two groups of pregnant womenwere studied. The first group of subjects (n = 35) was com-prised of normal women pregnant with a live fetus. Bloodsamples were obtained from these womenat - 2-wk intervalsfrom early in pregnancy until term. The levels of P andDOCin plasma of each of the blood samples were deter-mined. The second group of subjects (n = 47) of this studywere women pregnant with a dead fetus. Special studieswere conducted in five of these women (subjects 1-5) andin one other subject (no. 6) who was pregnant with a livefetus that was known to have abnormalities incompatiblewith extrauterine life. The infant died immediately afterbirth and at autopsy was found to have pituitary hypoplasiaand no adrenal glands.

Subject 1 was pregnant at term, had severe hypertension,and was in active labor. 30 min after fetal heart tones wereheard, fetal heart tones were not present. Blood was obtainedat this time and a number of times thereafter. P and DOCwere measured in plasma of each of the blood samples and17#-estradiol and estriol were quantified in selected samples.

In Subject 2 at 30-wk gestation, fetal heart tones werepresent, and blood was obtained for reasons unrelated to thisstudy. 1-2 wk later the fetus died. Approximately 1 wk later,this womanwas hospitalized for evaluation and study. Bloodsamples were collected twice daily for 5 d. At the end of thistime 25 mg diethylstilbestrol was given to this woman bymouth four times a day for 12 d. Blood sample collectiontwice daily was continued. Induction of labor by infusionof oxytocin was attempted several times unsuccessfully. Af-ter cessation of estrogen treatment, once-daily collection ofblood was continued for 15 d. After this time spontaneousrupture of the membranes occurred, and oxytocin inductionof labor at this time was successful. DOCand P in plasma

of each of the blood samples, including that which was col-lected before fetal death, were quantified. In addition 17,-estradiol and estriol also were measured in selected plasmasamples.

Subject 3 was pregnant at 35-36-wk gestation when fetaldeath occurred for unknown reasons. Approximately 1 wkafter fetal demise this woman was hospitalized for evalua-tion. Blood was collected twice daily for 5 d before com-mencing diethylstilbestrol treatment. On day 2 of estrogentreatment labor commenced spontaneously and delivery oc-curred after 18 h. Steroids in plasma of each of the bloodsamples were quantified.

Subject 4 was pregnant at 32-34-wk gestation when fetaldeath occurred. Near the time of fetal death there was sono-graphic evidence of hydrops fetalis, presumably due to non-immunologic causes. Approximately 1-2 wk after fetal de-mise blood was collected from this woman on 2 successived, and an intravenous infusion of a tracer dose of [3H]P(-50 1Ci) plus [14C]DOC (=0.2 uCi) was given. Urine wascollected for 48 h. The [p]ri5'f and the production rate (PR)of DOCwere computed from the 3H/'4C ratio and the spe-cific activity, respectively, of urinary tetrahydro-DOC asdescribed (14). After 2 d of diethylstilbestrol treatment (25mg, four times daily), a second infusion of [3H]P and ['IC]DOCwas given, and urine was collected again for 48 h. The[p]'-' PR-DOC during estrogen treatment were deter-mined. Estrogen treatment was continued for 5 d. Duringthis time oxytocin induction of labor was unsuccessful. 8 dafter discontinuation of estrogen treatment, oxytocin induc-tion of labor was successful. The placenta at delivery, evenafter several weeks of fetal death was large, viz., 785 g.

Subject 5, who was pregnant at 36 wk when fetal deathoccurred for unknown reasons, was hospitalized - 1 wk afterfetal death. Blood samples were collected intermittently for5 d before commencing estrogen treatment, during whichtime an intravenous infusion of [3H]P and ['4G]DOC wasgiven. 2 d after commencing estrogen treatment (25 mg di-ethylstilbestrol, four times daily), a second identical infu-sion was given. Urine was collected for 48 h after each tracerinfusion. Estrogen treatment was continued for 6 d. Oxytocininduction of labor was successful the day after estrogen treat-ment was discontinued.

