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Maternal betamethasone and fetal growth and development in the monkey
Maternal betamethasone and fetal growth and development in the monkey MICHAEL F. EPSTEIN, M.D. PHILIP M. FARRELL, M.D., PH.D. JOHN W. SPARKS, M.D. GERALD PEPE, PH.D. SHIRLEY G. DRISCOLL, M.D. RONALD A. CHEZ, M.D. Bethesda, Afaryland, and Boston, A1assachusetts
Betamrthasone was administered to pregnant rhesus monkeys of 134 to 150 days' gestation. At operative delivery, umbilical venous plosma cortisol concentrations n•eu significantly lower in the treated group than in the control group, indicating that the agn11 aossnl the placenta. In the treated group, accelPTatrd diffrrentiation was present in sel•l'ralfi,tal organs including lung, liver, kidney, and adrenal gland. Brain histologic changes suggestive of neuronal injury were found in some instances. There were no differences in the n•eights of these fetal organs except for liver. It was markedly increased in stnoid-treated fetuses and this was accompanied by a fowfold rise in total hepatic g/_1•cogm mntent. These obsavations suggest that in the subhuman fetal primate, the differmtiation of jdal organs in addition to lung is enhanced by short-term corticosteroid treatment H·hile grm1•th is not ajfected. (AM. J. 0BSTET. GYNECOL. 127: 261, 1977.)
STt:DIEs have convincingly demonstrated that fetal pulmonary development in several species (sheep. rats, rabbits. and monkeys) is accelerated by the administration of corticosteroids. 1 • 2 Liggins and Howie 3 found that administration of the synthetic glucocorticoid, betamethasone, to pregnant women early in the third trimester was associated with a lower incidence of the respiratory distress syndrome (RDS) at birth. Abnormalities of organ growth and neurologic development have been reported after prenatal steroid administration to rabbits and rats. 4 However, the relevance of these findings to primates, particularly human beings, is uncertain in view of the short gesta-
tions of these lower animal species, their different patterns of pre- and postnatal development compared to those of human beings, and the pharmacologic doses of steroids used in those studies. The question of similar side effects on fetal and neonatal growth and development is a potential deterrent to the use of glucocorticoid prophylaxis in human beings. We have examined this by administering betamethasone in doses and schedules approximating those of Liggins and Howie 3 to pregnant rhesus monkeys in the third trimester and examining fetal organ weights, histology. and various biochemical indices of development.
RECENT
Methods
Fourteen normal pregnant rhesus monkeys (Mamm mulatta) with accurately timed gestations ( ± 1 day) from the NIH primate colony (term = 161 ± 3 days) were utilized. Eight monkeys. the steroid-treated group, each received an intramuscular injection of 0.5 mi. of Celestone (1.5 mg. betamethasone sodium phosphate and 1.5 mg. of betamethasone acetate) 48 and 24 hours prior to operative delivery at 134 to 150 days' gestation (82 to 91 per cent of term). Six control animals, matched for gestational age. each received intramuscu-
From the Pregnancy Research Branch and Neonatal and Pediatric MediciYU! Branch, National Institute of Child Health and Human Development, National Institutes of Health, and the Department of Pathology, Boston Hospital for Women. Rw:ivedjilr publication April19, 1976.
Revised October I 3. 1976. AccPpted OrtobPr 15. 1976. Reprint requests: Dr. Ronald A. Chez., National Institutes
of Health, Building 10, Room l3N266, Bethesda, Afar.;land 20014.
261
262 Epstein et al.
February I. l97i
Am,
Table I. Corticosteroid treatment during rhesus pregnancy: Comparison of treated and control groups* Brtametha.1ot1R Control (N
Gestational age
140
administration fN ""8)
6j ::+.:
5.9
::+.:
1.56
13R
::+.:
5.0
::+.:
0.93
(days) Maternal weight (Kg.) Placental weight (Gm.) Fetal weight (Gm-l Fetal plasma Cortisol (Mgldl.) Liver glycogen (mg./Gm. wet weight)
6.93
7.45
138
::+.:
27
123
372 23.0
::+.:
86 14
364
23.0
::+.:
::+.:
9.5
24
!(>.:)
66.0
::+.: ::+.:
79 5.5+
::+.:
22t
*Mean± S.D. tp < 0.05. tp < 0.001.
