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Unconjugated estriol and 15α-hydroxyestriol in complicated pregnancies
Unconjugated estriol and 15a-hydroxyestriol in complicated pregnancies ALBERT D. NOTATION, PH.D. GEORGE E. TAGATZ, M.D. Minneapolis, Minnesota Simultaneous determinations of unconjugated estriol and 15a-hydroxyestriol (E.) levels in maternal serum were studied serially to ascertain the relative usefulness of these estrogens as indicators of fetal welfare. Complicated pregnancies included 16 patients with pre-eclampsia and/or hypertension, six patients with severe Rh-isoimmunization, 12 patients with diabetes mellitus, of which four had vascular disease, ~ree patients with fetal death in utero, and three twin pregnancies. Retrospective analysis failed to indicate a clinically useful role for serum E4 determinations in the evaluation of fetal welfare during high-risk pregnancies. (AM. J. 0BSTET. GYNECOL. 128: 747, 1977.)
EsTRIoL ( Ea) AssAYs are used extensively for the evaluation of fetoplacental function in high-risk pregnancies. Theoretically, 15a-hydroxyestriol (estetrol, f..t) concentrations might be a more sensitive indicator of fetoplacental function; cross-sectionai studies comparing E3 and E4 in serum 1 and studies evaluating L in serum 10 • 11 and urine 3 • 6 were promising. We investigated the possible usefulness of estetrol assays as a more sensitive indicator of intrauterine fetal distress 1 for the following reasons: I. Significant !Sa-hydroxylase activity, which is necessary for E4 formation, has been found regularly only in the fetus and the neonate. 2- 4 By contrast, although the majority of E3 production in late pregnancy originates in the fetoplacental unit, the maternal produciion of E3 is also increased during pregnancy. 2. The Et molecule is not as susceptible to further metabolism (hydroxylation) in vivo5 as is the less complex E3 molecule. 3. Preliminary cross-sectional studies demonstrated a relatively greater increase in Et concentration as com-
pared to E3 concentration as pregnancy progressed to term.L 6 In this studv. unconiu~:Tated F.. -----levels were o-- --- F.,... and -----------:. --determined by radioimmunoassay simultaneously in serial samples of maternal serum. In some patients, concomitant measurements of serum "total estrogens" (TE) by radioimmunoassay and urinary E3 by gasliquid chromatography were obtained to establish further the relative clinical usefulness of these estrogen assays. ,~
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Material and methods Patient selection and management. Patients were selected from the University of Minnesota and affiliated hospital obstetrics clinics. A total of 34 patients with various pregnancy complications were studied: 16 patients had pre-eclampsia and/or hypertension; six patients had severe Rh-isoimmunization; 12 patients had diabetes mellitus-eight patients had Class A, B, or C diabetes, and four patients had Class D, F, or T diabetes (White's classification). 7 Each of these pregnancies terminated with a live-born infant; there were no neonatal deaths. Additionally, three patients with fetal deaths in utero and also three twin pregnancies were studied. Most high-risk pregnancies were managed accordin~r ._. to the clinical course and the results --- of - - urinarv - -------I estriol determinations and oxytocin challenge tests (OCT's); serum estrogen data were analyzed retrospectively. Blood samples were obtained serially during the last trimester of pregnancy from all patients studied. Serum (5 to 10 ml.) was separated and frozen at -20° C. until assayed. Controls consisted of 57 samples ob-
From the Departments of Obstetrics and Gynecology and Biochemistry, University of Minnesota Medical School. Supported by The National Foundation, Grant CRBS-282.
Received for publication November 4, 1976. Revised February 24, 1977. Accepted March 3, 1977. Reprint requests: Dr. Aibert D. Notation, Department of Obstetrics and Gynecology, University of Minnesota Medical School, Box 395, Mayo Memorial Bldg., 420 Delaware St. S.E., Minneapolis, Minnesota 55455.
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Fig. 1. Serum TE levels obtained serially from nine patients with uncomplicated pregnancies. A normal range for each graph is represented by the shaded areas which outline the upper and lower boundaries of the 57 values measured. A regression line and its 95 per cent confidence limits have been calculated for each series of 57 values.(f)represents levels for twin pregnancies.
Fig. 2. Serum f.:J levels obtained serially from nine patients with uncomplicated pregnancies. A normal range for each graph is represented by the shaded areas which outline the upper and lower boundaries of the 57 values measured. A regression line and its 95 per cent confidence limits have been calculated for each series of 57 values.(f)represents levels for twin pregnancies.
tained from nine healthy patients with normal pregnancy outcomes. Reagents. Nonradioactive estriol was purchased from Schwarz/Mann, Orangeburg, New York; estetrol, from Steraloids; reagent grade ethyl acetate, benzene, and methanol from Fisher Scientific Co., Pittsburgh, Pennsylvania; and Sephadex LH-20, from Pharmacia Fine Chemicals, Inc., Piscataway, New Jersey. 6,7- 3 Hestetrol (3 H-E4), specific activity 40 Ci per miliimoie, and 2,4,6,7- 3 H-estriol eH-E3 ), specific activity 110 Ci per miiiimoie, were purchased from New Engiand Nuclear, Boston, Massachusetts. The antiserum was produced by Erlio Gurpide, Ph.D., of the Departments of Obstetrics and Gynecology and Biochemistry, Mount Sinai School of Medicine, New York, New York. Estriol-16, 17 -dihemisuccinate was conjugated to bovine serum albumin and used as the antigen to prepare ovine estrogen antiserum. Assay methods. Previous reports from this laboratory have provided detailed information on the assay methods used in this study. These include the specificity of the antiserum, the limits of error and sensitivity,8 the simultaneous determination of unconjugated E3 and E4 in maternal serum and amniotic fluid,' and the determination of TE in maternal serum, urine, or amniotic fluid. 9
TE were determined by precipitating proteins from serum with nine volumes of methanol; dried residues from aliquots of the crude methanolic extract were used for radioimmunoassay. The ovine antiserum utilized in the radioimmunoassay has very high binding affinities for ring-D conjugated estrogens but not ring-A conjugated estrogens or nonphenolic steroids. Estriol, estradiol-17{3, and estrone bind to the antibody with neariy equal efficiency, but ~ and epiestrioi bind with about one half the efficiency of E3 • The TE values are defined as an index of TE content; we previously found TE to be proportional to unconjugated E, in serum.' Simultaneous determination of unconjugated E3 and Lt in maternal serum was performed. Each sample of serum was placed in extraction tubes containing recovery standards (about 5,000 counts per minute (c.p,m,) for 3 H-E3 and about 2,000 c.p.m. for 3 H-Lt) and extracted three times with ethyl acetate. The dried residue from the combined ethyl acetate extracts of each sample was chromatographed on a Sephadex LH-20 (3 Gm.) column with benzene: methanol (80: 20) solvent. Midpeak fractions of each unconjugated estrogen, located by the presence of tritiated recovery standard, were separated into aliquots for radioimmunoassay and recovery measurements. Dried aiiquots were dis-
Unconjugated E3 and E4 in complicated pregnancies 749
Volume 128 Number 7
solved in 0.25 mi. of solution containing 3 H-E;; (45,000 c.p.m.) in O.OIM phosphate buffer containing 0.9 per cent sodium chloride at pH 7.4, then mixed with 0.5 ml. of buffered solution containing sheep antiserum ( 1 : 5,000 dilution) and 1 per cent human male serum. Samples were equilibrated in an ice bath for at least 30 minutes, then mixed with saturated ammonium sulfate solution (0.75 mi.) to precipitate protein. The precipitated protein fraction, which includes equilibrated estrogen antibody (sheep antiserum) saturated with bound estrogen, was isolated as a pellet at the bottom of the tube by centrifugation at 8,000 g for 30 minutes at 4° C. A 0.5 mi. aliquot of the supernatant which contained unbound estrogen was extracted with 10 mi. of toluene solution containing scintillation indicators, and the extracted estrogen was counted in a /1-scintillation counter. A calibration curve was constructed by plotting the per cent of unbound radioactive estrogen in the supernatant versus the amount of estrogen in the assay tube. Assay calculations include a correction for the radioactive recovery standard introduced into the serum sample prior to the initial extraction. An experienced technician required three days to pmcess each eight-sample batch.