Subject 6 was pregnant at term with a live fetus that wasknown to have congenital abnormalities incompatible withextrauterine life. By sonographic and radiographic exami-nation the diagnosis of severe hydrocephalus, omphalocele,and thantrophoric dwarfism was made. The metabolic clear-ance rate (MCR) of DOCwas measured in this womanbeforeand during diethylstilbestrol (25 mg, four times daily) treat-ment. Before estrogen treatment, 90 gCi [3H]DOC in 0.15M NaCl solution containing 6% ethanol was infused at aconstant rate for 199 min. 50 ml of blood was collected after145, 170, and 195 min of infusion. Urine was collected for48 h after beginning the infusion. 48 h after initiating es-trogen treatment the measurement of MCR-DOCwas re-peated. [3H]DOC (94 ,Ci) was infused at a constant rate for202 min. Blood was collected after 140, 170, and 200 minof infusion. Spontaneous labor commenced 20 h after thecompletion of the second infusion of radiolabeled tracers.An infant (Apgar 1, 3,300 g) was born that died within 15min of birth. At autopsy the pituitary was hypoplastic andadrenal glands were not found. The final diagnosis was short-rib polydactyly dwarfism with multiple congenital defects.Before delivery, the mean estriol level in maternal plasmawas very low, viz., 2.3 ng/ml. However, the mean plasmalevel of 17 #l-estradiol was 23.8 ng/ml.

Quantification of steroids in plasma. P (15), DOC(7),

470 MacDonald, Cutrer, MacDonald, Casey, and Parker

17#-estradiol, and estriol (16) were measured by radioim-munoassay procedures previously described in detail.

Steroids. [1,2,6,7-(N)-3H]P (98 Ci/mmol) and [4-'4C]DOC(40 mCi/mmol) (New England Nuclear Corp., Boston,Mass.) were purified as described (12, 14). NonradiolabeledDOCand tetrahydro-DOC were purchased from Steraloids(Wilton, N. H.). 513-Pregnan-3a-ol-20-one-21-acetate wassynthesized from 5,3-pregnan-3a,21-diol-20-one accordingto the procedure of Henbest et al. (17), and this product wastreated with pyridine/acetic anhydride (1:1) at 37°C for 2h to obtain tetrahydro-DOC-diacetate.

Determination of MCR, PR-DOC, and ([p]"J. TheMCR-DOCwas computed from the steady-state concentra-tion of [3H]DOC in plasma during the continuous infusionof a tracer dose of [3H]DOC in Subject 6. [(4C]DOC (-3,000dpm) was added to each plasma sample as an internal stan-dard. The plasma was extracted twice with dichloromethane.An aliquot (5%) of the extract was assayed for radioactivityand the 3H/'4C ratio of the steroid(s) in the extract wascomputed. The solvent was evaporated and the residue waspurified by thin-layer chromatography (TLC) using the sol-vent system methylene chloride/diethyl ether (7:3, vol/vol).The purified DOCwas eluted from the silica gel and treatedwith a mixture of pyridine and acetic anhydride for 2 h at37°C. The DOC-acetate was chromatographed by TLC us-ing the solvent system ethyl acetate/isooctane (1:1, vol/vol).The DOC-acetate was eluted from the chromatogram, 40mg nonradiolabeled DOC-acetate was added, and the mix-ture was crystallized three times. The 3H/'4C ratios in theplasma extract and after each purification step were similar(data not shown). The plasma PR-DOCwas computed as theproduct of the MCR-DOCand the plasma concentration ofDOC(Table II).

In Subjects 4 and 5, the [p]Pu was computed from therelationship between the 3H/'4C ratio of the infused tracers,[3H]P and [14G]DOC, and that of tetrahydro-DOC isolatedfrom a 48-h urine collection that was treated with ,B-gluc-uronidase. The procedures used for the purification of uri-nary tetrahydro-DOC have been described in detail (11).The production rate of DOCwas computed from the specificactivity of urinary tetrahydro-DOC with respect to carbon-14 and the quantity of ['4G]DOC infused (14). The specificactivity of tetrahydro-DOC was determined after acetyla-tion of an aliquot of the tetrahydro-DOC with [3H]aceticanhydride of known specific activity.

