Adrenocorticotropic hormone in human fetal blood at delivery KIYOSHI
ARAI,
M.D.
TAKUMI
YANAIHARA,
SHOICHI
OKINAGA.
M.D M.D.
Tokyo, Japan
Adre?tocorticotropic hormone (ACTH) concentrations in umbilical arterial and VPMIU.Y plasma were measured in newborn infants delivered both uaginally and br cesarean sectiotr. Mean ACTH was significantly higher in the arterial (602.0 pg. per milliliter) than ‘oeno1(s (261.5 pg. per milliliter) plasma in vaginal deliveries (p < 0.01). Arterial plasma oj‘ babies born by elective cesarean section before initiation of‘ labor contained lev.5 .4CTH (384.7 pg. per milliliter) nlhetl r~omparud to those delizvrrd aft?)- labor had .vtartetl (698.7 pg. per milliliter). Serial determinationr of ACTH in pooledjetal blood obtained during labor shouled that the azlerage hormone titers incretLsed signifirantfy irom jir\t stage (295.2 pg. per milliliter) to second stage of labor (440.5 pg. per mzllditer), and The pituitary gland of the fetus a~ reached a peak at delizle9 (645.4 pg. p tv milliliter). midtrimester conta.ined ACTH. Possible mechanisms,for~fetal secritiorr of.4CTi at delizjery aw discussed.
Material and methods
T H E ESTROGEN levels in the cord blood and maternal urine are elevated following adrenocorticotropic hormone (ACTH) injection to the fetus in utero, whereas they are markedly depressed after dexamethasone injection.’ With micromethods, estriol, dehydroepiandrosterone (DHA), 16a-hydroxy-DHA, and cortisol in fetal scalp blood increase during the course of delivery.‘, ’ These data suggest that the pituitary adrenal function of the human fetus has a probable effect upon the production of estriol precursor(s), such as DHA and/or 1Gn-hydroxy-DHA, during pregnancy. It was postulated that the fetal hypophyseal adrenal axis plays an important endocrinologic role during the enormous environmental changes and the stresses of delivery.3* I3 The following clinical study was undertaken to investigate fetal ACTH production during delivery and to attempt to correlate ACTH with steroid hormone secretion.
From the Department of Obstetrics Uniz~ersi~ School of Medicine.
and Gyr~~logy,
Thb Jtudy was supported in part by the Japanese of Education, Science and Culture. Received jar publication Accepted Frbruanb
October
All patients were seen in the Department of Ohstetrics and Gynecology of the ‘Teikyo University Hospital. Six women underwent elective cesarean section before the onset of labor for cephalopelvic dispropol,tion and a further six patients were sectioned for f’etai salvage after a prolonged labor of over 30 hours. ‘l’hr umbilical cord was clamped by two forceps immediately following delivery of the infant: the proximal site close to tht umbilicus and the distal site near the placenta. Rlootl samples were obtained from the umbilical arteries and veins with separate heparinized syringes. L’mbilical blood samples were collected in a similar \\a! from 12 primiparas immediately after normal \-aginal ticliveries. Fetal blood samples were collected from the scalp of 18 fetuses during labor according IO the. method of Saling.” It was not possible to do AC’I‘H determinations on the small volumes of plasma ohtained with individual scalp blood samples. Therefore, three individual plasma samples collected at identical stages of labor were pooled. The umbilical blood samples of those cases were also poolecl in UK same’ manner as the scalp blood; each plasma sample collected from a given individual case being adjusted 10 the same volume. The palienrs were informed about the study and gave their consent: there were no problems with any of the mothers or the infants. Radioimmunoassay of ACTH h2s prrf;,rmerl on Ihc
Teikyo Minist?
27, 1975.
3, 1976.
