Carter et al. BMC Medicine (2024) 22:338
https://doi.org/10.1186/s12916-024-03542-5

BMC Medicine

RESEARCH

Open Access

Antenatal steroids elicited
neurodegenerative‑associated transcriptionalchanges in the hippocampus of preterm fetal
sheep independent of lung maturation
Sean W. D. Carter1*, Erin L. Fee2, Haruo Usuda2,3, Gokce Oguz4, Adaikalavan Ramasamy4, Zubair Amin1,5,6,
Biswas Agnihotri5,6, Qin Wei1, Liu Xiawen1, Tsukasa Takahashi2,3, Yuki Takahashi2,3, Hideyuki Ikeda2,3,
Yusaku Kumagai1,3, Yuya Saito3, Masatoshi Saito2,3, Citra Mattar1, Mark I. Evans1,7,8, Sebastián E. Illanes1,9,10,
Alan H. Jobe11, Mahesh Choolani1 and Matthew W. Kemp1,2,3,12Abstract
Background Antenatal steroid therapy for fetal lung maturation is routinely administered to women at risk of preterm delivery. There is strong evidence to demonstrate benefit from antenatal steroids in terms of survival and respiratory disease, notably in infants delivered at or below 32 weeks’ gestation. However, dosing remains unoptimized
and lung benefits are highly variable. Current treatment regimens generate high-concentration, pulsatile fetal steroid
exposures now associated with increased risk of childhood neurodevelopmental diseases. We hypothesized that damage-associated changes in the fetal hippocampal transcriptome would be independent of preterm lung function.
Methods Date-mated ewes carrying a single fetus at 122 ± 2dGA (term = 150dGA) were randomized into 4 groups:(i) Saline Control Group, 4×2ml maternal saline intramuscular(IM) injections at 12hr intervals (n = 11); or (ii) Dex HighGroup, 2×12mg maternal IM dexamethasone phosphate injections at 12hr intervals followed by 2×2ml IM saline
injections at 12hr intervals (n = 12; representing a clinical regimen used in Singapore); or (iii) Dex Low Group, 4×1.5mg
maternal IM dexamethasone phosphate injections 12hr intervals (n = 12); or (iv) Beta-Acetate Group, 1×0.125mg/
kg maternal IM betamethasone acetate injection followed by 3×2ml IM sterile normal saline injections 12hr intervals (n = 8). Lambs were surgically delivered 48hr after first maternal injection at 122–125dGA, ventilated for 30min
to establish lung function, and euthanised for necropsy and tissue collection.
Results Preterm lambs from the Dex Low and Beta-Acetate Groups had statistically and biologically significant lungfunction improvements (measured by gas exchange, lung compliance). Compared to the Saline Control Group,
hippocampal transcriptomic data identified 879 differentially significant expressed genes (at least 1.5-fold change
and FDR < 5%) in the steroid-treated groups. Pulsatile dexamethasone-only exposed groups (Dex High and Dex Low)
had three common positively enriched differentially expressed pathways related in part to neurodegeneration (“Prion
Disease”, “Alzheimer’s Disease”, “Arachidonic Acid metabolism”). Adverse changes were independent of respiratory
function during ventilation.

*Correspondence:
Sean W. D. Cartere0983544@u.nus.edu
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Carter et al. BMC Medicine (2024) 22:338

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Conclusions Our data suggests that exposure to antenatal steroid therapy is an independent cause of damageassociated transcriptomic changes in the brain of preterm, fetal sheep. These data highlight an urgent need for carefulreconsideration and balancing of how antenatal steroids are used, both for patient selection and dosing regimens.
Keywords Preterm, Steroids, Sheep, Fetus, Brain injury, Hippocampus, Lung maturation

Background
Preterm birth (delivery before 37 weeks gestation)
occurs in ~ 11% of all pregnancies worldwide and is
the leading cause of mortality and morbidity for children under 5 years old [1]. When accurately targeted to
women at risk of preterm delivery, antenatal corticosteroid therapy (ACS) has been credited with reductionsin the risk of perinatal death and respiratory disease[2–4]. The largest benefits are seen in preterm infants
born at or below 32 weeks’ gestation. Thereafter, treatment effect magnitude tapers with proximity to term,reflected by large increases in the number of patientsneeded to be treated to prevent a single case of respiratory disease. However, despite over 50 years of clinicaluse, ACS therapy is still not optimized, and there is nointernational consensus regarding the ideal drug formulation, dosing regimen, or route of administration.
Three corticosteroids (betamethasone phosphate,dexamethasone phosphate, and betamethasone acetate) comprise most ACS dosing regimens worldwide[5–8]; these steroids readily cross the placenta andexert extensive genomic and non-genomic effects on
the developing fetus [9]. The two most widely studied
ACS formulations, dexamethasone phosphate and betamethasone phosphate (used alone or in combination
with betamethasone acetate), have significantly different pharmacokinetic profiles [10]. Unfortunately, therisk-benefits for the developing fetus remain poorly
understood which is critical as antenatal steroid use
has expanded over the past 20 years to include many
patients at considerably lower risks than for which
the therapy was originally designed [11]. Furthermore,
despite receiving ACS therapy, many babies do notdemonstrate any respiratory benefit from treatment(non-response) [12, 13]. Increased use persists, despite
evidence from population-level cohort studies concluding an association between exposure to ACS and anelevated risk of childhood neurological deficits/behavioral disorders, serious childhood infection [14, 15],and impaired academic performance [16]. Moreover,additional reports have described ACS exposure being
associated with an increased risk of neonatal hypoglycaemia [17], HPA axis dysregulation [18–22], reduced
birth weight, and head circumference [23, 24]. Eachof which is independently linked to diminished healthoutcomes.

The impact of ACS on the fetal brain is a topic of particular importance. Due to the high density of glucocorticoid receptor expression, the hippocampus could behighly susceptible to off-target, detrimental ACS effects[25–27]. These concerns are supported by evidence fromclinical studies in preterm neonates showing that a standard full-dose ACS exposure was associated with reduced
hippocampal neuron density [28] and bilateral cortex
thinning (maximally in areas of the brain related to emotional regulation) in pre-adolescents [29]. Animal studies
have also shown that ACS exposure increased neuronaldegeneration [25], reduced brain weight [30], delayed
central nervous system myelination [31], and suppressed
normal neurodevelopmental pathway differentiation
within the hippocampus [32, 33].
Based on the concept that current dosing regimens areprobably excessively high to achieve the expected lungmaturation and could unnecessarily increase the risk ofharm, several recent and ongoing efforts to optimize ACS
dosing have been proposed [34–36]. However, no study
has attempted to evaluate the effects of a high dose regimen in current clinical use (2 maternal intramuscular
injections of 12 mg dexamethasone phosphate spaced by
12 h, used clinically in Singapore) vs. lower dose pulsatileregimens of ACS on both fetal lung maturation and brain
development.
Study aim

We hypothesized that injury-associated changes in the
fetal hippocampal transcriptome would be independentof preterm lung function subsequent to antenatal steroid
exposure. We test this hypothesis using the sheep modelof pregnancy.

Methods
Animal model and antenatal corticosteroid treatment

Merino ewes with singleton fetuses received an intramuscular injection (IM) of 150 mg medroxyprogesterone acetate (Depo-Provera, Pfizer, New York, NY) on
117 ± 2 days gestational age (dGA) to decrease the risk ofsteroid-induced premature labor. Baseline maternal blood
samples were collected for analysis at 0800, 24–48 h prior
to allocation to treatment group. Animals were then randomly allocated to one of four groups (Fig. 1), with firsttreatment given at 122 ± 2 dGA (term = 150 dGA). Lung

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Fig. 1 Experimental design. Diagram of experimental design and intervention groups

maturation in the preterm fetal sheep at 124 days’ gestation approximates that of the human at 30–32 weeks’gestation. First injections occurred at 08:00 to control
for circadian effects: (i) Saline Control Group, 4 × 2 ml
maternal saline intramuscular (IM) injections at 12-hintervals (n = 11); or (ii) Dex High Group, 2 × 12 mg
maternal IM dexamethasone phosphate (DBL Dexamethasone sodium phosphate 4 mg/ml, Hospira NZ, New
Zealand) injections at 12-h intervals followed by 2 × 2 ml
IM saline injections at 12-h intervals (n = 12; this protocol simulates the standard ACS regimen used clinically in
Singapore); or (iii) Dex Low Group, 4 × 1.5 mg maternal
IM dexamethasone phosphate injections 12-h intervals(n = 12; this protocol represents a 75% reduction on the
Singapore clinical regimen); or (iv) Beta-Acetate Group,
1 × 0.125 mg/kg maternal IM betamethasone acetate
injection followed by 3 × 2 ml IM sterile normal saline
injections at 12-h intervals (n = 8; this protocol represents
a positive control group previously shown to elicit robust
preterm lung maturation in the preterm sheep equivalent
to standard courses of dexamethasone phosphate or betamethasone phosphate and acetate used worldwide).
Serial maternal plasma samples were collected at 6, 12,
18, 24, and 36 h after first maternal injection. All preterm lambs included in this study for ventilation analysis(122–125 days gestation) were surgically delivered 48 h
after first maternal injection at 122–125 dGA.
One ewe from the Saline Control group was removed
from the analysis as it was found not to be pregnant at
pre-delivery ultrasound. One lamb from the Beta-Acetate Group, which was incorrectly intubated and not
ventilated for 15 min, was removed from the ventilation
analysis.
Pharmacokinetics

