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Am J Transl Res 2019;11(3):1697-1710
www.ajtr.org /ISSN:1943-8141/AJTR0086365
Original Article
Combined antenatal and postnatal steroid effects on
fetal and postnatal growth, and neurological
outcomes in neonatal rats
Maria A Abrantes1,2,3#*, Arwin M Valencia1,2,4†*, Fayez Bany-Mohammed2, Jacob V Aranda5,6, Kay D
Beharry1,2,5,6Φ*
1Department of Pediatrics, Division of Neonatal-Perinatal Medicine, University of California, Irvine Medical Center,
Orange, CA, USA; 2Department of Pediatrics, Division of Neonatal-Perinatal Medicine, Miller Children’s Hospital,
Long Beach CA, USA; 3Department of Pediatrics, Division of Neonatal-Perinatal Medicine, Kaiser Permanente,
Irvine, CA, USA; 4Department of Pediatrics, Division of Neonatal-Perinatal Medicine, Saddleback Memorial
Medical Center, Laguna Hills, CA, USA; Departments of 5Pediatrics, 6Ophthalmology, Division of Neonatal-
Perinatal Medicine, State University of New York, Downstate Medical Center, Brooklyn, NY, USA. #Current Address:
Department of Pediatrics, Division of Neonatal-Perinatal Medicine, Kaiser Permanente, Irvine, CA, USA. †Current
Address: Department of Pediatrics, Division of Neonatal-Perinatal Medicine, Saddleback Memorial Medical
Center, Saddleback, CA, USA. ΦCurrent Address: Departments of Pediatrics & Ophthalmology, State University of
New York, Downstate Medical Center, Brooklyn, NY, USA. *Equal contributors.
Received October 5, 2018; Accepted January 16, 2019; Epub March 15, 2019; Published March 30, 2019
Abstract: Preterm infants are often exposed to both antenatal and postnatal glucocorticoids (GCs). We tested
the hypothesis that combined antenatal and postnatal GCs have long-lasting adverse effects on fetal and
neonatal growth, growth factors, and neurological outcomes. Pregnant rats were administered a single IM dose of
betamethasone (0.2 mg/Kg, AB), dexamethasone (0.2 mg/Kg, AD), or equivalent volumes of saline (AS) at 17 & 18
days gestation. Following delivery, pups from each treatment group were sacriced at P0, and the remainder was
treated with a single IM dose of either betamethasone (0.25 mg/Kg, PB), dexamethasone (0.25 mg/Kg, PD), or
equivalent volumes of saline (PS) on P5, P6, and P7. Somatic growth, neurological status, and growth factors were
determined at P14, P21, and P45. At birth, AD resulted in decreased somatic growth. AB advanced the hopping
reex associated with spinal rhythmic mechanisms. At P21, all GC groups were growth suppressed, but only the
AS/PD group had decits in brain weight and delayed plantar reex associated with brainstem function. By P45,
sustained reductions in body and brain weight occurred all combined antenatal and postnatal GC groups, as well as
elevated ACTH and corticosterone. Retardation in plantar reex occurred in all AD groups. IGF-I, GH and insulin levels
were elevated at all ages with dexamethasone. Combined antenatal and postnatal GCs has persistent detrimental
lasting effects on growth, growth factors, neurological outcomes, and HPA axis activity. Whether these effects persist
in adult life and are risk factors for insulin resistance, remains to be elucidated.
Keywords: Betamethasone, dexamethasone, growth hormone, insulin, insulin-like growth factor-I, neurological
outcomes
Introduction
Glucocorticoids (GCs) such as betamethasone
and dexamethasone are used antenatally to
reduce the risk of complications associated
with preterm delivery [1] and prevent or treat
chronic lung disease [2]. Dexamethasone and
betamethasone are the two most commonly
used corticosteroids for antenatal therapy.
These drugs are identical in biological activity
and readily cross the placenta in their active
forms. Treatment of 2 doses of 12 mg of beta-
methasone given intramuscularly (IM), every 24
hours, or four doses of 6 mg of dexamethasone
given IM every 12 hours has been shown to
deliver concentrations to the fetus that are
comparable to physiologic stress levels of corti-
sol after birth. These levels are estimated to
occupy 75% of available corticosteroid recep-
tors, which should induce maximal antenatal
Steroid inuence on growth and neurological outcomes
1698 Am J Transl Res 2019;11(3):1697-1710
corticosteroid receptor-mediated response in
the fetal target tissues [3]. The effects of an-
tenatal GCs are maximal at 48 hours after
administration, and decrease by 7 and 10 days
after administration therefore repeated cours-
es are given on a weekly basis when there is a
high risk of preterm birth [4]. Although a single
dose of antenatal GCs may provide levels
equivalent to cortisol levels, repeated doses
can and do result in supraphysiologic levels
that are harmful to the fetus [5, 6]. Clinical
studies demonstrate decreased head size and
visual memory with antenatal betamethasone,
and abnormal neurological outcomes such as
rate of neuromotor abnormalities and cerebral
palsy with postnatal dexamethasone [7].
