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Plasma estriol levels in normal and abnormal pregnancies: An index of fetal welfare
Plasma estriol levels in normal and abnormal pregnancies: An index of fetal welfare L.
NACHTIGALL,
M.
BASSETT,
U.
HOGSANDER
M.
LEVITZ,
New
York,
M.D.* B.S.
PH.D.*” New
York
Serial plasma estriol determinations were conducted in the third trimester of human pregnancy on 22 normal, 25 diabetic, and 16 toxemic patients. In addition, IO cases of suspected fetal death were evaluated. In normal pregnancy the levels rise in a generally regular fashion as pregnancy proceeds, increasing about fivefold in the last trimester. However, the individual curves vary considerably. The average plasma estriol level at term is about 27 pg per 100 ml. with a range between 10 and 55. The plasma estriol levels of the diabetic patients (Classes A, B, and C) in good control, were not statistically different from those of the normal patients. The toxemic patients could be classified into three groups according to their estriol levels. Those with severe symptoms and small fetuses had correspondingly low levels. The patients under good control had normal values while three patients with renal involvement superimposed on toxemia had surprisingly high levels. The values for patients with dead fetuses were extremely low. Although a larger number of cases involving fetal jeopardy must be studied before the contribution of plasma estriol determinations toward the improvement of fetal salvage rates can be assessed, the potential advantages of plasma assays over urine assays in this regard are discussed.
U R 1 N AR Y estriol excretion increases in a regular fashion during human pregnancy and at term the levels are some one thousand times greater than that found in the nonpregnant state. Most of the estriol of late pregnancy appears to be synthesized by the
From the Department of Obstetrics Gynecology, New York University of Medicine.
placenta from precursors which are of fetal origin. 1-3 Thus, it is reasonable that certain malfunctions of the fetus or placenta would be reflected in diminished rates of estriol excretion. Urinary estriol determinations have received acceptance as a valuable adjunct in the management of diabetes and toxemia of pregnancy and at times they have made a difference in the salvage of compromised fetuses.4-7 Plasma estriol determinations have also been used in the investigation of diabetes and toxemia but not in the routine study of patients with these complications.’ These plasma methods require large quantities of blood and are too lengthy to be practical for the periodic study of a large number of patients. A simplified, accurate, and convenient method for the determination of total plasma estriol in the second half of pregnancy was recently developed in this
and School
This investigation was supported in part by United States Public Health Service Grant CA-02071-I4 from the National Cancer Institute and Grant P-206H from the American Cancer Society. *Research Trainee,.United States Public Health Service Trazning Grant 2-TOl-HD-00011-06 from the National Institute of Child Health and Human Development. **United States Public Health Service Research Career Development Awardee 5-K3-HD-18,422-05 from the National Institute of Child Health and Human Development.
638
Plasma
laboratory.” The method requires only 1 ml. of plasma and multiple samples may be analyzed in one day. This paper records the results of our experiences with this method. Twenty-two normal, 25 diabetic, and 16 toxemic pregnant patients were followed regularly during the third trimester. In addition, 10 cases of suspected fetal death were e\,aluated. Material
and
methods
Plasma estriol method. The
assay proccdure has been described in detail.g Originally. the need to run samples in duplicate was emphasized. More recently, it was found that escept for occasional spot check, this has not been necessary and samples have been run singly. Clinical. A wide variety of patients have kern studied in our laboratory, including normal volunteers and patients with diabetes, toxemia. suspected dead fetuses, poor obstetric history of unknown etiology, and Rh incompatibility. Only the first four mentioned classifications will be considered in this report. A brief description of the patients studied is presented in this section, while further details including typical case histories are presented together with the results. .VornzaL. The 22 normal controls had an average age of 19. They were mostly primiparas and, except for 2 subjects, were nonclinic patients, who were seen regularly by the same obstetrician. The course of each pregnancy in this category was uncomplicated. Diabetcls. Twenty-five diabetics were studied of whom, according to White’s classification,l’ 6 were Class A, 8 were Class B, and 11 were Class C. Seven of these patients had one or more previous pregnancies end in fetal death and only 3 patients had living children. Except for 3 clinic cases, each patient was seen regularly by the same obstetrician. Toxemia. There were 16 patients with toxemia, 10 of whom exhibited severe symptoms, including three with serious renal involvement. The remaining 6 patients had
estrioi
levels
in pregnancy
639
mild symptoms which were controlled with drugs. All patients required some hospitalization. Only one subject had a previous history of the disease. Fetal death. Ten cases of suspected fetal death in the third trimester were studied. The fetal heartbeat was not heard, although in four of the cases, fetal movement was felt by the mother. In the 2 cases in which the fetal ECG’s were taken, the results were equivocal. Results
Normal. Table I shows the results of the estriol determinations of the 22 normal controls. Six of these cases, chosen at random, are presented in Fig. 1 to emphasize the progression of the estriol values as pregnancy proceeds. Fig. 1 also indicates averages, and approximate upper and lower limits of estriol levels in the third trimester for all normal subjects studied. It can be seen that on the average, the estriol levels rise about fivefold between the twenty-sixth week and term. There is an upward trend for each patient but the rise is not uniformly continuous. An occasional dip or spurt is observed. Diabetes. Class A. The course of each Class A diabetic was smooth throughout pregnancy. There were no pre- or postpartum comphcations and no fetal or neonatal deaths. Fig, 2 presents the estriol values for all the Class A diabetics studied. In general, the values were in the low normal range. A notable exception was M. R. whose values inexplicably rose significantly above normal. She was delivered spontaneously at 33 weeks. However, it is cautioned that the gestation time is calculated by the patient’s recall of her last menstrual period. R. D. was delivered by cesarean section several hours after the sample was taken in which a fall was noted. This single drop could not be correlated with any clinical condition in the mother or the baby. Class B. The plasma estriol values for all the patients in this class are presented in Fig. 3. The levels were not significantly different from those found in normal pa-
640
Nachtigall
et al.
Table I. Plasma estriol levels in normal
pregnancy*
I Patient A.
Week.c 2.5
/
26
27
A.
1
3.3
28 17
C. B. K.
29
30
32
13
15
16
12
12
25
21
25
/
32
B.
5.0
C. D. A.
j
F.
S. F.
12 6.4
17
8.3
6.1
9.6
9.4
14
14
18
14,
14 12
15
A. G. C.
H.
6.6
4.3
4.8
6.9
9.1
8.3
E.
