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BACKGROUND: In the fetus, the appropriate balance of prooxidants and antioxidants is essential to negate the detrimental effects of oxidative stress on lung maturation. Antioxidants improve respiratory function in postnatal life and adulthood. However, the outcomes and biological mechanisms of antioxidant action in the fetal lung are unknown. METHODS: We investigated the effect of maternal daily vitamin C treatment (200 mg/kg, intravenously) for a month in late gestation (105-138 days gestation, term ~145 days) on molecular regulation of fetal lung maturation in sheep. Expression of genes and proteins regulating lung development was quantified in fetal lung tissue. The number of surfactant-producing cells was determined by immunohistochemistry. RESULTS: Maternal vitamin C treatment increased fetal lung gene expression of the antioxidant enzyme SOD-1, hypoxia signaling genes (HIF-2α, HIF-3α, ADM, and EGLN-3), genes regulating sodium movement (SCNN1-A, SCNN1-B, ATP1-A1, and ATP1-B1), surfactant maturation (SFTP-B and ABCA3), and airway remodeling (ELN). There was no effect of maternal vitamin C treatment on the expression of protein markers evaluated or on the number of surfactant protein-producing cells in fetal lung tissue. CONCLUSIONS: Maternal vitamin C treatment in the last third of pregnancy in sheep acts at the molecular level to increase the expression of genes that are important for fetal lung maturation in a healthy pregnancy. IMPACT: Maternal daily vitamin C treatment for a month in late gestation in sheep increases the expression of gene-regulating pathways that are essential for normal fetal lung development. Following late gestation vitamin C exposure in a healthy pregnancy, an increase in lung gene but not protein expression may act as a mechanism to aid in the preparation for exposure to the air-breathing environment after birth. In the future, the availability/development of compounds with greater antioxidant properties than vitamin C or more specific targets at the site of oxidative stress in vivo may translate clinically to improve respiratory outcomes in complicated pregnancies at birth., National Health and Medical Research Council (NHMRC) of Australia

We are delighted to present the inaugural Frontiers in Pediatrics Women in Neonatology series. Gender inequity in health and medical research has been perpetuated by longstanding biases (including unconscious bias), stereotypes and structural barriers to the recruitment, retention, career progression and recognition of women’s achievements. While many structural changes are required to address these factors towards achieving equity, active allyship and visibility is also important to support and encourage the next generation of women and girls to pursue research careers. This article collection includes 5 original research papers, 2 brief research reports and 1 review article demonstrating a diversity of neonatology-related research led by women. Depicolzuane et al. reviewed and presented a summary of animal and translational studies, and clinical observations on the role of lung surfactant protein A in lung function and importantly in lung protection from both infectious and non-infectious agents. Surfactant protein A has a unique role in the lung through its interactions with lung macrophages where it impacts the activity of alveolar macrophages in response to injurious agents or stimuli. Interestingly, surfactant protein A also has a role in control of the in utero environment.

Advanced imaging techniques are enhancing research capacity focussed on the developmental origins of adult health and disease (DOHaD) hypothesis, and consequently increasing awareness of future health risks across various subareas of DOHaD research themes. Understanding how these advanced imaging techniques in animal models and human population studies can be both additively and synergistically used alongside traditional techniques in DOHaD-focussed laboratories is therefore of great interest. Global experts in advanced imaging techniques congregated at the advanced imaging workshop at the 2019 DOHaD World Congress in Melbourne, Australia. This review summarizes the presentations of new imaging modalities and novel applications to DOHaD research and discussions had by DOHaD researchers that are currently utilizing advanced imaging techniques including MRI, hyperpolarized MRI, ultrasound, and synchrotron-based techniques to aid their DOHaD research focus.

