British Journal of Obstetrics and Gynaecology November 1976. Vol83. pp 853-856
ACTH LEVE1,S IN AMNIOTIC FLUID DURING PREGNANCY BY
R. TUIMALA A. KAUPPILA AND
J. HAAPALAHTI Department of Obstetrics and Gynaecology, University Central Hospital of Oulu and Laboratory of the Deaconess Institute of Oulu, Oulu, Finland Summary The fetal pituita.ry-adrenal axis plays an important role in the regulation of fetal development. In order to obtain information about fetal ACTH secretion at different gestational ages, a total of 109 amniotic fluid ACTH determinations was performed by ra.dioimmunoassay. There was a significantly higher level of ACTH during 26 to 30 weeks of pregnancy (4291180.4 pg/ml) than in early (208-7&90.6 pg/ml) and in late (172-7197.4pg/ml) pregnancy; fetal sex, uterine contractions and maternal complications in pregnancy did not affect levels. The ACTH level in the first urine of six newborn infants (160.0f40.6 pg/ml) approximated to that in the amniotic fluid in late pregnancy. Our results support the assumption of a fetal origin for ACTH in amniotic fluid. The high secretion of ACTH at tht: beginning of the last trimester of pregnancy may stimulate the development of the adrenal cortex and result in the increased cortisol secretion necessary for fetal lung maturation.
measured by radioimmunoassay at least in umbilical cord blood from the 12th gestational week (Winters et al, 1974). It has also been shown that ACTH increases the steroidogenic capacity of fetal adrenal cortical cells in tissue culture (Milner and Villee, 1970). This close interaction in the fetal ACTH-cortisol axis led us to examine the pattern of fetal ACTH secretion throughout pregnancy in normal and complicated pregnancies by measuring ACTH levels in the amniotic fluid.
THE marked rise in the secretion of coitisol by the fetus in late pregnancy (Murphy, 1973) is probably responsible for the acceleration of the synthesis of pulmonary surfactant, and of lung maturation in the human fetus (Fencl and Tulchinsky, 1975). The riise of cortisol secretion is reflected in a rise in the cortisol level of the amniotic fluid (Murphy cpt al, 1975; Fencl and Tulchinsky, 1975). A good correlation exists between cortisol and the lecithin/sphingomyelin ratio in amniotic fluid (Fencl and Tulchinsky, 1975). These findings emphasize the importance of fetal adrenals in preparing the fetus for extrauterine life. Fetal adrenals may also be of significance in the initiation of labour (Murphy, 1973). The development of the definite zone of the fetal adrenals is regulated by fetal pituitary ACTH. This zone has a specific binding capacity for ACTH at least from the 12th gestational week (Seron-Ferre et al, 1975) and ACTH can be
METHODS A total of 109 amniotic fluid samples were taken from 97 women by 94 transabdominal and 15 transvaginal amniocenteses (Table I). The samples were taken into glass tubes and stored at --20 OC until assayed. Six samples of the first urine of newborn infants were also obtained for ACTH determination. ACTH was determined by radioimmunoassay 853
854
TUIMALA, KAUPPILA AND HAAPALAHTI TABLE I The clinical diagnosis of patients examined
approximated to that in the amniotic fluid during late pregnancy (Fig. 1).
Early termination of pregnancy Rhesus isoimmunization Hypertensive complications of pregnancy Diabetes Jaundice of pregnancy Hydramnios Placenta praevia Threatened premature labour Controls
15 20 17 11 5 4 2 19 16 I
Total 109
(Landon and Greenwood, 1968). ACTH, adsorbed from the amniotic fluid onto small glass particles, was liberated by acetone, and radioimmunoassay was performed after evaporation (Ratcliffe and Edwards, 1971) as duplicates at two dilutions. The intra-assay variation was 3 - 2 per cent and the interassay variation was 4 - 5 per cent. The reagents were supplied by the Radiochemical Centre, Amersham. Statistical differences were tested by Student’s ‘t’ test.