Liquid chromatography-mass spectrometry (LC–MS)
was performed on all maternal and fetal plasma samples to assess dexamethasone and betamethasone-acetate concentrations respectively as previously reported[37]. Betamethasone Acetate (Beta-Acetate Group)
pharmacokinetic parameters were estimated by noncompartmental analysis using Phoenix WinNonlin software (version 8.1, Certara, Inc.). Dexamethasone (Dex
Low and Dex High Groups) simulated pharmacokinetic
parameters were generated by Dr Mark Milad based on
existing ovine dexamethasone pharmacokinetic data [8].
Delivery and ventilatory assessment

Maternal plasma samples for analysis were taken immediately prior to delivery, i.e., 48 h after first maternal

Carter et al. BMC Medicine (2024) 22:338

injection. Fetal plasma samples for analysis were obtained
during operative delivery and immediately prior to ventilation commencing. Prior to delivery, ewes received
an intravenous bolus of midazolam (0.5 mg/kg) andketamine (10 mg/kg) for the deep induction of anaesthesia. The fetal head was delivered through abdominal
and uterine incisions, and a 4.5-mm endotracheal tube
was secured by tracheostomy. The fetus was then delivered, and the ewe euthanized under anaesthesia withpentobarbital. Lambs were weighed, dried, and placedin a radiant warmer (Cozy Cot, Fisher & Paykel Healthcare, New Zealand) bed under a plastic insulating wrap
(Neowrap, Fisher & Paykel, NZ). Mechanical ventilation(Fabian HFO, Accutronic Medical Systems AG, Switzerland) was immediately commenced with the following
settings: peak inspiratory pressure (PIP) of 35 ­cmH2O,
positive end expiratory pressure (PEEP) of 5 c­ mH2O, respiratory rate (RR) of 50 breaths per minute, inspiratory
time (iT) of 0.5 s, using 100% heated and humidified oxygen. We used 100% oxygen which allows comparison ofoxygenation through the partial arterial pressure of oxygen among the groups.
Investigators operating the ventilators were blinded tothe animal treatment groups. An umbilical artery wascatheterized for blood sampling and administration of
supplemental anaesthesia with ketamine (5 mg/kg) as
necessary. Tidal volume (VT) was continuously measured, and the PIP was adjusted to keep the VT between
7.0 and 8.0 ml/kg, but with PIP limited to 35 ­cmH2O.
At 10, 20, and 30 min of ventilation we measured temperature, blood pressure, ventilator data (PIP, VT, andcompliance), and performed blood gas measurements;pH, ­PCO2 (mmHg), P­ O2 (mmHg), O­ 2 saturation (­SO2,%), and total hemoglobin (Hb, g/dL) levels (Siemens
RapidPoint 500, Munich, Germany). Dynamic compliance (Cdyn, ml/cmH2O/kg) was recorded as measured by the ventilator. The ventilation efficiency index(VEI) was calculated using the formula VEI = 3800 /(RR × (PIP − PEEP) × ­PCO2 (mm Hg)) [38].
Definition of antenatal corticosteroid response

Steroid-treated lambs were defined as having responded
to ACS treatment if the ­PaCO2 level after 30 min of ventilation was two standard deviations below the mean of
the Saline group. The mean and standard deviation ofthe ­PaCO2 after 30 min of ventilation in the Saline Control group was 118.3 and 31.3 mmHg thus giving rise to
57.0 mmHg (= 118.3 − 2 × 31.3) as cutoff.
Lung functional assessments

After 30 min of ventilation, lambs were euthanized withpentobarbital, disconnected from the ventilator, and thePage 4 of 17

endotracheal tube was clamped for 2 min to achieve atelectasis by oxygen absorption. The lambs were weighed,and the chest was opened for visual evaluation of grosslung injury–pulmonary hemorrhage, pulmonary interstitial emphysema, gas pockets within the lung, or subpleural dissection. These were all performed by a single
investigator. A deflation pressure–volume curve wasmeasured after air inflation of the lungs to a pressure of
40 ­cmH2O. Volume at a pressure of 40 ­cmH2O was calculated using fetal weight after ventilation (kg) as V
­ 40 (ml/kg).
Hippocampal and lung mRNA extraction and RNA bulk
sequencing

Twenty-four animals (Six animals from each group-Saline Control, Dex High, Dex Low, Beta-Acetate) wereselected for RNA bulk sequencing based on the highest
maternal steroid plasma concentration prior to delivery. The same animals were used for both lung and hippocampal RNA extraction and analysis thereby allowing
for matched analysis with ventilation outcomes and ACS
responsiveness.
The ACS Responder Group for RNA bulk sequencing(total six animals) consisted of three animals from theDex Low and three animals from Beta-Acetate Groups;
ACS non-responder subgroup (total twelve animals)consisted of six animals from the Dex High Group,
three animals from the Dex Low Group, and three animals from Beta-Acetate Group. RNA was extracted fromsnap-frozen hippocampal (entire hippocampus) and lungtissue (inferior aspect of right lower lobe) using RNeasy
Plus Mini Kit (QIAGEN, Hilden, Germany) according to
the manufacturer’s instructions. The concentrations of
extracted RNA and RNA Integrity Number (RIN) were
determined using Agilent Technologies RNA Nano Chipas per manufacturer’s instructions. All RNA extracts
were diluted in nuclease-free water to yield a final RNA
concentration of 30 ng/μl. Hippocampal and lung tissue bulk RNA sequencing was performed by Novogene
Singapore.
Bulk RNA‑sequencing data processing and quality control

One hundred fifty-base pair paired-end sequenced reads
were processed using the nf-core/rnaseq v3.10.1 pipeline[39] with nextflow v22.10.4 [40]. Briefly, raw reads were
trimmed using Trim Galore! v0.6.7 [41] to remove lowquality bases and adapters. Trimmed reads were aligned
to the Ovis aries reference genome, ARS-UI_Ramb_v3.0
with STAR v2.6.1d aligner [42]. Finally, Salmon v1.9.0[43] was utilized for assigning reads. Principal component analysis was conducted for quality control assessment and exploratory analysis.

Carter et al. BMC Medicine (2024) 22:338

Differential gene expression and gene set enrichment
analysis

All downstream analyses were performed using R statistical software version 4.2.1 [44]. Differential gene
expression analysis was performed using the DESeq2
package v1.36.0 [45]. Genes with at least 1.5-fold change
in expression and FDR < 0.05 were considered significant.
The results were visualized using the Enhanced Volcano
v1.14.0 [46] and Complex Heatmap v2.12.1 [47]. Gene set
enrichment analysis (GSEA) was run on all genes ranked
by their signed log p-value using cluster Profiler v4.9.0[48] and KEGG database [49].
Hematological and biochemical data acquisition

Plasma isolated from maternal and fetal blood samples
was used for hematological analyses, including whiteblood cell counts (WBC,/μl), differential leukocyte
counts (%), neutrophil count (%), biochemical parameters; gamma-glutamyl transferase (GGT, U/l), alanine
aminotransferase (ALT, U/l), aspartate aminotransferase(AST, U/l), albumin (g/l), cortisol (nmol/L), adrenocorticotropic hormone (ACTH, pg/ml). Those analyses were
performed by an independent clinical pathology laboratory (Vetpath, Perth, Australia). Cortisol values underlimit of detection (< 5.52 nmol/l) were allocated a value of
5.52 nmol/l for statistical analyses. ACTH values underlimit of detection (< 5.0 pg/ml) were allocated a value of
5 pg/ml for statistical analyses.
Statistical analysis

Statistical analyses were performed using IBM SPSS Statistics for Windows, version 25.0 (IBM Corp, Armonk,
NY). Chi-square was used to test the differences of
nominal values between groups. All numerical data were
tested for normality with Shapiro–Wilk tests. In the comparison of the four intervention groups (Dex High, Dex
Low, Beta-Acetate, Saline Control Groups) betweengroup differences in parametric data were tested for significance with one-way ANOVA, while Kruskal Wallis
tests were used for non-parametric data. Multiple post
hoc comparisons were performed with Tukey’s and Dunnett t (2-sided) tests. p values < 0.05 being significant.
Repeated ANOVA test and pairwise comparisons wereused to analyze maternal Saline Control Group baseline
cortisol and ACTH compared to serial time points.

Results
Reduced dose antenatal steroid regimens accelerate fetallung maturation

There were no significant differences in delivery data
among treatment groups (Additional File 1 and Additional Table 1).