The PR-DOCin Subject 6 was computed from the specificactivity of [3H]tetrahydro-DOC isolated from a 48-h urinecollection that was treated with j3-glucuronidase. The[3H]tetrahydro-DOC was purified as described (11). The spe-cific activity of the purified [3H]tetrahydro-DOC was com-puted from the 3H/'4C ratio of tetrahydro-DOC-diacetateafter acetylation of the [3H]tetrahydro-DOC with ['4C]aceticanhydride of known specific activity (14). The tetrahydro-DOC-diacetate was purified by TLC and by recrystallizationto constant 3H/'4C ratios.

RESULTS

Plasma levels of DOCand P in women pregnantwith a dead fetus and in womenpregnant with a livefetus. The concentrations of DOCin plasma, as afunction of the concentrations of P in plasma, inwomenpregnant with a dead fetus are compared withthe concentrations of DOCand P in women pregnantwith a live fetus (Fig. 1). When the plasma levels of

40

20

E

N,

0J o 0I~0 0 00 0 0 0

0 0

L0%o0 01

I AI I I I II0 20 40 60 80 100 120 140 160 180 200 220

Progesterone (ng/ml)

FIGURE 1 The concentrations of DOCin plasma as a func-tion of the concentration of P in plasma (mean±SEM) inwomen pregnant with a live fetus (@) are compared withthose in women pregnant with a dead fetus and in whomplasma 17fB-estradiol levels are less than (0) or greater than(-) 6 ng/ml.

P were low (< 50 ng/ml), the plasma levels of DOCin women pregnant with a dead fetus were quite vari-able but in most instances were similar to those inwomen pregnant with a live fetus (Fig. 1). On theother hand, when P levels in plasma were high (>50ng/ml), the levels of DOCin plasma of most womenpregnant with a dead fetus were considerably lowerthan the levels of DOCin plasma of women pregnantwith a live fetus. In the case of women pregnant witha dead fetus with high levels of P and low levels ofDOC, the concentration of 17,3-estradiol usually waslow, i.e., <6 ng/ml. In five women pregnant with adead fetus the levels of both P and DOCwere high.In four of these five women the plasma levels of 17,B-estradiol were >6 ng/ml (Fig. 1).

The plasma levels of P and DOCin a woman soonafter fetal death and sequentially thereafter untiland after delivery. The levels of DOCand P in theplasma of a near-term pregnant woman (Subject 1)soon (within 30 min) after fetal death and every 30min thereafer until 3 h after delivery, hourly until 10h postpartum, and at 24, 36, and 60 h after deliveryare presented in Fig. 2. The levels of P and those ofDOC fluctuated somewhat before delivery, but theconcentration of both steroids remained high. Afterdelivery the levels of P and DOCin plasma declinedrapidly. These findings are consistent with the viewthat DOC in the maternal compartment of somewomen is derived primarily from the extraadrenal 21-hydroxylation of plasma P and not by transfer to themother of DOCproduced in the fetus.

Estrogen Stimulation of Extraadrenal Steroid 21-Hydroxylase Activity 471

IE

CP

0

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0

00E

CP

0-0

co

8.5:

0

x0

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0 2 4 6 8 10 12 ' 2 6 10 24 36 48 60Hours After Fetal Death Hours after Delivery

FIGURE 2 The levels of DOCand P in the plasma of a near-term pregnant woman (Subject1) within 30 min after fetal death and at various times thereafter.

