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Estimation of gestational age and maternal serum steroid concentrations: Total estriol
Estimation of gestational age and maternal serum steroid concentrations: Total estriol L. L. PENNEY El Paso, Texas Acute increases in maternal serum unconjugated estriol between 35 and 37 weeks of pregnancy have been reported to correlate predictably with gestational age. In this study total estriol was measured by radioimmunoassay in an effort to define maturational surges. Statistically significant variations from predicted regression lines were not demonstrated in any individual patient or in grouped data. (AM. J. 0BSTET. GYNECOL. 138:745, 1980.)
MuLTIPLE DIRECTLY and indirectly related events support an acceleration of fetal adrenal steroidogenesis in the third trimester of normal pregnancy. 1- 7 Additional data have been summarized in recent reviews. 8 • 9 A statistically significant surge in the maternal serum level of unconjugated estriol at 36 weeks ± 6 days has been reported to accurately predict gestational age. 10 • 11 The present study was undertaken to define the normal increase in serum total estriol from 5 to 3 weeks prior to term and to delineate any surge points.
Materia! end methods
Thirty-four patients were studied in a longitudinal manner following informed consent. All of the pregnancies were normal and gestational dates were accurate. Only patients whose menstrual cycles were 28 to 30 days in length and who had pelvic examinations to confirm uterine size in the first trimester were selected. Gestational age from quickening, first audible fetal heart tones. and newborn examinations were all consistent with the established estimated dates of confinement. Thirteen of the patients were studied weekly from 32 or 3~~ to 38 or 39 ·weeks" gestation. Twenty of the re-
From the Clinical Investigations Service and Department of Obstetrics and Gynecology, William Beaumont Army Medical Center. The opinions or assertions contained herein are the private views of the author and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. Received for publication June 9, 1980. Accepted julv 30, 1980. Reprint requests: L. L. Penney, Clinical investigations Service and Department o/ Obstetrics and Gynecology, William Beaumont Armv Medical Center, El Paso, Texas 79920. .
mammg 21 patients were studied daily beginning at week 35 and continuing for 12 to 15 days, and the last patient was studied for 8 days. Antecubital venous blood specimens were drawn between 0900 and 1500 hours for the group. Since diurnal variation is still controversial,12 individual patients were sampled within i hour of the same clock time at each visit. Dated specimens from ail patients were entered in a data book chronologically and assigned random numbers generated from a srandard tabie. The techmCian performed each determination sequentially by random numbers. The chronoiogic numbers were rematched after each assay. All samples from an individual patient were included in a single assay with the exception that unknowns with a coefficient of variation > 15% in the duplicate tubes were empirically rejected and repeated. The repeat rate was 4.6%. The median coefficient of variation between replicates for ~72 unknowns was 3.8%. Antiserum, estriol standards, and 125 1-labeled estriol were supplied by the Amersham Corporation, Arlington IIeights. Illinois. The enzyrne hydrolysis was performed according to the manufacturer\ directions, hut the assay was modified to inc~ude a total count iube and a nonspecific binding (NSB) tube of male serum with each run. The vveightcd log~t-log method \vas used for data reduction. 13 Two quality control sera were ineluded in each run and yielded means of .~ 1.6 ::t 7.6 and 147.4 ± 12.8 ng/ml. The within-assay coefficient of variation was 3.6% for the low control and 2.4% for the high control. 14 The corresponding figures between assays were 15.0o/t and 9.0%. Ten repetitions within a run of each control revealed no within-assav systematic drift. The logarithmic value of the serum total estriol' 0 • 15 as calculated by the above methods was used for further data reduction in this report. 745
746 Penney
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31.6(1.5) 32 33 34 35
36 3 7
38
39
WEEKS OF GESTATION
Fig. 1. Regression line (weekly data) at weeks 32 to 35 and 95% confidence interval.
3162(3.5)
1
00(20)1
~
I
tt[j
I
I 1Lllll I t I I
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316(2.51
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I
I
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~
1fffI~:·262+.059.
316(2.51
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I I I I I
Y= .034-t-.06ix
31.6(1.5JL,__..~......~--~~-::-::--::~-
32 33 34 35 36 ~7 38 WEEKS OF GESTATION
Fig. 2. Regression lines and 95% confidence intervals from 32 weeks in patient No.9. 0 =Point not used in computation.
Results Summary statistics from patients sampled weekly are shown in Table I and those from patients sampled daily are shown in Table II.