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Simulated neonatal steroid plasma concentrations foreach steroid-treated group highlight the pulsatile natureof the dexamethasone regimens compared to a constant
exposure Beta-Acetate group (Fig. 2A, B with pharmacokinetic profile illustrated in Additional Table 2) [8].
Maternal and cord (delivery) steroid plasma measurements and pharmacokinetic parameters are described in
Additional File 1.
Arterial umbilical cord gas ­PaCO2 values serve as arobust and well-established measure of acute preterm
functional lung maturation [50–53]. The Beta-Acetate
Group animals had significantly improved cord P­ aCO2
values compared to Saline Control Group animals at 10,
20, and 30 min of ventilation (p < 0.05) (Fig. 2C (Additional File 1 )). The Dex Low Group animals had significantly improved cord P­ aCO2 values compared to Saline
Control Group animals at 10 and 20 min (p < 0.05) ofventilation, but not significant at 30 min (p = 0.094/NS)(Fig. 2C). There were no statistically significant differences in cord ­PaCO2 between Dex High Group animals
and Saline Control Group at any ventilation timepoint.
Additional lung function data are shown in Additional
Figs. 1A-C,2. Our data show, for the first time, that thehigh-dose, pulsatile, and rapidly cleared dexamethasone
ACS regimen (Dex High Group) currently used clinically in Singapore did not convey statistically significant
improvements in preterm ovine neonatal lung function.
The greatest improvements in preterm neonatal lungfunction were actually associated with constant, lowconcentration and extended-duration fetal steroid exposures represented by the Positive Control (Beta-Acetate)Group.
The effects of different antenatal steroid regimens on fetallung transcriptome related to lung maturation

Principal component analysis (PCA) plots of the fetallung transcriptome profile showed that ACS-treatedanimals were distinct from Saline Control animals(Fig. 3A). Differential analysis identified 1533 significant genes (with at least 1.5-fold change and FDR < 5%)in the steroid-treated groups (Fig. 3B; Supplementary GSEA Table 1). Of these significant differentiallyexpressed genes (DEGs), 567 (37%) were upregulated
while 967 (63%) were downregulated (Fig. 3B). There
was a marked difference between upregulated anddownregulated genes. Upregulated genes common to
all steroid-treated groups related to inflammation andimmune regulation (IL1B, MARCO, C7, CXCL16, FGL1,TNFRSF13B). In comparison, downregulated genes
common to all steroid-treated groups were related tocellular division/ cell cycle (PIMREG, PRR11, FOXM1,
PRC1, KIF18B, ASPM, MKI67, CDCA3, CDCA8,
CDC20) (Fig. 4B).

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Fig. 2 Simulated fetal plasma ACS concentrations and arterial ­PCO2 at 10, 20, and 30 min of ventilation. A Simulated fetal plasma steroid
concentrations comparing Dex High, Dex Low, and Beta-Acetate dosing regimens based on maternal plasma concentration and known
pharmacokinetics of dexamethasone and betamethasone (Additional Table 2) dosing regimens [8]. B Simulated fetal plasma steroid concentrations
comparing Dex Low and Beta-Acetate dosing regimens based on maternal plasma concentration and known pharmacokinetics of dexamethasone
and betamethasone dosing regimens (Additional Table 2) [8]. C Umbilical ­PaCO2 at 10, 20, and 30 min of ventilation (Saline control, Dex High, Dex
Low, Beta-Acetate groups). D ACS Responder, ACS Non-responder and Saline Control group P­ aCO2 at 30 min of ventilation. The Asterix indicatesa significant difference among the groups. Error bars represent ± 1 standard deviation

Animals in the Positive Control Beta-Acetate Group(demonstrating the highest degree of lung maturation)
had the greatest number of group-specific significant
DEGs (708 genes), compared to Dex Low (170 genes) andDex High (58 genes) (Fig. 3B). Dex Low and Beta-Acetate
Group animals, which had the longest predicted durationof fetal ACS-exposure and functional lung maturation,

had upregulation of established markers of lung maturation including surfactant production and transport (SFTPAI, SFTPB, SFTPC, CTSH, LPCAT1, LPCAT3, SCNN1A[54]) genes related to immune response/inflammation(LAMP3, C5), and increased vasculature (HIF3A, EPAS1)(Figs. 3C and 4B). Dex Low and Beta-Acetate Groupanimals also had common downregulation of genes

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Fig. 3 Fetal lung RNA bulk sequencing for treatment groups. A Principal component analysis (PCA) plot of differential gene expression
for intervention groups Dex High / Dex Low / Beta-Acetate/ Saline Control Groups. B Venn diagram demonstrating differential up and down
gene regulation between Dex High/ Dex Low/ Beta Acetate group vs Saline Control group. C Heat map displaying differential gene expression
for twenty different genes related to fetal lung maturation [54] for each intervention group (Saline Control, Dex High, Dex Low, Beta-Acetate). D
Heatmap demonstrating the significant positively and negatively enriched KEGG Pathways (DEPs) (Fold change > 1.5, False Discovery Rate FDR < 5%)
between steroid treated groups (Dex High, Dex Low, Beta-Acetate) compared to Saline Control Grouprelated to extracellular matrix and cell adhesion (MFAP5,
CDH12, NFASC, FAP) (Fig. 4B). Consistent with functional studies, these markers of lung maturation were notsignificantly differentially expressed in Dex High Groupanimals (Figs. 3C and 4B).
To understand the biological themes associated with theDEGs, we conducted gene set enrichment analysis (GSEA)[48] with KEGG pathways [49] on all genes ranked by thesigned negative log p-value. Steroid-treated groups werecompared to Saline Control Group (Fig. 3D, Supplementary
GSEA Table 2). Overall, 93 (82.8%) KEGG pathways werepositively enriched while 16 (17.2%) KEGG pathways were
negatively enriched. We identified 22 positively enriched
KEGG pathways common to all steroid-treated groups
that are related to cellular metabolism, inflammation, andhuman disease (Fig. 3D). Six negatively enriched pathways
were common to all steroid-treated groups related to cellular homeostasis and reproduction (Fig. 3D).

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Table 1 Summary of the clinically relevant functional lung maturation status, differential gene and KEGG pathway expression for eachtreatment group compared to Saline Control group
Treatment group Functional lung maturation Lung tissue
Hippocampal tissue
Genes related to lung maturation Genes related toneurodegeneration

Hippocampal tissue
KEGG pathways
related toneurodegenerative
disordersDex HighNOT SIGNIFICANTLYIMPROVEDNOT SIGNIFICANTLY EXPRESSEDSIGNFICANTLY EXPRESSED- Prion disease- Alzheimer’s disease- AA metabolism- Spinocerebellar ataxia

Dex Low

VARIABLE IMPROVEMENTSIGNIFICANTLY EXPRESSEDSIGNIFICANTLY EXPRESSED- Prion disease- Alzheimer’s disease- AA metabolism- Parkinson disease- Huntington disease- Pathways of neurodegeneration – multiple
diseases- Amyotrophic lateralsclerosis

Beta-Acetate

SIGNIFCANTLY IMPROVEDSIGNIFICANTLY EXPRESSEDSIGNIFICANTLY EXPRESSED

Nil

Understanding the transcriptomic fetal lung maturation
response to ACS exposures

ACS treated animals were defined and grouped as ACS
Responder if the ­PaCO2 after 30 min of ventilation was
more extreme than two standard deviations below themean ­PaCO2 value for the Saline Control Group [12].
ACS response rates observed between steroid-treatedgroups were Dex High Group 8.33% (1/12), Dex Low
Group 25% (3/12), and Beta-Acetate Group 42.86% (3/7)(Fig. 2D). When compared to Saline Control Group animals, the ACS response rate was only significantly higher
in the Beta-Acetate Group animals (p = 0.048).
PCA plots revealed distinct clusters of Saline Control, ACS Responder, and ACS Non-Responder Groups(Fig. 4A). Comparative analysis comparing DEGs of ACS
Responders to Non-Responders showed 101 significant
DEGs (Supplementary GSEA Table 3). Responder Groupanimals had significant upregulation of genes known to be
associated with fetal lung maturation and surfactant production (SFTPA1, SFTPC, and SFTPB), inflammation andimmune response (CXCL8, CXCL17, C5, PLAUR​), and downregulation of genes related to extracellular matrix remodelling (COL12A1, NOG) (Fig. 4B). Analysis of differential
KEGG pathway expression between ACS Responders
and Non-Responders are presented in Fig. 4C (Supplementary GSEA Table 4). Responder Group animals had
positive enrichment of 50 KEGG pathways (related toinflammation, lipid, and sugar metabolism) and negativeenrichment of nine KEGG pathways (related to cell andDNA replication).

Antenatal steroid exposure causes
neurodegenerative‑associated changes to the fetal
hippocampal transcriptome

PCA plots for all treatment groups as well as ACS
Responder and Non-Responder Groups are shown in
Fig. 5A. When compared to Saline Control Group, differential analysis identified 879 significant DEGs (at least
1.5-fold change and FDR < 5%) in the steroid-treatedgroups (Fig. 5B, Supplementary GSEA Table 5). Common
to all steroid-treated groups were 41 upregulated DEGs
and 26 downregulated DEGs (Fig. 5B, Additional Fig. 3).
The Positive Control Beta-Acetate Group animals had
the greatest number of group specific, significant DEGs
(371 genes) compared to Dex Low (169 genes) and Dex
High (67 genes) (Fig. 5B).
Analyses were conducted for genes related to neurodevelopment and neurodegeneration. Upregulated
genes common to all steroid-treated groups included
NMB, CCN3, APOD, and NPPC, while downregulated
genes common to all steroid groups included ISLR2 andNTNG2 (Fig. 5C,D). Dex High and Beta Acetate Groupanimals had common upregulation of AZIN2 and C4A.
Dex Low and Positive Control Group animals had common upregulation of DRD5 and downregulation of SVB2.
Finally, Positive Control Group animals had downregulation of GLDN, which was not seen in the other steroidtreated groups.
Steroid-treated groups were compared to Saline Control Group animals in terms of differential KEGG pathway expression. Overall, the majority (75.8%) of KEGG