Results and comment Assay verification. Although the assays used in this study have been verified previously, 1• 8 the assay was continuously monitored by duplicate and repeat determinations. The lower limit for the measurement of either F-:~ or F-4 was considered to be about 0.1 ng. of estrogen per milliliter of serum. Measurements were obtained below this level, but the results became erratic. Independent verification of the serum E3 values was conducted on randomly selected duplicate samples by Dr. J. E. Christner at the Ames Co., Inc., in Elkhart, Indiana, where E;; was determined without previous chromatographic isolation with the estriol-specific antiserum of Lindner. The cross-reactivity of the Lindner antiserum with other estrogens present resulted in values consistently higher but linearly proportional to the Ea values obtained in this study. Normal values. Fifty-seven serial samples of maternal serum were obtained between 0730 and 0830 hours from nine patients with uncomplicated pregnancies during the thirtieth to the fortieth weeks of gestation. "Total radioimmunoassayable estrogens" (TEj are presented in Fig. I; unconjugated estriol (Ea), in Fig. 2; and unconjugated estetrol (E4 ), in Fig. 3: Each patient is identified by a single number. For each patient, there are apparent fluctuations with a general trend to increase in all estrogen values as pregnancy progresses. Sudden large increases in all estrogen levels often oc-
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Fig. 3. Serum E. levels obtained serially from nine patients with uncomplicated pregnancies. A normal range for each graph is represented by the shaded areas which outline the upper and lower boundaries of the 57 values measured. A regression line and its 95 per cent confidence limits have been calculated for each series of 57 values.QJrepresents levels for twin pregnancies.
Table I. Summary of estetrol levels in serum from patients with pregnancies having normal outcomes (complete data are represented in Fig. 3) E4 (ng.!mL of serum)
Weeks' gestation
No. of patients
31 33 35 36 37 38 39 40
4 7 9 5 8 7
7 8
Mean ±2 S.D. (95% confidence limits)
0.63 ± 1.37 ± 1.57 ± 2.58 ± 1.38 ± 1.97 ± 1.50 ± 2.02 ±
0.46 2.83 1.43 2.26 1.30 2.08 0.94 2.06
I Lowest value observed 0.46 0.59 0.69 1.37 0.92 1.02 0.76 1.02
curred after the thirty-fourth week of gestation. However, particularly large fluctuations in the concentration of serum ~ were observed which could not be related to the clinical course of pregnancy. Large individual variations in the concentration of estrogens were observed at a given gestational age; however, serial concentrations for a given patient tended to maintain a consistent (high or low) position within the range of values observed for all patients. Accepted statistical methods were applied to establish normal ranges: I. The regression line and its 95 per cent confidence limits were calculated for each set of values.
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Fig. 4. Maternal serum estrogen levels determined for samples taken at 0800 and 2000 hours for cornparison. Concotni~ tant urinary estriol values are presented. Patient L. H. had hypertension with superimposed severe pre-eclampsia; Patie~t S. W. had diabet~s meilitus, Class F. . .
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Fig. 6. Serum Ea levels obtained serially from four patients with diabetes mellitus, Class A, B, or C as designated, but no vascular disease. The upper and lower boundary (shaded areas) and the 95 per cent confidence limits of the regression line calculated for the corresponding normal values are superimposed for comparison.
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Fig. 5. Serum TE levels obtained serially from four patients with diabetes mellitus, Class A, B, or C as designated, but no vascular disease. The upper and lower boundary (shaded areas) and the 95 per cent confidence limits of the regression line calculated for the corresponding normal values are superimposed for comparison.
2. The mean value and its 95 per cent confidence limits were calculated for each time point in Figs. l, 2, and 3. Mean serum values at the fortieth week of pregnancy are 15.1 ± 1.4 (S.E.) p.g of TEper 100 mi. of serum; 14.3 ± 1.5 (S.E.) ng. of Ea per milliliter of serum; and 2.02 ± 0.36 (S.E.) ng. of E.t per milliliter of serum. Table I summarizes the serial changes in the serum concentration of E.t. The lower 95 per cent confidence limit (mean ± 2 S.D.) would give a value of nearly 0 ng. of E4 per miiiiiiter of serum, when, in fact, 0.46 ng. of
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Fig. 7. Serum E. levels obtained serially from four IYatients with diabetes mellitus, Class A, B, or C as designated, but no vascular disease. The upper and lower boundary (shaded areas) and the 95 per cent confidence limits of the regression line calculated for the corresponding normal values are superimposed for comparison.
E.t was the lowest value observed in normal pregnancy. The upper 95 per cent confidence limit more nearly approached the maximum values observed. Thus, for purposes of comparison, the range of normal values, depicted as the shaded area in these and subsequent figures, consists of smooth lines outlining the range of actuai vaiues observed in normai pregnancies.
Unconjugated E3 and E4 in complicated pregnancies
Volume 128 Number 7
751
Table II. Diabetes mellitus: Class A, B, or C Delivery Case No.* Al
TimF lapse between last serum estrogen assay and delivery
Urinary Ea excretion (mg.l24 hr.) 20.6, 2 days ptd:j: 13.4, 1 week ptd 9.4, 1 day ptd 38. I, 4 days ptd
2 days
Bl
I day
B2
1 week
B3
2 weeks
Cl C2 C3 C4
3 days 2 weeks 1 day 3 days
8.4, 30 weeks 30.3, 36 weeks Decreased Decreased ~.D.§
~ochange
Duration of pregnancy (weeks)
Modct CIS, mild pre-eclampsia,
Fetal weight (grams)
38
4,640
36
2,850
38¥.!
4,200
38
3,070
36 37¥.! 36 36¥.!
2,450 3,600 3,200 3,100
Duration of pregnancy (weeks)
Fetal weight (grams)
fetal macrosomia CIS, mild pre-eclampsia, positive OCT CIS, Type II fetal bradycardia, intrapartum hemorrhage Induction, NSVD, hydramnios CIS, decreased urinary Ea CIS, decreased urinary Ea CIS, positive OCT Induction, NSVD, elected to induce when LIS ratio indicated fetal maturity
*Letter designates diabetes class. tC/S =Cesarean section; NSVD =normal spontaneous vaginal delivery. tptd = Prior to delivery. §N.D. =Not determined.
Table III. Diabetes mellitus: Class D, F, or T
Case No.* Dl
Time lapse between last serum estrogen assay and delivery 1 day
Delivery Urinary£ 3 excretion (mg./24 hr.) F:j:
D2
1 day
N.D.§
F
2 days
Increased
T
I day
IJI
Mockt CIS, mature LIS and creatinine,
prior antepartum fetal death Repeat CIS, PSROM Spontaneous labor, Type II fetal bradycardia, emergency CIS CIS; mature LIS, biparietal
36 3 /7
3,160
35
2,680
344 /7 36
2,270 (mild RDS) 2,920
*Letter designates diabetes class. tC/S = Cesarean section; PSROM = premature spontaneous rupture of membranes. :j:F = fluctuating. §N.D. = Not determined. 11 L =Low.
The normal ranges for estrogen concentrations in maternal serum obtained in this study coincide with those reported previously from this laboratory and are in good agreement with other published values for TE, 12 E3 , 13 and E4 •10• u In high-risk pregnancies, many of the observed serum F4 concentrations approached the lower limit of detection. The variation between duplicate E.t assays increased significantly for values approaching the lowest detectabk concentration. Thus, with low values, the changes in serial E.t determinations must be relatively larger to be considered significant. In samples with low ~ concentrations, the Ea concen-
trations remained within the experimentally optimum range. Fluctuations in serial serum estrogen concentration and urinary estriol excretion during normal pregnancies are substantial. Urinary estriol excretion was found to be linearly proportional to the maternal serum concentrations ofT£9 and E3 •1 The day-to-day variation in urinary estriol excretion is such that a decrease of 35 per cent of the mean amount excreted during three previous days is clinically significant. In this study, maternal serum E.t levels decreased occasionally as much as 70 per cent without apparent cause and with
752 Notation and Tagatz Am.
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Fig. 8. Serum TE levels obtained serially from patients with pregnancies complicated by diabetes mellitus, Class D, F, or T as designated, and each with accompanying vascular disease. The upper and lower boundary (shaded areas) and the 95 per cent confidence limits of the regression line cakulated for the corresponding normal values are superimposed for comparison.