The relationship between antenatal GCs and
reduced fetal growth has been documented in
human and animal studies [8, 9]. Single cour-
ses of betamethasone treatment cause sup-
pression of the human fetal adrenal gland for
4 days [5]. A follow-up study for 3 years of pre-
term infants have shown that repeated corti-
costeroid courses were associated with decr-
eased size at birth [10]. The effects of antena-
tal corticosteroid treatment on body and brain
growth may be due to its effects on insulin-like
growth factors (IGFs-I and -II). Small for gesta-
tional age (SGA) babies who fail to demonst-
rate catch-up growth are at increased risk of
developing insulin resistance and cardiovas-
cular disease in later life [11]. In preterm lambs,
prenatal exposure to GCs given at mid-gesta-
tion resulted in growth retardation and chang-
es in lung structure [12]. Other studies have
demonstrated that short courses and low do-
ses of GCs can alter brain development and
cause growth failure [13], particularly growth
of the cerebellum [14-19]. Immature rats tre-
ated with a single dose of dexamethasone (1
mg/kg body weight, or 20 mg/kg body weight)
had impaired growth of the whole body, brain,
and thymus [20]. These negative effects on
fetal growth may be due to alterations in insu-
lin-like growth factor (IGF)-I, which plays a criti-
cal role in fetal and postnatal growth and devel-
opment [21-23].
IGFs are growth-promoting peptides that are
structurally homologous with insulin [24]. The
insulin-IGF family comprises insulin, insulin-like
growth factor-1 (IGF-1), IGF-II, and IGF binding
proteins [25]. IGFs exert their action by binding
to receptors. Availability to their receptors is
regulated by IGF binding proteins (IGFBP 1-6)
[26]. Binding proteins can cause either inhi-
bitory or stimulatory effects on IGF action. IGF
binding protein-3 (IGFBP-3) promotes IGF-I ac-
tivity, prolongs its half-life, and transports it to
target cells. IGFBP-3 is the predominant bind-
ing protein promoting growth in the early ne-
onatal period. Conversely, IGFBP-1 suppresses
IGF-I and is associated with decreased som-
atic growth and insulin resistance [24]. While
insulin is known to augment fetal growth by
stimulating the production of IGF-I, insulin de-
ciency is associated with high IGFBP-1 and low
IGF-I levels [24, 27, 28]. In intrauterine growth
restricted fetuses, marked elevations in serum
IGFBP-1 and decits in IGFBP-3 have been
noted [24, 29, 30]. Insulin is the major regula-
tor of IGFBP-1 and plays a key role in fetal and
early postnatal growth [24].
Postnatal steroids are used in preterm infants
to prevent or treat bronchopulmonary dyspla-
sia (BPD), for hemodynamic support, and to
facilitate early extubation [31, 32]. However,
early exposure to GCs cause many complica-
tions, such as high blood pressure and blood
glucose, gastrointestinal (GI) bleeding, sponta-
neous (GI) perforation, increased risk for cere-
bral palsy, and growth failure [32, 33]. Given
that a signicant number of premature infants
are exposed to both antenatal and postnatal
GCs, there is an imperative to determine whe-
ther dual exposure will further exacerbate post-
natal growth and neurological outcomes. We
therefore hypothesized that combined antena-
tal and postnatal GCs have long-lasting adverse
effects on fetal and neonatal growth, growth
factors, and neurological outcomes.
We examined and compared the effects of
antenatal and/or postnatal GCs on fetal and
postnatal growth, factors that inuence growth,
and neurological outcomes in newborn,
weaned, and adolescent rats.
Materials and methods
Animals
All experiments were approved by the Insti-
tutional Animal Care and Use Committee, Long
Beach Memorial Medical Center, Long Beach,
CA. Animals were cared for according to the
guidelines outlined by the Association for the
Assessment and Accreditation of Laboratory
Steroid inuence on growth and neurological outcomes
1699 Am J Transl Res 2019;11(3):1697-1710
Animal Care (AAALAC) and the Guide for the
Care and Use of Laboratory Animals (National
Research Council) (National Research Council).
Euthanasia of the animals was conducted ac-
cording to the guidelines of the American Vet-
erinary Medical Association (AVMA Panel). Ti-
med pregnant Sprague Dawley rats (200-300
grams body weight) were purchased from Ch-
arles River (Hollister, CA) at 15 days gestation.
The pregnant rats were allowed to stabilize for
48 hours under controlled environmental con-
ditions with free access to food and water.
Experimental design
At 17 days gestation (E17) and E18, the pre-
gnant dams were randomized to receive eith-
er a single injection of betamethasone (0.2
mg/kg) IM diluted in sterile 0.9% saline to a
volume of 0.25 mL (n=6), or single injections
of dexamethasone (0.2 mg/kg) IM diluted in
sterile 0.9% saline to a volume of 0.25 mL
(n=6). Controls animals were administered eq-
uivalent volume saline IM (n=6). The animals
remained undisturbed until delivery of their
pups. At birth (P0) 10 pups/group were euth-
anized and the remainder was pooled and ran-
domly distributed to matched treated dams. At
P5, the pups were randomly assigned to recei-
ve dexamethasone (D, 0.25 mg/Kg, IM), beta-
methasone (B, 0.25 mg/Kg), or equivalent vol-
ume saline (S) on P5, P6, and P7, resulting in
antenatal/postnatal treatment groups: 1) S/S;
2) S/D; 3) S/B; 4) D/S; 5) D/B; 6) D/D; 7) B/S; 8)
B/D; and 9) B/B (n=10 pups/group). Treatment
at P5 is in accord with the delayed dexamet-
hasone treatment of BPD [34]. The animals
were monitored for body weight, anthropom-
orphic growth, and neurological development.