RESULTS ACTH values at various gestational ages are shown in Table 11 and Figure 1. The ACTH level was significantly higher during the 26th to 30th weeks of pregnancy than at any time before or after this period (pr0.001). Between the 31st and 42nd weeks the mean ACTH (*SD) was 172.7k97.4 pg/ml. No other significant differences could be found between the periods examined. ACTH levels in the amniotic fluid were not significantly affected by the sex of the fetus, uterine activity before the amniocentesis or complications of pregnancy (Table 111). The ACTH level in the urine of the newborn infants
DISCUSSION The origin of ACTH in the amniotic fluid is not known. Many reports confirm that there is no placental transport of either endogenous (Allen et al, 1973; Miyakava et al, 1974; Dokumov et al, 1974; Winters et al, 1974) or exogenous (Miyakava et al, 1974) maternal ACTH. Simple filtration through the amniotic membrane from the maternal circulation into the amniotic sac is similarly unlikely, because the ACTH level in the maternal circulation of a patient with Nelson’s syndrome was about 100 times higher than in the amniotic fluid both in early and late pregnancy (Allen et al, 1973). There is also no indication that the placenta can synthesize ACTH (Genazzani et al, 1975). The fetal origin for the ACTH in the amniotic fluid is corroborated by our finding of quite a high ACTH level in the first urine of newborn infants, a level which approximated closely to that in the amniotic fluid. Reports of ACTH in the amniotic fluid are few (Allen et al, 1973), though umbilical cord ACTH has been a subject of many investigations, most of them dealing with fetuses at term. It has been reported that umbilical cord ACTH is significantly higher before the 34th gestational week than later (Winters et al, 1974). In our results, ACTH in the amniotic fluid remained almost constant from the 30th week of pregnancy until delivery. The most striking finding was the significantly higher level of ACTH during the 26th to 30th weeks of pregnancy, a period when there is a significant fall of amniotic fluid cortisol level (Turnbull, 1976). The explanation for this burst of ACTH secretion would be the ACTH-induced
TABLEI1 ACTH values in amniotic &id at different gestational ages Gestational week
No. ACTH (pg/ml)
-__
Mean SD
10-18
26-30
31-32
33-34
35-36
37-38
3940
4142
15
11
12
20
17
12
16
6
208.7
429.5
201.3
194.5
163.6
175.5
170.9
179.3
90.6
180.4
86.1
129.4
87.5
54.4
78.9
46.7
AMNIOTIC FLUID ACTH
855
TABLE 111 The effects of the sex of the fitus, uterine activity and complications of pregnancy on the ACTH level in amniotic fluid; values from patients before the 31st gestational week were excluded Sex Male
Female
52
27
18
10
17
11
4
5
16
176.7
166.9
196.8
158.7
153.1
208.6
156.0
145.4
143.3
X'9.8
112.2
125.0
78.6
56.2
154.1
45.1
38.5
49.7
No. ACTH Wml)
Mean
Rhesus Jaundice compliuterine isoimmuni- Hyper- Diabetes Hydraof cations zation tens*on pregnancy of pregnancy
SD
Differences between uncompli1:ated pregnancy and the various complications were not statistically significant
ACTH
t
Pglml
083C
0
'\
600
0
5 00 0
4 00 300
00 0
0
0 00
0
0
U-
0 0
0
0
"s" L
ooo
200
0
0
0
0
0
0 L
0 0
--
10-18 26-30 31-32
0
0
0
'8
bs %
ge 0 8
:Ti CEG
0
0
0
%
". P
0
0 0
D 0
00 0
0
-I 35-36 37-38 39-40 41-42 URINE OF NEWBORN
GESTATIONA L WEEKS Fro. 1
The ACTH values in amniotic fluid during different gestational ages and in the first urine of six newborn infants. The lines indicate the mean levels at each stage.