With the increasing incidence of obesity worldwide, the proportion of women entering pregnancy overweight or obese has increased dramatically. The fetus of an overnourished mother experiences numerous metabolic changes that may modulate lung development and hence successful transition to air breathing at birth. We used a sheep model of maternal late gestation overnutrition (LGON; from 115 days' gestation, term 147 ± 3 days) to determine the effect of exposure to an increased plane of nutrition in late gestation on lung development in the fetus (at 141 days' gestation) and the lamb (30 days after birth). We found a decrease in the numerical density of surfactant protein positive cells, as well as a reduction in mRNA expression of surfactant proteins (SFTP-A, -B and -C), a rate limiting enzyme in surfactant phospholipid synthesis (phosphate cytidylyltransferase 1, choline, α; PCYT1A), and glucose transporters (SLC2A1 and SLC2A4) in the fetal lung. In lambs at 30 days after birth, there were no differences between Control and LGON groups in the surfactant components that were downregulated in the LGON fetuses. However, mRNA expression of SFTP-A, PCYT1A, peroxisome proliferator activated receptor-γ, fatty acid synthase and fatty acid transport protein were increased in LGON lambs compared to controls. These results indicate a reduced capacity for surfactant production in late gestation. While these deficits are normalised by 30 days after birth, the lungs of LGON lambs exhibited altered glucose transport and fatty acid metabolism, which is consistent with an enhanced capacity for surfactant synthesis and restoration of surfactant maturity in these animals. Refereed/Peer-reviewed

Chronic fetal hypoxaemia is a common pregnancy complication that may arise from maternal, placental and/or fetal factors. Respiratory outcome of the infant at birth likely depends on the duration, timing and severity of the hypoxaemic insult. We have isolated the effect of maternal chronic hypoxia (MCH) for a month in late gestation on fetal lung development. Pregnant ewes were exposed to normoxia (21% O2) or hypoxia (10% O2) from 105 to 138 days of gestation (term ∼145 days). At 138 days, gene expression in fetal lung tissue was determined by quantitative RT-PCR. Cortisol concentrations were determined in fetal plasma and lung tissue. Numerical density of surfactant protein positive cells was determined by immunohistochemistry. MCH reduced maternal PaO2 (106 ± 2.9 vs. 47 ± 2.8 mmHg) and fetal body weight (4.0 ± 0.4 vs. 3.2 ± 0.9 kg). MCH increased fetal lung expression of the anti-oxidant marker CAT and decreased expression of the pro-oxidant marker NOX-4. MCH increased expression of genes regulating hypoxia signalling and feedback (HIF-3α, KDM3A, SLC2A1, EGLN-3). There was no effect of MCH on fetal plasma/lung tissue cortisol concentrations, nor genes regulating glucocorticoid signalling (HSD11B-1, HSD11B-2, NR3C1, NR3C2). MCH increased expression of genes regulating sodium (SCNN1-B, ATP1-A1, ATP1-B1) and water (AQP-4) movement in the fetal lung. MCH promoted surfactant maturation (SFTP-B, SFTP-D, ABCA3) at the molecular level, but did not alter the numerical density of surfactant positive cells in lung tissue. MCH in late gestation promotes molecular maturation of the fetal lung, which may be an adaptive response in preparation for the successful transition to air-breathing at birth. Refereed/Peer-reviewed

More women than not are entering pregnancy either overweight or obese. This presents a significant health care burden with respect to maternal morbidities and offspring complications at birth and in later life. In recent years it has also become clear that maternal obesity is an even greater global health problem than anticipated, because the effects are not limited to the mother but are also programmed in the fetus, known as the 'intergenerational cycle of obestiy'. Despite a large body of epidemiological evidence reporting outcomes of obese pregnancies, including offspring respiratory complications, much less is known about the molecular effects of maternal obesity on fetal lung development. This review focuses on the influence of altered substrate supply associated with the obesogenic intrauterine environment on fetal lung development. Understanding the molecular mechanisms contributing to altered fetal lung development will lead to improved respiratory outcomes for offspring at birth and in later life. Refereed/Peer-reviewed