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Fig. 4 Fetal lung RNA bulk sequencing for ACS responder vs non-responder groups. A Principal component analysis (PCA) plot of differential
gene expression for ACS Responder, ACS Non-Responder and Saline Control groups. B Heat map displaying differential gene expression for genes
related to fetal lung maturation, angiogenesis, inflammation, extra-cellular matrix, and cell cycle for ACS Responder vs ACS Non-Respondergroups and by specific treatment group (Dex High, Dex Low, Beta-Acetate) compared to Saline Control. C Wrap plot demonstrating the significant
positively and negatively enriched KEGG Pathways (DEPs) (Fold change > 1.5, False Discovery Rate FDR < 5%) for ACS Responder vs ACS
Non-responder

pathways in the steroid-treated groups were negatively
enriched (Fig. 6A, Additional File 1 and Supplementary
GSEA Table 6). We identified two positively enriched(“Non-Alcoholic Fatty Liver Disease” & “Ribosome”) and16 negatively enriched DEPs (pathways related to cellular
homeostasis & infection) common to all steroid-exposedgroups (Fig. 6B).
Pulsatile dexamethasone-only exposed groups (DexHigh and Dex Low) had three common positively

enriched DEPs related to neurodegeneration (“Prion Disease”, “Alzheimer’s Disease”, “Arachidonic Acid metabolism”) and two common negatively enriched DEPs(“Human T-cell leukemia virus 1 infection”, “Cellular
Senescence”) (Fig. 6B). Furthermore, in the pulsatile, highamplitude Dex High Group, we identified unique positive enrichment of the KEGG pathway “Spinocerebellar
Ataxia” (Fig. 6B) and group specific positive enrichment
in the low amplitude, pulsatile Dex Low Group of

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Fig. 5 Hippocampal bulk RNA sequencing data for differentially expressed genes. A Principal component analysis (PCA) plot of differential gene
expression for intervention groups Dex High / Dex Low / Beta Acetate/ Saline Control groups and ACS Responder / ACS Non-Responder subgroups.
B Venn diagram demonstrating differential up and down gene regulation (DEG) between Dex High/ Dex Low/ Beta Acetate group vs Saline
Control group. C Volcano plots of differential gene expression (DEG) for each treatment group Dex High, Dex Low, and Beta Acetate comparedto Saline Control group. D Heat map displaying significant differential gene expression of eight different genes related to neurodegenerative/
neuropsychiatric disorders for ACS Responder vs ACS Non-Responder and by specific treatment group (Dex High, Dex Low, Beta-Acetate) comparedto Saline Control“Parkinson Disease,” “Huntington disease,” “Pathwaysof neurodegeneration – multiple diseases,” and “Amyotrophic lateral sclerosis” (Fig. 6C).
Comparisons between Dex Low and Positive Control (Beta-Acetate) Group animals had five common

positively enriched DEPs (“Oxidative Phosphorylation”
and Chemical carcinogenesis – ROS”) and 11 common
negatively enriched DEPs (including “Wnt Signaling”)(Fig. 6B). Dex High and Beta-Acetate Group comparison had no common positively enriched and only fourCarter et al. BMC Medicine (2024) 22:338

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Fig. 6 Hippocampal bulk RNA sequencing data for differentially expressed KEGG pathways. A Venn diagram demonstrating differential
up and down KEGG pathway expression (DEP) between Dex High / Dex Low / Beta Acetate groups vs Saline Control group. B Heat map
demonstrating the significantly positively enriched and significantly negatively enriched differentially expressed KEGG Pathways (DEP) (Foldchange > 1.5, False Discovery Rate FDR < 5%) in common between all steroid-treated groups (Dex High/ Dex Low/ Beta Acetate), Dex High groupspecific and Beta Acetate group specific, compared to Saline Control. C Heat map demonstrating the Dex Low group specific 14 significantly
positively enriched and 65 significantly negatively enriched differentially expressed KEGG Pathways (DEP) (Fold change > 1.5, False Discovery Rate
FDR < 5%) compared to Saline Control groupcommon negatively enriched DEPs (“Motor proteins,”“DNA replication,” “African Trypanosomiasis,” “cellcycle”) (Fig. 6B). The lack of commonality between thesegroups may reflect the significant differences in pharmacokinetic profiles between these groups (high amplitude
pulsatile vs. low amplitude constant exposure) respectively (Fig. 2A). Surprisingly, the constant, low amplitude
exposure Beta-Acetate Group had only one additional
unique KEGG pathway (“Phagosome”), despite having
the greatest number of significant DEGs (Fig. 6B).
Neurodegenerative changes in the fetal hippocampal
transcriptome relate to steroid exposureand not ventilation outcome

Analysis of ACS Responder compared to ACS NonResponder Groups for significant DEGs surprisingly
revealed limited differential expression, none of which
related specifically to neurodegeneration (one uncharacterized upregulated gene and 37 downregulated genes)(Supplementary GSEA Table 7). By comparison, bothsteroid-exposed groups (Responder and Non-Responder
Groups) when compared to saline control respectively,
demonstrated the same pattern of upregulation in thegenes related to neurogenerative and neuropsychiatric disorders being NMB, CCN3, APOD, NPPC, AZIN2,
DRD5 and downregulation of NTNG2 (Fig. 5D, Supplementary GSEA Table 8). Notably, these genes were notsignificantly differentially expressed when ACS Responders were compared to Non-Responder Groups.
Finally, KEGG pathway analysis of ACS Responders vs.
Non-Responder subgroups did not demonstrate positive or negative enrichment for any pathways related toneurodegenerative disorders (Supplementary GSEA

Table 9 and Supplementary GSEA Table 10). These findings therefore suggest that the observed adverse effects of
ACS exposure on neurodevelopment are related to ACS
drug exposure and not impacted by lung maturation status and subsequent ventilation outcomes.
Cardiac, endocrine, immunological, and hepatic effects

Data describing the effects of different steroid dosing regimens on key homeostatic processes are shown in Additional Figs. 4–6. No outcomes likely to impact the lungmaturation or transcriptomic data presented herein wereidentified.

Discussion
Principal findings

The principal findings of this study are that:(i) An ACS dosing regimen used clinically in Singapore, conveying a high amplitude, pulsatile, andrapidly cleared dexamethasone ACS regimen (represented by the Dex High Group) did not demonstrate statistically significant improvements in fetallung maturation. Furthermore, consistent withearlier reports [50], a single dose of 0.125 mg/kg
Beta-Acetate induced robust fetal lung maturation
based on significantly improved ­PaCO2 (Fig. 2C),
dynamic lung compliance, VEI, and lung tidal volumes (Additional Fig. 1). The comparably poor
maturation effect observed in the high-dose dexamethasone group is consistent with our in silico
modelling and in vivo measurements of maternofetal dexamethasone exposures following two
12 mg maternal intramuscular injections given at a12-h interval [8, 11]. In this regimen, dexametha-

Carter et al. BMC Medicine (2024) 22:338

sone is rapidly cleared from the fetal compartment,with fetal plasma concentrations likely at sub-therapeutic levels within 24 h of treatment commencing. These data are consistent with our previous
findings, which showed that robust preterm lungmaturation in the sheep requires an uninterrupted
exposure of 1–2 ng/ml plasma betamethasone forat least ~ 30 h when delivering animals at a 48 htreatment to delivery interval [53, 55, 56]. The mostrobust maturation was seen in animals that had
this level of steroid exposure at the time of delivery and ventilation [56]. These data further support
optimizing of ACS therapy by utilizing a reduced
dose, extended-duration regimens;(ii) Significant patterns of gene expression corresponding with fetal lung maturation were only
observed in animals from the reduced dose, andextended duration ACS regimens (Dex Low andBeta-Acetate Groups). Examples of this pattern of gene expression include the upregulationof genes related to surfactant production/function (SFTPAI, SFTPB, SFTPC, CTSH, LPCAT1,
LPCAT3, SCNN1A) [57], and increased vasculature (HIF3A, EPAS1) as well as downregulation ofgenes related to extracellular remodelling (MFAP5,
CDH12, NFASC, FAP). This pattern of transcriptome changes relating to lung maturation was notobserved in the Dex High group, which was also
demonstrated at a physiological level, with poor
ventilation outcomes and therefore poor functionallung maturation. These data are especially relevant
since the Dex High regimen is recommended forclinical use in patients at risk of preterm birth inmany countries.
(iii) ACS-exposed animals had significantly dysregulated hippocampal RNA transcriptomes comparedto Saline Control Group animals, suggesting thepotential for altered neurodevelopment irrespective of the dose or pharmacokinetic profile of the
ACS regimen employed. Despite being exposed to
hypercapnia, hypoxia, and more pronounced acidosis, animals in the Saline Control Group from aneurodevelopmental injury perspective, demonstrated a far more benign transcriptome, compared
to both ACS-responding and non-responding animals; and(iv) The most concerning findings were seen in the
animals exposed to both high and low-dose, pulsatile dexamethasone regimens (Dex High and Dex
Low Groups). They had the greatest degree of significant enrichment of KEGG pathways associatedwith neurodegenerative disorders. For example,
both pulsatile dexamethasone exposed groups (DexPage 12 of 17

High and Dex Low) exhibited positive enrichmentof pathways related specifically to neurodegenerative disorders, including “Prion Disease,” “Alzheimer’s Disease,” and “Arachidonic Acid (AA) Metabolism” (Fig. 6B/C). By comparison, none of thesepathways were activated in the constant exposure,
low-dose Beta-Acetate Group (Table 1).
Clinical implications