The calculated regression lines were Y = 0.261 + 0.053X for the weekly data and Y = 0.466 + 0.048X for the daily data. They did not differ significantly in slope or intercept. 16 The regression line of the weekly data calculated through 35 weeks is shown in Fig. l. The values at 36 and 37 weeks did not exceed the upper limit of the predicted 95% confidence interval for the line. Including the data for 35 weeks, I day and 35 weeks, 2 days in the regression and expanding the comparisons to all daily and weekly points thereafter up to 38 weeks reveals that all points excluded from the regression still fall within the 95% confidence interval of the predicted lineY The apparent nadir in the data at 35 weeks, 2 days was marginally different from the apparent peak at 36 weeks, 2 days by Student's t test (t = - 1.99, 39 df, 0.025 > P < 0.05). However, comparisons of the slope from 32 to 35 weeks with the slope from 35 weeks to 37 weeks and the slope of combined daily and weekly data from 32 weeks to 35 weeks, 2 days with the slope from 35 weeks, 2 days to c~7 weeks revealed no difference. 16 Individual patients were studied by computing the regression line from the first three data points and iterating the search for an ensuing value which exceeded the 95% confidence interval predicted for that point in the sequence. 17 Defined in this manner, a surge occurred in none of the patients.
Comment Aithough an occasionai inflection point in the exponential increase was apparent between 35 and 37 weeks' gestation when individual maternal serum free estriol concentrations were studied, 18 no similar surges were noted with total estriol. With a small N of 3, minimal variability in the data from sample to sample produces very wide predicted confidence intervals, as illustrated by the patient example in Fig. 2. Note the change in scale of theY axis needed to fit N = 3. It is apparent that although an increasing N rapidly nar-
Gestational age and maternal serum steroids
Volume l:ll< !\umher 7. Pari
747
Table I. Summary statistics from weekly samples Week of gestation
N ~ean
(ng/ml)
S,
3'1
32
33
34
35
36
?7
18
7 95.1 26.9
12 117.1 36.9
13 121.3 44.0
13 136.1 38.2
13 161.2 45.2
13 179.4 45.9
2oo.2
2l:t3
54.3
47.1
u
X
Table II. Summary statistics from daily samples Length of gestation (weeks, days) 36,7
35, 1
N Mean (ng/ml) S,
21 154.8 65.6
21 145.3 53.9
21 159.7 59.2
20 162.7 64.2
21 158.4 63.0
21 154.4 55.4
rows the confidence interval, the week-to-week variabilitY still produces a relatively wide prediction. More frequent sampling and pooling of multiple specimens drawn several minutes apart at each sampling 19 could
21 158.3 63.0
20 182.2 64.5
20 175.6 67.3
20 181.5 71.0
20 179.0 82.5
19 170.1 t\6. 9
14 185.6 55.6
yield more reproducible data. Even it these techniques could be shown to yield reliable results. ttm would not seem to be a practical method of estimating gestational age.
REFERENCES I. Murphy, B. E. P.: Cortisol and cortisone levels in the cord blood at delivery of infants with and without the respiratory distress syndrome, AM. J. 0BSTET. G'>'NECOL. 119:1112, 1974. 2. Monsterrat, F. D., and Tulchinsky, D.: Total cortisol in amniotic fluid and fetal lung maturation, N. Engl. J. Med. 292:133, 1975. 3. Gluck, L., Kulovich, M. V., Borer, R. C., Jr., et al.: Diagnosis of the respiratory distress syndrome by amniocentesis, AM. J. OBSTET. GYNECOL. 109:441, 1971. 4. Liggins, G. C.: Adrenocortical-related maturational events in the fetus, AM. J. 0BSTET. GYNECOL. 126:931, 1976. 5. Winters, A. J ., Colsten, C., MacDonald, P. C., et al.: Fetal plasma prolactin levels, J. Clin. Endocrinol. Metab. 41:626, 1975. 6. Taylor, E. S., Hagerman, D. D., Betz, G., et al.: Estriol concentrations in blood during pregnancy, AM. J. 0BSTET. GYNECOL. 108:860, 1970. 7. Siiteri, P. K., and MacDonald, P. C.: Placental estrogen biosynthesis during human pregnancy, J. Clin. Endocrinol. Metab. 26:751. 1966. 8. Beling, C.: Estrogens, in Fuchs, F., and Klopper, A., editors: Endocrinology of Pregnancy, ed. 2, Hagerstown, 1977, Harper & Row, Publishers, chap. 5, pp. 76-98. 9. Peterson, R. E.: Cortisol, in Fuchs, F., and Klopper, A., editors: Endocrinology of Pregnancy, ed. 2, Hagerstown, 1977, Harper & Row. Publishers, chap. 8, pp. 157-176. 10. Buster, J. E., Sakakini, J., Jr., Killam, A. P., et al.: Serum unconjugated estriol levels in the third trimester and their relationship to gestational age, AM. ]. OssTET. GYNECOL. 125:672, 1976. II. Sakakini, J.. Jr., Buster, J. E., and Killam, A. P.: Serum
21 159.8 65.5
12.