Table II. Corticosteroid treatment during rhesus pregnancy: Comparison of fetal organ weights*
Brain weight (Gm.) ('*}t Lung weight (Gm.)
<'*>
Liver weight (Gm.) (%)
Adrenal weight (Gm.) (o/c)
Kidney weight (Gm.) (%)
Control
BetamethasonP administration
(N ""6)
(N = 8)
49.2 13.5 7.40 1.98
::+.:
6.3 ± 1.4
::+.: ::+.:
2.1 0.27
10.7 ± 2.9 2.88 ± 0.32
44.5 J9~.:J
::+.:
7.2 2.0
7.21 ::+.: 1.5 2.00 :t 0.27 18.1 ± .'i.5:j: 4.94 :t 0.68§
0.25 ± 0.06 0.066 ± 0.014
0.25 ::+.: 0.1 I 0.069 :t 0.029
2.25 ± 0.27 0.62 ± 0.11
2.05 :t 0.35 0.59 :t 0.14
*Mean± S.D. tPer cent of total body weight. :f:p < 0.02. §p < 0.001.
tar injections of 0.5 mi. of saline at the same times. At delivery by hysterotomy under N20-02-halothane anesthesia. the fetuses were bled and then killed by injecting 120 mg. of pentobarbital. The fetuses and their ext'ised lungs, liver, brain, kidneys. and adrenal glands were weighed on a Mettler balance accurate to I mg. Histologic sections were prepared from portions of fetal tissues placed in buffered formalin. These were stained with hematoxylin-eosin and were coded before
f. Obstet. 'GvnemL
examination hy a microscopist who was unaware olthe identity of the animals relatiYe to steroid treatment. The , remaining tissues were frozen for chemical analvse~. Fetal plasma cortisol concentrations \\Trc de~ tennined ln radioimrnunoassa:·. and lwpatit glvcognt concentrations bv the anthrone method.
Results Table I provides the mt>an gestational ages. maternal, placentaL and fetal weights and the fetal plasma cortisol concentrations for the six control and eight treated pregnancies. There were no significant differences in maternal, placentaL and fetal weights. The mean cortisol concentration of umbilical \ enous plasma was signif1cantly (p < (l.05) lower in the treated group than in the control subjects. Mean fetal oq~an weights expressed in absolme terms and as percentages of total body weight are listed in Table I I. Comparison of brain. lung, kidney. and adrenal weights in the two groups re\'ealcd no differences. In contrast, the mean liver weight of the treated fetuses was signiticantlv great· er (p < 0.02) than that of the control group. \Yhen the individual organ weights were expressed as 1wr cent of fetal bodr weight. the hepatic mass in the treated group remained signihcantlv increased (p < 0.001). Fetal hepatic glycogen wncentration (mg. per gram of' wet weight) was markedly higher at each gestational age among the n·eated animals compared to control fetuses. When all gestational ages were combined, the mean hepatic glycogen concentration in the treated group was significantly greater (p <
Volume 127 :\umber:>
normal. !\;one of the changes in nerve cell bodies was noted in the cerebrum of control fetuses.
Comment Administration of betamethasone to the pregnant rhesus monkey during the final 20 per cent of gestation resulted in significantly lower plasma cortisol concentrations in the fetus. Although the variable stress of anesthesia and surgery makes it impossible to view thes<: as basal cortisol levels. both the control and treated groups were subjected to the same procedures. It can therefore be posited that the betamethasone administered to the mother 48 and 24 hours prior to delivery crosst·d the plat:cnta, suppressed the fetal adrenal gland. and led to lower levels of endogenous fetal cortisol. This is in agreement with the recent hndings of· Ballard. Granberg. and Ballard" in human pregnan(-v. Total body weight, placental weight, and the weights of brain, lung. kidney, and adrenal gland were normal in treated fetuses. The finding of normal lung weight contrasts '' ith obsenations of decreased fetal lung mass after in utero steroid administration in shorter gestational species. 4 • 5 Fetal liver weight was significantly increased after exposure to betarnethasone. Associated with this was a fourfold elevation in liver glycogen content. The specific mechanism for the increase in glycogen stores is not known. Hvpotheses include: (1) glucocorticoids
Betamethasone and fetal growth and development
263
elevated maternal and fetal glucose levels providing increased glycogenic substrate to the fetus and (2) maternally administered betamethasone promoted increased glycogen production by enhancing total glycogen synthetase activity in the fetal liver. The administration of betamethasone to the pregnant monkey was also associated \\ ith histologic changes in fetal brain, lung, liver, kidney, and adrenal gland. Most of these changes are consistent with accelerated differentiation of the developing tissues. Brain histologic changes suggesting neuronal injury are of concern, but were not present consistently. Further comment on this observation will require a systematic examination of congruent tissue sections using morphometry as well as further analysis of tissue composition (e.g., DNA, RNA, protein). Some of the changes observed in this study indicating advanced organ differentiation in steroid-treated monkey fetuses have been looked for and found in other species. 4 • 5 For instance, histologic evidence of act:elerated lung development has been observed in fetal lambs and rabbits!. 2 • 4 ; fetal rats show increased pulmonary phospholipid metabolism and elevated hepatic glycogen levels. 4 ' 6 Thus, it appears that there is a consistency in effects of cortit:osteroids on the maturation of fetal organs. It remains to be determined whether the net result of all these changes is benefidal to the human fetus and infant.