At euthanasia (term, P21 and P45), blood sa-
mples were analyzed for insulin-like growth fa-
ctor (IGF)-I, growth hormone (GH), and insulin
levels. Due to the small blood volume at birth,
the expression of IGF-I, IGF-IR, IGFBP-1 and
IGFBP-3 was determined in the cerebral cortex
of term animals to correlate with neurodevelop-
ment outcomes.
Neurodevelopment testing
Rats were assessed for neurological develop-
ment at P0, P14, P21 and P45 using reex-
stimulus-responses as previously described
[35]. All neurological tests were conducted in a
masked manner by two individuals. The tests
were conducted on all animals in each group.
Responses were recorded as 0 (no response),
1 (mild response), or 2 (full response).
Assay of serum IGF-I and GH levels
Levels of IGF-I and GH in serum samples were
determined at P14, P21 and P45 (n=6/group)
using commercially available enzyme immuno-
assay kits from R&D Systems (Minneapolis,
MN, USA) according to the manufacturer’s pr-
otocol.
Assay of plasma insulin
Insulin levels in the plasma were determined at
P14, P21 and P45 (n=6/group) using enzyme
immunoassay kits purchased from Cayman
Chemicals (Ann Arbor, MI, USA) according to the
manufacturer’s protocol.
Assay of plasma corticosterone
Plasma corticosterone levels were determined
at P45 (n=6/group) using enzyme immunoas-
say kits purchased from Enzo Life Sciences
(Farmingdale, NY, USA) according to the manu-
facturer’s protocol.
Assay of plasma ACTH
Plasma ACTH levels were determined at P45
(n=5/group) using enzyme immunoassay kits
purchased from Sigma-Aldrich (St. Louis, MO,
USA) according to the manufacturer’s protocol.
Isolation of total RNA
Total cellular RNA in the cerebral cortex of term
animals (n=4/group; 2 males and 2 females)
was extracted by homogenization using a poly-
tron homogenizer (Brinkman Instruments, Inc.,
Westbury, N.J.) as previously described [36].
Reverse transcriptase-polymerase chain reac-
tion (RT-PCR)
RT-PCR was carried out using cDNA amplica-
tion kits purchased from Perkin Elmer, Norwalk,
CT, USA and sense and antisense primers for
rat GAPDH, IGF-I receptor, IGFBP-1 and IGFBP-3
(Life Technologies, Carlsbad CA, USA), and
according to previous reports [36-38].
Densitometric scanning
Gel electrophoresis of the PCR products was
performed on 1.5% agarose gels stained with
EtBr. The intensities of the bands were meas-
Steroid inuence on growth and neurological outcomes
1700 Am J Transl Res 2019;11(3):1697-1710
ured with the use of a GelDoc 1000 Darkroom
Imager and Molecular Analyst software (BioR-
ad Laboratories, Hercules, CA). The PCR frag-
ments were identied according to their mole-
cular mass using a DNA mass ladder (Perkin
Elmer, Norwalk, CT). The amount of DNA in
each specimen was quantitated by the integ-
rated density of the product bands within a cl-
osed rectangle, which was then normalized to
the density of the GAPDH bands. The data are
expressed as mean IGF-IR, IGFBP-1 and IGFBP-
3 to GAPDH ratio ± SEM (n=4 samples/group; 2
males and 2 females).
Statistical analyses
Analysis of variance was used to examine dif-
ferences among the treated and control grou-
ps for the each measured variable. Post hoc
test for signicance was determined using the
Student-Newman-Keuls test. Non-normally di-
stributed data was analyzed using Kruskall
Wallis test with Dunn’s multiple comparison te-
an body weight, tail length, and heart and pa-
ncreas weights compared to placebo saline.
Antenatal betamethasone improved the hop-
ping reex with 80% demonstrating mild re-
sponse and 20% exhibiting full responses co-
mpared to the placebo saline and dexameth-
asone groups.
Postnatal steroids, postnatal growth and neu-
rological outcomes
At P5 (before postnatal steroid treatment), ani-
mals that received antenatal steroids had
reduced body weight and linear growth com-
pared to placebo saline (data now shown). At
P14 (one week after postnatal steroid treat-
ment), there were no differences in anthropo-
morphic growth, but there were signicant
effects on neurological outcomes. In the ani-
mals that received postnatal steroids only, neu-
rological testing showed major reductions in
plantar, tactile, and negative geotaxis reexes
in the group AS/PB group; and reductions in the
Table 1. Effects of antenatal steroids on growth and neurological status
in rats at birth (P0)
Growth Parameters Saline Betamethasone Dexamethasone
Total Body Weight (g) 6.00 ± 0.22 6.55 ± 0.12** 5.73 ± 0.12*
Linear Growth
Tail length (cm) 1.74 ± 0.04 1.80 ± 0.03 1.57 ± 0.03**
Tibia length (cm) 1.15 ± 0.04 1.22 ± 0.02 1.19 ± 0.06
Organ Weights (g)
Brain 0.22 ± 0.01 0.26 ± 0.01** 0.23 ± 0.003
Heart 0.05 ± 0.005 0.038 ± 0.002 0.03 ± 0.002*
Lungs 0.13 ± 0.005 0.11 ± 0.006 0.12 ± 0.005
Liver 0.26 ± 0.02 0.24 ± 0.01 0.22 ± 0.011
Kidneys 0.07 ± 0.005 0.07 ± 0.003 0.07 ± 0.003
Pancreas 0.03 ± 0.004 0.02 ± 0.002 0.01 ± 0.002**
Spleen 0.02 ± 0.003 0.02 ± 0.003 0.02 ± 0.002
Neurological tests (% of animals responding)
None Mild Full None Mild Full None Mild Full
Palmar 10% 90% 0% 10% 60% 30% 10% 90% 0%
Plantar 70% 30% 0% 50% 50% 0% 80% 20% 0%
Hopping 80% 20% 0% 0%** 80%* 20% 50% 50% 0%
Tactile 50% 50% 0% 10% 60% 30% 20% 80% 0%
Negative geotaxis 20% 60% 20% 0% 80% 20% 10% 90% 0%
Freefall righting 80% 10% 10% 50% 0% 50% 100% 0% 0%
Pregnant rats were administered saline, betamethasone (0.2 mg/kg/day) or dexametha-
sone (0.2 mg/kg/day) intramuscularly (IM) on 17 and 18 days of gestation. Data are ex-
pressed as mean ± SEM where applicable (n=30/group). Data for neurological tests were
examined using the Fisher’s exact test (none =0; Mild =1; Full =2). *P<0.05, **P<0.01
vs. Saline.
