Intrauterine growth of chronically instrumented rhesus monkey fetuses

Harrod et al. Am.

2. Denkhaus H, Winsberg MD. Ultrasonic measurement of the fetal ventricular system. Radiology 1979;131:781. 3. Freeman RK, McQuown OS, Secrist LJ, et al. The diagnosis of fetal hydrocephalus before viability. Obstet GynecoI1977;49:109. 4. Dunne MD, Johnson ML. The ultrasonic demonstration of fetal abnormalities in utero. J Reprod Med 1979; 23: 195. 5. Birnholz JC, Frigoletto FD. Antenatal treatment of hydrocephalus. N Engl J Med 1981; 304: 1021. 6. Harrison MR, Golbus MS, Filly RA. Management of the fetus with a correctable congenital defect. JAMA 1981; 246:774. 7. Clewell WH, Johnson ML, Meier PR, et al. A surgical

8.

9. 10. 11.

September 1, 1984 Obstet. Gynecol.

J.

approach to the treatment of fetal hydrocephalus. N Engl J Med 1982;306:1320. Warkany J, Lemire Rj, Cohen MM. Hydrocephaly. In: Mental retardation and congenital malformations of the central nervous system. Chicago: Year Book, 1981: 48-82. Burton BK. Recurrence risk for congenital hydrocephalus. Clin Genet 1979;16:47. Salonen R, Herva R, Norio R. The hydrolethalus syndrome: delineation of a "new" lethal malformation syndrome based on 28 patients. Clin Genet 1981;19:321. Fryns JP, Van den Berghe K, Van Assche A, et al. Prenatal diagnosis of camptomelic dwarfism. Clin Genet 1981;19:199.

Intrauterine growth of chronically instrumented rhesus monkey fetuses Yuji Murata, M.D., Chester B. Martin, Jr., M.D., Tsuyomu Ikenoue, M.D., Michael L. Socol, M.D., and Maurice L. Druzin, M.D. Orange and Los Angeles, California The effect of surgical and experimental manipulations on intrauterine growth of the fetus was investigated in 61 rhesus monkey fetuses in which chronic preparations were attempted. The surgical procedure consisted of hysterotomy and insertion of vascular catheters and included unilateral ligation of the fetal carotid artery. The mother was kept in a restraining chair after the operation for the duration of the preparation (0 to 39 days). Thirty-four fetuses who died within 48 hours after operation served as the control group for the growth parameters. The remaining fetuses that survived 7 days or more after the operation were included in the experimental group. Body weight, crown-rump length, crown-heel length, and foot length of the fetus and placental weight were measured at the termination of the preparation. There were significant linear correlations between all parameters and gestational age. Comparison between the control and experimental groups revealed that none of the parameters from the experimental group differed significantly from those of the control group. No relationships were found between the duration of the preparation and any of the parameters. Total brain weights from 19 fetuses exhibited a significant increase with gestational age and these values were within the normal range reported previously. There were no significant differences in weight between right and left cerebral hemispheres. No evidence of unequal blood flow to the cerebral hemispheres was found with the radioactive microsphere technique. The data suggest that the surgery performed on both the mother and the fetus and prolonged maternal restraint did not alter intrauterine fetal development. (AM. J. OBSTET. GVNECOL. 150:40, 1984.)

The availability of subhuman primates for the study of fetoplacental physiology has contributed consid-

From...,the Department of Obstetrics and Gynecology, University of California (Irvine) School of Medicine, and the Department of Obstetrics and Gynecology, University of Southern California School of Medicine. Presented in part at the Twenty-sixth Annual Meeting of the Society for Gynecologic Investigation, San Dieg~, California, March 21-24,1979. Received for publication November 8, 1983; accepted March 9, 1984. Reprint requests: Yuji Murata, M.D., Department of Obstetrics and Gynecology, University of California (Irvine), 101 City Dr. S., Orange, CA 92668.