Weeks Gestation
Fig. 9. Serum E4 levels obtained serially from patients with pregnancies complicated by diabetes mellitus, Class D, F, or T as designated, and each with accompanying vascular disease. The upper and lower boundary (shaded areas) and the 95 per cent confidence limits of the regression line calculated for the corresponding normal values are superimposed for comparison.
no ill effect observed in the fetus. Similar large increases in f..tlevels occurred in an erratic manner. Such large spontaneous fluctuations in L concentration failed to provide the clinician with an interpretable trend. Twin pregnancies. Estrogen concentrations determined in 14 samples from three patients with twin pregnancies were generally within the ranges obtained
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Fig. 10. Serum E., levels obtained serially from patients with pregnancies complicated by diabetes mellitus, Class D, F. or T as designated, and each with acwmpanying vascular disease. The upper and lower boundary (shaded areas) and the 95 per cent confidence limits of the regression line calculated for the corresponding normal values are superimposed for comparison.
for normal singleton pregnancies: one patient bad a single determination at 31 weeks. These patients are identified by the symbol(f)in Figs. 1, 2. and 3. The values were not significantly different from those obtained for normal singleton pregnancies. Diurnal variation. There are conflicting reports regarding the existence of a circadian rhythm in plasma estriol levels. A rhythm with lowest levels at 0800 hours and highest at 2000 hours was reported. 14 However. the absence of regularity in morning-evening differences is well documented for plasma f.s 15 and plasma 1'4.10 Morning (0800 hours) and evening (2000 hours) samples were obtained in two high-risk patients depicted in Fig. 4. Serum E3 and E4 levels with concomitant daily urinary estriol values (by gas-liquid chromatography) at 35 to 36 weeks' gestation are shown for Patient L H. who had hypertension and superimposed severe pre-eclampsia; creatinine clearance was 103 mi. per minute per 1.73 sq. M. Serum TE. E3 , and L levels with concomitant daily urinary estriol values at about 34 weeks' gestation are shown for Patient S. W. with diabetes mellitus, Class F; creatinine clearance was 85 mi. per minute per 1.73 sq. M. A consistent relationship between morning-evening concentrations of £ 3 , E'..t, and TE was not observed. Diabetes mellitus with no vascular disease. Forty serum samples were obtained from eight pregnant patients with diabetes mellitus and no clinical evideme of vascular disease. TE values are presented in f'ig. 5: E3 .
Unconjugated E3 and E4 in complicated pregnancies 753
Volume 128 ~umber 7
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Fig. U. Serial serum concentrations of TE, ~.and ~ in relation to changes in AO.D-4 50 in amniotic fluid from a patient with pregnancy complicated with Rh-isoimmunization.
Fig. 12. Serial serum concentrations of TE, ~. and E4 in relation to changes of ~. E4 , and AO.D,.$0 in amniotic fluid from a patient with pregnancy complicated with Rhisoimmunization.
in Fig. 6; and E.&, in Fig. 7. The time interval elapsed from the last determination to the date of delivery, urinary estriol excretion data, and notes describing each delivery are presented in Table II. In two cases, B 1 and C3, OCT's were positive. In Case B 1, E4 and Ea remained high normal, although TE fell27 per cent and urinary estriol excretion fell 30 per cent. In Case C3, serum Ea decreased by 8 per cent, while TE fell 30 per cent; E4 was consistently below the normal range throughout pregnancy. Serial assays of E.& failed to correlate with the positive OCT in Case B I, but, in Case C3, persistently low E4 concentrations preceded a positive OCT. Diabetes mellitus with vascular disease. Forty-seven serum samples were obtained from four pregnant women with diabetes mellitus complicated by vascular disease. TE values are presented in Fig. 8; Ea. in Fig. 9; and E.&, in Fig. I 0. Additional clinical data for each patient are presented in Table III. All diabetic patients with vascular disease had serum estrogen determinations within two days of delivery. In Case D I, with serum TE and E3 levels indicating normal fetoplacental function prior to delivery, there was a persistently low E.& level. The fetus was delivered when mature and was not in distress. In Case D2, levels of serum estrogens were normal, but delivery was effected for a clinical indication, and the infant did well. In Case F, the persistently low serum Ea and E.t levels
Table IV. Serum estrogen levels for patients with pregnancies complicated with Rh-isoimmunization at
35 to 36 weeks' gestation Group
TE (~-tgllOO ml. of serum)
Ea (ng.lml. of serum)
E 4 (ng.lml. of serum)
0.24
Cases
1 2
10.8
11.8
ll.2
14.4
0.43
3 4
5.6 5.3
11.4
5 6*
4.0
6.4 l.O
0.88 0.33 0.20 0.25
8.5 ± 2.0
0.39 ± 0.23
Mean± 7.38 ± 0.96 S.E. (No. 5)
6.0
(No.= 6)
(No.
6)
Controls at 35 weeks
(No.= 9)
Mean ± 9. 79 ± 0.97
0.5
±
1.2
1.57 ± 0.70
S.E.
*AO.D. 450 = 0.94 in amniotic fluid. Fetus died after intrauterine transfusion.
may have indicated chronic placental insufficiency, since emergency cesarean section was performed for fetal bradycardia accompanying spontaneous labor. In Case T, serum Ea <1-nd TE correlated inversely with urinary Ea excretion as glomerular filtration decreased.7 However, serum~. in addition to fluctuating markedly, fell to low levels prior to delivery and may have indicated chronic placental insufficiency.
754
Notation and Tagatz ·\.m.
Weeks Gestation
Fig. 13. Serial serum concentrations of Ea in patients with pregnancies complicated with hypertension or pre-eclampsia. The upper and iower boundary (shaded areas) and che 95 per cent confidence limits of the regression line calculated for the corresponding normal values are superimposed for comparison.
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Fig. 14. Serial serum concentrations of E. in patients with pregnancies complicated with hypertension or pre-eclampsia. The upper and lower boundary (shaded areas) and the 95 per cent confidence limits of the regression line calculated for the corresponding normal values are superimposed for comparison.
Serum estrogen assays did not influence the timing of any delivery in t..~is study of 12 diabetic patients. In retrospect, the serum E4 measurements failed to provide clinically useful information for the management of these pregnancies. Rh-isoimmunization. The data presented in Table IV were obtained at approximately 35 weeks of gestation from six patients with pregnancies complicated with Rh-isoimmunization. The maternal serum TE and
1.
August I. l '177 Obstc!. c;, ncwl.
E3 levels were distributed within their respectiw normal ranges. However. the E~1 levels were consi~tentlv below the normal range. Two patients in whom the fetal condition improved greatly as pregnancy progressed still had low serum t~ levels. In one patient (Fig. 11 ), the maternal serum TE and £3 levels increased while the Ll optical density450 (LlO.D.45o) for concomitant amniotic fluid samples decreased, but serum £ 4 remained low and unchanged. In another patient (Fig. 12), both t~l and £ 4 levels in amniotic fluid increased significantlY as the llO.D.45o decreased. The matching TE and .1<~1 levels in maternal serum also increased, but no change 1ras observed for the accompanying £4. Four pregnancies in a previously reported group of nine patients with Rh-isoimmunization followed a irend in \vhich the concentration of Et in arnniotit fluid was inversely related to the Ll0.D.450 of the amniotic fluid. However, simultaneous maternal serum E 4 concentrations were not consistently related to either E 4 concentrations or Ll.O.D.-J,;o in atnniotic fluid.H The consistently low serum £ 4 is indicative of the liabilities inherent in assaying a product of fetal origin in maternal blood. When produced, fetal £ 4 undergoes at least two different types of distribution: It becomes distributed by further metabolism into wnjugated and unconjugated pools, and each of these pools is further distributed into various tissue compartments. Rates of entry into maternal blood and subsequent clearance rates vary for each metabolite and also for each compartment accessible to maternal peripheral circulation. It is beyond the scope of this study to identify which. if any, of these factors contribute to the low serum E4 values observed here. The fact remains that maternal serum E4 concentrations are not predictably related to the degree of sensitization in Rh-isoimmunized pregnancies. Hypertension and pre-eclampsia. Reports indicate that E4 determinations in maternal urine and serum may be useful in monitoring fetal welfare in patients with pre-eciampsia, eciampsia, andior hyperrensionH. 10 Fifty-seven serum samples were obtained from 16 patients with pregnancies complicated by hypertension or pre-eclampsia. Ea Yalues are presented in Fig. 13, and & levels arc given in Fig. 14. Solitary determinations were obtained in hve patients; all values \Vere \Yithin the norma! range. 1\vo of these patients had mild pre-eclampsia, and pregnancy was allowed to progress to the onset of spontaneous labor. Three patients had labor induced for di:nical indications of increasing hypertension or worsening pre-eclampsia. One determination was obtained four days prior to induction of labor, and two were obtained one day prior to labor induction; the values did not
Unconjugated E3 and E4 in complicated pregnancies 755
Volume 128 Number 7
Table V. Hypertension; pre-eclampsia Delivery Case
Time lapse between last serum estrogen
no.
assay and delivery
2 weeks 2
2 weeks
3 4 5 6
3 days 2 weeks 2 days 2 weeks
7
3 days
8
2 weeks
Urinary Ea
Duration of pregnancy (weeks)
Fetal weight (grams)
NSVD
40
2,670
Induction, NSVD
38
3,345
Induction Induction Induction Spontaneous labor, meconium staining, Type II fetal bradycardia, emergency CIS NSVD
37 38 38 40
2,190 3,270 3,420 3,200
41
3,170
Induction, (elected induction at40 weeks) NSVD Induction, Type II fetal bradycardia, emergency CIS Elective CIS
40
3,300
39 40
2,380 2,300
36
2,380
excretion (mg./24 hr.)