differentation of the adrenal cortex needed for increased steroid secretion (duringlate pregnancy. For example, a sharp increase in cortisol has been noted after the 34th to 36th weeks of pregnancy both in umbilical cord blood
(Murphy, 1973) and in the amniotic fluid (Murphy et al, 1975; Fencl and Tulchinsky, 1975). After the 30th week of pregnancy the secretion rates for ACTH and cortisol are not parallel; the secretion of cortisol increases
856
TUIMALA, KAUPPILA AND HAAPALAHTI
and that of ACTH remains constant, perhaps due to a negative feedback effect of fetal cortisol on fetal pituitary ACTH secretion. It is also possible that the sensitivity of human fetal adrenals to ACTH increases with gestational ages as has been recognized in lamb fetuses (Robinson and Thorburn, 1974). Moreover it has been shown that a switch from a corticotrophin-like intermediate lobe peptide to intact ACTH occurs before delivery (Silman et al, 1976). Uterine activity did not increase the ACTH level significantly, in agreement with earlier observations that the umbilical cord ACTH was not affected by the manner of delivery (Winters et al, 1974). Pre-eclampsia has been shown to increase maternal ACTH during pregnancy (Mukherjee and Swyer, 1972), though ACTH in the amniotic fluid, reflecting fetal ACTH secretion, is not influenced by this or any other complications of pregnancy, according to our results, which show that the autonomous secretion of fetal ACTH is not easily disturbed. Both female and male fetuses secreted ACTH at the same rate. This was not unexpected, since of all the pituitary hormones only FSH seems to be linked with the sex of the fetus, FSH of females being higher than that of males (Faiman et al, 1974). At present ACTH determinations in amniotic fluid are of no obvious clinical value, but when more information has been accumulated and methods are more specific, the ACTH in amniotic fluid may prove to be of importance in studying the development and well-being of the fetus in utero, in a manner similar to amniotic fluid cortisol.
REFERENCES Allen, P. J., Cook, D. M., Kendall, J. W., and McGilvra, R. (1973): Journal of Clinical Endocrinology and Metabolism, 37, 230. Dokumov, S. I., Milanov, S. C., and Trepetshov, S. P. (1974): Journal of Obstetrics and Gynaecology of the British Commonwealth, 81, 220. Faiman, G., Reyes, F. I., and Winters, J. S. D. (1974): Sexual Endocrinology of the Perinatal Period. Edited by M. G. Forest and C. Bertrand. Inserm Editions, Paris, p 281. Fend, M. de M., and Tulchinsky, D. (1975): New England Journal of Medicine, 292, 133. Genazzani, A. R., FraioIi, F., Hurliman, I., Fioretti, P., and Felber, J. P. (1975): Clinical Endocrinology, 4 , l . Landon, J., and Greenwood, F. C. (1968): Lancet, 1,273. Milner, A. J., and Villee, D. B. (1970): Endocrinology, 87, 596. Miyakava, I., Ikeda, I., and Maeyama, M. (1974): Journal of Clinical Endocrinofogy and Metabofism, 39, 440. Mukherjee, K., and Swyer, G. I. M. (1972): Journal of Obstetrics and Gynaecology of the British Commonwealth, 79, 504. Murphy, B. E. P. (1973): American Journal of Obstetrics and Gynecology, 115, 52 1 . Murphy, B. E. P., Patrick, J., and Denton, R. L. (1975): Journal of Clinical Endocrinology and Metabolism, 40, 164. Ratcliffe, J. G., and Edwards, C. R. W. (1971): Radioimmunoassay Methods. Edited by K. E. Kirkham and W. M. Hunter. E & S Livingstone, Edinburgh, p 502. Robinson, J. S., and Thorburn, G. D. (1974): British Journal of Hospital Medicine, 12, 15. Seron-Ferre, M., Lawrence, C. C., and Jaffe, R. B. (1975): Gynecologic Investigation, 6, 16. Silman, R. E., Chard, T., Lowry, P. J., Smith, I., and Young, I. M. (1976): Nature, 260, 716. Turnbull, A. C. (1976): Paper presented at the XIXth Nordic Congress of Obstetrics and Gynecology, Reykjavik. Winters, A. J., Oliver, C., Colston, C., MacDonald, P. C., and Porter, J. C. (1974): Journal of Clinical Endocrinology and Metabolism, 39, 269.