ACS therapy was introduced into clinical practice progressively over the past 50 years although there was initially much resistance to it. In fact, there was so muchconcern about failure to use it, that the NICHD held aconsensus conference to generate publications to warn
obstetricians that failure to use steroids for prematurelabor would be very problematic medicolegally [58, 59].
Recently there has been clinical concern about the overuse of such treatments and deleterious consequencesof such. In current practice, ACS is still very commonly
administered to women at risk of preterm delivery.
Although some 11% of all babies are born preterm, many
more pregnant women are judged to be at risk of preterm
delivery and are given ACS therapy. As such, a very large(perhaps as much as 20%) of mothers and fetuses are now
exposed to high-dose ACS.
Despite the very common usage, optimization of how
and when to use ACS has never been achieved, and theeffects of drug pharmacokinetics on the brain, lungs, andother vital fetal systems, remains poorly understood.
Understanding the molecular mechanisms and transcriptomic changes behind the fetal lung maturation
responses to ACS, as well as the risk of injury to thedeveloping fetal brain, are critical to deciding the proper
usage of such powerful pharmaceuticals [12].
We now have molecular tools capable of answering
many questions that were not possible when ACS was
introduced. In this study, we have highlighted the critical role of the surfactant genes SFTPA1, SFTPB, andSFTPC in the fetal lung maturation response by demonstrating significant upregulation of these genes in the
ACS Responder group compared to the non-respondergroup and Saline control. The late stages of fetal lungdevelopment require mesenchymal thinning to promote
gas exchange [60]. Correspondingly, we observed significant downregulation in genes related to extracellularmatrix (ECM) remodelling and collagen synthesis in the
fetal lungs of the Responder group compared to the nonresponder group and Saline control which fits with thisestablished process of mesenchymal thinning. Schmidt
et al., utilizing a non-human primate model of lung maturation, also observed downregulation in genes related
to ECM remodelling and mesenchymal growth factors

Carter et al. BMC Medicine (2024) 22:338[61]. These findings help inform on further targeted
approaches to inducing preterm fetal lung maturation to
improve the response rates to treatment.
Despite concerning associations observed in large
population cohort studies, indicating an elevated risk ofneurobehavioral/learning disorders in children exposed
to ACS in utero, the neurodevelopmental effects of various ACS exposures on the fetal brain are still not wellunderstood [14, 15]. In this study we have demonstrated
that all steroid-treated groups had common upregulationof genes related to dysregulation of neurodevelopment
including Neuromedin B (NMB), Cellular Communication Network Factor 3 (CCN3), Apolipoprotein D(APOD), Natriuretic Peptide C (NPPC). NMB is a neuropeptide that has been associated with the acute stressresponse and anxiety in animals [62], while animal models have demonstrated that elevated CCN3 impaired
hippocampal differentiation and normal hippocampal
neurogenesis [63]. Similarly, elevated levels of APOD
have been found in the CSF and hippocampus of patientswith Alzheimer’s disease [64], while elevated NPPC has
been shown to negatively affect hippocampal neuroplasticity and memory in rat models [65].
All steroid-treated groups also demonstrated downregulation of the genes Immunoglobulin superfamily
containing leucine rich repeat 2 (ISLR2) and Netrin G2(NTNG2). ILSR2 is a key factor for axon guidance andbrain development [66, 67]. Deficiency of ILSR2 is associated with the development of hydrocephalus in miceand clinical studies [66, 67]. Reduced mRNA levels of
NTNG2 have also been demonstrated in the brains ofpatients with bipolar disorder and schizophrenia, thereby
implicating NTNG2 in the pathophysiology of these diseases [68].
Further, groupwise comparisons of Dex High andBeta-Acetate Group animals revealed common significant upregulation of the genes Antizyme Inhibitor 2-like(AZIN2) and Complement Component 4A (C4A). C4A
is elevated in the CSF and brain tissue of patients withschizophrenia and is associated with increased synaptic
pruning [69, 70], while AZIN2 has been found in highlevels in the brains of Alzheimer’s patients [71]. Dex Low
and Beta-Acetate group animals both had significant
upregulation of Dopamine Receptor D5 (DRD5), which
has also been described in association with AttentionDeficit/Hyperactivity Disorder (ADHD) [72] and common downregulation of Synaptic Vesicle Glycoprotein 2B(SVB2) which is known to be downregulated in the brainsof patients with temporal lobe epilepsy (TLE) [73]. In
particular, SVB2 has been shown to be downregulated in
the brains of patients with pharmaco-resistant TLE which
paralleled increased synaptic loss [74]. Finally, although
overall of less concern from a developmental perspective,

Page 13 of 17

animals in the Beta Acetate group had unique downregulation of Gliomedin (GLDN) which is an important factor
in the development of nodes of Ranvier in the peripheral
nervous system [75].
Taken together, these data reveal the impact of ACS
treatment on the hippocampal transcriptome, with amaterial number of differentially regulated genes thatare associated with neurodegenerative disorders increasingly identified in large, population-level cohort studies[14–16]. Further research is needed to establish a causal
relationship of these findings with the epidemiologicalstudies that have shown the development of learning difficulties and neurobehavioral disorders in individuals
exposed to ACS [14, 15]. Data from our study and others
certainly suggest potential connections which warrant
further exploration in long-term clinical trial follow up
studies.
Research implications

Understanding the molecular pharmaco-molecular relationships between ACS exposure and brain injury isimportant to the optimisation of ACS intervention. In
addition to differential gene expression in the fetal hippocampus, all steroid exposed groups in the present
study had downregulation of the KEGG pathways “Gapjunction,” “Focal adhesion,” and “ECM -receptor activation” which are known to be involved in normal neuron
migration, gene expression, cell proliferation, differentiation, and survival [76–80]. Further analysis of thesenegatively enriched pathways revealed common downstream signaling processes including PI3K-AKT, RasMAPK-ERK, JAK-STAT, and NFҡB signaling. PI3K-AKT
signaling is known to be integral for normal brain development, sympathetic neuron growth, and survival [81]with clinical studies demonstrating that AKT gene deletion syndromes exhibit microcephaly and agenesis of
the corpus callosum phenotypes [82]. Furthermore, RasMAPK-ERK signaling is an established master regulator of neurodevelopment, important for normal neuraltissue proliferation and differentiation. Specifically, ERK
signaling is an integral component of learning, memory,and behavior [83], with animal studies demonstratingthat inhibition and downregulation of ERK signaling canresult in repressed long-term potentiation, memory consolidation, depression-like behavior, and impaired fear
memory consolidation [83, 84]. Additionally, JAK/STAT
signaling is important in CNS synaptic plasticity, memory, and learning [85], with downregulation in the JAK/STAT pathways (in particular JAK2/STAT3) being implicated in age-related memory decline and Alzheimer’s
disease [86]. Finally, NFҡB signaling is also important tonormal neurodevelopment, with deficits in neural plasticity and memory formation a consequence of negatively

Carter et al. BMC Medicine (2024) 22:338

enriched NFҡB functioning [87]. These relationships
need further clarification.
In summary, our data demonstrate that all ACS
exposed groups, including those with substantial dose
reductions, had significant downregulation within thepreterm hippocampus of crucial signaling pathways(PI3K-AKT, Ras-MAPK-ERK, JAK-STAT, and NFҡB)
for normal neurogenesis and development. These findings may also help explain a causal link between ACS
exposure and associated increased risks of learning deficits, mental, and behavioral disorders seen in population
cohort studies of ACS exposed children [14–16].
Limitations of this study

One key limitation for clinical application at this point is
our inability to differentiate between potential differencesin effect between betamethasone and dexamethasone.
Due to agent availability and the clinical regimen of interest, we assessed rapid-release dexamethasone phosphate(pulsatile exposures at high and low doses) against betamethasone acetate which has achieved a slow-release,constant exposure profile. Although both drugs act via
the glucocorticoid receptor, and the projected ­Cmax for
the Beta Acetate and Dex Low Groups were approximately equal, the differences in transcriptional effects
seen in this study may stem from differences in the activation profile of each drug. This is not a well-studied areaof glucocorticoid pharmacogenomics, and aside fromdifferences in receptor affinity, betamethasone and dexamethasone are viewed as being interchangeable from theperspective of signalling action. Whether or not dexamethasone and betamethasone elicit markedly different
transcriptional responses at identical drug exposures is
an intriguing question. However, as dexamethasone is
the front-line ACS agent for much of the world, our data
showing that dexamethasone exposure was associatedwith an increased risk of adverse transcriptional changes
in the fetal brain, may have serious implications for how
this drug is used in obstetric practice, as well as the supply of the less readily available betamethasone-based
preparations. It is also important to note that our studies relate to the acute transcriptomic effects of ACS on
the brain and lung. Although we have identified injuryassociated changes in the hippocampus independent of
respiratory function, it is not clear if these alterations
are maintained into childhood or contribute to a neurological phenotype consistent with that reported in theepidemiological and trials literature. It is also important
to note that all animals were delivered at a standardized
gestational age, following a fixed exposure interval. As
lung and brain responses to glucocorticoid exposure are
dynamic processes, it is important to note that the different findings may be observed following delivery atPage 14 of 17

different gestations or treatment to delivery intervals. We
also acknowledge that we did not do further analysis on
fetal cardiac function beyond heart rate and blood pressure (Additional File 1, Additional Figure 4); therefore,
we cannot exclude that undetected adverse cardiovascular instability due to ANS exposure may have impacted
other organ systems as well as respiratory and neurological development.