8.5, 39 weeks 42.6, delivery Ft 8.8-13.8, 36-38 weeks N.D.t 16,36 weeks 20.7, 2 days ptd§ 20.6, 38 weeks
9 10
216 weeks 2 days
11.7, 39 weeks 11.6, 40 weeks 11.9, 3716 weeks 6.5, 39 weeks N.D. Decreased ptd
ll
2 days
Widely F
Mode*
*CIS = Cesarean section; NSVD = normal spontaneous vaginal delivery. tF = Fluctuating. :j:N.D. Not determined. §ptd = Prior to delivery.
indicate impending fetal jeopardy, although the clinical course was ominous. Serial determinations were obtained from 11 patients. The time interval elapsed from the date of the last determination to the date of delivery, urinary estriol excretion data, and notes describing each delivery are presented in Table V. Five patients had final estrogen determinations performed within the three days prior to delivery. In Case 7, pregnancy continued to the spontaneous onset of labor; serum & was persistently low, although serum E4 was in the normal range. Three patients had induction of labor for increasing severity of pre-eclampsia; the serum .& level rose in Case 5, fell in Case 3, and fluctuated from a persistently low level in Case 10. In Case 10, cesarean section was performed for acute placental insufficiency during labor. The final patient (Case 11 with borderline normal ~ values) had an elective cesarean section for severe pre-eclampsia. Thus, serial determinations of E4 failed to correlate with the clinical course in three of these five pregnancies. Intrauterine fetal death. Serum samples were obtained from three patients in whom fetal death occurred. The first patient was initially seen at the thirty-ninth week of gestation. A fetogram revealed polyhydramnios and a hydrocephalic fetus with radiographic signs of fetal death. One week later, the fetal heart tones were still present, but the maternal serum
contained no detectable Ea or&. On the following day, fetal heart tones were not detected. At 40 weeks, decompression of the hydrocephalus was required to deliver a 3,400 gram fetus. Fetal adrenals weighed 2 grams, about one fifth of the normal weight. Multiple congenital anomalies of the fetus included hydrocephalus, deft palate, and bilateral deft lip with secondary deformation of the nose, patent ductus arteriosus, atrial septal defect, and polydactylism of the hands. A second patient with rheumatic heart disease, mitral stenosis (functional Class I), and idiopathic thrombocytopenia was carrying a dead fetus when examined at 25 weeks of gestation. At that time, 0.10 ng. of Ea and 0.14 ng. of & per milliliter of serum were detected, while corresponding urinary estriol excretion was 3.3 mg. per 24 hours. A 6 em. macerated fetus (25 weeks) was delivered following intra-amniotic injection of 90 Gm. of urea. The third patient had two stillbirths eight and 10 years prior to the current pregnancy. Gestational age was uncertain since this pregnancy followed oral contraceptive withdrawal. The first maternal serum sample was obtained during the second trimester and contained 2.6 ng. of F.-a and 0.43 ng. of.& per milliliter of serum. Three weeks later, when no fetal heart tones could be detected, three successive morning serum samples contained no detectable E3 and 0.37, 0.13, and
756
Notation and Tagatz Am.
0 ng. of E.t per milliliter, respectively. Intra-amniotic urea administration resulted in the expulsion of a 730 gram fetus at 26 weeks of gestation. Fetal adrenals weighed 0.4 grams, about one eighth of the normal weight. The other organs had weights which were generally about one half of the normal. The etiology of fetal death in utero could not be ascertained. In each of the three cases where fetal death occurred, the maternal serum concentrations of both E3 and E 4 were far below normal in the initial determination. When urinary excretion of E3 and E4 is compared, E.t is cleared less rapidly by the kidneys of both the postpartum mother and the neonate. 16 In one case of fetal death in utero, the maternal serum E3 was no longer detectable, while decreasing levels of & were
August l. 1977 Obstet. Gvnerol.
measured on three successive days. If the dead fetus no longer produces &. then I~ is retained much long-er than E3 in the various maternal and fetoplacental compartments. The determination of serial & concentrations in maternal serum has been evaluated as an indicator of fetal welfare during high-risk pregnancies. ~ was not consistently related to either ~ concentrations in maternal serum and urine or the clinical course and pregnancy outcome; no clinically useful trends were discernible. Thus, serial concentrations of maternal serum E4 do not appear to be clinically useful in the management of high-risk pregnancies. The authors acknowledge the helpful assistance of Mrs. Dianne Judd and Miss Jan McLeod.
REFERENCES I. Giebenhain, M. E., Tagatz, G. E., and Gurpide, E.: Serum levels of unconjugated estetrol during human pregnancy,]. Steroid. Biochem. 3: 707, 1972. 2. Hagen, A. A., Barr, M., and Diczfalusy, E.: Metabolism of 17,8-estradiol-4-"C in early infancy, Acta Endocrinol. 49: 207, 1965. 3. Adlercreutz, H., and Luukkainen, T.: Determinations of urinary estrogens by gas chromatography, in Lipsett, M. B., editor: Gas Chromatography of Steroids in Biological Fluids: Proceedings of an Airlie House Conference, New York, 1965, Plenum Press, Inc., p. 215. 4. Gurpide, E., Schwers, ]., Welch, M. T., Vande Wiele, R. L., and Lieberman, S.: Fetal and maternal metabolism of estradiol during pregnancy, .J. Clin. Endocrinol. Metab. . 26: 1355, 1966~ - 5. Fishman, J., Schut, H., and Solomon, S.: Metabolism, production, and excretion rates of 15a-hydroxyestriol in late pregnancy,]. Clin. Endocrinol. Meta b. 35: 339, 1972. 6. Heikkila, J., and Luukkainen, T.: Urinary excretion of estriol and 15a-hydroxyestriol in complicated pregnancies, AM.]. 0BSTET. GYNECOL. 110: 509, 1971. 7. Tagatz, G. E., Arnold, N. 1., Goetz, F. C., Najarian,]. S., and Simmons, R. L.: Pregnancy in ajuveniie diabetic after renal transplantation (Class T diabetes mellitus), Diabetes 24: 497. 1975. 8. Sciarra,].]., Tagatz, G. E., Notation, A. D., and Depp. R.:
J.
9.
10.
11.
12. 13. 14. 15.
16.
Estriol and estetrol in amniotic fluid. AM. J. OssTET. GYNECOL. 118: 626, 1974. Gurpide, E., Giebenhain, M. E., Tseng, L., and Kelly. W. G.: Radioimmunoassay for estrogens in human pregnancy urine, plasma, and amniotic fluid, AM. ]. Ossn:T. GYNECOL. 109: 897, 1971. Tnlrhimkv D .. Frivoletto. F. D .. Rvan. I. K .. and Fi~t;~~~~-.-]:: Pl~srn~"~s~trol as an index of fetal wellbeing,]. Clin. Endocrinol. Metab. 40: 560, 1975. Korda, A. R., Challis, ]. ]., and Anderson, A. B. M.: Plasma unconjugated 15a-hydroxyoestriol (oestetrol) levels in normal human pregnancy, Br. J. Obstet. Gynaecoi. 82:882,1975. Masson, G. M.: Plasma oestriol concentration during normal pregnancy, Br.]. Obstet. Gynaecol. 80: 201. 1973. Tulchinsky, D., and Abraham, G. E.: Radioimmunoassay of plasma estriol, ]. Clin. Endocrinol. Metab. 33: 775, 1971. Goebel, R., and Kuss, E.: Circadian rhythm of serum unconjugated estriol in late pregnancy, J. C1in. Encfocrinol. Metab. 39: 969, 1974. Levitz, M., Slyper, A.J., and Selinger, M.: On the lack of periodicity in plasma estriol in human pregnancv.J. Clin. Endot:rinul. ~fetab. 38: 698, 1974. Heikkila, J.: Excretion of 15a-hydroxyoestriol and oestriol in urine of newborn infants and in maternal urine before and after delivery, J. Endocrinol. 52: 119, 1972.