40

erably to our capability of exploring this field. Disadvantages of performing physiologic experiments on exteriorized fetuses even with the umbilical cords intact have been repeatedly discussed. l The effects of anesthesia, surgical stress, and artificial disturbances of maternal and fetoplacental homeostasis frequently invalidate the results of experiments. On the other hand, a chronically instrumented fetus in utero that has recovered from the initial insults of surgical manipulations and anesthesia and is documented to be in good health is apparently an ideal subject to use for this purpose. Because of the high degree of validity of the results, most investigators believe in the value of the

Chronically instrumented rhesus monkey fetuses

Volume 150 Number I

"chronic preparation" of subhuman primate fetuses, despite the difficulty in establishing them. The ultimate goal of this type of fetal preparation is to establish a normal physiologic state, even though, inevitably, the surgical procedure itself may disturb subsequent fetal physiology to some extent. The first report from our laboratory dealing mainly with the methodology of establishing the chronic preparation of the rhesus monkey fetuses described the techniques,2 which require hysterotomy, ligation of a unilateral fetal carotid artery and jugular vein, fetal tracheotomy, and postoperative placement of the mother in a restraining chair. These manipulations, either alone or together, could disturb subsequent fetoplacental development. In this report, based on our observations on 61 animals, an effort has been put forth to retrospectively evaluate the physiologic stability of our preparation by examinating fetal developmental parameters.

18

21

25

30

35

DURATION Of PREPARATION (dlys )

Fig. 1. Duration of preparation after operation in experimental animals.

rzzl

Material and methods

The pregnant rhesus monkeys (Macaca mulatta) were obtained from The National Primate Center at Davis, California, and all were known to be healthy mothers. Their pregnancy courses were normal. All fetuses were products of singleton pregnancies with known gestational ages, and operation was performed between III and 158 days' gestation (normal gestation, 165 days). The detailed surgical procedure was reported previously.2 With the pregnant monkey under phencyclidine-pentobarbital anesthesia, a midline incision was made on the abdomen. After the peritoneal cavity was entered , a small incision was made in the lower segment of the pregnant uterus without rupturing the amniotic membranes . The catheters and electrode leads for the fetal electrocardiogram were brought through the cervical canal. Then the membranes were ruptured and the uterine incision extended wide enough to deliver the fetal head, which was immediately covered with a surgical glove filled with warm saline solution in order to prevent fetal lung expansion. The electrodes for the electrocardiogram were implanted under the fetal chest skin. The fetal neck was incised and the fetal carotid artery, jugular vein, and trachea were catheterized. In the cases of breech presentation, either external version was performed and the head delivered or the fetal femoral artery and vein were catheterized. Upon return of the head into the uterus, an open-end catheter was placed in the amniotic cavity and then the membranes and myometrium were closed in two layers. Continuous monitoring of the tracheal pressure, intra-amniotic pressur~, fetal blood pressure, and fetal heart rate was begllfl immediately following the procedures and contiflued until pregnancy terminated.

15

41

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25

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110

120

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140

150

160

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Fig. 2. Distribution of gestational ages for all fetuses included in this study. ~, Control group; §, experimental group.

The animal was left on the surgical table in the lateral recumbent position until awakening, then was placed in the restraining chair. The mother and fetus were permitted to recover in the chair. The chair was positioned horizontally for at least 12 hours after operation to avoid maternal circulatory embarrassment. After maternal and fetal well-being was confirmed biophysically and biochemically, the chair was placed in an upright position. Food and water were given as desired. All cathethers were continuously infused with heparinized saline (0.008 mIl min). Blood samples were periodically drawn through maternal and fetal arterial catheters for examination of blood gas tensions and pH. An intramuscular injection of progesterone, an intravenous bolus injection of magnesium sulfate, an intravenous infusion of fenoprofen,3 or indomethacin

42 Murata et al.

September I, 1984 Am. J. Obstet. Gynecol.

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per os was given to the mother to obtain uterine quiescence, if necessary. The preparation was terminated by delivery of the fetus or intrauterine fetal death. In the latter case, delivery of the fetus was accomplished either by cesarean section or vaginally. Fetal body weight, placental weight, crown-rump length , crown-heel length, and foot length were measured at birth. In 19 fetuses with catheters in the carotid artery and jugular vein, the fetal brain was excised and right and left hemispheres of the cerebrum were weighed separately. Radioactive microspheres (chromiun 151 and cerium 141, 50 Mm in diameter, 3M Company) were injected into seven fetuses through the fetal jugular vein catheters under steady-state and also various experimental conditions. After the dissection of the fetus, radioactiv-

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Fig. 4. Relationship between fetal crown-rump length and gestational age in the control group (A) and in the experimental group (B). Symbols are as in Fig. 3.

ity was separately counted in the right and left hemispheres of the fetal cerebrum with a gamma counter. This study included 67 preparations in which the time of fetal death was known and the fetus was delivered within 24 hours. The unsuccessful preparations, in which the fetus died within the first 48 hours after the operation, consisted of 34 fetuses. An additional six fetuses were put to death immediately after the completion of experiments which were carried out during the surgical procedure. These fetuses were collected as the "control group" for the parameters of fetal development. The remaining fetuses, in which the preparations lasted 7 days or more, were included in the "experimental group." A histogram of the duration of preparations of the experimental group is shown in Fig. 1. The distribution of gestational ages in all fetuses in this study including both the control and experimental groups is shown in Fig. 2. The preparations which lasted between 2 and 6 days after operation were excluded from this study.