Reprint requests: Dr. Kiyoshi Arai, Department of Obst&ics and Gynecology, Teikyo University School oj Medicine, Kaga 2-l l-l, Itabashi-ku, Tokyo 173, Japan. 1136
Volume Number
I?.5 8
Table
I. ACTH
ACTH in fetal blood at delivery
concentrations
in the cord blood
of babies delivered
source of sa?npies
No. of infants
Umbilical artery
12
Umbilical vein
vaginally ACTH (pg.lml. 602.0
difference between arterial and venous concentrations
six groups of pooled samples. The heparinized blood samples were centrifuged immediately at 3,000 r.p.m. for 15 minutes at 4” C. The plasma was separated and kept at -20” C until assayed. A 10 mg. amount of silicic acid was added to each milliliter of plasma in a stoppered glass centrifuge tube, shaken for 20 minutes, and centrifuged at 3,000 r.p.m. for 10 minutes. After the supernatant had been discarded, the silicic acid precipitate was washed with 5 per cent horse plasma in water and the washing solution was discarded. Then 250 ~1 of a mixture of glacial acetic acid, acetone, and water (1 : 25 : 100) were added to the precipitate, mixed for 10 minutes, and separated by centrifugation at 3,000 r.p.m. for 10 minutes.’ The supernatant was ‘transferred to assay tubes and evaporated by a stream of nitrogen in a water bath kept at 50” C. The residue was dissolved in 0. I ml. of the standard diluent and was subjected to radioimmunoassay. The standard diluent consisted of 0.5 per cent human serum albumin, 0.5 per cent mercaptoethanol, and 500 U. Trasylol per milliliter in 0.075M phosphate saline buffer, pH 7.4. Synthetic ACTH standards were made up in horse plasma and were extracted in exactly the same way as plasma samples. The synthetic human ACTH standard was a gift of Dr. Noboru I’anaihara, Shizuoka. Japan. The antihuman ACTH serum, Wellcome Reagents, LTD., England, was diluted to 1 : 10,000 with the standard diluent, and 0.1 ml. was added to the assay tube containing extracts of the standard or sample. The radioiodination procedure of ACTH was performed with”“1 by the modified method of Greenwood and associates.6 Following purification on a Bio-gel P-10 column, the iodinated i\CTH was diluted with the standard diluent and the radioactivity in each milliliter was set to about 15,000 c.p.m. Then 0.1 ml. of this solution was added to the assay tube, mixed well, and incubated at 4” C. for 3 days, The separation of antibody bound and unbound ACTH was effected by adding 0.2 ml. of a suspension of dextran-coated charcoaL After centrifugation the supernatants were transfered to separate tubes, and the charcoal precipitate and supernatant were counted in an Aloka Auto-well Gammer System.
concrntratiom* r S.E.) lrangrl 2 106.4
(196.6 - 1592.0) 261.5 i- 34.7
12
( 64.4 *The
1137
- 467.9)
is statistically significant (p C 0.01). Pituitary glands of the fetuses removed for therapeutic abortions in the midtrimester were homogenized in 0.01N HCI and centrifuged in the cold, and the supernatant was diluted with the standard diluent. ACTH in the pituitary extract at various dilutions was assayed. The following substances were checked for crossreactivity in the ACTH radioimmunoassay: LER 907, hLH, hFSH, prolactin, synthetic oxytocin, vasopressin, synthetic LH-RH, HCG, and prostaglandins (FS and Es). Recovery experiments were carried out with 100 pg. of standard ACTH added to horse plasma.
Results Specificity of the ACTH radioimmunoassay system. The binding capacity of the antiserum was 48 per cent under the present experimental conditions, and it was possible to measure ACTH that ranged from 10 to 640 pg. per assay tube. Among the nine compounds tested, none demonstrated cross-reactivity when 1 to 9 ng. were assayed. Recovery and blank of the assay. Recoveries of 100 pg. of ACTH added to 1 ml. of horse plasma were 89.4 z 25.3 per cent. Extracts of blank plasma samples showed values close to zero. ACTH levels in cord blood. The average values of I2 normal vaginal deliveries are shown in Table I. The mean ACTH concentration of the umbilical artery blood was 602.0 pg. per milliliter (range, 196.6 to 1,592.O pg. per milliliter) and that of the vein was 261.5 pg. per milliliter (range, 64.4 to 467.9); the difference was statistically significant (p < 0.01). This trend was not found in babies delivered by elective cesarean section before the onset of labor; the average ACTH levels in the umbilical artery and vein were 384.7 pg. per milliliter (range, 187.5 to 992.0) and 333.0 (range, 67.9 to 912.0), respectively, and were not statistically different. The ACTH levels in the umbilical blood vessels were increased if cesarean section was performed after labor had begun; the mean arterial value was 698.7 pg. per milliliter (range, 255.0 to 1.176.0) as compared to the average vein concentration of 487.6
1138
Arai, Yanaihara, and Okinaga
Table II. ACTH onset of‘ labor
concentrations
in the cord blood
of babies delivered
by cesarean
section before and after I hc
*None of the differences is significant Table III. ACTH concentrations the course of deliver),.