EsTRIoL ( Ea) AssAYs are used extensively for the evaluation of fetoplacental function in high-risk pregnancies. Theoretically, 15a-hydroxyestriol (estetrol, f..t) concentrations might be a more sensitive indicator of fetoplacental function; cross-sectionai studies comparing E3 and E4 in serum 1 and studies evaluating L in serum 10 • 11 and urine 3 • 6 were promising. We investigated the possible usefulness of estetrol assays as a more sensitive indicator of intrauterine fetal distress 1 for the following reasons: I. Significant !Sa-hydroxylase activity, which is necessary for E4 formation, has been found regularly only in the fetus and the neonate. 2- 4 By contrast, although the majority of E3 production in late pregnancy originates in the fetoplacental unit, the maternal produciion of E3 is also increased during pregnancy. 2. The Et molecule is not as susceptible to further metabolism (hydroxylation) in vivo5 as is the less complex E3 molecule. 3. Preliminary cross-sectional studies demonstrated a relatively greater increase in Et concentration as com-
pared to E3 concentration as pregnancy progressed to term.L 6 In this studv. unconiu~:Tated F.. -----levels were o-- --- F.,... and -----------:. --determined by radioimmunoassay simultaneously in serial samples of maternal serum. In some patients, concomitant measurements of serum "total estrogens" (TE) by radioimmunoassay and urinary E3 by gasliquid chromatography were obtained to establish further the relative clinical usefulness of these estrogen assays. ,~
-
J
~
Material and methods Patient selection and management. Patients were selected from the University of Minnesota and affiliated hospital obstetrics clinics. A total of 34 patients with various pregnancy complications were studied: 16 patients had pre-eclampsia and/or hypertension; six patients had severe Rh-isoimmunization; 12 patients had diabetes mellitus-eight patients had Class A, B, or C diabetes, and four patients had Class D, F, or T diabetes (White's classification). 7 Each of these pregnancies terminated with a live-born infant; there were no neonatal deaths. Additionally, three patients with fetal deaths in utero and also three twin pregnancies were studied. Most high-risk pregnancies were managed accordin~r ._. to the clinical course and the results --- of - - urinarv - -------I estriol determinations and oxytocin challenge tests (OCT's); serum estrogen data were analyzed retrospectively. Blood samples were obtained serially during the last trimester of pregnancy from all patients studied. Serum (5 to 10 ml.) was separated and frozen at -20° C. until assayed. Controls consisted of 57 samples ob-
From the Departments of Obstetrics and Gynecology and Biochemistry, University of Minnesota Medical School. Supported by The National Foundation, Grant CRBS-282.
Received for publication November 4, 1976. Revised February 24, 1977. Accepted March 3, 1977. Reprint requests: Dr. Aibert D. Notation, Department of Obstetrics and Gynecology, University of Minnesota Medical School, Box 395, Mayo Memorial Bldg., 420 Delaware St. S.E., Minneapolis, Minnesota 55455.
747
748
Notation and Tagatz Am.
J.
August l. I Y77 Obstet. Gvne,ol.
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Fig. 1. Serum TE levels obtained serially from nine patients with uncomplicated pregnancies. A normal range for each graph is represented by the shaded areas which outline the upper and lower boundaries of the 57 values measured. A regression line and its 95 per cent confidence limits have been calculated for each series of 57 values.(f)represents levels for twin pregnancies.
Fig. 2. Serum f.:J levels obtained serially from nine patients with uncomplicated pregnancies. A normal range for each graph is represented by the shaded areas which outline the upper and lower boundaries of the 57 values measured. A regression line and its 95 per cent confidence limits have been calculated for each series of 57 values.(f)represents levels for twin pregnancies.
tained from nine healthy patients with normal pregnancy outcomes. Reagents. Nonradioactive estriol was purchased from Schwarz/Mann, Orangeburg, New York; estetrol, from Steraloids; reagent grade ethyl acetate, benzene, and methanol from Fisher Scientific Co., Pittsburgh, Pennsylvania; and Sephadex LH-20, from Pharmacia Fine Chemicals, Inc., Piscataway, New Jersey. 6,7- 3 Hestetrol (3 H-E4), specific activity 40 Ci per miliimoie, and 2,4,6,7- 3 H-estriol eH-E3 ), specific activity 110 Ci per miiiimoie, were purchased from New Engiand Nuclear, Boston, Massachusetts. The antiserum was produced by Erlio Gurpide, Ph.D., of the Departments of Obstetrics and Gynecology and Biochemistry, Mount Sinai School of Medicine, New York, New York. Estriol-16, 17 -dihemisuccinate was conjugated to bovine serum albumin and used as the antigen to prepare ovine estrogen antiserum. Assay methods. Previous reports from this laboratory have provided detailed information on the assay methods used in this study. These include the specificity of the antiserum, the limits of error and sensitivity,8 the simultaneous determination of unconjugated E3 and E4 in maternal serum and amniotic fluid,' and the determination of TE in maternal serum, urine, or amniotic fluid. 9
TE were determined by precipitating proteins from serum with nine volumes of methanol; dried residues from aliquots of the crude methanolic extract were used for radioimmunoassay. The ovine antiserum utilized in the radioimmunoassay has very high binding affinities for ring-D conjugated estrogens but not ring-A conjugated estrogens or nonphenolic steroids. Estriol, estradiol-17{3, and estrone bind to the antibody with neariy equal efficiency, but ~ and epiestrioi bind with about one half the efficiency of E3 • The TE values are defined as an index of TE content; we previously found TE to be proportional to unconjugated E, in serum.' Simultaneous determination of unconjugated E3 and Lt in maternal serum was performed. Each sample of serum was placed in extraction tubes containing recovery standards (about 5,000 counts per minute (c.p,m,) for 3 H-E3 and about 2,000 c.p.m. for 3 H-Lt) and extracted three times with ethyl acetate. The dried residue from the combined ethyl acetate extracts of each sample was chromatographed on a Sephadex LH-20 (3 Gm.) column with benzene: methanol (80: 20) solvent. Midpeak fractions of each unconjugated estrogen, located by the presence of tritiated recovery standard, were separated into aliquots for radioimmunoassay and recovery measurements. Dried aiiquots were dis-
Unconjugated E3 and E4 in complicated pregnancies 749
Volume 128 Number 7
solved in 0.25 mi. of solution containing 3 H-E;; (45,000 c.p.m.) in O.OIM phosphate buffer containing 0.9 per cent sodium chloride at pH 7.4, then mixed with 0.5 ml. of buffered solution containing sheep antiserum ( 1 : 5,000 dilution) and 1 per cent human male serum. Samples were equilibrated in an ice bath for at least 30 minutes, then mixed with saturated ammonium sulfate solution (0.75 mi.) to precipitate protein. The precipitated protein fraction, which includes equilibrated estrogen antibody (sheep antiserum) saturated with bound estrogen, was isolated as a pellet at the bottom of the tube by centrifugation at 8,000 g for 30 minutes at 4° C. A 0.5 mi. aliquot of the supernatant which contained unbound estrogen was extracted with 10 mi. of toluene solution containing scintillation indicators, and the extracted estrogen was counted in a /1-scintillation counter. A calibration curve was constructed by plotting the per cent of unbound radioactive estrogen in the supernatant versus the amount of estrogen in the assay tube. Assay calculations include a correction for the radioactive recovery standard introduced into the serum sample prior to the initial extraction. An experienced technician required three days to pmcess each eight-sample batch.
Results and comment Assay verification. Although the assays used in this study have been verified previously, 1• 8 the assay was continuously monitored by duplicate and repeat determinations. The lower limit for the measurement of either F-:~ or F-4 was considered to be about 0.1 ng. of estrogen per milliliter of serum. Measurements were obtained below this level, but the results became erratic. Independent verification of the serum E3 values was conducted on randomly selected duplicate samples by Dr. J. E. Christner at the Ames Co., Inc., in Elkhart, Indiana, where E;; was determined without previous chromatographic isolation with the estriol-specific antiserum of Lindner. The cross-reactivity of the Lindner antiserum with other estrogens present resulted in values consistently higher but linearly proportional to the Ea values obtained in this study. Normal values. Fifty-seven serial samples of maternal serum were obtained between 0730 and 0830 hours from nine patients with uncomplicated pregnancies during the thirtieth to the fortieth weeks of gestation. "Total radioimmunoassayable estrogens" (TEj are presented in Fig. I; unconjugated estriol (Ea), in Fig. 2; and unconjugated estetrol (E4 ), in Fig. 3: Each patient is identified by a single number. For each patient, there are apparent fluctuations with a general trend to increase in all estrogen values as pregnancy progresses. Sudden large increases in all estrogen levels often oc-
E
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Fig. 3. Serum E. levels obtained serially from nine patients with uncomplicated pregnancies. A normal range for each graph is represented by the shaded areas which outline the upper and lower boundaries of the 57 values measured. A regression line and its 95 per cent confidence limits have been calculated for each series of 57 values.QJrepresents levels for twin pregnancies.