The plasma levels of P and DOCin women preg-nant with a dead fetus before and during estrogentreatment. The levels of P and DOCin plasma ofSubject 2 at 30-wk gestation, when her fetus was alive,as well as the levels of these hormones after fetal deathbefore and during estrogen treatment are presentedin Fig. 3A. The ratios of the concentrations of DOCto those of P in this subject are presented in Fig. 3B.In this woman the concentration of 17f3-estradiol inplasma before fetal death was 7.8 ng/ml and 1-2 wkafter fetal death it was 4.3 ng/ml. The plasma estriollevel was 3.1 ng/ml before fetal death and was <0.5ng/ml after fetal death. Before fetal death the plasmalevel of DOC(0.952 ng/ml) and that of P (78 ng/ml)were similar to those found in women pregnant witha live fetus at comparable stages of pregnancy. Afterfetal death the levels of DOC, but not those of P, de-clined. However, after the institution of estrogen treat-ment, the concentration of DOC, but not that of Prose. The level of DOCbefore estrogen treatment,0.24±0.023 ng/ml (mean±SEM), was significantly lessthan that during estrogen treatment, 0.54±0.053 ng/ml, P <0.001. Importantly the mean ratio of theplasma concentrations of DOCto those of P duringestrogen treatment, 0.010±0.0007, was similar to theratio found before fetal death, 0.012, and significantlygreater (P < 0.001) than the mean ratio foundafter fetal death but before estrogen treatment,0.003±0.0004. After the discontinuation of estrogentreatment the ratio of the concentration of DOCtothat of P remained high. However, by this time pla-cental function had deteriorated to such an extent thatP levels had declined appreciably to levels similar to

or less than those found during the luteal phase of theovarian cycle.

Similar studies were conducted in Subjects 3-6. Theresults of these studies are presented in Figs. 4-7. Theplasma 17f3-estradiol level in Subject 3 was low, 5.2ng/ml, as was the mean of the estriol levels in plasmaof several blood samples, 0.93 ng/ml. Within 12 h ofcommencement of estrogen treatment, the concentra-tion of DOCin plasma increased (Fig. 4A). Within 24h the maximum concentration of DOCas well as ahigh ratio of the concentration of DOCto P duringestrogen treatment (Fig. 4B) was attained. The plasmaconcentration of DOC before estrogen treatment,0.48±0.026 ng/ml, was significantly less than duringestrogen treatment, 1.05±0.11 ng/ml, P < 0.001. Sim-ilarly the ratios of the plasma concentration of DOCto that of P before estrogen, 0.0047±0.0003 was sig-nificantly less than during estrogen treatment,0.0133±0.0023, P < 0.001.

The plasma concentration of 17#B-estradiol in Subject4 was high, viz., 24 ng/ml, whereas the concentrationof estriol was low, viz., 3 ng/ml. After instituting es-trogen treatment, the levels of DOC, which were highbefore estrogen treatment, increased to very high, al-though fluctuating levels (Fig. 5A). Similarly the highratio of the plasma concentration of DOCto that ofP before estrogen treatment was increased even higherduring estrogen treatment (Fig. 5B). Indeed, on oneoccasion during estrogen treatment, a ratio of the con-centrations of DOCto P of 0.1 was found. The meanof the plasma concentrations of DOCbefore estrogentreatment, 4.11±0.38 ng/ml, was significantly less thanduring estrogen treatment, 7.22±0.85 ng/ml, P < 0.01.

472 MacDonald, Cutrer, MacDonald, Casey, and Parker

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0020

.Qa: 0.015

0.0100

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Days+24 +30

before estrogen treatment, 19.1±1.1 ng/ml, P < 0.05.1.0 Nonetheless the [p]pDoc in this woman, as discussed

below, did rise during estrogen treatment.08 In Subject 6, pregnant with a deformed but live fetus

: with adrenal aplasia, the plasma concentration of 17,B-c estradiol was high, viz., 24 ng/ml, whereas the plasma

06 > concentration of estriol was low, viz., 2.3 ng/ml. Dur-0 ing estrogen treatment the levels of DOC(Fig. 7A) as0 well as the ratios of the plasma concentrations of

04 8 DOCto those of P (Fig. 7B) increased appreciably.Ot The mean concentration of DOCbefore estrogen treat-

02 ment, 1.63±0.10 ng/ml, was significantly less than thatduring estrogen treatment, 2.44±0.14 ng/ml, P < 0.001.The mean of the ratios of the plasma concentrations

0 of DOC to those of P before estrogen treatment,0.015±0.0007, was significantly less than during estro-gen treatment, 0.0254±0.0023, P < 0.01.