F&d
in pooled
fetal blood collected
from the scalp and the umbilical
vessels during
mdp:
First stage of labor
295.2 2
6
(54.4
-
133.0 622.0) 143.7
I OO.(~
ti
440.5 + (109.9 - 1.OOO.O)
i44,tit
Umbilical artery-
6
L’47.5:;:
Umbilical vein
6
645.4 2 202.4 (214.0 .- I,.iY2.0) 300.2 + il.6
Second stage of’ labor Cord blood at drlivrf-y.~
(88.2
-
137.0
583.8)
*The increments as compared to the first stage valrie are statistically significant (+ 1) < O.O.?,+ p ‘: 0.01). Table IV. ACTH at midtrimester
16-20
23
contents
16.08 (3.6
in the fetal pituitary
-t 1.98 - 38.8)
pg. per milliliter (range, 58.9 to 960.0). difference was not significant (Table II).
gland
2.01 t 0.34 t 5.71)
(0.44
though
the
ACTH in fetal blood during labor. The average ACTH levels of the fetuses during delivery as measured in the pooled samples at identical stages are shown in Table III. In the first stage of labor, the mean value was 295.2 pg. per milliliter (range, 54.4 to 622.0). During the second stage of labor the average titer of ACTH was 440.5 pg. per tnilliliter (range, 109.9 to 1,OOO.O): this value was 44.6 per cent greater than that of the titer for the first stage samples and the increment vvas statistically significant (p < 0.05). At delivery of the
same cases, the ACT’H concentration in the umbilical artery v\as 645.4 pg. per milliliter (range, 2 14.0 to 1,592.0): 147.5 per cent tnore than the comparable fetal titer in the first stage of’ labor (p ‘:: 0.01). The ACTH titer in the utnbilical vein blood obtained at delivery was 300.2 pg. per milliliter (range, 58.2 to 583.8). This was similar to the hormone concentrations found at the first stage of labor. ACTH in the fetal pituitary gland. The average ACTH content in 23 anterior pituitary ghr~ds of fetuses at midtrimester was 2.01 ng, per nrilligratn (range, 0.44 to j.71) of wet tissue weight (Table IV). The extracts of the anterior hypophysis showed a parallel curve with standard ACTH when serial dilutions of the extract were made and assayed.
Comment In an earlier study utilizing a radic,immu31~)ass.~1y technique, Berson and Yallown reported that plasrna ACTH concentrations were significantly higher in the cord blood (16 1 + 29 pg. per milliliter, mean and SD.) than the maternal venous blood. Similar results were
Volume Number
125 8
reported by Allen and associates’ (mean, 226 pg. milliliter) and Miyakawa and associates” per (261.98 ? 81.65 pg. per milliliter, mean and S.D.), who emphasized the fact that ACTH did not cross the placental barrier. Those investigators also suggested that ACTH might be secreted by the fetus. Winters and associates’7 showed the absence of elevated ACTH levels in the cord blood of infants born after spontaneous labor (143 2 7 pg. per milliliter, mean value for term infants at delivery). The absolute values of ACTH reported in the present communication are higher than those of other investigators, probably due to different sampling techniques or to the assay method in which an extraction loss is considered. In experimental animals, the pituitary adrenal relationship in fetal life is well established; decapitation of fetuses in utero resulted in marked atrophy of the adrenal gland.” Our results demonstrate that ACTH concentration was significantly higher in the blood from umbilical arteries than from umbilical veins in normal vaginal deliveries. The onset of labor seemed to alter ACTH levels in the fetus. It may be postulated from these results that ACTH is actively secreted in the fetus and that the rate of its secretion is augmented by the stress of labor. In sampling the cord blood, a larger amount of blood is easily collected from the vein than the arteries. Previous reports on ACTH concentration in the cord blood represent the hormone levels in the mixed cord blood, the greater part of which might have consisted of blood returned from the placenta.8-10’ I7 A relatively low ACTH level in the umbilical vein blood may partly, if not entirely, be due to the effect of aminopeptidase of the placenta. l2 In order to further investigate the effect of labor upon the ACTH secretion in the fetus, serial samples were obtained from fetuses, plasma was pooled, and ACTH levels were determined. Since ACTH does not cross the placental barrier, the fetus actively secretes ACTH during the course of delivery with increasing titers as delivery nears. The increasing concentrations of ACTH could be related to uterine