13.
14. 15.
16. 17.
18. 19.
unconjugated estriol levels in the third trimester and their relationship to gestational age. 11. A~1. J. OssTET. GYNECOL. 127:452, 1977. Allen, E. T., and Lachelin, G. E. L.: A comparison of plasma levels of progesterone, oestradiol. unconjugated oestriol and total oestriol with urinary total oestrogen levels in clinical obstetric practice, Br. J. Obstet. Gynaecol. 85:278, 1978. Faden, F. B., and Rodbard, D.: The "logit-log" method and Scatchard plot, in Radioimmunoassay Data Processing: Listings and Documentation, ed. 3, PB246 223, Na· tional Technical Information Service, United States Department of Commerce, Springfield, September, 1975. Rodbard, D.: Statistical qualitv control and routine data processing for radioimmunoassays and immunoradiometric assays, Clin. Chern. 20:1255, 1974. Hagerman, D. D.: Clinical use of plasma total estriol measurements in late pregnancy, J. Reprod. \led. 23:179. 1979. Dixon, W. J., and Massey. F. J., Jr.: Introduction to Statistical Analysis, New York, 1969, \kGraw-Hill Book Company, Inc. pp. 200-202. Remington, R. D., and Schork, M.A.: Statistics with Applications to the Biological and Health Sciences, En· glewood Cliffs. New Jersey, 1970, Prentice-Hall, pp 265275. Penney, L. L.: Estimation of gestational age and maternal serum steroid concentrations. Unconjugated estriol. t'npublished data. Buster, J. E., Meis, P. J., Hobel, C. J.. et a!.: Subhourlv variability of circulating third trimester maternal steroid concentrations as a source of sampling crro1. J Clin. Endocrinol. Metab. 46:907, 1978.
MuLTIPLE DIRECTLY and indirectly related events support an acceleration of fetal adrenal steroidogenesis in the third trimester of normal pregnancy. 1- 7 Additional data have been summarized in recent reviews. 8 • 9 A statistically significant surge in the maternal serum level of unconjugated estriol at 36 weeks ± 6 days has been reported to accurately predict gestational age. 10 • 11 The present study was undertaken to define the normal increase in serum total estriol from 5 to 3 weeks prior to term and to delineate any surge points.
Materia! end methods
Thirty-four patients were studied in a longitudinal manner following informed consent. All of the pregnancies were normal and gestational dates were accurate. Only patients whose menstrual cycles were 28 to 30 days in length and who had pelvic examinations to confirm uterine size in the first trimester were selected. Gestational age from quickening, first audible fetal heart tones. and newborn examinations were all consistent with the established estimated dates of confinement. Thirteen of the patients were studied weekly from 32 or 3~~ to 38 or 39 ·weeks" gestation. Twenty of the re-
From the Clinical Investigations Service and Department of Obstetrics and Gynecology, William Beaumont Army Medical Center. The opinions or assertions contained herein are the private views of the author and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. Received for publication June 9, 1980. Accepted julv 30, 1980. Reprint requests: L. L. Penney, Clinical investigations Service and Department o/ Obstetrics and Gynecology, William Beaumont Armv Medical Center, El Paso, Texas 79920. .