st. Data for neurologic-
al development were an-
alyzed using the Fisher
exact test. Data are exp-
ressed as mean ± SEM.
A p-value of less than or
equal to 0.05 was con-
sidered signicant. Stat-
istical analyses were ac-
complished with the use
of SPSS, version 21; and
all graphs were prepar-
ed with the use of Gr-
aphPad Prism, version 7.
Results
Antenatal steroids, fetal
growth and neurological
outcomes
Table 1 shows the effe-
ct of antenatal steroids
on fetal growth and neu-
rological tests. All anim-
als were born at E22 (te-
rm). Exposure to betam-
ethasone resulted in in-
creased mean body wei-
ght and brain weights,
while exposure to dexa-
methasone reduced me-
Steroid inuence on growth and neurological outcomes
1701 Am J Transl Res 2019;11(3):1697-1710
negative geotaxis and freefall righting reexes
in the AS/PD group. None of the animals exhib-
ited 0 responses (Table 2).
In the animals that received antenatal beta-
methasone, reductions in palmar, plantar, hop-
ping, tactile and negative geotaxis reexes oc-
Table 2. Effect of combined antenatal and postnatal steroids on neurological status at P14
AS/PS AS/PB AS/PD AB/PB AB/PS AB/PD AD/PD AD/PS AD/PB
(Mild response) % of animals responding
Palmar 0 0 0 60%* 0 0 0 0 0
Plantar 10% 30% 0* 40%* 10% 10% 30%* 0* 40%*
Hopping 0 0 0 10% 010% 0 0 10%
Tactile 20% 40%* 0* 30% 0* 20% 0* 10% 20%
Negative geotaxis 10% 20% 30%* 100%* 40%* 40%* 10% 60%* 40%*
Freefall righting 0 0 20%* 0 0 60%* 20%* 20%* 0
(Full response) % of animals responding
Palmar 100% 100% 100% 40% 100% 100% 100% 100% 100%
Plantar 90% 70%* 100% 60% 90% 90% 70%* 100% 60%
Hopping 100% 100% 100% 90% 100% 90% 100% 100% 90%
Tactile 80% 60%* 100% 70% 100% 80% 100% 90% 80%
Negative geotaxis 90% 80% 70%* 0* 60%* 60%* 90% 40%* 60%*
Freefall righting 100% 100% 80%* 100% 0* 40%* 80%* 80%* 100%
For antenatal steroid exposure, pregnant rats were administered antenatal saline, betamethasone (0.2 mg/kg/day) or dexa-
methasone (0.2 mg/kg/day) intramuscularly (IM) on 17 and 18 days of gestation. For postnatal steroids, pups were admin-
istered saline, betamethasone (0.25 mg/kg) or dexamethasone (0.25 mg/kg on postnatal day 5, 6, and 7. AS/PS (antenatal
saline+postnatal saline); AS/PB (antenatal saline+postnatal betamethasone); AS/PD (antenatal saline+postnatal dexametha-
sone); AB/PB (antenatal betamethasone+postnatal betamethasone); AB/PS (antenatal betamethasone+postnatal saline); AB/
PD (antenatal betamethasone+postnatal dexamethasone); AD/PD (antenatal dexamethasone+postnatal dexamethasone);
AD/PS (antenatal dexamethasone+postnatal saline); and AD/PB (antenatal dexamethasone+postnatal betamethasone). None
of the pups exhibited 0 response. *P<0.05 vs. AS/PS (Fisher’s exact test).