ACTH LEVE1,S IN AMNIOTIC FLUID DURING PREGNANCY BY
R. TUIMALA A. KAUPPILA AND
J. HAAPALAHTI Department of Obstetrics and Gynaecology, University Central Hospital of Oulu and Laboratory of the Deaconess Institute of Oulu, Oulu, Finland Summary The fetal pituita.ry-adrenal axis plays an important role in the regulation of fetal development. In order to obtain information about fetal ACTH secretion at different gestational ages, a total of 109 amniotic fluid ACTH determinations was performed by ra.dioimmunoassay. There was a significantly higher level of ACTH during 26 to 30 weeks of pregnancy (4291180.4 pg/ml) than in early (208-7&90.6 pg/ml) and in late (172-7197.4pg/ml) pregnancy; fetal sex, uterine contractions and maternal complications in pregnancy did not affect levels. The ACTH level in the first urine of six newborn infants (160.0f40.6 pg/ml) approximated to that in the amniotic fluid in late pregnancy. Our results support the assumption of a fetal origin for ACTH in amniotic fluid. The high secretion of ACTH at tht: beginning of the last trimester of pregnancy may stimulate the development of the adrenal cortex and result in the increased cortisol secretion necessary for fetal lung maturation.
measured by radioimmunoassay at least in umbilical cord blood from the 12th gestational week (Winters et al, 1974). It has also been shown that ACTH increases the steroidogenic capacity of fetal adrenal cortical cells in tissue culture (Milner and Villee, 1970). This close interaction in the fetal ACTH-cortisol axis led us to examine the pattern of fetal ACTH secretion throughout pregnancy in normal and complicated pregnancies by measuring ACTH levels in the amniotic fluid.
THE marked rise in the secretion of coitisol by the fetus in late pregnancy (Murphy, 1973) is probably responsible for the acceleration of the synthesis of pulmonary surfactant, and of lung maturation in the human fetus (Fencl and Tulchinsky, 1975). The riise of cortisol secretion is reflected in a rise in the cortisol level of the amniotic fluid (Murphy cpt al, 1975; Fencl and Tulchinsky, 1975). A good correlation exists between cortisol and the lecithin/sphingomyelin ratio in amniotic fluid (Fencl and Tulchinsky, 1975). These findings emphasize the importance of fetal adrenals in preparing the fetus for extrauterine life. Fetal adrenals may also be of significance in the initiation of labour (Murphy, 1973). The development of the definite zone of the fetal adrenals is regulated by fetal pituitary ACTH. This zone has a specific binding capacity for ACTH at least from the 12th gestational week (Seron-Ferre et al, 1975) and ACTH can be
METHODS A total of 109 amniotic fluid samples were taken from 97 women by 94 transabdominal and 15 transvaginal amniocenteses (Table I). The samples were taken into glass tubes and stored at --20 OC until assayed. Six samples of the first urine of newborn infants were also obtained for ACTH determination. ACTH was determined by radioimmunoassay 853
854
TUIMALA, KAUPPILA AND HAAPALAHTI TABLE I The clinical diagnosis of patients examined
approximated to that in the amniotic fluid during late pregnancy (Fig. 1).
Early termination of pregnancy Rhesus isoimmunization Hypertensive complications of pregnancy Diabetes Jaundice of pregnancy Hydramnios Placenta praevia Threatened premature labour Controls
15 20 17 11 5 4 2 19 16 I
Total 109
(Landon and Greenwood, 1968). ACTH, adsorbed from the amniotic fluid onto small glass particles, was liberated by acetone, and radioimmunoassay was performed after evaporation (Ratcliffe and Edwards, 1971) as duplicates at two dilutions. The intra-assay variation was 3 - 2 per cent and the interassay variation was 4 - 5 per cent. The reagents were supplied by the Radiochemical Centre, Amersham. Statistical differences were tested by Student’s ‘t’ test.