Chronically instrumented rhesus monkey fetuses

Volume 150 Number 1

43

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Fig. 5. Relationship between crown-heel length of the fetus and gestational age in the control group (A) and in the experimental group (B). Symbols are as in Fig. 3.

Fig. 6. Fetal foot length and gestational age in the control group (A) and in the experimental group (B) . Symbols are as in Fig. 3.

Twenty-seven pregnant animals (17 control, 10 experimental) were placed in the restraining chair for 1 to 10 days prior to the operation, in an attempt to acclimatize the mother to the chair.

sexes at comparative gestational age periods. None of the parameters were found significantly different between male and female fetuses either in the control group or in the experimental group (p > 0.05, MannWhitney U test). Relationship to gestational age in the control group. Significant positive linear correlations were found between all parameters and gestational ages of the fetuses (Figs. 3, A, 4, A, 5, A, 6, A, and 7, A).

Results Chairing prior to operation. The duration of "prechairing" ranged from 1 to 10 days, the average being 4.2 days. There was no significant difference in any fetal development parameter between the "prechaired group" and "not prechaired group" at equivalent gestational age periods, (p > 0.05, Mann-Whitney U test) ; nor did the duration of prechairing and any of these parameters show a significant relationship (p > 0.05, Kendall rank correlation test). Consequently, the "prechaired" animals were treated equally with the "not prechaired" animals in the following statistics. Fetal sex. The fetal body weight, crown-rump length, crown-heel length, foot length , and placental weight were compared between the different fetal

Fetal body weight = 3.7X (gestational age) - 157 (r = 0.71, n = 40, P < 0.0 1) Crown-rump length = 0.06X (gestational age) + 9.4 (r = 0.58, n = 37, P < 0.01) Crown-heel length = 0.15X (gestational age) + 5.4 (r = 0.85, n = 37, P < 0.01) Foot length = 0.057X (gestational age) - 1.3 (r = 0.77, n = 36, P < 0.0 1) Placental weight = 1.5X (gestational age) - 79 (r = 0.55, n = 38, P < 0.01)

44 Murata et al.

September 1, 1984

Am . J. Obstet. Gynecol.

Brain weight Total brain weight. Total brain weight from 19 freshly

•. Control Group 200

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Total brain weight = 0.34X (gestational age) - 1.5 (r = 0.71, n = 19, P < 0.01)

120

The duration of the preparations ranged from 0 to 24 days, the mean being 6.7 days. None of the mothers were chaired prior to the operation. Because of the small population, all values of total brain weight were calculated to those of 120 days' gestation according to the slope obtained from the regression line in Fig. 8. The calculated total brain weight at 120 days of gestation again did not show any correlation with the duration of the preparation (p > 0.05, Kendall rank correlation test) . In Fig. 8, the mean and 2 SD of the mean at 125, ISO, and 175 days of gestation represent the noninstrumen ted normal fetal brain weight ranges previously reported by Kerr et al. 4 Cerebral hemispheres. The right and left hemispheres were separately weighed and compared immediately after birth. Despite the permanent ligation of the left fetal common carotid artery during the operation, the weights of the right and left hemispheres did not differ significantly (p > 0.05, paired t test). Cerebral bloodflow. Seven fetuses in which ligation had been performed on the left common carotid artery during the initial operation received radioactive microspheres on two different occasions. The first injection of 5lCr was given during a control observation prior to an experiment. At the second injection, microspheres tagged with 14lCe were given under various experimental conditions. The counts of radioactivity between right and left hemispheres in an individual animal indicate an equal amount of blood flow to both hemispheres in both the control and the experimental period (p > 0.05, paired t test) (Table II).

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The fifth and ninety-fifth percentiles of these parameters in relation to the gestational age were determined in the control group and are shown as the shaded areas in Figs. 3, B, 4, B, 5, B, 6, B, and 7, B. Control versus experimental groups. All values from the preparations that lasted 7 days or more after operation are shown in relation to their gestational ages in Figs. 3, B, 4, B, 5, B, 6, B, and 7, B. An open circle indicates a preparation that lasted between 7 and 13 days, and a closed circle indicates a fetus that survived 14 days or more. None of the developmental parameters from the experimental group differed significantly from those of the control group at each comparative gestational age period of 10 days (p > 0.05, MannWhitney U test). Moreover, among the experimental animals, no relationships were found between the duration of the preparation and any of the fetal developmental parameters (p > 0.05, Kendall rank correlation test). Values obtained from 10 preparations that lasted more than 13 days are listed in Table 1.