H.
3.6
3.7
5.3
5.5
6.3
6.1
J. J.
15
J. K.
9.7
11 6.7 13
9.6
K. K.
11
10
8.4
M. M.
R. R. B. S.
6.0
4.5
4.5
5.4
6.2
A. T. C. T.
7.8
12
10
13
L. T.
4.8
4.7
7.4
6.9
6.9
17
15
20
22
10
16
12
16
14
14
17
c. w.
5.2
F. W.
4.9
s. w.
7.8
7.1
6.9
11
6.1
9.2
4.3
11
11
w. w. “Values
are in micrograms
per
100 ml.
plasma.
tients. AIthough many of the patients were frequently out of control necessitating reevaluation of insulin requirements, all patients delivered live babies. However, one neonate born to C. F. died within 24 hours. The case history is as follows: C. F. was a 24-year-old white, para 0, Class B diabetic patient, whose diabetes was diagnosed for the first time during pregnancy. During the fifth month, she exhibited glycosuria, 4+ acetonuria, and an FBS of 360 mg. per 100 ml. She was hospitalized for 2 weeks and the diabetes brought under control with diet, 70 units of NPH, and 45 units of regular insulin.. The other aspects of her pregnancy were uneventful except that a true fetal heartbeat was never heard, only a placental souffle. Labor was induced at 38
weeks and a 2,250 gram baby with an Apgar score of 3 was delivered. The baby died 24 hours later of multiple congenital anomalies, including tetralogy of Fallot. The estriol levels can be seen in Fig. 3. The vaues rose impressively from the thirty-second to the thirty-seventh week. A drop of about 20 per cent was observed in the final 2 weeks of pregnancy; however, a decrease of this magnitude is seen in normal patients. Class C. Table II presents the values of the estriol determinations performed on the plasma of 11 Class C diabetic patients. The values obtained in 6 of these cases are shown in Fig. 4. Except for S. G. and M. O., the estriol levels were indistinguishable from normal. All patients were delivered of live
9.7
Plasma
estriol
levels
in pregnancy
641
gestation 33 14
34 21
16
22
8.0 17
6.7 21
35
8.5
36 17
37 17
38 11
49
42
46
40
10
13
7.7 12
12 3.3
15
\
19
39 11
40 24
25
18
17
29
35
32
28
17
27
15
17
24
27
33
15
12
17
16
25
13
25
26
“4
20
15
25
17
12 21
(
11 7.7 12
16 9.0 12
24
16
19
11
10
16
15
8.1
7.7
22
22
26
31
10
11
13
9
18
24
26
11
14
14
17
27
25
23
6.6
14
16
17
25
23
26
7.5
12
10
13
13
34
63
63
20
19
19
20
31
13
31
4.7 10 14
7.8 9.5 14
22
23
7.1
33
14
17
15
10
16
17
20
43
24
27
27
24
25
30
babies. However, S. G.‘s baby died 8 hours after delivery. S. G. was a 25-year-old white female, para 0, with Class C diabetes. She had diabetes since the age of 15. She was hospitalized twice during her pregnancy, once for multiple hypoglycemic episodes and once for an increase of blood pressure from 110/80 to 170/110. There was no edema or albuminuria. She went into spontaneous labor in the thirty-fifth week and was delivered of a 1,450 gram baby with an Apgar score of 3 and whose condition became increasingly poor until death. The falling estriol values in the latter part of her pregnancy to a level well below normal can be seen in Fig. 4. M. 0. was a para O-l-O-0 patient with Class C diabetes in poor control throughout pregnancy.
42
29
32
22 45
11
9.6
i
19
13
27 38
38
42
------
A 2,350 gram baby was delivered by cesarean section in the thirty-ninth week shortly after the last estriol determination. Although the plasma estriols were declining, the baby was normal but small.
Although 6 of the other Class C diabetics had histories of previous fetal or neonatal deaths, the course of pregnancy in each case was reasonably good. A typical case is presented. L. G., a 30-year-old, para O-l-O-0, Class C diabetic patient had onset of diabetes at the age of 13 and it had been fairly well controlled on 30 units of NPH ins&n. She conceived one year prior to the pregnancy reported here and suffered an intrauterine death at 32 weeks. Dur-
642
Nachtigall
et al.
60-
/’ /
50 -
/
40 -
20 -
0”
I 26
’
’ 28
’
’ 30
’
’ 32
’
Gestation Fig. 1. Plasma average values. pregnancy.
estriol levels in The broken lines
normal indicate
’ 34
’
1 36
’
1 38
’
J 40
(weeks)
pregnancy.. The. the approximate
dashed lower
line indicates approximate and upper limits in normal
72r 50 -
40 -
20-
RD
10-
O'&
' 26
'
' 28
'
' 30
'
' 32’ Gestation
Fig.
2. Plasma
estriol
levels
in Class A diabetes
'
' 34
'
' 36
(weeks)
of pregnancy.
-
I
' 38
I
' 40
'
Plasma
esbiol
levels
in
pregnancy
643
,MY / I I
I
0
28
30
32
34
Gestation
Fig. 3. Plasma
estriol
levels
in Class
I
I
I
26
B diabetes
I
36
38
40
(weeks)
of pregnancy.
Table II. Plasma estriol levels in Class C diabetic
patients”
..~- -___~-Weeks
--______ 25 / 26
Patient
j
27
\ 28
) 29
I. A.
7.9
1 30
/ 31
20
19
B. c.
of gestation / 32
33
18
12
L. G.t
2.8
6.4
5.5
K. M.
6.1
5.0
T. N.
6.5
7.0
8.4
11
12
8.8
9.1
5.4
6.4
7.8
3.1
M. 0.t
4.8
5.1
s. s.t
7.6
3.4
iq
are
several in Fig.
in
micrograms
instances, 4.
per
more
the present pregnancy,
crease
‘her
insulin
io
45
100 ml.
plasma.
one
determination
than
per
week
it was necessary to inunits
of
NPH
plus
10
units of regular insulin. She h’ad no episodes of acidosis. Cesarean section was performed at 36 to 37 weeks, 2,750 gram
resulting in baby. The
the birth of a normal rising profile of the
estriol kvels is shown in Fig. 4.