The [p]-"c and PR-DOCbefore and during estrogentreatment in womenpregnant with a dead fetus. After

120

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aE80

40

FIGURE 3 The levels of P and DOCin plasma (panel A) andthe ratios of the concentrations of DOCto those of P (panelB) in Subject 2 at 30-wk gestation, when her fetus was alive,and after fetal death both before and during estrogen treat-ment.

0.016

The mean of the ratios of the plasma concentrationsof DOC to those of P before estrogen treatment,0.020±0.0024, was significantly less than during estro-gen treatment, 0.043±0.0073, P < 0.02. In this subjectthe plasma levels of P were quite high, possibly dueto the hyperplacentosis that is associated with hydropsfetalis. After discontinuing estrogen treatment theplasma concentration of DOC, as well as the ratio ofthe concentration of DOCto that of P, declined.

In Subject 5 the plasma concentrations of 17,B-es-tradiol and estriol were low, viz., <1 ng/ml. After theinitiation of estrogen treatment, the levels of DOCdidnot rise appreciably (Fig. 6A), nor did the ratio of theconcentration of DOCto that of P (Fig. 6B). However,placental function had declined appreciably and themean concentration of plasma P during estrogen treat-ment, 14.6±1.5 ng/ml, was significantly less than that

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FIGURE 4 The levels of P and DOCin plasma (panel A) andthe ratio of the concentrations of DOCto those of P (panelB) in Subject 3 before and during estrogen treatment.

Estrogen Stimulation of Extraadrenal Steroid 21-Hydroxylase Activity

-BEstrogen Treatment

rato DeathiF

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13 Nolten et al. (18), viz., -1,600 liter/d, we can estimatethe plasma PR-DOC. Doing so we computed that the

° estimated value for plasma PR-DOCwas similar to that° measured by the isotope dilution technique (Table I).N The MCR-DOCand plasma PR-DOCin a woman

9 -0 pregnant with a severely deformed fetus with adrenalaplasia before and during estrogen treatment. After

7 f purification to radiochemical homogeneity of DOCin.° plasma from blood samples obtained during the in-° fusion of [3H]DOC to Subject 6, the MCR-DOCwas

5 o computed. The MCR-DOCin Subject 6 was 1,596 li-0 ter/24 h before estrogen treatment and was 1,583 liter/

24 h during estrogen treatment (Table II). The6° plasma PR-DOCbefore and during estrogen treatment

were 2.75 and 4.31 mg/24 h, respectively, in thiswoman (Table II).

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4FIGURE 5 The levels of P and DOCin plasma (panel A) andthe ratio of the concentrations of DOCto those of P (panelB) in Subject 4 before and during estrogen treatment.

0.5

purification to radiochemical homogeneity of radiola-beled tetrahydro-DOC isolated from the urine of Subjects4 and 5 (who were given an intravenous infusion of [3H]Pand [14C]DOC), the [pkuDoc in each subject was computed.The [pkff in Subject 4 was 0.011 before estrogen treat-ment and was 0.024 during estrogen treatment (TableI). Similarly, the [p]eoc in Subject 5 rose from 0.005before to 0.013 during estrogen treatment (Table I).