Comment The rhesus monkey has been the species of nonhuman primate most commonly used in research mainly because of its availability. This particular species has also been popularly used for fetal and placental research because of its close anatomic and physiologic similarity to humans. From an economical and practical point of view, the pregnant ewe is more easily available and also easier to manipulate surgically. However, because of anatomic and physiologic differences between ovine and subhuman species, some results experimentally obtained from sheep models are not readily applicable to human physiology. Difference in placental structure is one of the most typical examples. The sheep placenta, being epitheliochorial, is different from that of the rhesus monkey

Chronically instrumented rhesus monkey fetuses

Volume 150 Number 1

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GESTATIONAL AGE (DAYS)

Fig. 8. Relationship between total brain weight and gestational age of the fetus in days. Br.W. . Total brain weight of the fetus; G.A., gestational age in days; 'Y (r). correlation coefficient; n, number of fetuses. Means and 2 SD. (Fro m Kerr et al")

Table I. Developmental parameters from chronically catheterized rhesus monkey fetuses (duration of 14 days or more)

Experiment No.

1 2 3 4 5 6 7 8 9

10

Duration of model (days)

14 14 17 15 17 39 20 23 29 35

Gestational age (days)

Fetal body weight (gm)

Crownrump length

Crownheel length

(C1ll)

133 138 144 145 146 150 156 157 157 158

440 290 340 410 450 450 480 430 430 480

19.3 17.5 18.5 19.3 18.5 19.5 21.0 21.0 20.0 22.0

which is hemochorial. In addition to the anatomic differences. the difference in blood flow pattern inside the placenta has been known to affect placental transfer not o nly quantitatively but also qualitatively. The placental passage of cortisol5 and antibiotics6 has been proved to be different between these two species. The rhesus monkey is more similar to the human. In addition to hormonal studies. cardiovasc ular and pulmonary physiology of the fetal rhes us monkey has been used as a valuable source of information. Lecithin/sphingom yelin ratio determinations have been carried out in pregnant monkeys. The monkey is considered to be an appropriate model for such studies/' 8 whereas the pregnant ewe is known to be a poor model. 9 Because of the degree of validity of results obtained from the chronically catheterized pregnant rhesus monkey model. this type of preparation will remain important for studies of perinatal pathophysiology.

(e1ll)

Foot length (em)

Placental weight (g)

Sex

Prechaired

27.8 25.0 25.0 24.5 27.0 28.0 27.5 31.0 27.0 31.0

7.0 5.5 6.5 6.0 5.0 7.0 7.0 7.5 7.5 7.4

140 115 100 140 140 170 100 11 5 121 150

M M M M M F F M M F

Yes No No No No Yes No No No No

The reported success ra tes in establishing the chronic preparation of rhesus monkey fetuses, defined by fetal survival under good postoperative conditions for 48 hours or greater. range from 10% to 85%. depending on the investigators and the type of surgical procedure performed. 10 Our experience in the last 9 years revealed an overall success rate of 45%. Excluding the cases in which the cause of the failure could have been avoided with our present knowledge for appropriate perioperative care and in which failure was not directly related to the operation we performed, the corrected success rate becomes approximately 70% to 75%. Determination of the degree of normal physiology which both maternal and fetal animals can regain after surgical manipulations and can maintain during prolonged restraining has been of paramount importance in the pursuit of physiologic studies of pregnancy. In regard to maternal responses. the published evidence l l

46

Murata et al.

September 1. 1984 Am. J. Obstet. Gynecol.

Table II. Ratio of blood flow in right and left hemispheres of fetal monkey brain

Experiment No.