Toxemia. The toxemic patients could be
20
25
18
24
40
11
14
20
12
21
20
8.1 13
15
19
5.4
4.4
7.8
11
8.1
21
7.8
45 17
‘Values
20
16
11
7.0
27
D.---__S. tin plotted
17
6.3
19
/ 3.9
3.7
12
8.7
6.9
24
7.2
J. H.t I,. L.
/341/-.75m-i-38
23
17
S. G.
j
was
15 --___ performed
17 on
19
---.-___-~-..~ these
patients.
18
..--. All
the
valurs
-. --
ohtainr~d
arc
divided into three groups, according to their estriol levels (Fig. 5). The first group had severe symptoms of edema, hypertension, and albuminuria, and there was difficulty in controlling these symptoms. Of the 7 patients in Group I, .only 1 patient (E. IL) was delivered, at term. Five went into spontaneous
px-emature labor and in one (F. S.) labor
644
Nachtigall
et al.
July I, 1968 Am. J. Obst. & Gymc,
E
a” 9
Gestation (weeks) Fig. 4. Plasma estriol levels in Class C diabetes of pregnancy.
was induced at 39 weeks. All had extremely small babies for their period of gestation, while H. T. was delivered of a dead fetus. Subnormal estriol values were noted for each patient. Two case histories follow: C. K., a 24-year-old white female, para 0, with toxemia, was noted to have a blood pressure of 160/130, marked edema, and proteinuria at the twenty-sixth week of pregnancy. She was hospitalized from the twenty-ninth week on, but in spite of bed rest, severe sodium restriction, and the use of various diuretics, her blood pressure remained elevated and fluid retention increased. She had multiple allergic reactions to drugs. In the thirty-fifth week of gestation, she went into spontaneous labor and was delivered of a 1,260 gram baby with an Apgar score of 4. It took 4 months for her blood pressure to return to normal. Her plasma estriol values which were significantly below normal are shown in Fig. 5, Group 1. H. T., a 33-year-old Negro female, had a blood pressure of 180/130, a 3+ albuminuria, and edema in the thirty-second week of pregnancy. She was hospitalized from the thirtysecond week of pregnancy with uncontrollable symptoms. The plasma estriol levels declined sharply (Fig. 5, Group 1). She was delivered of a dead fetus in the thirty-fifth week of gestation.
There was no precise record heartbeat was lost.
of when the fetal
The second group of toxemic patients exhibited only mild symptoms. The blood pressure was high but not excessively so. The edema was controlled with drugs and albuminuria was intermittent. Of the 6 patients in this group, M. P., G. G., and B. M. were delivered normally at term; 2 patients, B. R. and D. D., were delivered prematurely, while one (D. T.) was sectioned. All babies were of normal size. The estriol levels for the patients in this group were indistinguishable from normal (Fig. 5, Group 2), The third group of 3 toxemic patients, 2 of whom presented with a nephrotic syndrome, had severe symptoms. Abnormally high estriol levels were obtained for all the patients
in
this
group
(Fig.
5, Group
3).
Two patients (L. M. and A. B.) were delivered spontaneously and in the third (C. M.) labor was induced. A casehistory which is illustrative
of
the
2 nephrotic
patients
(L. M. and A. B.) follows: A. B., a 29-year-old, para 4-O-O-4, Puerto Rican female, with no known previous illnesses presented at clinic in her twenty-fourth week of
Plasma estriol levels in pregnancy
645
* Group 2
Gestation (weeks) Fig. 5. Plasma estriol levels in toxemia of pregnancy.
gestation with edema, a blood pressure of 160/ 110, a cholesterol level of 510, 3+ albuminuria, and a BUN level of 52. She was hospitalized from the twenty-fifth week on and was maintained on diuretics, MgSO,, severe sodium restriction, and bed rest. On this regimen the edema decreased and her proteinuria diminished, but the blood pressure remained elevated as did the BUN and cholesterol levels. She went into spontaneous labor at 36 weeks and was delivered of a 2,185 gram baby with an Apgar score of 7. Three days post partum the patient’s BUN level was 15. The plasma estriol values which are abnormally high are shown in Fig. 5, Group 3. The third patient in this group in that she had a previous history disease.
differed of renal
C. M., a 23-year-old, para 0, toxemic female, had been treated for glomerulonephritis at the age of 18 with no known follow-up until she presented in the twenty-fourth week of gestation with a blood pressure of 210/120, BUN 23, cholesterol 280, 2+ albuminuria, anemia, and weakness. She was hospitalized and the edema
and albuminuria improved considerably on treatment. Her blood pressure fluctuated between 170/100 and 210/120. The BUN level was never below the initial value of 23 and on two occasions it was above 30. Renal biopsy revealed chronic pyelonephritis. In the thirty-second week, she began to have nausea and vomiting and the BUN level rose to 35. At 33 weeks labor was induced and she was delivered of a 1,820 gram infant in fair condition with an Apgar score of 7. The patient’s BUN continued to rise, and 6 weeks after delivery she was stiI1 hospitalized,
Suspected fetal death. Plasma estriol Ievels were determined in 10 cases of suspected fetal death. In each case the fetal heartbeat was not detectable by fetoscope although in some cases the mother claimed to feel fetal movement. The values obtained are shown in Table III. It can be seen that in the 9 cases in which extremely low levels were obtained the patients subsequently were delivered of dead fetuses. In the remaining study, the values were in the low normal range and a live baby was subsequently de-
646
Nachtigall
et al.
Table III. Plasma estriol levels in relation to outcome of pregnancy in cases of suspected fetal death
Patient
1
M. D. D. A. F. G. P.L. Z.N. C.N. P.D. A. R. J. 0. B. J. *The ground ternal
~ZlL$
34, 34, 25, 28 32 27 20 36 30 35,
1 (,u&%%Zd.)
35 35 29
36, 37
2.7, 2.6, 4.8, 1.1 0.3 1.8 1.7 1.0 0.7 6.1,
1.0 1.4 2.6
11, 13
1 ;:Z’
Dead Dead Dead Dead Dead Dead Dead Dead Dead Live
values are not corrected for the calculated of 0.7 fig per 100 ml. which is attributed to isotopic
backthe in-
standards.
livered. The pertinent as follows:
aspects of this case are
B. J. was admitted in the thirty-fifth week of pregnancy with greater than 30 per cent of her body including the abdomen severely burned. The sterile dressing limited the careful evaluation of the fetal heart either by fetoscope or ECG. In addition, the mother felt no fetal movement. Because of her poor condition, it was felt that her toxic state might be improved by inducing labor and removing the “dead” fetus. The plasma estriol level indicated a live fetus, and the value continued to rise (Table III). The patient went into spontaneous labor at the end of the thirty-seventh week and was delivered of a normal male infant with an Apgar score of 8. Comment Plasma estriol determinations have at least three advantages over the analysis of urine in the evaluation of human pregnancy. First, the need for the troublesome 24 hour urine collection is eliminated. Second, if necessary, 2 or more plasma samples may be drawn for analysis in a single day. Finally, in view of the short plasma estriol half-life,gl I1 a decreasing rate of estriol synthesis would be reflected more rapidly in a single plasma sample than in a pooled urine collection which reflects the average of the preceding 24 hours.