RESULTS ACTH values at various gestational ages are shown in Table 11 and Figure 1. The ACTH level was significantly higher during the 26th to 30th weeks of pregnancy than at any time before or after this period (pr0.001). Between the 31st and 42nd weeks the mean ACTH (*SD) was 172.7k97.4 pg/ml. No other significant differences could be found between the periods examined. ACTH levels in the amniotic fluid were not significantly affected by the sex of the fetus, uterine activity before the amniocentesis or complications of pregnancy (Table 111). The ACTH level in the urine of the newborn infants
DISCUSSION The origin of ACTH in the amniotic fluid is not known. Many reports confirm that there is no placental transport of either endogenous (Allen et al, 1973; Miyakava et al, 1974; Dokumov et al, 1974; Winters et al, 1974) or exogenous (Miyakava et al, 1974) maternal ACTH. Simple filtration through the amniotic membrane from the maternal circulation into the amniotic sac is similarly unlikely, because the ACTH level in the maternal circulation of a patient with Nelson’s syndrome was about 100 times higher than in the amniotic fluid both in early and late pregnancy (Allen et al, 1973). There is also no indication that the placenta can synthesize ACTH (Genazzani et al, 1975). The fetal origin for the ACTH in the amniotic fluid is corroborated by our finding of quite a high ACTH level in the first urine of newborn infants, a level which approximated closely to that in the amniotic fluid. Reports of ACTH in the amniotic fluid are few (Allen et al, 1973), though umbilical cord ACTH has been a subject of many investigations, most of them dealing with fetuses at term. It has been reported that umbilical cord ACTH is significantly higher before the 34th gestational week than later (Winters et al, 1974). In our results, ACTH in the amniotic fluid remained almost constant from the 30th week of pregnancy until delivery. The most striking finding was the significantly higher level of ACTH during the 26th to 30th weeks of pregnancy, a period when there is a significant fall of amniotic fluid cortisol level (Turnbull, 1976). The explanation for this burst of ACTH secretion would be the ACTH-induced
TABLEI1 ACTH values in amniotic &id at different gestational ages Gestational week
No. ACTH (pg/ml)
-__
Mean SD
10-18
26-30
31-32
33-34
35-36
37-38
3940
4142
15
11
12
20
17
12
16
6
208.7
429.5
201.3
194.5
163.6
175.5
170.9
179.3
90.6
180.4
86.1
129.4
87.5
54.4
78.9
46.7
AMNIOTIC FLUID ACTH
855
TABLE 111 The effects of the sex of the fitus, uterine activity and complications of pregnancy on the ACTH level in amniotic fluid; values from patients before the 31st gestational week were excluded Sex Male
Female
52
27
18
10
17
11
4
5
16
176.7
166.9
196.8
158.7
153.1
208.6
156.0
145.4
143.3
X'9.8
112.2
125.0
78.6
56.2
154.1
45.1
38.5
49.7
No. ACTH Wml)
Mean
Rhesus Jaundice compliuterine isoimmuni- Hyper- Diabetes Hydraof cations zation tens*on pregnancy of pregnancy
SD
Differences between uncompli1:ated pregnancy and the various complications were not statistically significant
ACTH
t
Pglml
083C
0
'\
600
0
5 00 0
4 00 300
00 0
0
0 00
0
0
U-
0 0
0
0
"s" L
ooo
200
0
0
0
0
0
0 L
0 0
--
10-18 26-30 31-32
0
0
0
'8
bs %
ge 0 8
:Ti CEG
0
0
0
%
". P
0
0 0
D 0
00 0
0
-I 35-36 37-38 39-40 41-42 URINE OF NEWBORN
GESTATIONA L WEEKS Fro. 1
The ACTH values in amniotic fluid during different gestational ages and in the first urine of six newborn infants. The lines indicate the mean levels at each stage.