Administration of glucocorticoids (GCs) to women at risk of preterm delivery reduces the newborn's risk of respiratory distress syndrome (RDS) by 35% to 40%; however, not all infants respond to this treatment. Fetal growth restriction (FGR) increases the risk of prematurity, perinatal morbidity, and mortality. This review aims to synthesize current evidence reporting the difference in RDS risk between FGR and normally grown infants (Question 1) and whether antenatal GC administration reduces the risk of RDS morbidity in FGR infants (Question 2). Systematic searches were performed, and after screening, a total of 27 and 9 citations were eligible for inclusion for Questions 1 and 2, respectively. In order to answer the two questions, odds ratios and 95% confidence intervals were calculated for all studies. The evidence was equivocal for a difference in risk of RDS in FGR compared with normally grown infants. Despite antenatal GC administration, there was evidence suggesting that the risk of RDS persists in FGR infants. The range of risk of RDS morbidity observed between studies is likely influenced by the definitions (RDS and FGR), gestational age, and small sample sizes of FGR infants evaluated. In addition, RDS morbidity may be related to the heterogeneous nature of FGR etiologies (including maternal, placental, and/or fetal factors). Further understanding of RDS morbidity and responsiveness to current treatments in FGR infants at a range of gestational ages, larger sample sizes, and stratification according to the specific etiology of FGR, may lead to improved respiratory outcomes at birth in this obstetric subpopulation. Refereed/Peer-reviewed

Exposure to altered intrauterine conditions during pregnancy influences both fetal growth and organ development. Chronic fetal hypoxaemia is a common pregnancy complication associated with intrauterine growth restriction (IUGR) that may influence the risk of infants experiencing respiratory complications at birth. There are a variety of signalling pathways that contribute to normal fetal lung development at the molecular level. The specific molecular effects of chronic hypoxaemia associated with IUGR on lung development are likely to be dependent on the specific aetiology (maternal, placental and/or fetal factors) that can alter hormone concentrations, oxygen and nutrient transport to the fetus. This review discusses molecular pathways that may contribute to altered fetal lung maturation following exposure to chronic hypoxaemia. Importantly, these studies highlight that the heterogeneity in respiratory outcomes at birth in this obstetric subpopulation are likely determined by the timing, severity and duration of chronic hypoxaemia encountered by the fetus during pregnancy. Refereed/Peer-reviewed

Intrauterine growth restriction (IUGR) induced by placental restriction (PR) in sheep leads to chronic hypoxemia and reduced surfactant maturation. The underlying molecular mechanism involves altered regulation of hypoxia signaling by increased prolyl hydroxylase domain (PHD) expression. Here, we evaluated the effect of intratracheal administration of the PHD inhibitor, dimethyloxalylglycine (DMOG), on functional, molecular and structural determinants of lung maturation in the Control and PR sheep fetus. There was no effect of DMOG on fetal blood pressure or fetal breathing movements. DMOG reduced lung expression of genes regulating hypoxia signaling (HIF-3α, ACE1), anti-oxidant defence (CAT), lung liquid reabsorption (SCNN1-A, ATP1-A1, AQP-1, AQP-5) and surfactant maturation (SFTP-A, SFTP-B, SFTP-C, PCYT1A, LPCAT, ABCA3, LAMP3) in Control fetuses. There were very few effects of DMOG on gene expression in the PR fetal lung (reduced lung expression of angiogenic factor ADM, water channel AQP-5 and increased expression of glucose transporter SLC2A1). DMOG administration in Controls reduced total lung lavage phosphatidylcholine to the same degree as in PR fetuses. These changes appear to be regulated at the molecular level as there was no effect of DMOG on the percent tissue, air space or numerical density of SFTP-B positive cells in the Control and PR lung. Hence, DMOG administration mimics the effects of PR in reducing surfactant maturation in the lung of Control fetuses. The limited responsiveness of the PR fetal lung suggests a potential biochemical limit or reduced plasticity to respond to changes in regulation of hypoxia signaling following exposure to chronic hypoxemia in utero. Refereed/Peer-reviewed