The validity of the method with regard to the precision, accuracy, specificity, and senaitiveity had been established previously.” In the present study which included 73 cases of normal and pathologic pregnancies, the potential advantage of plasma estriol determinations as a valuable adjunct in gaining insight into the course of difficult pregnancies has been demonstrated. The major feature of the normal plasma estriol values is that they continue to rise in a generally regular fashion as pregnancy progresses. Although the curve is not always smooth for each individual, the variations are no greater than those obtained with urine.’ The absolute values vary widely from patient to patient. The average plasma estrio1 value at term is about 27 pg per 100 ml., while the range is between 10 and 55 pg per 100 ml. This emphasizes the need for obtaining serial determinations on each patient studied. An occasional fall in the values has been observed at random times of gestation. However, the report of Smith” that plasma estriol levels fall significantly in the days prior to labor was not confirmed in this study. In the 22 normal patients studied, only 4 exhibited a decline of greater than 15 per cent at term. The plasma estriol values of the diabetic patients were not statistically different from those of the normal patients. Thus, even Class C diabetic patients in good control could be expected to have plasma estriol levels indistinguishable from normal provided fetal and placental functions are not impaired. An interesting case was that of S. G. This Class C diabetic patient exhibited an obvious decline in estriol values and in the thirty-fifth week of gestation she was spontaneously delivered of a fetus with a poor Apgar score who died shortly after birth. The significant point is that an estriol value of 3.7 pg per 100 ml., although well below normal for the thirty-fifth week of gestation, is still compatible with a live fetus. A Class B diabetic patient, C. F., was delivered of a live baby in the thirty-eighth week of gestation who died 24 hours after birth. She had normal estriol values. The autopsy report
Plasma
revealed a fetal heart-lung anomaly of the type which is compatible only with fetal life. It is apparent that these plasma estriol studies would have to be extended before a delinite statement could be made concerning the value of the determinations in improving the fetal salvage rate in diabetes of pregnancy. The toxemic patients displayed a good degree of consistency between the plasma cstriol levels and the clinical picture. The classification of these patients into three groups according to their estriol profiles may prove to be useful. The cases in Group 1 were in poor control and in each case development of the fetus and placenta was markedly retarded. The estriol levels were severely depressed and they appeared to decline even further at the time of delivery which was premature. One of the patients (H. T.) suffered an intrauterine death which, according to the declining estriol le\,els to a final value of 1.1, was not surprising. The toxemic patients in Group 2 were under good control and the fetuses apparently developed normally. The plasma estriol values of these patients were in the normal good correlation berange, demonstrating tween the plasma estriol values and the clinical picture. However, the steadily declining estriol pattern of M. P. toward term was the only one of this type in the entire study which resulted in a normal outcome. The strikingly elevated plasma estriol l(~vcls in the Group 3 toxemic patients are puzzling. There were only 3 patients in this 2 of whom presented with the group, nrphrotic syndrome and 1 with chronic
estriol
levels
in
pregnancy
647
renal disease superimposed on toxemia. It would be premature to speculate on the origin of the elevated estriol but an att,ractive working hypothesis is that these patients have high estriol kidney thresholds. To test this hypothesis in the future, the plasma clearance rates will be measured in patirrkts of this type. It was shown recently in I)UI laboratory that with only minor changes the plasma estriol method may be applied to the analysis of urine.‘” Unequivocal results have been obtained in studies of suspected fetal death. ‘i’he plasma estriol values were less than 2.6 rug per 100 ml. in each case of fetal death. However, the length of time that the fetus was dead could not be accurately judged in any patient. At this stage of the study it is not possible to cite a level below wllich the life of the fetus is in immediate jeopard). Although 33 patients have been studickd. a precise assessment of the value of plasma estriol determinations in the managcmrnt of Complications in pregnancy cannot as y
REFERENCES
1. Siiteri, P. K., and MacDonald, P. C.: J. Clin. Endocrinol. 26: 751, 1966. 2. Diczfalusy, E., and Benagiano, G.: Research on Steroids, Transactions of Second Meeting International Study Croup for Steroid Hormones, Rome, 1965, p. 27. 3. Magendantz, H. G., and Ryan, K. J.: J. Clin. Endocrinol. 24: 1155, 1964. 4. Kellar, R.. Matthew, G. D., MacKay, R., Brown, J. B., and Roy, E. J.: J. Obst. & Gynaec. Brit. Emp. 66: 804, 1959.
5. Greene, J. W., Jr., Smith, K., Kyle, G. C.. Touchstone, J. C., and Duhring. J. I,.: .41u. J. OBST. & GYNEC. 91: 684, 1965. 6. Taylor, E. S., Bruns, P. D., Drose, V. E., and Kartchner, M. J.: AM. J. ORST. & GYTEC. 83: 194, 1962. 7. Frandsen, A., and Stakemann, G.: Danish M. Bull. 7: 98, 1960. 8. Schwers, J.: Les oestroghnes au tours de la seconde moitiC de Ia grossesse, Brussels~ 1965, Editions Arscia S.A.
648
9.
10.
Nachtigall
et al.
Nachtigall, L., Bassett, M., Hogsander, U., Slagle, S., and Levitz, M.: J. Clin. Endocrinol. 26: 941, 1966. White, P.: Pregnancy Complicating Diabetes in Jo&n, E. P., Root, H. F., White, P., and Marble, A., editors: The Treatment of Diabetes Mellitus, Philadelphia, 1952, Lea & Febiger.
11.
12. !3.
Roy, E. J., Harkness, R. A., and Kerr, M. G.: J. Obst. & Gynaec. Brit. Comm. 70: 1034, 1963. Smith, 0. W.: Acta endocrinol. 51: Suppl. 104, 1966. Jaffe, S., and Levitz, M.: AM. J. OBST. & GYNEC. 98: 992, 1967.