Table 3. Effect of combined antenatal and postnatal steroids on neurological status at P21
AS/PS AS/PB AS/PD AB/PB AB/PS AB/PD AD/PD AD/PS AD/PB
(Mild response) % of animals responding
Palmar 0 0 0 0 0 0 10% 0 0
Plantar 0 20%* 10% 0 0 0 30%* 10% 40%*
Hopping 0 0 0 0 0 0 0 0 0
Tactile 0 0 0 0 0 0 10% 0 0
Negative geotaxis 30% 20% 10%* 0 0 0 10% 10% 0
Freefall righting 0 0 0 0 0 0 0 0 0
(Full response) % of animals responding
Palmar 100% 100% 100% 100% 100% 100% 90% 100% 100%
Plantar 100% 80%* 90% 100% 100% 100% 70%* 90% 60%*
Hopping 100% 100% 100% 100% 100% 100% 100% 100% 100%
Tactile 100% 100% 100% 100% 100% 100% 90% 100% 100%
Negative geotaxis 70% 80% 90%* 100%* 100%* 100%* 90%* 90%* 100%*
Freefall righting 100% 100% 100% 100% 100% 100% 100% 100% 100%
For antenatal steroid exposure, pregnant rats were administered antenatal saline, betamethasone (0.2 mg/kg/day) or dexa-
methasone (0.2 mg/kg/day) intramuscularly (IM) on 17 and 18 days of gestation. For postnatal steroids, pups were admin-
istered saline, betamethasone (0.25 mg/kg) or dexamethasone (0.25 mg/kg on postnatal day 5, 6, and 7. AS/PS (antenatal
saline+postnatal saline); AS/PB (antenatal saline+postnatal betamethasone); AS/PD (antenatal saline+postnatal dexametha-
sone); AB/PB (antenatal betamethasone+postnatal betamethasone); AB/PS (antenatal betamethasone+postnatal saline); AB/
PD (antenatal betamethasone+postnatal dexamethasone); AD/PD (antenatal dexamethasone+postnatal dexamethasone); AD/
PS (antenatal dexamethasone+postnatal saline); and AD/PB (antenatal dexamethasone+postnatal betamethasone). None of
the pups exhibited 0 response. *P<0.05 vs. AS/PS (Fisher’s exact test).
Steroid inuence on growth and neurological outcomes
1702 Am J Transl Res 2019;11(3):1697-1710
curred in the AB/PB group; reductions in ne-
gative geotaxis and freefall righting reexes
occurred in the AB/PS group; and reductions in
hopping, negative geotaxis and freefall righting
occurred in the AB/PD group. In the animals
that received antenatal dexamethasone, reduc-
tions in plantar and freefall righting occurred in
the AD/PD group; reductions in negative geo-
taxis and freefall right occurred in the AD/PS
group; and reductions in plantar, hopping and
negative geotaxis occurred in the AD/PB group.
At P21, a time of weaning from the dams and
when the pups begin eating solid food, animals
in the AS/PB (41.6 ± 0.8, P<0.01) AS/PD (43.4
± 1.5, P<0.01), AB/PS (45.0 ± 0.81, P<0.01),
AD/PD (45.3 ± 3.0, P<0.01) and AD/PS (42.1 ±
0.52, P<0.01) groups had lower body weights
while animals exposed to AB/PB (53.5 ± 0.9,
P<0.05) had higher body weight compared to
placebo saline (49.7 ± 0.7). There were no dif-
ferences in body weight noted in the AB/PD and
AD/PB groups. None of the groups exhibited
differences in body length. Brain weight was
reduced in the AS/PD (1.31 ± 0.02 vs. 1.47 ±
0.03, P<0.01) group, while midbrain (0.34 ±
0.02 vs. 0.2 ± 0.01, P<0.01) and cerebellum
(0.21 ± 0.006 vs. 0.17 ± 0.01, P<0.01) we-
ights were increased with AB/PB and AD/PB
groups, respectively, compared to controls. Ne-
urological outcomes showed sustained plant-
ar reex reductions in the AS/PB, AS/PD, AD/
PD, AD/PS, and ADPB groups; reduced palmar
reex in the AD/PD group; and reduced tactile
reex in the AD/PD group (Table 3).
At P45, lower body weight was noted in the AB/
PS (175.4 ± 6.0 vs. 216 ± 12.1, P<0.05) group
as was lower tail length in the AS/PB (14.7 ±
0.3, P<0.01), AB/PS (14.0 ± 0.1, P<0.01), AD/
PD (14.8 ± 0.4, P<0.01), AD/PS (15.2 ± 0.2,
P<0.05), and AD/PB (14.7 ± 0.3, P<0.01) gr-
oups compared to control (16.6 ± 0.4). Lower
tibia length was noted in the AS/PB (4.1 ± 0.08,
P<0.05) and AD/PB (4.2 ± 0.05, P<0.05) grou-
ps compared to control (4.5 ± 0.1). Brain weight
was lower in the AS/PB (1.7 ± 0.02, P<0.01),
AB/PB (1.6 ± 0.05, P<0.01), AB/PS (1.6 ± 0.15,
P<0.01), AB/PD (1.56 ± 0.04, P<0.01), AD/PD
(1.7 ± 0.03, P<0.01), and AD/PB (1.7 ± 0.03,
P<0.01) groups compared to control (1.9 ±
0.02). Cerebellum and lung weights were lower
in the AB/PD (0.18 ± 0.03 vs. 0.29 ± 0.01) and
Figure 1. Messenger RNA expression of IGF-I (A), IGFIR (B), IGFBP-1 (C) and IGFBP-3 (D) in the newborn rat brain
at birth (P0). Pups were exposed antenatally to 0.25 mL betamethasone or dexamethasone (0.2 mg/kg in
sterile normal saline), injected intramuscularly to the pregnant dam on E17 and E18. Control pups were exposed to
equivalent volume sterile normal saline. Data are expressed as mean ratio of genes/b-actin (n=4 samples/group; 2
males and 2 females). *P<0.05; **P<0.01 vs. placebo saline.