Table I. Summary of estetrol levels in serum from patients with pregnancies having normal outcomes (complete data are represented in Fig. 3) E4 (ng.!mL of serum)
Weeks' gestation
No. of patients
31 33 35 36 37 38 39 40
4 7 9 5 8 7
7 8
Mean ±2 S.D. (95% confidence limits)
0.63 ± 1.37 ± 1.57 ± 2.58 ± 1.38 ± 1.97 ± 1.50 ± 2.02 ±
0.46 2.83 1.43 2.26 1.30 2.08 0.94 2.06
I Lowest value observed 0.46 0.59 0.69 1.37 0.92 1.02 0.76 1.02
curred after the thirty-fourth week of gestation. However, particularly large fluctuations in the concentration of serum ~ were observed which could not be related to the clinical course of pregnancy. Large individual variations in the concentration of estrogens were observed at a given gestational age; however, serial concentrations for a given patient tended to maintain a consistent (high or low) position within the range of values observed for all patients. Accepted statistical methods were applied to establish normal ranges: I. The regression line and its 95 per cent confidence limits were calculated for each set of values.
750 Notation and Tagatz Am.
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Fig. 4. Maternal serum estrogen levels determined for samples taken at 0800 and 2000 hours for cornparison. Concotni~ tant urinary estriol values are presented. Patient L. H. had hypertension with superimposed severe pre-eclampsia; Patie~t S. W. had diabet~s meilitus, Class F. . .
I
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32
34
36
38
40
\Veeks Gestation
Fig. 6. Serum Ea levels obtained serially from four patients with diabetes mellitus, Class A, B, or C as designated, but no vascular disease. The upper and lower boundary (shaded areas) and the 95 per cent confidence limits of the regression line calculated for the corresponding normal values are superimposed for comparison.
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Fig. 5. Serum TE levels obtained serially from four patients with diabetes mellitus, Class A, B, or C as designated, but no vascular disease. The upper and lower boundary (shaded areas) and the 95 per cent confidence limits of the regression line calculated for the corresponding normal values are superimposed for comparison.
2. The mean value and its 95 per cent confidence limits were calculated for each time point in Figs. l, 2, and 3. Mean serum values at the fortieth week of pregnancy are 15.1 ± 1.4 (S.E.) p.g of TEper 100 mi. of serum; 14.3 ± 1.5 (S.E.) ng. of Ea per milliliter of serum; and 2.02 ± 0.36 (S.E.) ng. of E.t per milliliter of serum. Table I summarizes the serial changes in the serum concentration of E.t. The lower 95 per cent confidence limit (mean ± 2 S.D.) would give a value of nearly 0 ng. of E4 per miiiiiiter of serum, when, in fact, 0.46 ng. of
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36
38
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Fig. 7. Serum E. levels obtained serially from four IYatients with diabetes mellitus, Class A, B, or C as designated, but no vascular disease. The upper and lower boundary (shaded areas) and the 95 per cent confidence limits of the regression line calculated for the corresponding normal values are superimposed for comparison.
E.t was the lowest value observed in normal pregnancy. The upper 95 per cent confidence limit more nearly approached the maximum values observed. Thus, for purposes of comparison, the range of normal values, depicted as the shaded area in these and subsequent figures, consists of smooth lines outlining the range of actuai vaiues observed in normai pregnancies.
Unconjugated E3 and E4 in complicated pregnancies
Volume 128 Number 7
751
Table II. Diabetes mellitus: Class A, B, or C Delivery Case No.* Al
TimF lapse between last serum estrogen assay and delivery
Urinary Ea excretion (mg.l24 hr.) 20.6, 2 days ptd:j: 13.4, 1 week ptd 9.4, 1 day ptd 38. I, 4 days ptd
2 days
Bl
I day
B2
1 week
B3
2 weeks
Cl C2 C3 C4
3 days 2 weeks 1 day 3 days
8.4, 30 weeks 30.3, 36 weeks Decreased Decreased ~.D.§
~ochange
Duration of pregnancy (weeks)
Modct CIS, mild pre-eclampsia,
Fetal weight (grams)
38
4,640
36
2,850
38¥.!
4,200
38
3,070
36 37¥.! 36 36¥.!
2,450 3,600 3,200 3,100
Duration of pregnancy (weeks)
Fetal weight (grams)
fetal macrosomia CIS, mild pre-eclampsia, positive OCT CIS, Type II fetal bradycardia, intrapartum hemorrhage Induction, NSVD, hydramnios CIS, decreased urinary Ea CIS, decreased urinary Ea CIS, positive OCT Induction, NSVD, elected to induce when LIS ratio indicated fetal maturity
*Letter designates diabetes class. tC/S =Cesarean section; NSVD =normal spontaneous vaginal delivery. tptd = Prior to delivery. §N.D. =Not determined.
Table III. Diabetes mellitus: Class D, F, or T
Case No.* Dl
Time lapse between last serum estrogen assay and delivery 1 day
Delivery Urinary£ 3 excretion (mg./24 hr.) F:j:
D2
1 day
N.D.§
F
2 days
Increased
T
I day
IJI
Mockt CIS, mature LIS and creatinine,
prior antepartum fetal death Repeat CIS, PSROM Spontaneous labor, Type II fetal bradycardia, emergency CIS CIS; mature LIS, biparietal
36 3 /7
3,160
35
2,680
344 /7 36
2,270 (mild RDS) 2,920
*Letter designates diabetes class. tC/S = Cesarean section; PSROM = premature spontaneous rupture of membranes. :j:F = fluctuating. §N.D. = Not determined. 11 L =Low.
The normal ranges for estrogen concentrations in maternal serum obtained in this study coincide with those reported previously from this laboratory and are in good agreement with other published values for TE, 12 E3 , 13 and E4 •10• u In high-risk pregnancies, many of the observed serum F4 concentrations approached the lower limit of detection. The variation between duplicate E.t assays increased significantly for values approaching the lowest detectabk concentration. Thus, with low values, the changes in serial E.t determinations must be relatively larger to be considered significant. In samples with low ~ concentrations, the Ea concen-
trations remained within the experimentally optimum range. Fluctuations in serial serum estrogen concentration and urinary estriol excretion during normal pregnancies are substantial. Urinary estriol excretion was found to be linearly proportional to the maternal serum concentrations ofT£9 and E3 •1 The day-to-day variation in urinary estriol excretion is such that a decrease of 35 per cent of the mean amount excreted during three previous days is clinically significant. In this study, maternal serum E.t levels decreased occasionally as much as 70 per cent without apparent cause and with
752 Notation and Tagatz Am.
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August l. 1977 Obstf>L (;ynerol.
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32
33
34
35
36
37
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Fig. 8. Serum TE levels obtained serially from patients with pregnancies complicated by diabetes mellitus, Class D, F, or T as designated, and each with accompanying vascular disease. The upper and lower boundary (shaded areas) and the 95 per cent confidence limits of the regression line cakulated for the corresponding normal values are superimposed for comparison.