REFERENCES l. Epstein, M. F .. and Farrell, P. M.: The choline incorporation pathway: Primary mechanism for de novo lecithin synthesis in fetal primate lung, Pediatr. Res. 9: 558, 1975. 2. Farrell, P. M., and Zachman, R. D.: Induction of choline phosphotransferase and lecithin synthesis by corticosteroids, Science 179: 297, 1973. 3. Liggins, G. C .. and Howie, R. N.: A controlled trial of antepartum glucocorticoid treatment for prevention of the respiratory distress syndrome in premature infanL-;, Pediatrics 50: 515, 1972.
4. Taeusch, H. W.: Glucocorticoid prophylaxis for respiratory distress syndrome: A review of potential toxicity, J. Pediatr. 87: 617, 1975. 5. Ballard, P. L., Granberg, P., and Ballard, R. A.: Glucocorticoid levels in maternal and cord serum after prenatal betamethasone therapy to prevent respiratory distress syndrome,]. Clin. Invest. 56: 1548, 1975. 6. Greengard, 0., and Dewey, K. H.: The premature deposition or lysis of glycogen in livers of fetal rats injected with hydrocortisone or glucagon, Dev. Bioi. 21: 452, 1970.
Betamrthasone was administered to pregnant rhesus monkeys of 134 to 150 days' gestation. At operative delivery, umbilical venous plosma cortisol concentrations n•eu significantly lower in the treated group than in the control group, indicating that the agn11 aossnl the placenta. In the treated group, accelPTatrd diffrrentiation was present in sel•l'ralfi,tal organs including lung, liver, kidney, and adrenal gland. Brain histologic changes suggestive of neuronal injury were found in some instances. There were no differences in the n•eights of these fetal organs except for liver. It was markedly increased in stnoid-treated fetuses and this was accompanied by a fowfold rise in total hepatic g/_1•cogm mntent. These obsavations suggest that in the subhuman fetal primate, the differmtiation of jdal organs in addition to lung is enhanced by short-term corticosteroid treatment H·hile grm1•th is not ajfected. (AM. J. 0BSTET. GYNECOL. 127: 261, 1977.)
STt:DIEs have convincingly demonstrated that fetal pulmonary development in several species (sheep. rats, rabbits. and monkeys) is accelerated by the administration of corticosteroids. 1 • 2 Liggins and Howie 3 found that administration of the synthetic glucocorticoid, betamethasone, to pregnant women early in the third trimester was associated with a lower incidence of the respiratory distress syndrome (RDS) at birth. Abnormalities of organ growth and neurologic development have been reported after prenatal steroid administration to rabbits and rats. 4 However, the relevance of these findings to primates, particularly human beings, is uncertain in view of the short gesta-
tions of these lower animal species, their different patterns of pre- and postnatal development compared to those of human beings, and the pharmacologic doses of steroids used in those studies. The question of similar side effects on fetal and neonatal growth and development is a potential deterrent to the use of glucocorticoid prophylaxis in human beings. We have examined this by administering betamethasone in doses and schedules approximating those of Liggins and Howie 3 to pregnant rhesus monkeys in the third trimester and examining fetal organ weights, histology. and various biochemical indices of development.