Inhibition of hypoxia signalling leads to respiratory distress syndrome (RDS), whereas administration of vascular endothelial growth factor (VEGF), the most widely characterized hypoxia responsive factor, protects from RDS. In the lung of the chronically hypoxaemic placentally restricted (PR) fetus, there is altered regulation of hypoxia signalling. This leads to reduced surfactant maturation in late gestation and provides evidence for the increased risk of RDS in growth restricted neonates at birth. We evaluated the effect of recombinant human VEGF administration with respect to bypassing the endogenous regulation of hypoxia signalling in the lung of the normally grown and PR sheep fetus. There was no effect of VEGF administration on fetal blood pressure or fetal breathing movements. We examined the effect on the expression of genes regulating VEGF signalling (FLT1 and KDR), angiogenesis (ANGPT1, AQP1, ADM), alveolarization (MMP2, MMP9, TIMP1, COL1A1, ELN), proliferation (IGF1, IGF2, IGF1R, MKI67, PCNA), inflammation (CCL2, CCL4, IL1B, TNFA, TGFB1, IL10) and surfactant maturation (SFTP-A, SFTP-B, SFTP-C, SFTP-D, PCYT1A, LPCAT, LAMP3, ABCA3). Despite the effects of PR on the expression of genes regulating airway remodelling, inflammatory signalling and surfactant maturation, there were very few effects of VEGF administration on gene expression in the lung of both the normally grown and PR fetus. There were, however, positive effects of VEGF administration on percentage tissue, air space and numerical density of SFTP-B positive alveolar epithelial cells in fetal lung tissue. These results provide evidence for the stimulatory effects of VEGF administration on structural maturation in the lung of both the normally grown and PR fetus.C Refereed/Peer-reviewed

Experimental placental restriction (PR) by carunclectomy in fetal sheep results in intrauterine growth restriction (IUGR), chronic hypoxemia, increased plasma cortisol, and decreased lung surfactant protein (SP) expression. The mechanisms responsible for decreased SP expression are unknown but may involve decreased glucocorticoid (GC) action or changes in hypoxia signaling. Endometrial caruncles were removed from nonpregnant ewes to induce PR. Lungs were collected from control and PR fetuses at 130-135 (n = 19) and 139-145 (n = 28) days of gestation. qRT-PCR and Western blotting were used to quantify lung mRNA and protein expression, respectively, of molecular regulators and downstream targets of the GC and hypoxia-signaling pathways. We confirmed a decrease in SP-A, -B, and -C, but not SP-D, mRNA expression in PR fetuses at both ages. There was a net downregulation of GC signaling with a reduction in GC receptor (GR)-α and -β protein expression and a decrease in the cofactor, GATA-6. GC-responsive genes including transforming growth factor-β1, IL-1β, and β2-adrenergic receptor were not stimulated. Prolyl hydroxylase domain (PHD)2 mRNA and protein and PHD3 mRNA expression increased with a concomitant increase in hypoxia-inducible factor-1α (HIF-1α) and HIF-1β mRNA expression. There was an increase in mRNA expression of several, but not all, hypoxia-responsive genes. Hence, both GC and hypoxia signaling may contribute to reduced SP expression. Although acute hypoxia normally inactivates PHDs, chronic hypoxemia in the PR fetus increased PHD abundance, which normally prevents HIF signaling. This may represent a mechanism by which chronic hypoxemia contributes to the decrease in SP production in the IUGR fetal lung. Refereed/Peer-reviewed

Glucocorticoids are administered to pregnant women at risk of preterm labour to promote fetal lung surfactant maturation. Intrauterine growth restriction (IUGR) is associated with an increased risk of preterm labour. Hence, IUGR babies may be exposed to antenatal glucocorticoids. The ability of the placenta or blood brain barrier to remove glucocorticoids from the fetal compartment or the brain is compromised in the IUGR fetus, which may have implications for lung, brain, and heart development. There is conflicting evidence on the effect of exogenous glucocorticoids on surfactant protein expression in different animal models of IUGR. Furthermore, the IUGR fetus undergoes significant cardiovascular adaptations, including altered blood pressure regulation, which is in conflict with glucocorticoid-induced alterations in blood pressure and flow. Hence, antenatal glucocorticoid therapy in the IUGR fetus may compromise regulation of cardiovascular development. The role of cortisol in cardiomyocyte development is not clear with conflicting evidence in different species and models of IUGR. Further studies are required to study the effects of antenatal glucocorticoids on lung, brain, and heart development in the IUGR fetus. Of specific interest are the aetiology of IUGR and the resultant degree, duration, and severity of hypoxemia. Refereed/Peer-reviewed