NACHTIGALL,
M.
BASSETT,
U.
HOGSANDER
M.
LEVITZ,
New
York,
M.D.* B.S.
PH.D.*” New
York
Serial plasma estriol determinations were conducted in the third trimester of human pregnancy on 22 normal, 25 diabetic, and 16 toxemic patients. In addition, IO cases of suspected fetal death were evaluated. In normal pregnancy the levels rise in a generally regular fashion as pregnancy proceeds, increasing about fivefold in the last trimester. However, the individual curves vary considerably. The average plasma estriol level at term is about 27 pg per 100 ml. with a range between 10 and 55. The plasma estriol levels of the diabetic patients (Classes A, B, and C) in good control, were not statistically different from those of the normal patients. The toxemic patients could be classified into three groups according to their estriol levels. Those with severe symptoms and small fetuses had correspondingly low levels. The patients under good control had normal values while three patients with renal involvement superimposed on toxemia had surprisingly high levels. The values for patients with dead fetuses were extremely low. Although a larger number of cases involving fetal jeopardy must be studied before the contribution of plasma estriol determinations toward the improvement of fetal salvage rates can be assessed, the potential advantages of plasma assays over urine assays in this regard are discussed.
U R 1 N AR Y estriol excretion increases in a regular fashion during human pregnancy and at term the levels are some one thousand times greater than that found in the nonpregnant state. Most of the estriol of late pregnancy appears to be synthesized by the
From the Department of Obstetrics Gynecology, New York University of Medicine.
placenta from precursors which are of fetal origin. 1-3 Thus, it is reasonable that certain malfunctions of the fetus or placenta would be reflected in diminished rates of estriol excretion. Urinary estriol determinations have received acceptance as a valuable adjunct in the management of diabetes and toxemia of pregnancy and at times they have made a difference in the salvage of compromised fetuses.4-7 Plasma estriol determinations have also been used in the investigation of diabetes and toxemia but not in the routine study of patients with these complications.’ These plasma methods require large quantities of blood and are too lengthy to be practical for the periodic study of a large number of patients. A simplified, accurate, and convenient method for the determination of total plasma estriol in the second half of pregnancy was recently developed in this
and School
This investigation was supported in part by United States Public Health Service Grant CA-02071-I4 from the National Cancer Institute and Grant P-206H from the American Cancer Society. *Research Trainee,.United States Public Health Service Trazning Grant 2-TOl-HD-00011-06 from the National Institute of Child Health and Human Development. **United States Public Health Service Research Career Development Awardee 5-K3-HD-18,422-05 from the National Institute of Child Health and Human Development.
638
Plasma
laboratory.” The method requires only 1 ml. of plasma and multiple samples may be analyzed in one day. This paper records the results of our experiences with this method. Twenty-two normal, 25 diabetic, and 16 toxemic pregnant patients were followed regularly during the third trimester. In addition, 10 cases of suspected fetal death were e\,aluated. Material
and
methods
Plasma estriol method. The
assay proccdure has been described in detail.g Originally. the need to run samples in duplicate was emphasized. More recently, it was found that escept for occasional spot check, this has not been necessary and samples have been run singly. Clinical. A wide variety of patients have kern studied in our laboratory, including normal volunteers and patients with diabetes, toxemia. suspected dead fetuses, poor obstetric history of unknown etiology, and Rh incompatibility. Only the first four mentioned classifications will be considered in this report. A brief description of the patients studied is presented in this section, while further details including typical case histories are presented together with the results. .VornzaL. The 22 normal controls had an average age of 19. They were mostly primiparas and, except for 2 subjects, were nonclinic patients, who were seen regularly by the same obstetrician. The course of each pregnancy in this category was uncomplicated. Diabetcls. Twenty-five diabetics were studied of whom, according to White’s classification,l’ 6 were Class A, 8 were Class B, and 11 were Class C. Seven of these patients had one or more previous pregnancies end in fetal death and only 3 patients had living children. Except for 3 clinic cases, each patient was seen regularly by the same obstetrician. Toxemia. There were 16 patients with toxemia, 10 of whom exhibited severe symptoms, including three with serious renal involvement. The remaining 6 patients had
estrioi
levels
in pregnancy
639
mild symptoms which were controlled with drugs. All patients required some hospitalization. Only one subject had a previous history of the disease. Fetal death. Ten cases of suspected fetal death in the third trimester were studied. The fetal heartbeat was not heard, although in four of the cases, fetal movement was felt by the mother. In the 2 cases in which the fetal ECG’s were taken, the results were equivocal. Results
Normal. Table I shows the results of the estriol determinations of the 22 normal controls. Six of these cases, chosen at random, are presented in Fig. 1 to emphasize the progression of the estriol values as pregnancy proceeds. Fig. 1 also indicates averages, and approximate upper and lower limits of estriol levels in the third trimester for all normal subjects studied. It can be seen that on the average, the estriol levels rise about fivefold between the twenty-sixth week and term. There is an upward trend for each patient but the rise is not uniformly continuous. An occasional dip or spurt is observed. Diabetes. Class A. The course of each Class A diabetic was smooth throughout pregnancy. There were no pre- or postpartum comphcations and no fetal or neonatal deaths. Fig, 2 presents the estriol values for all the Class A diabetics studied. In general, the values were in the low normal range. A notable exception was M. R. whose values inexplicably rose significantly above normal. She was delivered spontaneously at 33 weeks. However, it is cautioned that the gestation time is calculated by the patient’s recall of her last menstrual period. R. D. was delivered by cesarean section several hours after the sample was taken in which a fall was noted. This single drop could not be correlated with any clinical condition in the mother or the baby. Class B. The plasma estriol values for all the patients in this class are presented in Fig. 3. The levels were not significantly different from those found in normal pa-
640
Nachtigall
et al.
Table I. Plasma estriol levels in normal
pregnancy*
I Patient A.
Week.c 2.5
/
26
27
A.
1
3.3
28 17
C. B. K.
29
30
32
13
15
16
12
12
25
21
25
/
32
B.
5.0
C. D. A.
j
F.
S. F.
12 6.4
17
8.3
6.1
9.6
9.4
14
14
18
14,
14 12
15
A. G. C.
H.
6.6
4.3
4.8
6.9
9.1
8.3
E.
H.