The PR-DOC(computed from the specific activityof urinary tetrahydro-DOC) in Subject 4 was 7.7 mg/24 h before and 12.2 mg/24 h during estrogen treat-ment. In Subject 5 the PR-DOC before and duringestrogen treatment were 0.75 and 0.63 mg/24 h, re-spectively. However, in Subject 5 plasma P levels werelow and falling. Thus despite an increase in [pke° dur-ing estrogen treatment, PR-DOCdid not rise. The MCR-DOCwas not measured in Subjects 4 and 5. Howeverif we assume that MCR-DOCin these womenwas similarto that in Subject 6 of this study and to those found by

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DaysFIGURE 6 The levels of P and DOCin plasma (panel A) andthe ratio of the concentrations of DOCto those of P (panelB) in Subject 5 before and during estrogen treatment.

474 MacDonald, Cutrer, MacDonald, Casey, and Parker

0

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Estrogen Treatment

Estrogen Treatment

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FIGURE 7 The levels of P and DOCin plasma (panel A) andthe ratio of the concentrations of DOCto those of P (panelB) in Subject 6 before and during estrogen treatment.

DISCUSSION

This investigation was designed originally to ascertainwhether significant quantities of DOCwere trans-

ferred from the fetal to the maternal compartment.Wewere of the view that this was not the case. Thisconclusion was based primarily upon the finding thatmuch of the DOCin pregnant women can be ac-

counted for by the extraadrenal production of DOCfrom plasma P (11). Thus, we were surprised to findthat when plasma P levels were high the plasma levelsof DOCwere lower than expected in some women

pregnant with a dead fetus. However, it also was ob-served that when P levels were low the plasma con-

centrations of DOCwere similar among most pregnantwomen irrespective of whether the fetus was alive or

dead. In five womenpregnant with a dead fetus plasmaP levels were high and DOClevels also were high. Infour of these five women the plasma 17,-estradiol lev-els also were high. These findings were suggestive thatthe rate of formation of DOCfrom circulating P may

be regulated by estrogen, which is produced in largeamounts in pregnancies in which the fetus is alive.

Estriol is synthesized in the human placenta from16a-OH-dehydroisoandrosterone sulfate derived pri-marily from the fetus (19, 20) and 17,-estradiol is syn-

thesized in the placenta from dehydroisoandrosteronesulfate and other circulating CG9-steroids derived fromboth fetal and maternal plasma (19, 21-22). P is syn-

thesized in the placenta almost exclusively from ma-

ternal plasma low-density lipoprotein-cholesterol (24).Therefore after fetal death the rate of estrogen syn-

thesis, particularly that of estriol, falls precipitouslywhereas P synthesis persists until placental functiondeteriorates. If estrogen stimulates extraadrenal ste-roid 21-hydroxylase activity, it could be envisionedthat a decline in steroid 21-hydroxylase activity afterfetal death would be sufficient to cause a reduction in

TABLE ITransfer Constant of Conversion of Plasma Progesterone to DOCffpj-jC)and the Production Rate of DOCbefore and during Estrogen Treatment

of WomenPregnant Near Term with a Dead Fetus

Plasma PR-

[4IJOu PR-DOC DOGt

mg/24 h mg/24 h

Subject 4Before estrogen treatment 0.011 7.7 7.2

Subject 4During estrogen treatment 0.024 12.2 12.7

Subject 5Before estrogen treatment 0.005 0.75 0.88

Subject 5During estrogen treatment 0.013 0.63 0.68

Computed from the specific activity of urinary tetrahydro-DOCComputed from plasma concentration of DOC(average of plasma DOCconcentration

in four blood samples obtained during 2 d of urine collection) assuming an MCR-DOC= 1,600/24 h.

Estrogen Stimulation of Extraadrenal Steroid 21-Hydroxylase Activity

'~~~~~~-e' 2lI/~~~~~o

p- 2

/ 26810 12 14

Estrogen Treatment 0.