RECENT
Methods
Fourteen normal pregnant rhesus monkeys (Mamm mulatta) with accurately timed gestations ( ± 1 day) from the NIH primate colony (term = 161 ± 3 days) were utilized. Eight monkeys. the steroid-treated group, each received an intramuscular injection of 0.5 mi. of Celestone (1.5 mg. betamethasone sodium phosphate and 1.5 mg. of betamethasone acetate) 48 and 24 hours prior to operative delivery at 134 to 150 days' gestation (82 to 91 per cent of term). Six control animals, matched for gestational age. each received intramuscu-
From the Pregnancy Research Branch and Neonatal and Pediatric MediciYU! Branch, National Institute of Child Health and Human Development, National Institutes of Health, and the Department of Pathology, Boston Hospital for Women. Rw:ivedjilr publication April19, 1976.
Revised October I 3. 1976. AccPpted OrtobPr 15. 1976. Reprint requests: Dr. Ronald A. Chez., National Institutes
of Health, Building 10, Room l3N266, Bethesda, Afar.;land 20014.
261
262 Epstein et al.
February I. l97i
Am,
Table I. Corticosteroid treatment during rhesus pregnancy: Comparison of treated and control groups* Brtametha.1ot1R Control (N
Gestational age
140
administration fN ""8)
6j ::+.:
5.9
::+.:
1.56
13R
::+.:
5.0
::+.:
0.93
(days) Maternal weight (Kg.) Placental weight (Gm.) Fetal weight (Gm-l Fetal plasma Cortisol (Mgldl.) Liver glycogen (mg./Gm. wet weight)
6.93
7.45
138
::+.:
27
123
372 23.0
::+.:
86 14
364
23.0
::+.:
::+.:
9.5
24
!(>.:)
66.0
::+.: ::+.:
79 5.5+
::+.:
22t
*Mean± S.D. tp < 0.05. tp < 0.001.
Table II. Corticosteroid treatment during rhesus pregnancy: Comparison of fetal organ weights*
Brain weight (Gm.) ('*}t Lung weight (Gm.)
<'*>
Liver weight (Gm.) (%)
Adrenal weight (Gm.) (o/c)
Kidney weight (Gm.) (%)
Control
BetamethasonP administration
(N ""6)
(N = 8)
49.2 13.5 7.40 1.98
::+.:
6.3 ± 1.4
::+.: ::+.:
2.1 0.27
10.7 ± 2.9 2.88 ± 0.32
44.5 J9~.:J
::+.:
7.2 2.0
7.21 ::+.: 1.5 2.00 :t 0.27 18.1 ± .'i.5:j: 4.94 :t 0.68§
0.25 ± 0.06 0.066 ± 0.014
0.25 ::+.: 0.1 I 0.069 :t 0.029
2.25 ± 0.27 0.62 ± 0.11
2.05 :t 0.35 0.59 :t 0.14
*Mean± S.D. tPer cent of total body weight. :f:p < 0.02. §p < 0.001.
tar injections of 0.5 mi. of saline at the same times. At delivery by hysterotomy under N20-02-halothane anesthesia. the fetuses were bled and then killed by injecting 120 mg. of pentobarbital. The fetuses and their ext'ised lungs, liver, brain, kidneys. and adrenal glands were weighed on a Mettler balance accurate to I mg. Histologic sections were prepared from portions of fetal tissues placed in buffered formalin. These were stained with hematoxylin-eosin and were coded before
f. Obstet. 'GvnemL
examination hy a microscopist who was unaware olthe identity of the animals relatiYe to steroid treatment. The , remaining tissues were frozen for chemical analvse~. Fetal plasma cortisol concentrations \\Trc de~ tennined ln radioimrnunoassa:·. and lwpatit glvcognt concentrations bv the anthrone method.