Steroid inuence on growth and neurological outcomes
1703 Am J Transl Res 2019;11(3):1697-1710
AS/PB (1.02 ± 0.05 vs. 1.37 ± 0.07, P<0.01)
groups, respectively. Neurological tests at P45
showed sustained reductions in full plantar
reex in the AS/PB (80%, P<0.05), AD/PD (70%,
P<0.05) and AD/PB (60%, P<0.05) groups, and
a non-signicant lower negative geotaxis reex
in the AS/PD (90%).
Antenatal steroids, fetal brain IGF-I signaling
Figure 1 represents the expression of IGF-I,
IGF-IR, IGFBP-1 and IGFBP-3 in the neonatal
rat brain at birth. Brain IGF-I mRNA was elev-
ated in the betamethasone-treated group (Fi-
gure 1A). Treatment with dexamethasone sig-
Figure 2. Effects of combined antenatal and
postnatal steroids on serum IGF-I levels at P14,
P21, and P45. The doses are as described in
Figure 1. Drug or placebo saline injections were
administered IM on 17 and 18 days gestation
(n=6 pregnant rats per group). The postnatal
doses of betamethasone and dexamethasone
were 0.25 mg/kg IM diluted in sterile normal
saline. Pups were injected on P5, P6, and P7. The
control group received equivalent normal saline.
Data are represented as mean ± SEM (10 pups/
group). **P<0.01 vs. combined antenatal saline
(AS)/postnatal saline (PS).
Figure 3. Effects of antenatal and postnatal steroid
exposure on serum GH levels at P14, P21, and P45.
Groups are as described in Figures 1 and 2. Data
are represented as mean ± SEM (10 pups/group).
*P<0.05; **P<0.01 vs. combined antenatal saline
(AS)/postnatal saline (PS).
Steroid inuence on growth and neurological outcomes
1704 Am J Transl Res 2019;11(3):1697-1710
nicantly downregulated IGF-IR mRNA (2-fold),
and IGFBP-3 (2-fold), but elevated IGFBP-1 mR-
NA (3-fold) with no change in IGF-I expression
(Figure 1B-D).
Combined antenatal and postnatal steroids,
serum IGF-I
Figure 2 shows the serum levels of IGF-I at P14,
P21 and P45. At P14, IGF-I levels were signi-
cantly higher in the AS/PD and AD/PD groups.
At P21, serum IGF-I levels were higher in the
AB/PB group. By P45, no differences were
noted among the groups.
Combined antenatal and postnatal steroids,
serum GH
Figure 3 shows serum GH levels at P14, P21,
and P45. At P14, only the AD/PS group had
higher GH levels, but by P21 the levels incr-
eased in the AS/PD group and decreased in the
AB/PS group. By P45, higher GH levels were
noted in AS/PD, AB/PB, AB/PS, AB/PD and AD/
PS groups compared to the AS/PS group.
Combined antenatal and postnatal steroids,
plasma insulin
Figure 4 shows plasma insulin levels at P14,
P21 and P45. Insulin levels declined at P14 in
the AB/PB and AB/PS groups, but increased at
P21 in the AS/PB, AB/PS, AB/PD, and AD/PD
groups. By P45 only treatment with AS/PD had
sustained higher insulin levels.
Combined antenatal and postnatal steroids,
plasma ACTH and corticosterone
Plasma ACTH and corticosterone levels at P45
are presented in Figure 5. Treatment with AB/
PS and AD/PB resulted in higher ACTH levels
while only AD/BP resulted in a robust increase
in corticosterone levels.
Discussion
The present study tested the hypothesis that
the adverse effects of antenatal steroids on
fetal and neonatal growth are mediated in part,
by their inuence on the IGF system. The rst
test of our hypothesis was demonstrated by the
effects of dexamethasone on brain IGF-IR and
IGFBP-3 mRNA expression, with negligible eff-
ects on IGF-I mRNA expression and brain wei-
ght. The results of our ndings demonstrated
that decits in IGF-IR and IGFBP-3 mRNA ap-
proached 50% in the dexamethasone group
compared to brain/body weight decits of only
10%, whereas actual brain weight remained
Figure 4. Effects of antenatal and postnatal steroid
exposure on plasma insulin levels at P14, P21, and
P45. Groups are as described in Figures 1 and 2.
Data are represented as mean ± SEM (10 pups/
group). *P<0.05; **P<0.01 vs. combined antenatal
saline (AS)/postnatal saline (PS).
Steroid inuence on growth and neurological outcomes
1705 Am J Transl Res 2019;11(3):1697-1710
unchanged. This is also in contrast to the mini-
mal effects of betamethasone.
IGFs are potent mitogens that promote cell
growth and anabolism in many tissues inclu-
ding the central nervous system [39-41]. Sti-
mulation of neurite growth including the neur-
otrophic actions has been attributed to IGF-I
activity. Considering the important role of IGF-I
in brain development, and growth promoting
role of IGFBP-3 (the most abundant binding
protein in the late fetal and early postnatal age)
[24] in facilitating IGF-I bioactivity, these nd-
ings may suggest that dexamethasone-induc-
ed brain growth suppression can occur even
when the actual brain weight is spared. It is
important to note that IGF-I protein levels in the
brain were not measured, but the mRNA expres-
sion level which does not necessarily reect
corresponding protein levels. Moreover, decrea-
sed brain to body weight ratio by treatment with
dexamethasone, suggests that the brain cells
might have been targeted. Our ndings on IGF-I
mRNA are in contrast to a previous in-vitro st-
udy which showed that dexamethasone reduc-
ed IGF-I mRNA levels in the primary cultures of
neuronal and glial cells from rat brain [42].