ACTH in fetal blood at delivery
1139
contraction, variations in oxygen, the trauma of delivery, or result from other causes. A possibility must also be considered that the samples obtained by Saling’s technique had less ACTH because the samples were collected in glass capillaries. One may also postulate that the fetal pituitary secretes ACTH in reponse to the tremendous environmental changes occurring in delivery. To our knowledge this is the first report to demonstrate that the ACTH concentration in fetal blood significantly increases as labor progresses. We have previously demonstrated that the fetal blood levels of estrogen, dehydroepiandrosterone, 16aOH-dehydroepiandrosterone, and cortisol tended to increase during the course of delivery.** 3 Fetal cortisol levels at term are reported elsewherer3. I4 and correlate with the present sutdy. Although the contents were lower when compared to the adult pituitary,” radioimmunoreactive ACTH was found in the fetal pituitary at midtrimester. Previous’* r3, I5 and present studies add further evidence to the theory that the fetal pituitary adrenal axis is functioning during delivery and steroid output is increased responding to the secretion of fetal ACTH. However, as already suggested, hormonal changes in fetal blood might be the result of labor and the hormones studied may not trigger the onset of labor. The authors wish to thank Dr. Kaoru Abe and Dr. Noboru Yanaihara for their valuable advice and for supplying us with standard materials. The skillful technical assistance of Miss Motomi Kanazawa is gratefully acknowledged. We are indebted to Dr. Howard R. Nankin, Pittsburgh, Pennsylvania, for reading the manuscript and correcting English. LER 907, hLH, hFSH, and h-prolactin were supplied to us by the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, ‘Maryland. Oxytocin and vasopressin were made by Sandoz, Switzerland. Synthetic LH-RH was prepared by Dr. N. Yanaihara, Shizuoka, Japan. HCG was a product of Teikokuzoki Co., Kanagawa, Japan. Prostglandins were supplied to us by Ono Pharm. Co., Tokyo, Japan.
REFERENCES
1. Arai. K.. Kuwabara, Y., and Okinaga, S.: The Effect of adrenocorticotropic hormone and dexamethasone, administered to the fetus in utero, upon maternal and fetal estrogens, AM. J. OBSTET. GYNECOL. 113: 316, 1972. 2. Arai, K., Kuwabara, Y., Kihara, K., Okinaga, S., and Sakamoto, S.: Steroid hormone levels in human fetal blood during delivery, AM. J. OBSTET. GYNECOL. 114: 812, 1972. 3. Arai, K., Kuwabara, Y.. Yanaihara, T., and Okinaga, S.: Levels of steroid hormones in fetal blood during delivery, VII World Congress of Obstetrics and Gynecology, Aug.
1973, Moscow, Excerpta Medica International Congress Series No. 279, p.75. Saling, E.: Neues Vorgehen zur Untersuchung des Kindes unter der Geburt-Einfiihrung, Technik und Grundlagen, Arch. Gynakol 197: 108, 1962. Donald, R. A.: A rapid method for extracting corticotrophin from plasma, J. Endocrinol 39: 451, 1967. Greenwood, F. C., Hunter, W. M., and Glover, J. S.: The human growth hormone of preparation of ‘3’I-labelled high specific radioactivity, Biochem. J. 89: 114, 1963. Donald, R. A.: Application of the coated charcoal
I140
8. 9.
10.
11. 12.
13.
Arai,
Yanaihara,
August Am. J. Obstet.
and Okinaga
separation method to the radioimmunoassay of plasma corticotrophin, J. Endocrinol. 41: 499, 1968. Berson, S. A., and Yalow, R. S.: Radioimmunoassay of ACTH in plasma, J. Clin. Invest. 47: 2725, 1968. Allen, J. P., Cook, D. M., Kendall, J. W., and McGilvra, R.: Maternal-fetal ACTH relationship in man, J, Clin. Endocrinol. Metab. 37: 230, 1973. Miyakawa, I., Ikeda, I., and Maeyama, M.: Transport of ACTH across human placenta, J, Clin. Endocrinol. Metab. 39: 440, 1974. Jest, A.: Problems of fetal endocrinology; the adrenal glands, Rec. Prog. Horm. Res. 22: 541, 1966. Hooper, K. C., and lessup, D. C.: The distribution of enzymes destroying cxytoc’in and vasopressin, J. Physiol. 148: 283, 1959. Murphy, B. E. P.: Does the human fetal adrenal play a
14. 15.