I I I I 00 1 6 8 10 12 14

B

-r

Estrogen Treatrnnt

I 1 Jf- I I a I I I I I I

475

TABLE IIMetabolic Clearance Rate and Plasma Production Rate of DOCbefore and during Estrogen Treatment

of a WomanPregnant Near Term

[1"CDOC/liter plasmaPlasma

Blood sample Blood sample Blood sample concentrationt PlasmaTreatment No. 1 No. 2 No. 3 MCR-DOC DOC PR-DOC

dpm X 10' liters plasma/24 h ng/mn mg/24 h

Subject 6Before estrogen treatment 891 876 897 1,596 1.72 2.75

Subject 6During estrogen treatment 923 807 932 1,583 2.72 4.31

MCRcomputed from concentration of [3H]DOC in plasma of blood sample No. 3.Mean plasma DOCconcentration in blood samples obtained on four occasions, viz., the day before, the day of, and each of 2 d after

infusion of [3H]DOC.

the fractional conversion of plasma P to DOC. Thispossibility seemed reasonable in view of the similaritybetween the concentration of P in plasma of womenlate in pregnancy and the concentration of P that isknown to saturate the 21-hydroxylase enzyme in hu-man kidney tissue microsomes in vitro (12). If in thelatter part of pregnancy extraadrenal steroid 21-hy-droxylase enzyme were saturated by P, a decline inenzyme activity would eventuate in a decrease in thefractional conversion of plasma P to DOC. On theother hand, if P levels do become saturating for ex-traadrenal steroid 21-hydroxylase late in pregnancy,an estrogen-induced increase in enzyme activity inwomen pregnant with a live fetus could provide amechanism for maintaining the fractional conversionof plasma P to DOCreasonably constant despite risinglevels of P in plasma.

Wesought to evaluate this possibility in a study ofwomen pregnant, near term, with a dead fetus. Suchsubjects were considered to be ideal naturally occur-ring models for this study for the following reasons.(a) In some such women the plasma levels of P remainhigh whereas those of estrogen, particularly estriol,decline precipitously after fetal death. (b) In suchwomen, DOCcannot arise from the fetus. (c) Theo-retically estrogen treatment of such women could betherapeutically beneficial because similar treatmentof animals brings about an increase in oxytocin recep-tor concentration in myometrium (25, 26). Commonly,women pregnant with a dead fetus are refractory tothe induction of labor with oxytocin as were thewomenof the present study. Moreover, estrogen treat-ment of pregnant women, even those in whom thefetus is dead, does not produce nausea (27), and in a25-yr follow-up study of women who ingested di-ethylstilbestrol during pregnancy, there was no evi-dence of ill effects (28).

Several studies were conducted. First we evaluated,

sequentially, the levels of P and DOCin plasma of awoman commencing within 30 min after fetal death.The levels of the two hormones remained reasonablyconstant until after delivery of the placenta. There-after the levels of the two hormones declined precip-itously. These findings are suggestive that in somepregnancies significant quantities of DOC are nottransferred from fetus to mother.

In a second study we sought to ascertain if the ad-ministration of estrogen to women pregnant with afetus that had been dead from some time would giverise to an increase in the levels of DOCin plasma,presumably as the consequence of stimulation of extra-adrenal steroid 21-hydroxylase activity. Wefound thatwithin 12 h after the initiation of estrogen treatmentof four womenpregnant with a dead fetus, DOClevelshad risen appreciably in three of the four. Perhapsmore importantly, the ratio of the concentration ofDOCto that of P (which was depressed markedly afterfetal death) had risen strikingly in these three women.Indeed in one of these women the ratio of the con-centrations of DOCto those of P during estrogen treat-ment was similar to that found when her fetus wasalive. The ratio of the plasma concentration of DOCto that of P (corrected for difference in the metabolicclearance rates of the two steroids) should be similarto the transfer constant of conversion of plasma P toDOCprovided that the proportion of DOCin plasmathat arose by secretion from the maternal adrenal orby transfer from the fetus is small compared to thatwhich is formed from plasma P when plasma P levelsare high. In one subject (no. 5), DOClevels did notrise appreciably during estrogen treatment. However,the plasma levels of P in this woman were very lowand were falling during estrogen treatment. Even inthis woman the [ptuD°c increased 2.6-fold during es-trogen treatment.