These discrepancies may be attributed to dif-
ferences between the in-vivo and in-vitro mod-
els and inuence of IGFBPs.
Although IGF-IR mRNA has been found in the
fetal rat at day 14, and is expressed at high
levels through perinatal age [43], its mRNA
expression is maximal at fetal day 15 and 20,
whereas the maximal mRNA expression for
the insulin receptor is at fetal day 20 and the
day of birth. It is well known that the biological
effects of IGF-I, 2 and insulin are mediated
through their interactions with specic cell me-
mbrane receptors. In a study by Yamamoto et
al. [44], a decrease in specic IGF-I binding was
directly related to the ligand concentration and
was dependent on the duration of pituitary cell
exposure to IGF-I. Chronic exposure as well as
high levels caused a down regulation of the
IGF-I receptor number with no change in recep-
tor afnity. IGFBP-3 increases the half-life of
IGFs in circulation [43]. When IGF-I and -II are
injected into normal rats, they bind to IGFBP-3
increasing their stability and half-life to 4 hours
compared to 20 minutes in hypophysectomized
rats [24, 45]. Guler et al. [46] determined the
half-lives of free and IGFBP bound IGF-I and -II,
and their ndings further support the role of IGF
binding proteins in augmenting the bioactivity
of IGFs. The most signicant nding is the effect
of antenatal dexamethasone on IGF-IR, IGFBP-
1 and IGFBP-3 mRNA expression in the brain. In
a previous study by Villafuerte et al. [47], dexa-
methasone reduced the production of IGFBP-3
on cultured hepatocytes by inhibition of IGFBP-
3 gene transcription, which may be the same
mechanism in our study. However, this could
not explain the unchanged mRNA expression
levels of brain IGF-I, and may suggest that IGF-I
must be produced at higher rates in the ante-
natal dexamethasone group due to a decrea-
sed bioavailability of IGFBP-3. Nevertheless, it
seems that the effect of antenatal dexame-
thasone on IGF signaling (i.e. reduced mRNA
expression levels of IGF-IR and IGFBP-3) in the
brain did not adversely affect the actual brain
size.
Rat neurons have the same composition and
electrical properties as human neurons [7]. In
rats, the brain growth spurt occurs after term.
Figure 5. Effects of antenatal and postnatal steroid exposure on plasma ACTH and corticosterone levels at P45.
Groups are as described in Figures 1 and 2. Data are represented as mean ± SEM (10 pups/group). *P<0.05;
**P<0.01 vs. combined antenatal saline (AS)/postnatal saline (PS).
Steroid inuence on growth and neurological outcomes
1706 Am J Transl Res 2019;11(3):1697-1710
In humans, neuronal division except for the
cerebellum and dentate gyrus, is completed
before the 24th week of gestation and the peak
brain growth spurt occurs around term. Thus a
rat at 8 days of age is roughly equivalent to a
full term infant in terms of growth, periventri-
cular germinal matrix, neurochemical data, el-
ectroencephalographic pattern, and synapse
formation [7, 48]. It was interesting to note th-
at animals that were antenatally exposed to
betamethasone had higher brain weights and
IGF-I mRNA expression, and exhibited advanc-
ed hopping reexes in a greater percentage of
the rats compared to placebo saline and dex-
amethasone, suggesting maturation of spinal
mechanisms involved in rhythmic stepping
responses [35].
At P14, all groups exposed to antenatal and/or
postnatal steroids exhibited retarded negative
geotaxis and free-fall righting reexes both of
which have been attributed to vestibular func-
tion [35]. Vestibular function is associated with
cerebellar development which has been shown
to be negatively affected by GCs [49]. At P14,
the cerebellum plays an important role in the
regulation of complex movement patterns [50],
therefore, the delayed effect of GCs on nega-
tive geotaxis and free-fall righting reexes sug-
gest abnormal cerebellum function [35]. This
delayed effect was not seen at P21. Instead,
delayed plantar and palmar reexes were noted
in the postnatal GC and antenatal dexametha-
sone groups, suggesting delayed sensory thre-
sholds and signaling from the brainstem [51].
At P45, brain weight was lower predominantly
in the animals exposed to combined antenatal
and postnatal GCs. Delayed plantar reexes
persisted in the AS/PB, AD/PD and AD/PB
groups, while delayed negative geotaxis reex-
es persisted in the AS/PD group. These ndings
provide evidence that the combined use of
antenatal and postnatal GCs have lasting detri-
mental effects on neurological outcomes.
In the suckling rats at P14, it was interesting
to note the higher serum IGF-I levels in the AS/
PD and AD/PD groups compared to all other
groups despite no change in body weight or
linear growth. The GH/IGF-I/insulin system is
important for postnatal growth and develop-
ment. Particularly, IGF-I is a major stimulus for
postnatal growth [52]. It is induced by pituita-
ry GH in the liver to promote growth, along with
its binding proteins [53], and accounts for
about 75% of all circulating IGFs [54]. Together
with its receptor, it is expressed in almost all
tissues for autocrine/paracrine purposes [55].