3.6
3.7
5.3
5.5
6.3
6.1
J. J.
15
J. K.
9.7
11 6.7 13
9.6
K. K.
11
10
8.4
M. M.
R. R. B. S.
6.0
4.5
4.5
5.4
6.2
A. T. C. T.
7.8
12
10
13
L. T.
4.8
4.7
7.4
6.9
6.9
17
15
20
22
10
16
12
16
14
14
17
c. w.
5.2
F. W.
4.9
s. w.
7.8
7.1
6.9
11
6.1
9.2
4.3
11
11
w. w. “Values
are in micrograms
per
100 ml.
plasma.
tients. AIthough many of the patients were frequently out of control necessitating reevaluation of insulin requirements, all patients delivered live babies. However, one neonate born to C. F. died within 24 hours. The case history is as follows: C. F. was a 24-year-old white, para 0, Class B diabetic patient, whose diabetes was diagnosed for the first time during pregnancy. During the fifth month, she exhibited glycosuria, 4+ acetonuria, and an FBS of 360 mg. per 100 ml. She was hospitalized for 2 weeks and the diabetes brought under control with diet, 70 units of NPH, and 45 units of regular insulin.. The other aspects of her pregnancy were uneventful except that a true fetal heartbeat was never heard, only a placental souffle. Labor was induced at 38
weeks and a 2,250 gram baby with an Apgar score of 3 was delivered. The baby died 24 hours later of multiple congenital anomalies, including tetralogy of Fallot. The estriol levels can be seen in Fig. 3. The vaues rose impressively from the thirty-second to the thirty-seventh week. A drop of about 20 per cent was observed in the final 2 weeks of pregnancy; however, a decrease of this magnitude is seen in normal patients. Class C. Table II presents the values of the estriol determinations performed on the plasma of 11 Class C diabetic patients. The values obtained in 6 of these cases are shown in Fig. 4. Except for S. G. and M. O., the estriol levels were indistinguishable from normal. All patients were delivered of live
9.7
Plasma
estriol
levels
in pregnancy
641
gestation 33 14
34 21
16
22
8.0 17
6.7 21
35
8.5
36 17
37 17
38 11
49
42
46
40
10
13
7.7 12
12 3.3
15
\
19
39 11
40 24
25
18
17
29
35
32
28
17
27
15
17
24
27
33
15
12
17
16
25
13
25
26
“4
20
15
25
17
12 21
(
11 7.7 12
16 9.0 12
24
16
19
11
10
16
15
8.1
7.7
22
22
26
31
10
11
13
9
18
24
26
11
14
14
17
27
25
23
6.6
14
16
17
25
23
26
7.5
12
10
13
13
34
63
63
20
19
19
20
31
13
31
4.7 10 14
7.8 9.5 14
22
23
7.1
33
14
17
15
10
16
17
20
43
24
27
27
24
25
30
babies. However, S. G.‘s baby died 8 hours after delivery. S. G. was a 25-year-old white female, para 0, with Class C diabetes. She had diabetes since the age of 15. She was hospitalized twice during her pregnancy, once for multiple hypoglycemic episodes and once for an increase of blood pressure from 110/80 to 170/110. There was no edema or albuminuria. She went into spontaneous labor in the thirty-fifth week and was delivered of a 1,450 gram baby with an Apgar score of 3 and whose condition became increasingly poor until death. The falling estriol values in the latter part of her pregnancy to a level well below normal can be seen in Fig. 4. M. 0. was a para O-l-O-0 patient with Class C diabetes in poor control throughout pregnancy.
42
29
32
22 45
11
9.6
i
19
13
27 38
38
42
------
A 2,350 gram baby was delivered by cesarean section in the thirty-ninth week shortly after the last estriol determination. Although the plasma estriols were declining, the baby was normal but small.
Although 6 of the other Class C diabetics had histories of previous fetal or neonatal deaths, the course of pregnancy in each case was reasonably good. A typical case is presented. L. G., a 30-year-old, para O-l-O-0, Class C diabetic patient had onset of diabetes at the age of 13 and it had been fairly well controlled on 30 units of NPH ins&n. She conceived one year prior to the pregnancy reported here and suffered an intrauterine death at 32 weeks. Dur-
642
Nachtigall
et al.
60-
/’ /
50 -
/
40 -
20 -
0”
I 26
’
’ 28
’
’ 30
’
’ 32
’
Gestation Fig. 1. Plasma average values. pregnancy.
estriol levels in The broken lines
normal indicate
’ 34
’
1 36
’
1 38
’
J 40
(weeks)
pregnancy.. The. the approximate
dashed lower
line indicates approximate and upper limits in normal
72r 50 -
40 -
20-
RD
10-
O'&
' 26
'
' 28
'
' 30
'
' 32’ Gestation
Fig.
2. Plasma
estriol
levels
in Class A diabetes
'
' 34
'
' 36
(weeks)
of pregnancy.
-
I
' 38
I
' 40
'
Plasma
esbiol
levels
in
pregnancy
643
,MY / I I
I
0
28
30
32
34
Gestation
Fig. 3. Plasma
estriol
levels
in Class
I
I
I
26
B diabetes
I
36
38
40
(weeks)
of pregnancy.
Table II. Plasma estriol levels in Class C diabetic
patients”
..~- -___~-Weeks
--______ 25 / 26
Patient
j
27
\ 28
) 29
I. A.
7.9
1 30
/ 31
20
19
B. c.
of gestation / 32
33
18
12
L. G.t
2.8
6.4
5.5
K. M.
6.1
5.0
T. N.
6.5
7.0
8.4
11
12
8.8
9.1
5.4
6.4
7.8
3.1
M. 0.t
4.8
5.1
s. s.t
7.6
3.4
iq
are
several in Fig.
in
micrograms
instances, 4.
per
more
the present pregnancy,
crease
‘her
insulin
io
45
100 ml.
plasma.
one
determination
than
per
week
it was necessary to inunits
of
NPH
plus
10
units of regular insulin. She h’ad no episodes of acidosis. Cesarean section was performed at 36 to 37 weeks, 2,750 gram
resulting in baby. The
the birth of a normal rising profile of the
estriol kvels is shown in Fig. 4.
Toxemia. The toxemic patients could be
20
25
18
24
40
11
14
20
12
21
20
8.1 13
15
19
5.4
4.4
7.8
11
8.1
21
7.8
45 17
‘Values
20
16
11
7.0
27
D.---__S. tin plotted
17
6.3
19
/ 3.9
3.7
12
8.7
6.9
24
7.2
J. H.t I,. L.
/341/-.75m-i-38
23
17
S. G.
j
was
15 --___ performed
17 on
19
---.-___-~-..~ these
patients.