Weeks Gestation
Fig. 9. Serum E4 levels obtained serially from patients with pregnancies complicated by diabetes mellitus, Class D, F, or T as designated, and each with accompanying vascular disease. The upper and lower boundary (shaded areas) and the 95 per cent confidence limits of the regression line calculated for the corresponding normal values are superimposed for comparison.
no ill effect observed in the fetus. Similar large increases in f..tlevels occurred in an erratic manner. Such large spontaneous fluctuations in L concentration failed to provide the clinician with an interpretable trend. Twin pregnancies. Estrogen concentrations determined in 14 samples from three patients with twin pregnancies were generally within the ranges obtained
L......L.--'-----'"-'"-----__L__. ---"-----34 32 36
..L.___.L__~ 38
40
Weeks Gestation
Fig. 10. Serum E., levels obtained serially from patients with pregnancies complicated by diabetes mellitus, Class D, F. or T as designated, and each with acwmpanying vascular disease. The upper and lower boundary (shaded areas) and the 95 per cent confidence limits of the regression line calculated for the corresponding normal values are superimposed for comparison.
for normal singleton pregnancies: one patient bad a single determination at 31 weeks. These patients are identified by the symbol(f)in Figs. 1, 2. and 3. The values were not significantly different from those obtained for normal singleton pregnancies. Diurnal variation. There are conflicting reports regarding the existence of a circadian rhythm in plasma estriol levels. A rhythm with lowest levels at 0800 hours and highest at 2000 hours was reported. 14 However. the absence of regularity in morning-evening differences is well documented for plasma f.s 15 and plasma 1'4.10 Morning (0800 hours) and evening (2000 hours) samples were obtained in two high-risk patients depicted in Fig. 4. Serum E3 and E4 levels with concomitant daily urinary estriol values (by gas-liquid chromatography) at 35 to 36 weeks' gestation are shown for Patient L H. who had hypertension and superimposed severe pre-eclampsia; creatinine clearance was 103 mi. per minute per 1.73 sq. M. Serum TE. E3 , and L levels with concomitant daily urinary estriol values at about 34 weeks' gestation are shown for Patient S. W. with diabetes mellitus, Class F; creatinine clearance was 85 mi. per minute per 1.73 sq. M. A consistent relationship between morning-evening concentrations of £ 3 , E'..t, and TE was not observed. Diabetes mellitus with no vascular disease. Forty serum samples were obtained from eight pregnant patients with diabetes mellitus and no clinical evideme of vascular disease. TE values are presented in f'ig. 5: E3 .
Unconjugated E3 and E4 in complicated pregnancies 753
Volume 128 ~umber 7
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Fig. U. Serial serum concentrations of TE, ~.and ~ in relation to changes in AO.D-4 50 in amniotic fluid from a patient with pregnancy complicated with Rh-isoimmunization.
Fig. 12. Serial serum concentrations of TE, ~. and E4 in relation to changes of ~. E4 , and AO.D,.$0 in amniotic fluid from a patient with pregnancy complicated with Rhisoimmunization.
in Fig. 6; and E.&, in Fig. 7. The time interval elapsed from the last determination to the date of delivery, urinary estriol excretion data, and notes describing each delivery are presented in Table II. In two cases, B 1 and C3, OCT's were positive. In Case B 1, E4 and Ea remained high normal, although TE fell27 per cent and urinary estriol excretion fell 30 per cent. In Case C3, serum Ea decreased by 8 per cent, while TE fell 30 per cent; E4 was consistently below the normal range throughout pregnancy. Serial assays of E.& failed to correlate with the positive OCT in Case B I, but, in Case C3, persistently low E4 concentrations preceded a positive OCT. Diabetes mellitus with vascular disease. Forty-seven serum samples were obtained from four pregnant women with diabetes mellitus complicated by vascular disease. TE values are presented in Fig. 8; Ea. in Fig. 9; and E.&, in Fig. I 0. Additional clinical data for each patient are presented in Table III. All diabetic patients with vascular disease had serum estrogen determinations within two days of delivery. In Case D I, with serum TE and E3 levels indicating normal fetoplacental function prior to delivery, there was a persistently low E.& level. The fetus was delivered when mature and was not in distress. In Case D2, levels of serum estrogens were normal, but delivery was effected for a clinical indication, and the infant did well. In Case F, the persistently low serum Ea and E.t levels
Table IV. Serum estrogen levels for patients with pregnancies complicated with Rh-isoimmunization at
35 to 36 weeks' gestation Group
TE (~-tgllOO ml. of serum)
Ea (ng.lml. of serum)
E 4 (ng.lml. of serum)
0.24
Cases
1 2
10.8
11.8
ll.2
14.4
0.43
3 4
5.6 5.3
11.4
5 6*
4.0
6.4 l.O
0.88 0.33 0.20 0.25
8.5 ± 2.0
0.39 ± 0.23
Mean± 7.38 ± 0.96 S.E. (No. 5)
6.0
(No.= 6)
(No.
6)
Controls at 35 weeks
(No.= 9)
Mean ± 9. 79 ± 0.97
0.5
±
1.2
1.57 ± 0.70
S.E.
*AO.D. 450 = 0.94 in amniotic fluid. Fetus died after intrauterine transfusion.
may have indicated chronic placental insufficiency, since emergency cesarean section was performed for fetal bradycardia accompanying spontaneous labor. In Case T, serum Ea <1-nd TE correlated inversely with urinary Ea excretion as glomerular filtration decreased.7 However, serum~. in addition to fluctuating markedly, fell to low levels prior to delivery and may have indicated chronic placental insufficiency.
754
Notation and Tagatz ·\.m.
Weeks Gestation
Fig. 13. Serial serum concentrations of Ea in patients with pregnancies complicated with hypertension or pre-eclampsia. The upper and iower boundary (shaded areas) and che 95 per cent confidence limits of the regression line calculated for the corresponding normal values are superimposed for comparison.
§ a;
V)
0
E
.
'g'
2.0
w
Weeks Gestation
Fig. 14. Serial serum concentrations of E. in patients with pregnancies complicated with hypertension or pre-eclampsia. The upper and lower boundary (shaded areas) and the 95 per cent confidence limits of the regression line calculated for the corresponding normal values are superimposed for comparison.
Serum estrogen assays did not influence the timing of any delivery in t..~is study of 12 diabetic patients. In retrospect, the serum E4 measurements failed to provide clinically useful information for the management of these pregnancies. Rh-isoimmunization. The data presented in Table IV were obtained at approximately 35 weeks of gestation from six patients with pregnancies complicated with Rh-isoimmunization. The maternal serum TE and
1.
August I. l '177 Obstc!. c;, ncwl.
E3 levels were distributed within their respectiw normal ranges. However. the E~1 levels were consi~tentlv below the normal range. Two patients in whom the fetal condition improved greatly as pregnancy progressed still had low serum t~ levels. In one patient (Fig. 11 ), the maternal serum TE and £3 levels increased while the Ll optical density450 (LlO.D.45o) for concomitant amniotic fluid samples decreased, but serum £ 4 remained low and unchanged. In another patient (Fig. 12), both t~l and £ 4 levels in amniotic fluid increased significantlY as the llO.D.45o decreased. The matching TE and .1<~1 levels in maternal serum also increased, but no change 1ras observed for the accompanying £4. Four pregnancies in a previously reported group of nine patients with Rh-isoimmunization followed a irend in \vhich the concentration of Et in arnniotit fluid was inversely related to the Ll0.D.450 of the amniotic fluid. However, simultaneous maternal serum E 4 concentrations were not consistently related to either E 4 concentrations or Ll.O.D.-J,;o in atnniotic fluid.H The consistently low serum £ 4 is indicative of the liabilities inherent in assaying a product of fetal origin in maternal blood. When produced, fetal £ 4 undergoes at least two different types of distribution: It becomes distributed by further metabolism into wnjugated and unconjugated pools, and each of these pools is further distributed into various tissue compartments. Rates of entry into maternal blood and subsequent clearance rates vary for each metabolite and also for each compartment accessible to maternal peripheral circulation. It is beyond the scope of this study to identify which. if any, of these factors contribute to the low serum E4 values observed here. The fact remains that maternal serum E4 concentrations are not predictably related to the degree of sensitization in Rh-isoimmunized pregnancies. Hypertension and pre-eclampsia. Reports indicate that E4 determinations in maternal urine and serum may be useful in monitoring fetal welfare in patients with pre-eciampsia, eciampsia, andior hyperrensionH. 10 Fifty-seven serum samples were obtained from 16 patients with pregnancies complicated by hypertension or pre-eclampsia. Ea Yalues are presented in Fig. 13, and & levels arc given in Fig. 14. Solitary determinations were obtained in hve patients; all values \Vere \Yithin the norma! range. 1\vo of these patients had mild pre-eclampsia, and pregnancy was allowed to progress to the onset of spontaneous labor. Three patients had labor induced for di:nical indications of increasing hypertension or worsening pre-eclampsia. One determination was obtained four days prior to induction of labor, and two were obtained one day prior to labor induction; the values did not
Unconjugated E3 and E4 in complicated pregnancies 755
Volume 128 Number 7
Table V. Hypertension; pre-eclampsia Delivery Case
Time lapse between last serum estrogen
no.
assay and delivery
2 weeks 2
2 weeks
3 4 5 6
3 days 2 weeks 2 days 2 weeks
7
3 days
8
2 weeks
Urinary Ea
Duration of pregnancy (weeks)
Fetal weight (grams)
NSVD
40
2,670
Induction, NSVD
38
3,345
Induction Induction Induction Spontaneous labor, meconium staining, Type II fetal bradycardia, emergency CIS NSVD
37 38 38 40
2,190 3,270 3,420 3,200
41
3,170
Induction, (elected induction at40 weeks) NSVD Induction, Type II fetal bradycardia, emergency CIS Elective CIS
40
3,300
39 40
2,380 2,300
36
2,380
excretion (mg./24 hr.)