Although IGF-I is predominantly produced by
the liver, studies have shown that liver-derived
IGF-I is not required for postnatal growth, sug-
gesting that local production of IGF-I may be
more important than liver-derived circulating
IGF-I for body growth [56]. IGF-I availability is
tightly regulated by its binding proteins which
increase IGF-I half-life from minutes to hours,
and shuttles IGF-I to specic target tissues
[57]. IGF-I is present in high concentrations in
serum, and is mostly protein bound [58]. Ap-
proximately 90% of IGF-I is bound to IGFBP-3,
the primary hepatic-derived IGFBP [59]. In rats,
the fetal serum prole, characterized by high
IGF-II and IGFBP-2, is replaced around the thi-
rd week of life by the adult-type prole of high
IGF-I and IGFBP-3, with a dramatic reduction in
IGF-II and IGFBP-2 [60]. Evidence suggest th-
at dexamethasone causes marked increases
in serum IGF-I and insulin levels, whereas IGF-I
bioactivity was signicantly decreased [61, 62].
Thus, the high serum levels noted in this study
with dexamethasone is in agreement with th-
ose previous ndings and may suggest redu-
ced bioavailability. This effect of dexametha-
sone was long lasting and remained sustain-
ed until adolescent, despite normal food inta-
ke as rats are weaned at P21, and may acco-
unt for the lower brain weight as well as the
sustained retardation in plantar and palmar
reexes which are representative of delayed
sensory thresholds and signaling from the
brainstem [51].
The responses of serum GH to dexamethas-
one were similar to IGF-I at P14 and P21.
Dexamethasone has been shown to augment
the insensitivity to GH and IGF-I by reducing
GHR and IGF-IR expression [63]. Although we
did not measure GHR expression, our data
showed reductions in IGF-IR in the brain of
term rats with dexamethasone (Figure 1B).
Dexamethasone-induced elevations in serum
IGF-I and GH concurrent with reduced brain
and body weight concur with previous reports
of GH insensitivity. Similar elevations in serum
GH levels and growth retardation were also
noted at P45 in the AS/PB, AB/PB, and AB/PS
groups suggesting that betamethasone may
also promote GH insensitivity, but the effect is
Steroid inuence on growth and neurological outcomes
1707 Am J Transl Res 2019;11(3):1697-1710
latent. Studies suggest that during the perina-
tal period, GH does not appear to play a major
role in growth and IGF-I secretion but may play
a more important role in regulation of gluco-
se metabolism [64]. Ovine fetuses antenatally-
treated with dexamethasone had higher glu-
cose and insulin levels than controls, suggest-
ing adverse metabolic effects due to hypergly-
cemia and hyperinsulinemia [65].
In our study, plasma insulin levels also in-
creased with dexamethasone at P14, and wi-
th combined antenatal and postnatal dexa-
methasone at P21. The effect of antenatal de-
xamethasone on plasma insulin remained sus-
tained until P45, providing further evidence
that combined antenatal and postnatal GCs
result in hormonal changes that may have lo-
ng-lasting detrimental effects. We also obse-
rved elevated levels of ACTH and corticoste-
rone in the AB/PS and AD/PB animals at P45.
In these same groups, body weight, brain we-
ight, and tail length were reduced. Studies in
human newborns show similar elevations in
cord blood ACTH and cortisol with reduced fetal
growth [66]. We now show sustained elevations
in serum ACTH and corticosterone levels with
combined antenatal and postnatal GCs sug-
gesting permanent programming of hypotha-
lamic-pituitary-adrenal (HPA) axis activity and
function.
While this study provides important and clin-
ically-relevant information regarding, one lim-
itation was the use of semi-quantitative RT-
PCR to assess the mRNA expression of IGFs
and IGFBPs in the brain. Real-time PCR wou-
ld have provided more quantitative assess-
ments. However, the biomolecular and neuro-
logical outcomes, taken together, point to the
signicance of combined exposure to antenatal
and postnatal GCs. While caution is necessary
when extrapolating data from animals to the
human situation, there are still developmental
similarities between species, that can provide
valuable information.
In conclusion, betamethasone and dexame-
thasone are both synthetic GCs that are wi-
dely used during the perinatal period. The pr-
esent study showed differential benecial ef-
fects of antenatal betamethasone over dexa-
methasone with respect to body growth and
the IGF-I system in the brain. Antenatal beta-
methasone produced no signicant effects on
the IGF system in the brain or neurological de-
cits at birth or adolescence compared to the
antenatal dexamethasone. Combined antena-
tal and postnatal GCs has lasting effects on
body and brain growth, factors that inuence
growth and glucose homeostasis, neurological
outcomes, and HPA axis activity. Whether the-
se effects persist in adult life and are risk fa-
ctors for insulin resistance, remains to be el-
ucidated.
Acknowledgements
This work was supported through a grant from
Memorial Health Services Research Founda-
tion, Long Beach, CA, USA (Grant #007-010).
Disclosure of conict of interest
None.
Address correspondence to: Kay D Beharry, De-
partment of Pediatrics/Div. Neonatal-Perinatal Me-
dicine & Ophthalmology, Director, Neonatal-Perina-
tal Medicine Clinical & Translational Laboratories,
State University of New York, Downstate Medical
Center, 450 Clarkson Avenue, Box 49, Brooklyn, NY,
11203, USA. Tel: 718-270-1475; Fax: 718-270 -
1985; E-mail: kbeharry@downstate.edu
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