16.
17.
15, 1976 Gynecol.
role in parturition? AM. J. OBSTET. GYNECOL. 115: 521, 1973. Pokoly, T. B.: The role of cortisol in human parturition, AM. J. OBSTET. GYNECOL. 117: 549, 1973. Simmer, H. H., Frankland, M., and Greipel, M.: On the regulation of fetal and maternal 16a-hydroxydehydroepiandrosterone and its sulfate by cortisol and ACTH in human pregnancy at term, AM. J. OBSTET. GYNECOL. 121: 646, 1975. Yalow, R. S., and Berson, S. A.: Characteristics of “big ACTH” in human plasma and pituitary extracts, J. Clin. Endocrinol. Metab. 36: 415, 1973. Winters, A. J., Oliver, C., Colston, C., MacDonald, P. C., and Porter, J. C.: Plasma ACTH levels in the human fetus and neonate as related to age and parturition, J. Clin. Endocrinol. Metab. 39: 269, 1974.
ARAI,
M.D.
TAKUMI
YANAIHARA,
SHOICHI
OKINAGA.
M.D M.D.
Tokyo, Japan
Adre?tocorticotropic hormone (ACTH) concentrations in umbilical arterial and VPMIU.Y plasma were measured in newborn infants delivered both uaginally and br cesarean sectiotr. Mean ACTH was significantly higher in the arterial (602.0 pg. per milliliter) than ‘oeno1(s (261.5 pg. per milliliter) plasma in vaginal deliveries (p < 0.01). Arterial plasma oj‘ babies born by elective cesarean section before initiation of‘ labor contained lev.5 .4CTH (384.7 pg. per milliliter) nlhetl r~omparud to those delizvrrd aft?)- labor had .vtartetl (698.7 pg. per milliliter). Serial determinationr of ACTH in pooledjetal blood obtained during labor shouled that the azlerage hormone titers incretLsed signifirantfy irom jir\t stage (295.2 pg. per milliliter) to second stage of labor (440.5 pg. per mzllditer), and The pituitary gland of the fetus a~ reached a peak at delizle9 (645.4 pg. p tv milliliter). midtrimester conta.ined ACTH. Possible mechanisms,for~fetal secritiorr of.4CTi at delizjery aw discussed.
Material and methods
T H E ESTROGEN levels in the cord blood and maternal urine are elevated following adrenocorticotropic hormone (ACTH) injection to the fetus in utero, whereas they are markedly depressed after dexamethasone injection.’ With micromethods, estriol, dehydroepiandrosterone (DHA), 16a-hydroxy-DHA, and cortisol in fetal scalp blood increase during the course of delivery.‘, ’ These data suggest that the pituitary adrenal function of the human fetus has a probable effect upon the production of estriol precursor(s), such as DHA and/or 1Gn-hydroxy-DHA, during pregnancy. It was postulated that the fetal hypophyseal adrenal axis plays an important endocrinologic role during the enormous environmental changes and the stresses of delivery.3* I3 The following clinical study was undertaken to investigate fetal ACTH production during delivery and to attempt to correlate ACTH with steroid hormone secretion.
From the Department of Obstetrics Uniz~ersi~ School of Medicine.
and Gyr~~logy,
Thb Jtudy was supported in part by the Japanese of Education, Science and Culture. Received jar publication Accepted Frbruanb
October
All patients were seen in the Department of Ohstetrics and Gynecology of the ‘Teikyo University Hospital. Six women underwent elective cesarean section before the onset of labor for cephalopelvic dispropol,tion and a further six patients were sectioned for f’etai salvage after a prolonged labor of over 30 hours. ‘l’hr umbilical cord was clamped by two forceps immediately following delivery of the infant: the proximal site close to tht umbilicus and the distal site near the placenta. Rlootl samples were obtained from the umbilical arteries and veins with separate heparinized syringes. L’mbilical blood samples were collected in a similar \\a! from 12 primiparas immediately after normal \-aginal ticliveries. Fetal blood samples were collected from the scalp of 18 fetuses during labor according IO the. method of Saling.” It was not possible to do AC’I‘H determinations on the small volumes of plasma ohtained with individual scalp blood samples. Therefore, three individual plasma samples collected at identical stages of labor were pooled. The umbilical blood samples of those cases were also poolecl in UK same’ manner as the scalp blood; each plasma sample collected from a given individual case being adjusted 10 the same volume. The palienrs were informed about the study and gave their consent: there were no problems with any of the mothers or the infants. Radioimmunoassay of ACTH h2s prrf;,rmerl on Ihc
Teikyo Minist?