8.5, 39 weeks 42.6, delivery Ft 8.8-13.8, 36-38 weeks N.D.t 16,36 weeks 20.7, 2 days ptd§ 20.6, 38 weeks
9 10
216 weeks 2 days
11.7, 39 weeks 11.6, 40 weeks 11.9, 3716 weeks 6.5, 39 weeks N.D. Decreased ptd
ll
2 days
Widely F
Mode*
*CIS = Cesarean section; NSVD = normal spontaneous vaginal delivery. tF = Fluctuating. :j:N.D. Not determined. §ptd = Prior to delivery.
indicate impending fetal jeopardy, although the clinical course was ominous. Serial determinations were obtained from 11 patients. The time interval elapsed from the date of the last determination to the date of delivery, urinary estriol excretion data, and notes describing each delivery are presented in Table V. Five patients had final estrogen determinations performed within the three days prior to delivery. In Case 7, pregnancy continued to the spontaneous onset of labor; serum & was persistently low, although serum E4 was in the normal range. Three patients had induction of labor for increasing severity of pre-eclampsia; the serum .& level rose in Case 5, fell in Case 3, and fluctuated from a persistently low level in Case 10. In Case 10, cesarean section was performed for acute placental insufficiency during labor. The final patient (Case 11 with borderline normal ~ values) had an elective cesarean section for severe pre-eclampsia. Thus, serial determinations of E4 failed to correlate with the clinical course in three of these five pregnancies. Intrauterine fetal death. Serum samples were obtained from three patients in whom fetal death occurred. The first patient was initially seen at the thirty-ninth week of gestation. A fetogram revealed polyhydramnios and a hydrocephalic fetus with radiographic signs of fetal death. One week later, the fetal heart tones were still present, but the maternal serum
contained no detectable Ea or&. On the following day, fetal heart tones were not detected. At 40 weeks, decompression of the hydrocephalus was required to deliver a 3,400 gram fetus. Fetal adrenals weighed 2 grams, about one fifth of the normal weight. Multiple congenital anomalies of the fetus included hydrocephalus, deft palate, and bilateral deft lip with secondary deformation of the nose, patent ductus arteriosus, atrial septal defect, and polydactylism of the hands. A second patient with rheumatic heart disease, mitral stenosis (functional Class I), and idiopathic thrombocytopenia was carrying a dead fetus when examined at 25 weeks of gestation. At that time, 0.10 ng. of Ea and 0.14 ng. of & per milliliter of serum were detected, while corresponding urinary estriol excretion was 3.3 mg. per 24 hours. A 6 em. macerated fetus (25 weeks) was delivered following intra-amniotic injection of 90 Gm. of urea. The third patient had two stillbirths eight and 10 years prior to the current pregnancy. Gestational age was uncertain since this pregnancy followed oral contraceptive withdrawal. The first maternal serum sample was obtained during the second trimester and contained 2.6 ng. of F.-a and 0.43 ng. of.& per milliliter of serum. Three weeks later, when no fetal heart tones could be detected, three successive morning serum samples contained no detectable E3 and 0.37, 0.13, and
756
Notation and Tagatz Am.
0 ng. of E.t per milliliter, respectively. Intra-amniotic urea administration resulted in the expulsion of a 730 gram fetus at 26 weeks of gestation. Fetal adrenals weighed 0.4 grams, about one eighth of the normal weight. The other organs had weights which were generally about one half of the normal. The etiology of fetal death in utero could not be ascertained. In each of the three cases where fetal death occurred, the maternal serum concentrations of both E3 and E 4 were far below normal in the initial determination. When urinary excretion of E3 and E4 is compared, E.t is cleared less rapidly by the kidneys of both the postpartum mother and the neonate. 16 In one case of fetal death in utero, the maternal serum E3 was no longer detectable, while decreasing levels of & were
August l. 1977 Obstet. Gvnerol.
measured on three successive days. If the dead fetus no longer produces &. then I~ is retained much long-er than E3 in the various maternal and fetoplacental compartments. The determination of serial & concentrations in maternal serum has been evaluated as an indicator of fetal welfare during high-risk pregnancies. ~ was not consistently related to either ~ concentrations in maternal serum and urine or the clinical course and pregnancy outcome; no clinically useful trends were discernible. Thus, serial concentrations of maternal serum E4 do not appear to be clinically useful in the management of high-risk pregnancies. The authors acknowledge the helpful assistance of Mrs. Dianne Judd and Miss Jan McLeod.
REFERENCES I. Giebenhain, M. E., Tagatz, G. E., and Gurpide, E.: Serum levels of unconjugated estetrol during human pregnancy,]. Steroid. Biochem. 3: 707, 1972. 2. Hagen, A. A., Barr, M., and Diczfalusy, E.: Metabolism of 17,8-estradiol-4-"C in early infancy, Acta Endocrinol. 49: 207, 1965. 3. Adlercreutz, H., and Luukkainen, T.: Determinations of urinary estrogens by gas chromatography, in Lipsett, M. B., editor: Gas Chromatography of Steroids in Biological Fluids: Proceedings of an Airlie House Conference, New York, 1965, Plenum Press, Inc., p. 215. 4. Gurpide, E., Schwers, ]., Welch, M. T., Vande Wiele, R. L., and Lieberman, S.: Fetal and maternal metabolism of estradiol during pregnancy, .J. Clin. Endocrinol. Metab. . 26: 1355, 1966~ - 5. Fishman, J., Schut, H., and Solomon, S.: Metabolism, production, and excretion rates of 15a-hydroxyestriol in late pregnancy,]. Clin. Endocrinol. Meta b. 35: 339, 1972. 6. Heikkila, J., and Luukkainen, T.: Urinary excretion of estriol and 15a-hydroxyestriol in complicated pregnancies, AM.]. 0BSTET. GYNECOL. 110: 509, 1971. 7. Tagatz, G. E., Arnold, N. 1., Goetz, F. C., Najarian,]. S., and Simmons, R. L.: Pregnancy in ajuveniie diabetic after renal transplantation (Class T diabetes mellitus), Diabetes 24: 497. 1975. 8. Sciarra,].]., Tagatz, G. E., Notation, A. D., and Depp. R.:
J.
9.
10.
11.
12. 13. 14. 15.
16.
Estriol and estetrol in amniotic fluid. AM. J. OssTET. GYNECOL. 118: 626, 1974. Gurpide, E., Giebenhain, M. E., Tseng, L., and Kelly. W. G.: Radioimmunoassay for estrogens in human pregnancy urine, plasma, and amniotic fluid, AM. ]. Ossn:T. GYNECOL. 109: 897, 1971. Tnlrhimkv D .. Frivoletto. F. D .. Rvan. I. K .. and Fi~t;~~~~-.-]:: Pl~srn~"~s~trol as an index of fetal wellbeing,]. Clin. Endocrinol. Metab. 40: 560, 1975. Korda, A. R., Challis, ]. ]., and Anderson, A. B. M.: Plasma unconjugated 15a-hydroxyoestriol (oestetrol) levels in normal human pregnancy, Br. J. Obstet. Gynaecoi. 82:882,1975. Masson, G. M.: Plasma oestriol concentration during normal pregnancy, Br.]. Obstet. Gynaecol. 80: 201. 1973. Tulchinsky, D., and Abraham, G. E.: Radioimmunoassay of plasma estriol, ]. Clin. Endocrinol. Metab. 33: 775, 1971. Goebel, R., and Kuss, E.: Circadian rhythm of serum unconjugated estriol in late pregnancy, J. C1in. Encfocrinol. Metab. 39: 969, 1974. Levitz, M., Slyper, A.J., and Selinger, M.: On the lack of periodicity in plasma estriol in human pregnancv.J. Clin. Endot:rinul. ~fetab. 38: 698, 1974. Heikkila, J.: Excretion of 15a-hydroxyoestriol and oestriol in urine of newborn infants and in maternal urine before and after delivery, J. Endocrinol. 52: 119, 1972.