18
..--. All
the
valurs
-. --
ohtainr~d
arc
divided into three groups, according to their estriol levels (Fig. 5). The first group had severe symptoms of edema, hypertension, and albuminuria, and there was difficulty in controlling these symptoms. Of the 7 patients in Group I, .only 1 patient (E. IL) was delivered, at term. Five went into spontaneous
px-emature labor and in one (F. S.) labor
644
Nachtigall
et al.
July I, 1968 Am. J. Obst. & Gymc,
E
a” 9
Gestation (weeks) Fig. 4. Plasma estriol levels in Class C diabetes of pregnancy.
was induced at 39 weeks. All had extremely small babies for their period of gestation, while H. T. was delivered of a dead fetus. Subnormal estriol values were noted for each patient. Two case histories follow: C. K., a 24-year-old white female, para 0, with toxemia, was noted to have a blood pressure of 160/130, marked edema, and proteinuria at the twenty-sixth week of pregnancy. She was hospitalized from the twenty-ninth week on, but in spite of bed rest, severe sodium restriction, and the use of various diuretics, her blood pressure remained elevated and fluid retention increased. She had multiple allergic reactions to drugs. In the thirty-fifth week of gestation, she went into spontaneous labor and was delivered of a 1,260 gram baby with an Apgar score of 4. It took 4 months for her blood pressure to return to normal. Her plasma estriol values which were significantly below normal are shown in Fig. 5, Group 1. H. T., a 33-year-old Negro female, had a blood pressure of 180/130, a 3+ albuminuria, and edema in the thirty-second week of pregnancy. She was hospitalized from the thirtysecond week of pregnancy with uncontrollable symptoms. The plasma estriol levels declined sharply (Fig. 5, Group 1). She was delivered of a dead fetus in the thirty-fifth week of gestation.
There was no precise record heartbeat was lost.
of when the fetal
The second group of toxemic patients exhibited only mild symptoms. The blood pressure was high but not excessively so. The edema was controlled with drugs and albuminuria was intermittent. Of the 6 patients in this group, M. P., G. G., and B. M. were delivered normally at term; 2 patients, B. R. and D. D., were delivered prematurely, while one (D. T.) was sectioned. All babies were of normal size. The estriol levels for the patients in this group were indistinguishable from normal (Fig. 5, Group 2), The third group of 3 toxemic patients, 2 of whom presented with a nephrotic syndrome, had severe symptoms. Abnormally high estriol levels were obtained for all the patients
in
this
group
(Fig.
5, Group
3).
Two patients (L. M. and A. B.) were delivered spontaneously and in the third (C. M.) labor was induced. A casehistory which is illustrative
of
the
2 nephrotic
patients
(L. M. and A. B.) follows: A. B., a 29-year-old, para 4-O-O-4, Puerto Rican female, with no known previous illnesses presented at clinic in her twenty-fourth week of
Plasma estriol levels in pregnancy
645
* Group 2
Gestation (weeks) Fig. 5. Plasma estriol levels in toxemia of pregnancy.
gestation with edema, a blood pressure of 160/ 110, a cholesterol level of 510, 3+ albuminuria, and a BUN level of 52. She was hospitalized from the twenty-fifth week on and was maintained on diuretics, MgSO,, severe sodium restriction, and bed rest. On this regimen the edema decreased and her proteinuria diminished, but the blood pressure remained elevated as did the BUN and cholesterol levels. She went into spontaneous labor at 36 weeks and was delivered of a 2,185 gram baby with an Apgar score of 7. Three days post partum the patient’s BUN level was 15. The plasma estriol values which are abnormally high are shown in Fig. 5, Group 3. The third patient in this group in that she had a previous history disease.
differed of renal
C. M., a 23-year-old, para 0, toxemic female, had been treated for glomerulonephritis at the age of 18 with no known follow-up until she presented in the twenty-fourth week of gestation with a blood pressure of 210/120, BUN 23, cholesterol 280, 2+ albuminuria, anemia, and weakness. She was hospitalized and the edema
and albuminuria improved considerably on treatment. Her blood pressure fluctuated between 170/100 and 210/120. The BUN level was never below the initial value of 23 and on two occasions it was above 30. Renal biopsy revealed chronic pyelonephritis. In the thirty-second week, she began to have nausea and vomiting and the BUN level rose to 35. At 33 weeks labor was induced and she was delivered of a 1,820 gram infant in fair condition with an Apgar score of 7. The patient’s BUN continued to rise, and 6 weeks after delivery she was stiI1 hospitalized,
Suspected fetal death. Plasma estriol Ievels were determined in 10 cases of suspected fetal death. In each case the fetal heartbeat was not detectable by fetoscope although in some cases the mother claimed to feel fetal movement. The values obtained are shown in Table III. It can be seen that in the 9 cases in which extremely low levels were obtained the patients subsequently were delivered of dead fetuses. In the remaining study, the values were in the low normal range and a live baby was subsequently de-
646
Nachtigall
et al.
Table III. Plasma estriol levels in relation to outcome of pregnancy in cases of suspected fetal death
Patient
1
M. D. D. A. F. G. P.L. Z.N. C.N. P.D. A. R. J. 0. B. J. *The ground ternal
~ZlL$
34, 34, 25, 28 32 27 20 36 30 35,
1 (,u&%%Zd.)
35 35 29
36, 37
2.7, 2.6, 4.8, 1.1 0.3 1.8 1.7 1.0 0.7 6.1,
1.0 1.4 2.6
11, 13
1 ;:Z’
Dead Dead Dead Dead Dead Dead Dead Dead Dead Live
values are not corrected for the calculated of 0.7 fig per 100 ml. which is attributed to isotopic
backthe in-
standards.
livered. The pertinent as follows:
aspects of this case are
B. J. was admitted in the thirty-fifth week of pregnancy with greater than 30 per cent of her body including the abdomen severely burned. The sterile dressing limited the careful evaluation of the fetal heart either by fetoscope or ECG. In addition, the mother felt no fetal movement. Because of her poor condition, it was felt that her toxic state might be improved by inducing labor and removing the “dead” fetus. The plasma estriol level indicated a live fetus, and the value continued to rise (Table III). The patient went into spontaneous labor at the end of the thirty-seventh week and was delivered of a normal male infant with an Apgar score of 8. Comment Plasma estriol determinations have at least three advantages over the analysis of urine in the evaluation of human pregnancy. First, the need for the troublesome 24 hour urine collection is eliminated. Second, if necessary, 2 or more plasma samples may be drawn for analysis in a single day. Finally, in view of the short plasma estriol half-life,gl I1 a decreasing rate of estriol synthesis would be reflected more rapidly in a single plasma sample than in a pooled urine collection which reflects the average of the preceding 24 hours.