Conclusions
In summary, our findings show that, in the preterm fetal
sheep, a therapeutically beneficial (for lung maturation)dose of glucocorticoids delivered via the maternal compartment cannot be accomplished without materiallydisrupting key homeostatic systems in both mother andfetus.
In this study, we show for the first time that a high-dose
regimen currently in clinical use in Singapore, fails toachieve its primary goal of improving functional maturation of the preterm fetal sheep lung, but causes marked,
neurodegenerative/psychiatric disease-associated transcriptional changes in the preterm hippocampus, andseverely disrupts the materno-fetal HPA axis. We additionally show, also for the first time, that adverse changes
in the hippocampal transcriptome were not affected bysignificant dose reduction in the dexamethasone ACS
groups, and that these changes occurred independentof functional lung maturation status. These indicate thatadministering ACS at a dose necessary for functional
maturation of the preterm lung may not be achievable
without an elevated risk of adverse impacts on the developing brain.
Additionally, although transcriptional changes seen in
the hippocampal tissue of Beta-Acetate Group animals
were less extreme (to pulsatile ACS-exposures), they still
represented material changes consistent with increased
risk of harm. Together with additional findings of poor
lung maturation in animals exposed to very brief, highamplitude, pulsatile dexamethasone treatments, thesedata strongly underscore the importance of judicious
patient selection, appropriately treatment targeting, andthe need to develop constant, extended-duration glucocorticoid regimens to maximize benefit and minimize
risk of harm deriving from ACS therapy.
Both the magnitude (dose) of materno-fetal antenatal steroid exposure, and the pharmacokinetic profileof that exposure (i.e., constant vs. pulsatile) contribute
quantitatively and qualitatively to transcriptomic perturbation in the preterm fetal hippocampus. Since ACS
dosing worldwide is based on pulsatile dosing regimens
and high-dose dexamethasone is widely utilized as theantenatal steroid of choice, our data have significant
implications for on-going efforts to optimize the safety

Carter et al. BMC Medicine (2024) 22:338

Page 15 of 17

and efficacy of this important obstetric intervention.
Just as the dosing of oxytocin for labor induction andaugmentation underwent a complete rethink a few decades ago with dramatic reduction in dosage, these datastrongly suggest a need to conceptualize a similar process for antenatal steroids.

gov/geo/query/acc.cgi?acc = GSE248786). All supplementary GSEA tables,
physiological and haematological data presented in figures and text within
this manuscript is available for review in the Supplementary GSEA Tables andSupporting Data Values Files at https://doi.org/https://​doi.​org/​10.​6084/​m9.​
figsh​are.​25532​740.​v1.

Abbreviations
ACS	Antenatal corticosteroids
DEG	Differentially expressed genes
DEP	Differentially expressed pathways
Dex P	Dexamethasone phosphatedGA	Days gestations age
FDR	False Discovery Rate
GSEA	Gene set enrichment analysis
HPA	Hypothalamic pituitary adrenal
IM	Intramuscular
LC-MS	Liquid chromatography-mass spectrometry
PEEP	Positive end expiratory pressure
PIP	Peak inspiratory pressure
PTB	Preterm birth
RR	Respiratory rate
VEI	Ventilation Efficiency Index
VT	Tidal volume

Ethics approval and consent to participate
Our studies were approved by The University of Western Australia’s Animal
Ethics Committee (2021/ET000974) and adhered to the NIH Guide for the Careand Use of Laboratory Animals.

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12916-​024-​03542-5.
Additional File 1. (Additional Tables 1-2, Additional Figures 1-6): Maternal
and neonatal plasma steroid concentrations, Neonatal delivery data,
Neonatal respiratory physiological data at 10, 20 and 30 minutes ofventilation,Neonatal ventilation data lung maturation analysis, Fetal
Hippocampal transcriptome changes after antenatal steroid exposure,
Maternal and fetal plasma endocrine assessments, Maternal and fetal
immune modulation, Fetal Liver Function.  Additional Table 1: Summary of delivery data. Additional Table 2: Pharmacokinetic data for
Betamethasone Acetate following single 0.125mg/ kg dose. Additional
Figure 1: Preterm neonatal respiratory physiological data at 10, 20 and 30
minutes of ventilation.  Additional Figure 2: Preterm neonatal ventilation
data lung maturation analysis. Additional Figure 3: Hippocampal bulk RNA
sequencing data for differentially expressed genes common to all steroid
treated groups. Additional Figure 4: Preterm neonatal cardiovascular data
at 10, 20, 30 minutes of ventilation. Additional Figure 5: Maternal and fetal
plasma endocrine data. Additional Figure 6: Plasma Immune and liverfunction data.
Authors’ contributions
Study design and conceptualisation: SC, MK Animal studies SC, HU, EF, YT, TT, YK,
HI, YS, ZA, BA, MS, MK Laboratory studies SC, MK, QW, XL, Data analysis SC, MK, AR,
GO, AHJ Manuscript preparation SC Manuscript review SC, SI, MK, ME, AR, CM, MC,
AHJ Manuscript approval SC, MK, HU, EF, YT, TT, CM, BA, ZA, GO, AR, QW, XL, YK, YS,
HI, MS, MC, SI, AHJ, ME. All authors read and approved the final manuscript.
Funding
This work was supported by grants to MWK from The Channel 7 Telethon
Trust Western Australia, The Department of Health, Government of Western
Australia, The Stan Perron Charitable Foundation, The National Health andMedical Research Council (GNT1162572), The National University of Singapore
NUHSRO/2021/075, The Ministry of Education, Government of Singapore,NUHSRO/2021/109/T1/Seed-Sep/02.
Availability of data and materials
The raw data and processed data for RNA-sequencing experiments discussed
in this publication have been deposited in the NCBI’s Gene Expression
Omnibus [88] and are publicly available as of the date of publication throughthe GEO Series accession number GSE248786 (https://www.ncbi.nlm.nih.

Declarations

Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Author details
1
Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, 1E Kent Ridge Road NUHS Tower Block,
Level 12, Singapore 119228, Singapore. 2 Division of Obstetrics and Gynaecology, University of Western Australia, Perth, Australia. 3 Centre for Perinatal
and Neonatal Medicine, Tohoku University Hospital, Sendai, Japan. 4 Genome
Institute of Singapore, Agency for Science, Technology and Research (A*STAR),
60 Biopolis Street, Genome #02‑01, Singapore 138632, Republic of Singapore.
5
Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore. 6 Department of Neonatology Khoo
Teck Puat, National University Children’s Medical Institute, National University
Hospital, Singapore, Singapore. 7 Icahn School of Medicine at Mount Sinai,
New York, NY, USA. 8 Fetal Medicine Foundation of America, New York, NY, USA.
9
Reproductive Biology Program, Center for Biomedical Research and Innovation, Universidad de los Andes, Santiago, Chile. 10 IMPACT​, Center of Interventional Medicine for Precision and Advanced Cellular Therapy, Santiago, Chile.
11
Centre for Pulmonary Biology, Cincinnati Children’s Hospital Medical Centre,
Cincinnati, OH, USA. 12 Women and Infants Research Foundation, Perth, WA,
Australia.
Received: 12 May 2024 Accepted: 25 July 2024
Published: 26 August 2024

References
1. Blencowe H, Cousens S, Chou D, Oestergaard M, Say L, Moller AB, et al.
Born too soon: the global epidemiology of 15 million preterm births.
Reprod Health. 2013;10 Supp 1(Suppl 1):S2.
2. McGoldrick E, Stewart F, Parker R, Dalziel SR. Antenatal corticosteroids foraccelerating fetal lung maturation for women at risk of preterm birth.
Cochrane Database Syst Rev. 2020;12:CD004454.
3. Roberts D, Dalziel S. Antenatal corticosteroids for accelerating fetal lungmaturation for women at risk of preterm birth. Cochrane Database Syst
Rev. 2007;4:1–144.
4. Roberts D, Brown J, Medley N, Dalziel SR. Antenatal corticosteroids foraccelerating fetal lung maturation for women at risk of preterm birth.
Cochrane Database Syst Rev. 2017;3:1–191.
5. Norman J, Shennan A, Jacobsson B, Stock SJ, Birth FWGfP. FIGO goodpractice recommendations on the use of prenatal corticosteroids toimprove outcomes and minimize harm in babies born preterm. Int J
Gynaecol Obstet. 2021;155(1):26–30.
6. Practice CoO. Committee Opinion No. 713: antenatal corticosteroid
therapy for fetal maturation. Obstet Gynecol. 2017;130(2):e102-e9.
7. Panel ACCPG. Antenatal corticosteroids given to women prior to birth toimprove fetal, infant, child and adult health: Clinical Practice Guidelines. 2015.
8. Gosavi A, Amin Z, Carter SWD, Choolani MA, Fee EL, Milad MA, et al. Antenatal corticosteroids in Singapore: A clinical and scientific assessment.
Singapore Med J. 2023(0):09. Kemp MW, Newnham JP, Challis J, Jobe AH, Stock S. The clinical use ofcorticosteroids in pregnancy. Hum Reprod Update. 2016;22(2):240–59.