Results Table I provides the mt>an gestational ages. maternal, placentaL and fetal weights and the fetal plasma cortisol concentrations for the six control and eight treated pregnancies. There were no significant differences in maternal, placentaL and fetal weights. The mean cortisol concentration of umbilical \ enous plasma was signif1cantly (p < (l.05) lower in the treated group than in the control subjects. Mean fetal oq~an weights expressed in absolme terms and as percentages of total body weight are listed in Table I I. Comparison of brain. lung, kidney. and adrenal weights in the two groups re\'ealcd no differences. In contrast, the mean liver weight of the treated fetuses was signiticantlv great· er (p < 0.02) than that of the control group. \Yhen the individual organ weights were expressed as 1wr cent of fetal bodr weight. the hepatic mass in the treated group remained signihcantlv increased (p < 0.001). Fetal hepatic glycogen wncentration (mg. per gram of' wet weight) was markedly higher at each gestational age among the n·eated animals compared to control fetuses. When all gestational ages were combined, the mean hepatic glycogen concentration in the treated group was significantly greater (p <
Volume 127 :\umber:>
normal. !\;one of the changes in nerve cell bodies was noted in the cerebrum of control fetuses.
Comment Administration of betamethasone to the pregnant rhesus monkey during the final 20 per cent of gestation resulted in significantly lower plasma cortisol concentrations in the fetus. Although the variable stress of anesthesia and surgery makes it impossible to view thes<: as basal cortisol levels. both the control and treated groups were subjected to the same procedures. It can therefore be posited that the betamethasone administered to the mother 48 and 24 hours prior to delivery crosst·d the plat:cnta, suppressed the fetal adrenal gland. and led to lower levels of endogenous fetal cortisol. This is in agreement with the recent hndings of· Ballard. Granberg. and Ballard" in human pregnan(-v. Total body weight, placental weight, and the weights of brain, lung. kidney, and adrenal gland were normal in treated fetuses. The finding of normal lung weight contrasts '' ith obsenations of decreased fetal lung mass after in utero steroid administration in shorter gestational species. 4 • 5 Fetal liver weight was significantly increased after exposure to betarnethasone. Associated with this was a fourfold elevation in liver glycogen content. The specific mechanism for the increase in glycogen stores is not known. Hvpotheses include: (1) glucocorticoids
Betamethasone and fetal growth and development
263
elevated maternal and fetal glucose levels providing increased glycogenic substrate to the fetus and (2) maternally administered betamethasone promoted increased glycogen production by enhancing total glycogen synthetase activity in the fetal liver. The administration of betamethasone to the pregnant monkey was also associated \\ ith histologic changes in fetal brain, lung, liver, kidney, and adrenal gland. Most of these changes are consistent with accelerated differentiation of the developing tissues. Brain histologic changes suggesting neuronal injury are of concern, but were not present consistently. Further comment on this observation will require a systematic examination of congruent tissue sections using morphometry as well as further analysis of tissue composition (e.g., DNA, RNA, protein). Some of the changes observed in this study indicating advanced organ differentiation in steroid-treated monkey fetuses have been looked for and found in other species. 4 • 5 For instance, histologic evidence of act:elerated lung development has been observed in fetal lambs and rabbits!. 2 • 4 ; fetal rats show increased pulmonary phospholipid metabolism and elevated hepatic glycogen levels. 4 ' 6 Thus, it appears that there is a consistency in effects of cortit:osteroids on the maturation of fetal organs. It remains to be determined whether the net result of all these changes is benefidal to the human fetus and infant.
REFERENCES l. Epstein, M. F .. and Farrell, P. M.: The choline incorporation pathway: Primary mechanism for de novo lecithin synthesis in fetal primate lung, Pediatr. Res. 9: 558, 1975. 2. Farrell, P. M., and Zachman, R. D.: Induction of choline phosphotransferase and lecithin synthesis by corticosteroids, Science 179: 297, 1973. 3. Liggins, G. C .. and Howie, R. N.: A controlled trial of antepartum glucocorticoid treatment for prevention of the respiratory distress syndrome in premature infanL-;, Pediatrics 50: 515, 1972.
4. Taeusch, H. W.: Glucocorticoid prophylaxis for respiratory distress syndrome: A review of potential toxicity, J. Pediatr. 87: 617, 1975. 5. Ballard, P. L., Granberg, P., and Ballard, R. A.: Glucocorticoid levels in maternal and cord serum after prenatal betamethasone therapy to prevent respiratory distress syndrome,]. Clin. Invest. 56: 1548, 1975. 6. Greengard, 0., and Dewey, K. H.: The premature deposition or lysis of glycogen in livers of fetal rats injected with hydrocortisone or glucagon, Dev. Bioi. 21: 452, 1970.