1 2 3 4 5 6 7

Mean 1 SO

Gestational age (days)

142 133 157 120 140 134 123 135.6

Days after operation

I

1 22 0 8 7 0 5.7

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I

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1.0 1.2 0.9 1.2 1.3 1.3 0.8

0.9 1.0 1.0 0.9

l.l

1.0 0.7

0.2

l.l

1.3 l.l

Two different radioactive microspheres were separately injected into the fetus. No alteration of fetal cerebral blood flow between right and left hemispheres was noted despite a permanent surgical ligation of the left common carotid artery of the fetal monkey. suggests that the operation was stressful but that acclimation to the restraining chair occurred. After the preparation was established, the biochemical and biophysical data indicated that these animals were minimally stressed. Our present observations have reassured us that chronic instrumentation of both mother and fetus plus maternal restraining did not alter intrauterine fetal and placental development within the range of gestational ages observed. The control group in this study consisted of the fetuses that received the surgical stress and failed to survive more than 48 hours. This may become a point of discussion regarding the reliability of the control animals. It is, however, hard to believe that the developmental parameter can be affected within this short time period, especially as 68% (27 of 40 fetuses) of the control group died within 24 hours. Moreover, all of the parameters from the control group in this study matched well with the values from non instrumented animals reported previously by other investigators. 12. 13 Fujikura and Niemann 12 presented a large series of observations on rhesus monkey fetuses at various gestational ages, stating that the mean birth weight of male newborn animals is consistently higher than that of female animals at each gestational interval. However, they failed to substantiate this relationship statistically at any gestational age, with either vaginal delivery or cesarean section. Our observation demonstrated there was no significant difference. It is also important to document that this type of preparation did not interfere with brain development

or disturb the circulation to the hemisphere despite unilateral ligation of the carotid artery. Johnson et alP have demonstrated that daily intramuscular injections of betamethasone to maternal monkeys for 13 days, starting at 119 days' gestation , clearly produced low-birth weight fetuses without affecting crown-rump or crown-heel length. Had the stresses which the maternal and fetal monkeys received in our populations elicited a strong and persistent adrenal response, reduction of the fetal body weight could have been reasonably expected during the gestational age we observed . Since this was not the case in our population, we feel that this is important evidence in support of the validity of our physiologic model of the pregnant rhesus monkey.

REFERENCES

I. Assali NS, Brinkman CR III, Nuwayhid B. Comparison of maternal and fetal cardiovascular function in acute and chronic experiment in sheep. AM J OBSTET GYNECOL 1974;120:411. 2. Martin CB Jr, Murata Y, Parer JT. A method for obtaining biochemical and biophysical measurements for rhesus monkey fetuses for prolonged periods. AM J OBSTET GYNECOL 1973;117: 126. 3. Johnson W, Harbert G, Martin CB Jr. Pharmacologic control of uterine contractility. AM J OBSTET GYNECOL 1975; 123:364. 4. Kerr GR, Kennan AL, Waismann HA, Allen JR. Growth 1969;33:201. 5. Beitins IZ, Kowaiski A, Shermeta OW, DeLenos RA, Migeon Cj. Fetal and maternal secretion rate of cortisol in sheep. Pediatr Res 1970;4: 129. 6. Murata Y, Miller RD, Ikenoue T, Hashimoto T, Taira S, Sagawa T. Pharmacokinetics of maternal-fetal antibiotic transfer. Presented at the Annual Meeting of the Infectious Disease Society for Obstetrics and Gynecology, Charleston, South Carolina, May, 198\. 7. Gluck L, Chez RA, Kulovich MV, Hutchinson DL, Niemann WHo Comparison of phospholipid indicators of fetal lung maturity in the amniotic fluid of the monkey (Macaw mulatta) and baboon (Papio papio). AM J OBSTET GYNECOL 1974; 120:524. 8. Epstein MF, Farrell PM, Chez RA. Fetal lung lecithin metabolism and the amniotic fluid LIS ratio in rhesus monkey gestations. AMJ OBSTET GYNECOL 1976;125:545. 9. Bethmeier HB, Egberts H. Transversal and longitudinal lecithin-sphingomyelin ratios in amniotic and lung fluid of fetal lambs. AMJ OBSTET GYNECOL 1975;122:593. 10. Chez RA. Chronic catheterization of the pregnant nonhuman primate (Workshop report). Bethesda: National Institutes of Health, December 17, 1976. 11. Socol ML, Manning FA, Murata Y, Challis J, Martin CB Jr. Plasma cortisol in the chronic rhesus monkey preparation (Macaca mulatta). AM J OBSTET GYNECOL 1978; 132:421. 12. Fujikura T, Niemann WHo Birth weight, gestational age, and type of delivery in rhesus monkeys. AM J OBSTET GYNECOL 1967;97:76. 13. Johnson JW, Mitzner W, London WT, Palmer AE, Scott RP. Betamethasone and the rhesus fetus: multisystemic effect. AM J OBSTET GYNECOL 1979;833:677.