The validity of the method with regard to the precision, accuracy, specificity, and senaitiveity had been established previously.” In the present study which included 73 cases of normal and pathologic pregnancies, the potential advantage of plasma estriol determinations as a valuable adjunct in gaining insight into the course of difficult pregnancies has been demonstrated. The major feature of the normal plasma estriol values is that they continue to rise in a generally regular fashion as pregnancy progresses. Although the curve is not always smooth for each individual, the variations are no greater than those obtained with urine.’ The absolute values vary widely from patient to patient. The average plasma estrio1 value at term is about 27 pg per 100 ml., while the range is between 10 and 55 pg per 100 ml. This emphasizes the need for obtaining serial determinations on each patient studied. An occasional fall in the values has been observed at random times of gestation. However, the report of Smith” that plasma estriol levels fall significantly in the days prior to labor was not confirmed in this study. In the 22 normal patients studied, only 4 exhibited a decline of greater than 15 per cent at term. The plasma estriol values of the diabetic patients were not statistically different from those of the normal patients. Thus, even Class C diabetic patients in good control could be expected to have plasma estriol levels indistinguishable from normal provided fetal and placental functions are not impaired. An interesting case was that of S. G. This Class C diabetic patient exhibited an obvious decline in estriol values and in the thirty-fifth week of gestation she was spontaneously delivered of a fetus with a poor Apgar score who died shortly after birth. The significant point is that an estriol value of 3.7 pg per 100 ml., although well below normal for the thirty-fifth week of gestation, is still compatible with a live fetus. A Class B diabetic patient, C. F., was delivered of a live baby in the thirty-eighth week of gestation who died 24 hours after birth. She had normal estriol values. The autopsy report
Plasma
revealed a fetal heart-lung anomaly of the type which is compatible only with fetal life. It is apparent that these plasma estriol studies would have to be extended before a delinite statement could be made concerning the value of the determinations in improving the fetal salvage rate in diabetes of pregnancy. The toxemic patients displayed a good degree of consistency between the plasma cstriol levels and the clinical picture. The classification of these patients into three groups according to their estriol profiles may prove to be useful. The cases in Group 1 were in poor control and in each case development of the fetus and placenta was markedly retarded. The estriol levels were severely depressed and they appeared to decline even further at the time of delivery which was premature. One of the patients (H. T.) suffered an intrauterine death which, according to the declining estriol le\,els to a final value of 1.1, was not surprising. The toxemic patients in Group 2 were under good control and the fetuses apparently developed normally. The plasma estriol values of these patients were in the normal good correlation berange, demonstrating tween the plasma estriol values and the clinical picture. However, the steadily declining estriol pattern of M. P. toward term was the only one of this type in the entire study which resulted in a normal outcome. The strikingly elevated plasma estriol l(~vcls in the Group 3 toxemic patients are puzzling. There were only 3 patients in this 2 of whom presented with the group, nrphrotic syndrome and 1 with chronic
estriol
levels
in
pregnancy
647
renal disease superimposed on toxemia. It would be premature to speculate on the origin of the elevated estriol but an att,ractive working hypothesis is that these patients have high estriol kidney thresholds. To test this hypothesis in the future, the plasma clearance rates will be measured in patirrkts of this type. It was shown recently in I)UI laboratory that with only minor changes the plasma estriol method may be applied to the analysis of urine.‘” Unequivocal results have been obtained in studies of suspected fetal death. ‘i’he plasma estriol values were less than 2.6 rug per 100 ml. in each case of fetal death. However, the length of time that the fetus was dead could not be accurately judged in any patient. At this stage of the study it is not possible to cite a level below wllich the life of the fetus is in immediate jeopard). Although 33 patients have been studickd. a precise assessment of the value of plasma estriol determinations in the managcmrnt of Complications in pregnancy cannot as y
REFERENCES
1. Siiteri, P. K., and MacDonald, P. C.: J. Clin. Endocrinol. 26: 751, 1966. 2. Diczfalusy, E., and Benagiano, G.: Research on Steroids, Transactions of Second Meeting International Study Croup for Steroid Hormones, Rome, 1965, p. 27. 3. Magendantz, H. G., and Ryan, K. J.: J. Clin. Endocrinol. 24: 1155, 1964. 4. Kellar, R.. Matthew, G. D., MacKay, R., Brown, J. B., and Roy, E. J.: J. Obst. & Gynaec. Brit. Emp. 66: 804, 1959.
5. Greene, J. W., Jr., Smith, K., Kyle, G. C.. Touchstone, J. C., and Duhring. J. I,.: .41u. J. OBST. & GYNEC. 91: 684, 1965. 6. Taylor, E. S., Bruns, P. D., Drose, V. E., and Kartchner, M. J.: AM. J. ORST. & GYTEC. 83: 194, 1962. 7. Frandsen, A., and Stakemann, G.: Danish M. Bull. 7: 98, 1960. 8. Schwers, J.: Les oestroghnes au tours de la seconde moitiC de Ia grossesse, Brussels~ 1965, Editions Arscia S.A.
648
9.
10.
Nachtigall
et al.
Nachtigall, L., Bassett, M., Hogsander, U., Slagle, S., and Levitz, M.: J. Clin. Endocrinol. 26: 941, 1966. White, P.: Pregnancy Complicating Diabetes in Jo&n, E. P., Root, H. F., White, P., and Marble, A., editors: The Treatment of Diabetes Mellitus, Philadelphia, 1952, Lea & Febiger.
11.
12. !3.
Roy, E. J., Harkness, R. A., and Kerr, M. G.: J. Obst. & Gynaec. Brit. Comm. 70: 1034, 1963. Smith, 0. W.: Acta endocrinol. 51: Suppl. 104, 1966. Jaffe, S., and Levitz, M.: AM. J. OBST. & GYNEC. 98: 992, 1967.