mammg 21 patients were studied daily beginning at week 35 and continuing for 12 to 15 days, and the last patient was studied for 8 days. Antecubital venous blood specimens were drawn between 0900 and 1500 hours for the group. Since diurnal variation is still controversial,12 individual patients were sampled within i hour of the same clock time at each visit. Dated specimens from ail patients were entered in a data book chronologically and assigned random numbers generated from a srandard tabie. The techmCian performed each determination sequentially by random numbers. The chronoiogic numbers were rematched after each assay. All samples from an individual patient were included in a single assay with the exception that unknowns with a coefficient of variation > 15% in the duplicate tubes were empirically rejected and repeated. The repeat rate was 4.6%. The median coefficient of variation between replicates for ~72 unknowns was 3.8%. Antiserum, estriol standards, and 125 1-labeled estriol were supplied by the Amersham Corporation, Arlington IIeights. Illinois. The enzyrne hydrolysis was performed according to the manufacturer\ directions, hut the assay was modified to inc~ude a total count iube and a nonspecific binding (NSB) tube of male serum with each run. The vveightcd log~t-log method \vas used for data reduction. 13 Two quality control sera were ineluded in each run and yielded means of .~ 1.6 ::t 7.6 and 147.4 ± 12.8 ng/ml. The within-assay coefficient of variation was 3.6% for the low control and 2.4% for the high control. 14 The corresponding figures between assays were 15.0o/t and 9.0%. Ten repetitions within a run of each control revealed no within-assav systematic drift. The logarithmic value of the serum total estriol' 0 • 15 as calculated by the above methods was used for further data reduction in this report. 745
746 Penney
Decemhn L i'IHI! Am . .J. Ohstet. C; tltTol
w
3
"'>
316(2.5)1
-' 0
a:
~ U-
0 :;;
,...r
'ovv""'"..' v.l"J
a:
"g"'
~ Y=
.522
+
.046X
31.6(1.5) 32 33 34 35
36 3 7
38
39
WEEKS OF GESTATION
Fig. 1. Regression line (weekly data) at weeks 32 to 35 and 95% confidence interval.
3162(3.5)
1
00(20)1
~
I
tt[j
I
I 1Lllll I t I I
316(0.5)1
N=3 Y = i.506 + .Oi7x
316(2.
100(2.0 N=4 Y= .716+.041x 31.6(1.5~1'.__._..__...._......~..~----
316(2.51
IOO~.j 31.6(1.5
I
I
I
I
I ./
~
1fffI~:·262+.059.
316(2.51
100(2.0)1
J
I I I I I
Y= .034-t-.06ix
31.6(1.5JL,__..~......~--~~-::-::--::~-
32 33 34 35 36 ~7 38 WEEKS OF GESTATION
Fig. 2. Regression lines and 95% confidence intervals from 32 weeks in patient No.9. 0 =Point not used in computation.
Results Summary statistics from patients sampled weekly are shown in Table I and those from patients sampled daily are shown in Table II.
The calculated regression lines were Y = 0.261 + 0.053X for the weekly data and Y = 0.466 + 0.048X for the daily data. They did not differ significantly in slope or intercept. 16 The regression line of the weekly data calculated through 35 weeks is shown in Fig. l. The values at 36 and 37 weeks did not exceed the upper limit of the predicted 95% confidence interval for the line. Including the data for 35 weeks, I day and 35 weeks, 2 days in the regression and expanding the comparisons to all daily and weekly points thereafter up to 38 weeks reveals that all points excluded from the regression still fall within the 95% confidence interval of the predicted lineY The apparent nadir in the data at 35 weeks, 2 days was marginally different from the apparent peak at 36 weeks, 2 days by Student's t test (t = - 1.99, 39 df, 0.025 > P < 0.05). However, comparisons of the slope from 32 to 35 weeks with the slope from 35 weeks to 37 weeks and the slope of combined daily and weekly data from 32 weeks to 35 weeks, 2 days with the slope from 35 weeks, 2 days to c~7 weeks revealed no difference. 16 Individual patients were studied by computing the regression line from the first three data points and iterating the search for an ensuing value which exceeded the 95% confidence interval predicted for that point in the sequence. 17 Defined in this manner, a surge occurred in none of the patients.
Comment Aithough an occasionai inflection point in the exponential increase was apparent between 35 and 37 weeks' gestation when individual maternal serum free estriol concentrations were studied, 18 no similar surges were noted with total estriol. With a small N of 3, minimal variability in the data from sample to sample produces very wide predicted confidence intervals, as illustrated by the patient example in Fig. 2. Note the change in scale of theY axis needed to fit N = 3. It is apparent that although an increasing N rapidly nar-
Gestational age and maternal serum steroids
Volume l:ll< !\umher 7. Pari
747
Table I. Summary statistics from weekly samples Week of gestation
N ~ean
(ng/ml)
S,
3'1
32
33
34
35
36
?7
18
7 95.1 26.9
12 117.1 36.9
13 121.3 44.0
13 136.1 38.2
13 161.2 45.2
13 179.4 45.9
2oo.2
2l:t3
54.3
47.1
u
X
Table II. Summary statistics from daily samples Length of gestation (weeks, days) 36,7
35, 1
N Mean (ng/ml) S,
21 154.8 65.6
21 145.3 53.9
21 159.7 59.2
20 162.7 64.2
21 158.4 63.0
21 154.4 55.4
rows the confidence interval, the week-to-week variabilitY still produces a relatively wide prediction. More frequent sampling and pooling of multiple specimens drawn several minutes apart at each sampling 19 could
21 158.3 63.0
20 182.2 64.5
20 175.6 67.3
20 181.5 71.0
20 179.0 82.5
19 170.1 t\6. 9
14 185.6 55.6
yield more reproducible data. Even it these techniques could be shown to yield reliable results. ttm would not seem to be a practical method of estimating gestational age.