27, 1975.
3, 1976.
Reprint requests: Dr. Kiyoshi Arai, Department of Obst&ics and Gynecology, Teikyo University School oj Medicine, Kaga 2-l l-l, Itabashi-ku, Tokyo 173, Japan. 1136
Volume Number
I?.5 8
Table
I. ACTH
ACTH in fetal blood at delivery
concentrations
in the cord blood
of babies delivered
source of sa?npies
No. of infants
Umbilical artery
12
Umbilical vein
vaginally ACTH (pg.lml. 602.0
difference between arterial and venous concentrations
six groups of pooled samples. The heparinized blood samples were centrifuged immediately at 3,000 r.p.m. for 15 minutes at 4” C. The plasma was separated and kept at -20” C until assayed. A 10 mg. amount of silicic acid was added to each milliliter of plasma in a stoppered glass centrifuge tube, shaken for 20 minutes, and centrifuged at 3,000 r.p.m. for 10 minutes. After the supernatant had been discarded, the silicic acid precipitate was washed with 5 per cent horse plasma in water and the washing solution was discarded. Then 250 ~1 of a mixture of glacial acetic acid, acetone, and water (1 : 25 : 100) were added to the precipitate, mixed for 10 minutes, and separated by centrifugation at 3,000 r.p.m. for 10 minutes.’ The supernatant was ‘transferred to assay tubes and evaporated by a stream of nitrogen in a water bath kept at 50” C. The residue was dissolved in 0. I ml. of the standard diluent and was subjected to radioimmunoassay. The standard diluent consisted of 0.5 per cent human serum albumin, 0.5 per cent mercaptoethanol, and 500 U. Trasylol per milliliter in 0.075M phosphate saline buffer, pH 7.4. Synthetic ACTH standards were made up in horse plasma and were extracted in exactly the same way as plasma samples. The synthetic human ACTH standard was a gift of Dr. Noboru I’anaihara, Shizuoka. Japan. The antihuman ACTH serum, Wellcome Reagents, LTD., England, was diluted to 1 : 10,000 with the standard diluent, and 0.1 ml. was added to the assay tube containing extracts of the standard or sample. The radioiodination procedure of ACTH was performed with”“1 by the modified method of Greenwood and associates.6 Following purification on a Bio-gel P-10 column, the iodinated i\CTH was diluted with the standard diluent and the radioactivity in each milliliter was set to about 15,000 c.p.m. Then 0.1 ml. of this solution was added to the assay tube, mixed well, and incubated at 4” C. for 3 days, The separation of antibody bound and unbound ACTH was effected by adding 0.2 ml. of a suspension of dextran-coated charcoaL After centrifugation the supernatants were transfered to separate tubes, and the charcoal precipitate and supernatant were counted in an Aloka Auto-well Gammer System.
concrntratiom* r S.E.) lrangrl 2 106.4
(196.6 - 1592.0) 261.5 i- 34.7
12
( 64.4 *The
1137
- 467.9)
is statistically significant (p C 0.01). Pituitary glands of the fetuses removed for therapeutic abortions in the midtrimester were homogenized in 0.01N HCI and centrifuged in the cold, and the supernatant was diluted with the standard diluent. ACTH in the pituitary extract at various dilutions was assayed. The following substances were checked for crossreactivity in the ACTH radioimmunoassay: LER 907, hLH, hFSH, prolactin, synthetic oxytocin, vasopressin, synthetic LH-RH, HCG, and prostaglandins (FS and Es). Recovery experiments were carried out with 100 pg. of standard ACTH added to horse plasma.