After cessation of estrogen treatment of Subject 2,

476 MacDonald, Cutrer, MacDonald, Casey, and Parker

the ratio of the concentration of DOCin plasma tothat of P remained high. However, by this time thelevels of P in her plasma had fallen to levels similarto those found in the luteal phase of the ovarian cycle.Wehave shown previously that the [plu in a givenwoman is similar in the follicular and luteal phase ofher cycle (29). Thus despite the discontinuation of es-trogen treatment it is likely that the level of steroid21-hydroxylase activity was sufficient to maintain thetransfer constant of conversion of plasma P to DOCsufficient for these lower levels of P.

To test directly whether the increase in the plasmaconcentration of DOCduring estrogen treatment wasdue to increased 21-hydroxylation of plasma P, the[p]ki'?f and PR-DOCwere determined before and dur-ing estrogen treatment in two subjects pregnant witha dead fetus. The [p]'jDoc increased during estrogentreatment in both subjects. In Subject 4 the PR-DOCbefore estrogen treatment was 7.7 mg/24 h whereasduring estrogen treatment it was 12.2 mg/24 h. ThePR-DOCbefore and during estrogen treatment of Sub-ject 5 were similar. However as noted before theplasma P levels in this woman during estrogen treat-ment were low and falling. Thus the increase in[p]P-DO may have allowed for maintenance of the pre-treatment PR-DOCdespite falling levels of P.

These findings are suggestive that the rise in plasmaconcentration of DOC, as well as the increase in thePR-DOC, during estrogen treatment was due to in-creased conversion of circulating progesterone to DOCand was not due to increased maternal adrenal secre-tion of DOC. In addition it was found that in onewoman pregnant with a live but deformed fetus withadrenal aplasia, the MCR-DOCdid not decrease dur-ing estrogen treatment and the values for MCR-DOCin this woman were similar to those found previouslyin nonpregnant women (11, 18). In this subject theplasma PR-DOC was 2.75 mg/24 h before estrogentreatment and was 4.31 mg/24 h during estrogen treat-ment. These several findings are supportive of the con-clusion that increased levels of cortisol binding glob-ulin did not account for the increased levels of DOCobserved during estrogen treatment of women preg-nant with a dead fetus.

Based on the findings of the present and previous(7, 11) investigations we conclude that the DOCpres-ent in blood of most near-term pregnant women isderived primarily from the extraadrenal 21-hydrox-ylation of plasma P. The extraadrenal 21-hydroxyl-ation of plasma P may take place, in part, in kidney(12, 13), a tissue site of DOCaction. Moreover, steroid21-hydroxylase activity in extraadrenal tissue wasstimulated by estrogen. From the results of this studywe cannot deduce whether estrogen acts directly orindirectly, e.g., through an alteration in the rate of

formation of a substance (possibly a pituitary hor-mone) that stimulates extraadrenal steroid 21-hydrox-ylase activity. The kidney, however is believed to bea tissue of estrogen responsiveness (30, 31). In anyevent it seems likely that estrogen, acting directly orindirectly, stimulates extraadrenal steroid 21-hydrox-ylase activity in a manner such that the transfer con-stant of conversion of plasma progesterone to DOCremains reasonably constant as pregnancy advancesand plasma levels of P rise. In this manner the pro-duction rate of DOCin most women pregnant witha live fetus remains proportional to the plasma con-centration of P.

ACKNOWLEDGMENTS

Wethank Messrs. Frank Hereford, Jesse Smith, and SheltonRagland for expert technical assistance and Ms. BeckyMcKinney-Reese and Vicki Rankin for valuable editorialassistance.

This investigation was supported, in part, by U. S. PublicHealth Service grants no. 5-P50-HD11149, 5-ROI-HD14513,and 5-POl-AG00306. Dr. Casey is a postdoctoral trainee sup-ported, in part, by U. S. Public Health Service training grantno. 1-T32-HD07190.

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