differentation of the adrenal cortex needed for increased steroid secretion (duringlate pregnancy. For example, a sharp increase in cortisol has been noted after the 34th to 36th weeks of pregnancy both in umbilical cord blood
(Murphy, 1973) and in the amniotic fluid (Murphy et al, 1975; Fencl and Tulchinsky, 1975). After the 30th week of pregnancy the secretion rates for ACTH and cortisol are not parallel; the secretion of cortisol increases
856
TUIMALA, KAUPPILA AND HAAPALAHTI
and that of ACTH remains constant, perhaps due to a negative feedback effect of fetal cortisol on fetal pituitary ACTH secretion. It is also possible that the sensitivity of human fetal adrenals to ACTH increases with gestational ages as has been recognized in lamb fetuses (Robinson and Thorburn, 1974). Moreover it has been shown that a switch from a corticotrophin-like intermediate lobe peptide to intact ACTH occurs before delivery (Silman et al, 1976). Uterine activity did not increase the ACTH level significantly, in agreement with earlier observations that the umbilical cord ACTH was not affected by the manner of delivery (Winters et al, 1974). Pre-eclampsia has been shown to increase maternal ACTH during pregnancy (Mukherjee and Swyer, 1972), though ACTH in the amniotic fluid, reflecting fetal ACTH secretion, is not influenced by this or any other complications of pregnancy, according to our results, which show that the autonomous secretion of fetal ACTH is not easily disturbed. Both female and male fetuses secreted ACTH at the same rate. This was not unexpected, since of all the pituitary hormones only FSH seems to be linked with the sex of the fetus, FSH of females being higher than that of males (Faiman et al, 1974). At present ACTH determinations in amniotic fluid are of no obvious clinical value, but when more information has been accumulated and methods are more specific, the ACTH in amniotic fluid may prove to be of importance in studying the development and well-being of the fetus in utero, in a manner similar to amniotic fluid cortisol.
REFERENCES Allen, P. J., Cook, D. M., Kendall, J. W., and McGilvra, R. (1973): Journal of Clinical Endocrinology and Metabolism, 37, 230. Dokumov, S. I., Milanov, S. C., and Trepetshov, S. P. (1974): Journal of Obstetrics and Gynaecology of the British Commonwealth, 81, 220. Faiman, G., Reyes, F. I., and Winters, J. S. D. (1974): Sexual Endocrinology of the Perinatal Period. Edited by M. G. Forest and C. Bertrand. Inserm Editions, Paris, p 281. Fend, M. de M., and Tulchinsky, D. (1975): New England Journal of Medicine, 292, 133. Genazzani, A. R., FraioIi, F., Hurliman, I., Fioretti, P., and Felber, J. P. (1975): Clinical Endocrinology, 4 , l . Landon, J., and Greenwood, F. C. (1968): Lancet, 1,273. Milner, A. J., and Villee, D. B. (1970): Endocrinology, 87, 596. Miyakava, I., Ikeda, I., and Maeyama, M. (1974): Journal of Clinical Endocrinofogy and Metabofism, 39, 440. Mukherjee, K., and Swyer, G. I. M. (1972): Journal of Obstetrics and Gynaecology of the British Commonwealth, 79, 504. Murphy, B. E. P. (1973): American Journal of Obstetrics and Gynecology, 115, 52 1 . Murphy, B. E. P., Patrick, J., and Denton, R. L. (1975): Journal of Clinical Endocrinology and Metabolism, 40, 164. Ratcliffe, J. G., and Edwards, C. R. W. (1971): Radioimmunoassay Methods. Edited by K. E. Kirkham and W. M. Hunter. E & S Livingstone, Edinburgh, p 502. Robinson, J. S., and Thorburn, G. D. (1974): British Journal of Hospital Medicine, 12, 15. Seron-Ferre, M., Lawrence, C. C., and Jaffe, R. B. (1975): Gynecologic Investigation, 6, 16. Silman, R. E., Chard, T., Lowry, P. J., Smith, I., and Young, I. M. (1976): Nature, 260, 716. Turnbull, A. C. (1976): Paper presented at the XIXth Nordic Congress of Obstetrics and Gynecology, Reykjavik. Winters, A. J., Oliver, C., Colston, C., MacDonald, P. C., and Porter, J. C. (1974): Journal of Clinical Endocrinology and Metabolism, 39, 269.