Results Specificity of the ACTH radioimmunoassay system. The binding capacity of the antiserum was 48 per cent under the present experimental conditions, and it was possible to measure ACTH that ranged from 10 to 640 pg. per assay tube. Among the nine compounds tested, none demonstrated cross-reactivity when 1 to 9 ng. were assayed. Recovery and blank of the assay. Recoveries of 100 pg. of ACTH added to 1 ml. of horse plasma were 89.4 z 25.3 per cent. Extracts of blank plasma samples showed values close to zero. ACTH levels in cord blood. The average values of I2 normal vaginal deliveries are shown in Table I. The mean ACTH concentration of the umbilical artery blood was 602.0 pg. per milliliter (range, 196.6 to 1,592.O pg. per milliliter) and that of the vein was 261.5 pg. per milliliter (range, 64.4 to 467.9); the difference was statistically significant (p < 0.01). This trend was not found in babies delivered by elective cesarean section before the onset of labor; the average ACTH levels in the umbilical artery and vein were 384.7 pg. per milliliter (range, 187.5 to 992.0) and 333.0 (range, 67.9 to 912.0), respectively, and were not statistically different. The ACTH levels in the umbilical blood vessels were increased if cesarean section was performed after labor had begun; the mean arterial value was 698.7 pg. per milliliter (range, 255.0 to 1.176.0) as compared to the average vein concentration of 487.6
1138
Arai, Yanaihara, and Okinaga
Table II. ACTH onset of‘ labor
concentrations
in the cord blood
of babies delivered
by cesarean
section before and after I hc
*None of the differences is significant Table III. ACTH concentrations the course of deliver),.
F&d
in pooled
fetal blood collected
from the scalp and the umbilical
vessels during
mdp:
First stage of labor
295.2 2
6
(54.4
-
133.0 622.0) 143.7
I OO.(~
ti
440.5 + (109.9 - 1.OOO.O)
i44,tit
Umbilical artery-
6
L’47.5:;:
Umbilical vein
6
645.4 2 202.4 (214.0 .- I,.iY2.0) 300.2 + il.6
Second stage of’ labor Cord blood at drlivrf-y.~
(88.2
-
137.0
583.8)
*The increments as compared to the first stage valrie are statistically significant (+ 1) < O.O.?,+ p ‘: 0.01). Table IV. ACTH at midtrimester
16-20
23
contents
16.08 (3.6
in the fetal pituitary
-t 1.98 - 38.8)
pg. per milliliter (range, 58.9 to 960.0). difference was not significant (Table II).
gland
2.01 t 0.34 t 5.71)
(0.44
though
the
ACTH in fetal blood during labor. The average ACTH levels of the fetuses during delivery as measured in the pooled samples at identical stages are shown in Table III. In the first stage of labor, the mean value was 295.2 pg. per milliliter (range, 54.4 to 622.0). During the second stage of labor the average titer of ACTH was 440.5 pg. per tnilliliter (range, 109.9 to 1,OOO.O): this value was 44.6 per cent greater than that of the titer for the first stage samples and the increment vvas statistically significant (p < 0.05). At delivery of the
same cases, the ACT’H concentration in the umbilical artery v\as 645.4 pg. per milliliter (range, 2 14.0 to 1,592.0): 147.5 per cent tnore than the comparable fetal titer in the first stage of’ labor (p ‘:: 0.01). The ACTH titer in the utnbilical vein blood obtained at delivery was 300.2 pg. per milliliter (range, 58.2 to 583.8). This was similar to the hormone concentrations found at the first stage of labor. ACTH in the fetal pituitary gland. The average ACTH content in 23 anterior pituitary ghr~ds of fetuses at midtrimester was 2.01 ng, per nrilligratn (range, 0.44 to j.71) of wet tissue weight (Table IV). The extracts of the anterior hypophysis showed a parallel curve with standard ACTH when serial dilutions of the extract were made and assayed.