Carter et al. BMC Medicine (2024) 22:338

10. Jobe AH, Milad MA, Peppard T, Jusko WJ. Pharmacokinetics andpharmacodynamics of intramuscular and oral betamethasone anddexamethasone in reproductive age women in India. Clin Transl Sci.
2020;13(2):391–9.
11. Jobe AH, Kemp M, Schmidt A, Takahashi T, Newnham J, Milad M. Antenatal corticosteroids: a reappraisal of the drug formulation and dose. Pediatr
Res. 2021;89(2):318–25.
12. Takahashi T, Jobe AH, Fee EL, Newnham JP, Schmidt AF, Usuda H, et al. The
Complex Challenge of Antenatal Steroid Therapy Non-Responsiveness.
Am J Obstetr Gynecol. 2022;227(5):96–704.
13. Rodriguez A, Wang Y, Ali Khan A, Cartwright R, Gissler M, Järvelin M-R.
Antenatal corticosteroid therapy (ACT) and size at birth: A populationbased analysis using the Finnish Medical Birth Register. PLoS Med.
2019;16(2): e1002746.
14. Räikkönen K, Gissler M, Kajantie E. Associations between maternal antenatal corticosteroid treatment and mental and behavioral disorders inchildren. JAMA. 2020;323(19):1924–33.
15. Lin Y-H, Lin C-H, Lin M-C, Hsu Y-C, Hsu C-T. Antenatal Corticosteroid
Exposure Is Associated with Childhood Mental Disorders in Late Preterm
and Term Infants. J Pediatr. 2022;253:245–51.
16. Stutchfield PR, Whitaker R, Gliddon AE, Hobson L, Kotecha S, Doull IJ.
Behavioural, educational and respiratory outcomes of antenatal betamethasone for term caesarean section (ASTECS trial). Arch Dis Child Fetal
Neonatal Ed. 2013;98(3):F195–200.
17. Gyamfi-Bannerman C, Thom EA. Antenatal Betamethasone for Women at
Risk for Late Preterm Delivery. N Engl J Med. 2016;375(5):486–7.
18. Niwa F, Kawai M, Kanazawa H, Iwanaga K, Matsukura T, Shibata M, et al.
Limited response to CRH stimulation tests at 2 weeks of age in preterm
infants born at less than 30 weeks of gestational age. Clin Endocrinol.
2013;78(5):724–9.
19. Waffarn F, Davis EP. Effects of antenatal corticosteroids on the
hypothalamic-pituitary-adrenocortical axis of the fetus and newborn:
experimental findings and clinical considerations. Am J Obstet Gynecol.
2012;207(6):446–54.
20. Schäffer L, Luzi F, Burkhardt T, Rauh M, Beinder E. Antenatal betamethasone administration alters stress physiology in healthy neonates. Obstet
Gynecol. 2009;113(5):1082–8.
21. Tegethoff M, Pryce C, Meinlschmidt G. Effects of intrauterine exposure tosynthetic glucocorticoids on fetal, newborn, and infant hypothalamicpituitary-adrenal axis function in humans: a systematic review. Endocr
Rev. 2009;30(7):753–89.
22. Weiss SJ, Keeton V, Richoux S, Cooper B, Niemann S. Exposure to antenatal corticosteroids and infant cortisol regulation. Psychoneuroendocrinology. 2023;147: 105960.
23. Davis E, Waffarn F, Uy C, Hobel C, Glynn L, Sandman C. Effect of prenatal
glucocorticoid treatment on size at birth among infants born at term
gestation. J Perinatol. 2009;29(11):731–7.
24. Asztalos E, Willan A, Murphy K, Matthews S, Ohlsson A, Saigal S, et al.
Association between gestational age at birth, antenatal corticosteroids,
and outcomes at 5 years: multiple courses of antenatal corticosteroids
for preterm birth study at 5 years of age (MACS-5). BMC Pregnancy Childbirth. 2014;14(1):272.
25. Uno H, Lohmiller L, Thieme C, Kemnitz JW, Engle MJ, Roecker EB, et al.
Brain damage induced by prenatal exposure to dexamethasone in fetal
rhesus macaques I Hippocampus. Dev Brain Res. 1990;53(2):157–67.
26. Velísek L. Prenatal corticosteroid impact on hippocampus: implications
for postnatal outcomes. Epilepsy Behav. 2005;7(1):57–67.
27. Whitelaw A, Thoresen M. Antenatal steroids and the developing brain.
Arch Dis Child Fetal Neonatal Ed. 2000;83(2):F154–7.
28. Tijsseling D, Wijnberger LDE, Derks JB, van Velthoven CTJ, de Vries WB, van
Bel F, et al. Effects of antenatal glucocorticoid therapy on hippocampal
histology of preterm infants. PLoS ONE. 2012;7(3):e33369.
29. Davis EP, Sandman CA, Buss C, Wing DA, Head K. Fetal glucocorticoid
exposure is associated with preadolescent brain development. Biol
Psychiat. 2013;74(9):647–55.
30. Huang W, Beazley L, Quinlivan J, Evans S, Newnham J, Dunlop S. Effectof corticosteroids on brain growth in fetal sheep. Obstet Gynecol.
1999;94(2):213–8.
31. Dunlop SA, Archer MA, Quinlivan JA, Beazley LD, Newnham JP. Repeatedprenatal corticosteroids delay myelination in the ovine central nervous
system. J Matern Fetal Med. 1997;6(6):309–13.

Page 16 of 17

32. Schmidt AF, Kannan PS, Bridges JP, Filuta A, Lipps D, Kemp M, et al. Dosing
and formulation of antenatal corticosteroids for fetal lung maturationand gene expression in rhesus macaques. Sci Rep. 2019;9(1):1–10.
33. Schmidt AF, Schnell DJ, Eaton KP, Chetal K, Kannan PS, Miller LA, et al.
Fetal maturation revealed by amniotic fluid cell-free transcriptome inrhesus macaques. JCI Insight. 2022;7(18):1–19.
34. Usuda H, Fee EL, Carter S, Furfaro L, Takahashi T, Takahashi Y, et al. Lowdose antenatal betamethasone treatment achieves preterm lung maturation equivalent to that of the World Health Organization dexamethasone
regimen but with reduced endocrine disruption in a sheep model ofpregnancy. Am J Obstetr Gynecol. 2022;227(6):903–e1.
35. Organization WH. ACTION III: A multi-country, multi-centre, three-arm,parallel group, double-blind, placebo-controlled, randomized trial of two
doses of antenatal corticosteroids for women with a high probability ofbirth in the late preterm period in hospitals in low-resource countries toimprove newborn outcomes: WHO; 2021 [Available from: https://​cdn.​
who.​int/​media/​docs/​defau​lt-​source/​mca-​docum​ents/​nbh/​action-​iii---a-​multi-​count​r y-​multi-​centre-​three-​arm-​paral​lel-​group-​double-​blind-​place​bo-​contr​olled-​rando​mized-​trial-​of-​two-​doses-​of-​anten​atal-​corti​coste​
riods.​pdf?​sfvrsn=​c255b​253_​1&​downl​oad=​true.
36. Takahashi T, Fee EL, Takahashi Y, Saito M, Yaegashi N, Usuda H, et al.
Betamethasone phosphate reduces the efficacy of antenatal steroid
therapy and is associated with lower birthweights when administeredto pregnant sheep in combination with betamethasone acetate. Am J
Obstetr Gynecol. 2022;226(4):5641.e1-e14.
37. Kemp MW, Saito M, Usuda H, Molloy TJ, Miura Y, Sato S, et al. Maternofetal
pharmacokinetics and fetal lung responses in chronically catheterized
sheep receiving constant, low-dose infusions of betamethasone phosphate. Am J Obstetr Gynecol. 2016;215(6):775 (e1- e12).
38. Pillow JJ, Musk GC, McLean CM, Polglase GR, Dalton RGB, Jobe AH, et al.
Variable ventilation improves ventilation and lung compliance in pretermlambs. Intensive Care Med. 2011;37(8):1352–9.
39. Ewels PA, Peltzer A, Fillinger S, Patel H, Alneberg J, Wilm A, et al. The
nf-core framework for community-curated bioinformatics pipelines. Nat
Biotechnol. 2020;38(3):276–8.
40. Di Tommaso P, Chatzou M, Floden EW, Barja PP, Palumbo E, Notredame C.
Nextflow enables reproducible computational workflows. Nat Biotechnol.
2017;35(4):316–9.
41. Krueger F. Trim Galore!: A wrapper around Cutadapt and FastQC toconsistently apply adapter and quality trimming to FastQ files, with extra
functionality for RRBS data. Babraham Institute. 2015.
42. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR:ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29(1):15–21.
43. Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C. Salmon provides fastand bias-aware quantification of transcript expression. Nat Methods.
2017;14(4):417–9.
44. R Core Team R. R: A language and environment for statistical computing. 2013.
45. Love MI, Huber W, Anders S. Moderated estimation of fold change anddispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):1–21.
46. Blighe K, Rana S, Lewis M. EnhancedVolcano: Publication-ready volcano
plots with enhanced colouring and labeling. R package version.
2019;1:10–18129.
47. Gu Z, Eils R, Schlesner M. Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics. 2016;32(18):2847–9.
48. Wu T, Hu E, Xu S, Chen M, Guo P, Dai Z, et al. clusterProfiler 4.0: A
universal enrichment tool for interpreting omics data. Innovation.
2021;2(3):100141.
49. Kanehisa M, Goto S. KEGG: Kyoto Encyclopedia of Genes and Genomes.
Nucleic Acids Res. 2000;28(1):27–30.
50. Schmidt AF, Kemp MW, Rittenschober-Böhm J, Kannan PS, Usuda H, Saito
M, et al. Low-dose betamethasone-acetate for fetal lung maturation inpreterm sheep. Am J Obstetr Gynecol. 2018;218(1):132 (e1-e9).
51. Usuda H, Fee EL, Carter S, Furfaro L, Takahashi T, Takahashi Y, et al. Lowdose antenatal betamethasone treatment achieves preterm lung maturation equivalent to that of the World Health Organization dexamethasone
regimen but with reduced endocrine disruption in a sheep model ofpregnancy. Am J Obstetr Gynecol. 2022;227(6):903 (e1-e16).
52. Takahashi T, Saito M, Schmidt AF, Usuda H, Takahashi Y, Watanabe S, et al.
Variability in the efficacy of a standardized antenatal steroid treatment
was independent of maternal or fetal plasma drug levels: evidence from

Carter et al. BMC Medicine (2024) 22:338

53.