REFERENCES I. Murphy, B. E. P.: Cortisol and cortisone levels in the cord blood at delivery of infants with and without the respiratory distress syndrome, AM. J. 0BSTET. G'>'NECOL. 119:1112, 1974. 2. Monsterrat, F. D., and Tulchinsky, D.: Total cortisol in amniotic fluid and fetal lung maturation, N. Engl. J. Med. 292:133, 1975. 3. Gluck, L., Kulovich, M. V., Borer, R. C., Jr., et al.: Diagnosis of the respiratory distress syndrome by amniocentesis, AM. J. OBSTET. GYNECOL. 109:441, 1971. 4. Liggins, G. C.: Adrenocortical-related maturational events in the fetus, AM. J. 0BSTET. GYNECOL. 126:931, 1976. 5. Winters, A. J ., Colsten, C., MacDonald, P. C., et al.: Fetal plasma prolactin levels, J. Clin. Endocrinol. Metab. 41:626, 1975. 6. Taylor, E. S., Hagerman, D. D., Betz, G., et al.: Estriol concentrations in blood during pregnancy, AM. J. 0BSTET. GYNECOL. 108:860, 1970. 7. Siiteri, P. K., and MacDonald, P. C.: Placental estrogen biosynthesis during human pregnancy, J. Clin. Endocrinol. Metab. 26:751. 1966. 8. Beling, C.: Estrogens, in Fuchs, F., and Klopper, A., editors: Endocrinology of Pregnancy, ed. 2, Hagerstown, 1977, Harper & Row, Publishers, chap. 5, pp. 76-98. 9. Peterson, R. E.: Cortisol, in Fuchs, F., and Klopper, A., editors: Endocrinology of Pregnancy, ed. 2, Hagerstown, 1977, Harper & Row. Publishers, chap. 8, pp. 157-176. 10. Buster, J. E., Sakakini, J., Jr., Killam, A. P., et al.: Serum unconjugated estriol levels in the third trimester and their relationship to gestational age, AM. ]. OssTET. GYNECOL. 125:672, 1976. II. Sakakini, J.. Jr., Buster, J. E., and Killam, A. P.: Serum
21 159.8 65.5
12.
13.
14. 15.
16. 17.
18. 19.
unconjugated estriol levels in the third trimester and their relationship to gestational age. 11. A~1. J. OssTET. GYNECOL. 127:452, 1977. Allen, E. T., and Lachelin, G. E. L.: A comparison of plasma levels of progesterone, oestradiol. unconjugated oestriol and total oestriol with urinary total oestrogen levels in clinical obstetric practice, Br. J. Obstet. Gynaecol. 85:278, 1978. Faden, F. B., and Rodbard, D.: The "logit-log" method and Scatchard plot, in Radioimmunoassay Data Processing: Listings and Documentation, ed. 3, PB246 223, Na· tional Technical Information Service, United States Department of Commerce, Springfield, September, 1975. Rodbard, D.: Statistical qualitv control and routine data processing for radioimmunoassays and immunoradiometric assays, Clin. Chern. 20:1255, 1974. Hagerman, D. D.: Clinical use of plasma total estriol measurements in late pregnancy, J. Reprod. \led. 23:179. 1979. Dixon, W. J., and Massey. F. J., Jr.: Introduction to Statistical Analysis, New York, 1969, \kGraw-Hill Book Company, Inc. pp. 200-202. Remington, R. D., and Schork, M.A.: Statistics with Applications to the Biological and Health Sciences, En· glewood Cliffs. New Jersey, 1970, Prentice-Hall, pp 265275. Penney, L. L.: Estimation of gestational age and maternal serum steroid concentrations. Unconjugated estriol. t'npublished data. Buster, J. E., Meis, P. J., Hobel, C. J.. et a!.: Subhourlv variability of circulating third trimester maternal steroid concentrations as a source of sampling crro1. J Clin. Endocrinol. Metab. 46:907, 1978.