Comment In an earlier study utilizing a radic,immu31~)ass.~1y technique, Berson and Yallown reported that plasrna ACTH concentrations were significantly higher in the cord blood (16 1 + 29 pg. per milliliter, mean and SD.) than the maternal venous blood. Similar results were
Volume Number
125 8
reported by Allen and associates’ (mean, 226 pg. milliliter) and Miyakawa and associates” per (261.98 ? 81.65 pg. per milliliter, mean and S.D.), who emphasized the fact that ACTH did not cross the placental barrier. Those investigators also suggested that ACTH might be secreted by the fetus. Winters and associates’7 showed the absence of elevated ACTH levels in the cord blood of infants born after spontaneous labor (143 2 7 pg. per milliliter, mean value for term infants at delivery). The absolute values of ACTH reported in the present communication are higher than those of other investigators, probably due to different sampling techniques or to the assay method in which an extraction loss is considered. In experimental animals, the pituitary adrenal relationship in fetal life is well established; decapitation of fetuses in utero resulted in marked atrophy of the adrenal gland.” Our results demonstrate that ACTH concentration was significantly higher in the blood from umbilical arteries than from umbilical veins in normal vaginal deliveries. The onset of labor seemed to alter ACTH levels in the fetus. It may be postulated from these results that ACTH is actively secreted in the fetus and that the rate of its secretion is augmented by the stress of labor. In sampling the cord blood, a larger amount of blood is easily collected from the vein than the arteries. Previous reports on ACTH concentration in the cord blood represent the hormone levels in the mixed cord blood, the greater part of which might have consisted of blood returned from the placenta.8-10’ I7 A relatively low ACTH level in the umbilical vein blood may partly, if not entirely, be due to the effect of aminopeptidase of the placenta. l2 In order to further investigate the effect of labor upon the ACTH secretion in the fetus, serial samples were obtained from fetuses, plasma was pooled, and ACTH levels were determined. Since ACTH does not cross the placental barrier, the fetus actively secretes ACTH during the course of delivery with increasing titers as delivery nears. The increasing concentrations of ACTH could be related to uterine
ACTH in fetal blood at delivery
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contraction, variations in oxygen, the trauma of delivery, or result from other causes. A possibility must also be considered that the samples obtained by Saling’s technique had less ACTH because the samples were collected in glass capillaries. One may also postulate that the fetal pituitary secretes ACTH in reponse to the tremendous environmental changes occurring in delivery. To our knowledge this is the first report to demonstrate that the ACTH concentration in fetal blood significantly increases as labor progresses. We have previously demonstrated that the fetal blood levels of estrogen, dehydroepiandrosterone, 16aOH-dehydroepiandrosterone, and cortisol tended to increase during the course of delivery.** 3 Fetal cortisol levels at term are reported elsewherer3. I4 and correlate with the present sutdy. Although the contents were lower when compared to the adult pituitary,” radioimmunoreactive ACTH was found in the fetal pituitary at midtrimester. Previous’* r3, I5 and present studies add further evidence to the theory that the fetal pituitary adrenal axis is functioning during delivery and steroid output is increased responding to the secretion of fetal ACTH. However, as already suggested, hormonal changes in fetal blood might be the result of labor and the hormones studied may not trigger the onset of labor. The authors wish to thank Dr. Kaoru Abe and Dr. Noboru Yanaihara for their valuable advice and for supplying us with standard materials. The skillful technical assistance of Miss Motomi Kanazawa is gratefully acknowledged. We are indebted to Dr. Howard R. Nankin, Pittsburgh, Pennsylvania, for reading the manuscript and correcting English. LER 907, hLH, hFSH, and h-prolactin were supplied to us by the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, ‘Maryland. Oxytocin and vasopressin were made by Sandoz, Switzerland. Synthetic LH-RH was prepared by Dr. N. Yanaihara, Shizuoka, Japan. HCG was a product of Teikokuzoki Co., Kanagawa, Japan. Prostglandins were supplied to us by Ono Pharm. Co., Tokyo, Japan.
REFERENCES
1. Arai. K.. Kuwabara, Y., and Okinaga, S.: The Effect of adrenocorticotropic hormone and dexamethasone, administered to the fetus in utero, upon maternal and fetal estrogens, AM. J. OBSTET. GYNECOL. 113: 316, 1972. 2. Arai, K., Kuwabara, Y., Kihara, K., Okinaga, S., and Sakamoto, S.: Steroid hormone levels in human fetal blood during delivery, AM. J. OBSTET. GYNECOL. 114: 812, 1972. 3. Arai, K., Kuwabara, Y.. Yanaihara, T., and Okinaga, S.: Levels of steroid hormones in fetal blood during delivery, VII World Congress of Obstetrics and Gynecology, Aug.
1973, Moscow, Excerpta Medica International Congress Series No. 279, p.75. Saling, E.: Neues Vorgehen zur Untersuchung des Kindes unter der Geburt-Einfiihrung, Technik und Grundlagen, Arch. Gynakol 197: 108, 1962. Donald, R. A.: A rapid method for extracting corticotrophin from plasma, J. Endocrinol 39: 451, 1967. Greenwood, F. C., Hunter, W. M., and Glover, J. S.: The human growth hormone of preparation of ‘3’I-labelled high specific radioactivity, Biochem. J. 89: 114, 1963. Donald, R. A.: Application of the coated charcoal
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