54.

55.

56.

57.
58.

59.
60.

61.
62.
63.

64.
65.
66.
67.

68.
69.
70.

71.
72.

a sheep model of pregnancy. Am J Obstetr Gynecol. 2020;223(6):921(e1-e10).
Takahashi T, Takahashi Y, Fee EL, Saito M, Yaegashi N, Usuda H, et al. Continuous but not pulsed low-dose fetal betamethasone exposures extend
the durability of antenatal steroid therapy. Am J Physiol Lung Cell Mole
Physiol. 2022;322(6):L784–93.
Kamath-Rayne BD, Du Y, Hughes M, Wagner EA, Muglia LJ, DeFranco EA,
et al. Systems biology evaluation of cell-free amniotic fluid transcriptomeof term and preterm infants to detect fetal maturity. BMC Med Genomics.
2015;8:67.
Fee EL, Takahashi T, Takahashi Y, Carter S, Furfaro L, Clarke MW, et al. One
percent of the clinical dose used for antenatal steroid therapy is sufficient
to induce lung maturation when administered directly to the preterm
ovine fetus. Am J Physiol Lung Cell Mole Physiol. 2022;322(6):L853–65.
Fee EL, Takahashi T, Takahashi Y, Carter SW, Clarke MW, Milad MA,
et al. Respiratory benefit in preterm lambs is progressively lost when
the concentration of fetal plasma betamethasone is titrated below
two nanograms per milliliter. Am J Physiol Lung Cell Mole Physiol.
2023;325(5):L628–37.
Polin RA, Carlo WA, Fetus Co, Newborn, Papile L-A, Polin RA, et al.
Surfactant replacement therapy for preterm and term neonates withrespiratory distress. Pediatrics. 2014;133(1):156–63.
Gilstrap LC, Christensen R, Clewell WH, D’Alton ME, Davidson EC,
Escobedo MB, et al. Effect of corticosteroids for fetal maturation on
perinatal outcomes: NIH consensus development panel on the effectof corticosteroids for fetal maturation on perinatal outcomes. JAMA.
1995;273(5):413–8.
Evans MI, Britt DW. Resistance to change. Reprod Sci. 2023;30(3):835–53.
Willet KE, McMenamin P, Pinkerton KE, Ikegami M, Jobe AH, Gurrin L, et al.
Lung morphometry and collagen and elastin content: changes during
normal development and after prenatal hormone exposure in sheep.
Pediatr Res. 1999;45(5):615–25.
Schmidt AF, Kannan PS, Bridges J, Presicce P, Jackson CM, Miller LA, et al.
Prenatal inflammation enhances antenatal corticosteroid–induced fetallung maturation. JCI insight. 2020;5(24):e139452.
Merali Z, Kent P, Anisman H. Role of bombesin-related peptides in
the mediation or integration of the stress response. Cell Mole Life Sci.
2002;59:272–87.
Luan Y, Zhang H, Ma K, Liu Y, Lu H, Chen X, et al. CCN3/NOV Regulates
Proliferation and Neuronal Differentiation in Mouse Hippocampal Neural
Stem Cells via the Activation of the Notch/PTEN/AKT Pathway. Int J Mol
Sci. 2023;24(12):10324.
Terrisse L, Poirier J, Bertrand P, Merched A, Visvikis S, Siest G, et al.
Increased levels of apolipoprotein D in cerebrospinal fluid and hippocampus of Alzheimer’s patients. J Neurochem. 1998;71(4):1643–50.
Decker JM, Wójtowicz AM, Bartsch JC, Liotta A, Braunewell KH, Heinemann U, et al. C-type natriuretic peptide modulates bidirectional plasticity in hippocampal area CA1 in vitro. Neuroscience. 2010;169(1):8–22.
Abudureyimu S, Asai N, Enomoto A, Weng L, Kobayashi H, Wang X, et al.
Essential Role of Linx/Islr2 in the Development of the Forebrain AnteriorCommissure. Sci Rep. 2018;8(1):7292.
Alazami AM, Maddirevula S, Seidahmed MZ, Albhlal LA, Alkuraya FS.
A novel ISLR2-linked autosomal recessive syndrome of congenitalhydrocephalus, arthrogryposis and abdominal distension. Hum Genet.
2019;138(1):105–7.
Eastwood SL, Harrison PJ. Decreased mRNA Expression of Netrin-G1 andNetrin-G2 in the Temporal Lobe in Schizophrenia and Bipolar Disorder.
Neuropsychopharmacology. 2008;33(4):933–45.
Gracias J, Orhan F, Hörbeck E, Holmén-Larsson J, Khanlarkani N, Malwade
S, et al. Cerebrospinal fluid concentration of complement component 4A
is increased in first episode schizophrenia. Nat Commun. 2022;13(1):6427.
Yilmaz M, Yalcin E, Presumey J, Aw E, Ma M, Whelan CW, et al. Overexpression of schizophrenia susceptibility factor human complement C4A
promotes excessive synaptic loss and behavioral changes in mice. Nat
Neurosci. 2021;24(2):214–24.
Mäkitie LT, Kanerva K, Polvikoski T, Paetau A, Andersson LC. Brain neurons
express ornithine decarboxylase-activating antizyme inhibitor 2 withaccumulation in Alzheimer’s disease. Brain Pathol. 2010;20(3):571–80.
Li D, Sham PC, Owen MJ, He L. Meta-analysis shows significant association between dopamine system genes and attention deficit hyperactivity
disorder (ADHD). Hum Mol Genet. 2006;15(14):2276–84.

Page 17 of 17

73. Van Vliet EA, Aronica E, Redeker S, Boer K, Gorter JA. Decreased expression of synaptic vesicle protein 2A, the binding site for levetiracetam, during epileptogenesis and chronic epilepsy. Epilepsia. 2009;50(3):422–33.
74. Crèvecœur J, Kaminski RM, Rogister B, Foerch P, Vandenplas C, Neveux
M, et al. Expression pattern of synaptic vesicle protein 2 (SV2) isoforms inpatients with temporal lobe epilepsy and hippocampal sclerosis. Neuropathol Appl Neurobiol. 2014;40(2):191–204.
75. Eshed Y, Feinberg K, Poliak S, Sabanay H, Sarig-Nadir O, Spiegel I, et al.
Gliomedin mediates Schwann cell-axon interaction and the molecularassembly of the nodes of Ranvier. Neuron. 2005;47(2):215–29.
76. Berman A, Kozlova N, Morozevich G. Integrins: structure and signaling.
Biochem Mosc. 2003;68:1284–99.
77. van der Flier A, Sonnenberg A. Function and interactions of integrins. Cell
Tissue Res. 2001;305(3):285–98.
78. Milner R, Campbell IL. The integrin family of cell adhesion molecules has
multiple functions within the CNS. J Neurosci Res. 2002;69(3):286–91.
79. Benson DL, Schnapp LM, Shapiro L, Huntley GW. Making memories
stick: cell-adhesion molecules in synaptic plasticity. Trends Cell Biol.
2000;10(11):473–82.
80. Roerig B, Feller MB. Neurotransmitters and gap junctions in developing
neural circuits. Brain Res Rev. 2000;32(1):86–114.
81. Virdee K, Xue L, Hemmings BA, Goemans C, Heumann R, Tolkovsky AM.
Nerve growth factor-induced PKB/Akt activity is sustained by phosphoinositide 3-kinase dependent and independent signals in sympathetic
neurons. Brain Res. 1999;837(1–2):127–42.
82. Wang L, Zhou K, Fu Z, Yu D, Huang H, Zang X, et al. Brain development
and Akt signaling: the crossroads of signaling pathway and neurodevelopmental diseases. J Mol Neurosci. 2017;61(3):379–84.
83. Iroegbu JD, Ijomone OK, Femi-Akinlosotu OM, Ijomone OM. ERK/MAPK
signalling in the developing brain: Perturbations and consequences.
Neurosci Biobehav Rev. 2021;131:792–805.
84. Giese KP, Friedman E, Telliez J-B, Fedorov NB, Wines M, Feig LA, et al.
Hippocampus-dependent learning and memory is impaired in mice lacking the Ras-guanine-nucleotide releasing factor 1 (Ras-GRF1). Neuropharmacology. 2001;41(6):791–800.
85. Nicolas CS, Peineau S, Amici M, Csaba Z, Fafouri A, Javalet C, et al.
The Jak/STAT pathway is involved in synaptic plasticity. Neuron.
2012;73(2):374–90.
86. Chiba T, Yamada M, Aiso S. Targeting the JAK2/STAT3 axis in Alzheimer’s
disease. Expert Opin Ther Targets. 2009;13(10):1155–67.
87. Dresselhaus EC, Meffert MK. Cellular Specificity of NF-κB Function in the
Nervous System. Front Immunol. 2019;10:1043.
88. Edgar R, Domrachev M, Lash AE. Gene Expression Omnibus: NCBI gene
expression and hybridization array data repository. Nucleic Acids Res.
2002;30(1):207–10.

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