Antenatal steroids elicited neurodegenerative-associated transcriptional changes in the hippocampus of preterm fetal sheep independent of lung maturation.

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 High Group, 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 lung function 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. CONCLUSIONS: Our data suggests that exposure to antenatal steroid therapy is an independent cause of damage- associated transcriptomic changes in the brain of preterm, fetal sheep. These data highlight an urgent need for careful reconsideration and balancing of how antenatal steroids are used, both for patient selection and dosing regimens.

Antenatal corticosteroids: an updated assessment of anticipated benefits and potential risks.

Antenatal steroid therapy is increasingly central to the obstetrical management of women at imminent risk of preterm birth. For women likely to deliver between 24 and 34 weeks' gestation, antenatal steroid therapy is the standard of care, conferring sizable benefits and few risks in high-resource environments when appropriately targeted. Recent studies have focused on antenatal steroid use in periviable and late preterm populations, and in term cesarean deliveries. As a result, antenatal steroid therapy has now been applied from 22 to 39+6 weeks of estimated gestational age. There is also an increased appreciation that the vast majority of randomized control data informing the use of antenatal steroids are derived from predominantly high-resource, White populations. Accordingly, a sizable amount of work has recently been undertaken to test how to safely use antenatal steroids in low- and middle-resource environments, wherein the often high rates of preterm birth make these low-cost, easily administered interventions an attractive proposition. It is likely underappreciated by the obstetrical and neonatal communities that the overall efficacy of antenatal steroid therapy is highly variable (including when preterm risk is accurately assessed), the treatment regimens used are largely arbitrary, dosing is suprapharmacologic for effect, and the benefit-risk balance is significantly and differentially modified by gestation. It is also very likely that the patients consenting to receive these treatments are similarly unaware of the complex balance of potential benefits and harms. Although a small number of follow-up studies present a generally benign picture of long-term antenatal steroid risk, several large, population-based retrospective studies have identified associations between antenatal steroid use, childhood mental disease, and newborn infections that warrant urgent attention. Of particular contemporary importance are emergent efforts to optimize antenatal steroid regimens on the basis of the pharmacokinetics and pharmacodynamics of the agents themselves, the need for better targeting of these potent drugs, and clear articulation of the potential benefits and harms of antenatal steroid use at differing stages of pregnancy and in different delivery contexts.

Postnatal budesonide improved lung function in preterm lambs exposed to antenatal steroids and chorioamnionitis.

BACKGROUND: A combination of budesonide and surfactant decreases the rates of BPD in infants and lung injury in preterm sheep. Whether this combination will show benefit in the setting of chorioamnionitis and antenatal steroids is not known. METHODS: Ewes at 123 ± 1 day gestational age received intra-amniotic (IA) injections of 10 mg LPS before being randomized to receive either 0.25 mg/kg maternal betamethasone phosphate and acetate or saline by intramuscular (IM) injection at 48 and 24 h prior to delivery at 125 ± 1 day. Lambs (N = 6-9/group) underwent intentionally injurious ventilation for 15 min, then lambs received surfactant mixed with either: (1) saline; or (2) Budesonide 0.25 mg/kg and were ventilated for 4 h. RESULTS: Compared with LPS-exposed animals that received no IM steroid treatment, betamethasone exposed fetuses had improved hemodynamic stability, lung compliance, and ventilation efficiency. The addition of budesonide to surfactant further improved markers of injury and pro-inflammatory cytokine mRNA in both betamethasone IM or no IM lambs exposed to LPS IA. Antenatal betamethasone and IA LPS exposures decreased budesonide levels in the fetal lung and plasma. CONCLUSION: Antenatal betamethasone stabilizes physiologic parameters in LPS treated lambs. Budesonide mixed with surfactant further decreases injury and improves respiratory physiology in betamethasone treated animals. IMPACT: Antenatal betamethasone improved lung and systemic physiology in the setting of intra-amniotic LPS. The addition of budesonide to the surfactant further improved lung function. Budesonide levels in the plasma and lung were lower in lambs exposed to either LPS or LPS and Betamethasone animals, and these findings were not explained by increased esterification in the lungs. The combination of antenatal steroids and budesonide with surfactant had the lowest markers of pro-inflammatory cytokines in the lung of LPS exposed animals.

Preterm ovine respiratory epithelial cell responses to mechanical ventilation, lipopolysaccharide, and interleukin-13.

Mechanical ventilation causes airway injury, respiratory epithelial cell proliferation, and lung inflammation in preterm sheep. Whether preterm epithelial cells respond similarly to adult epithelial cells or are altered by mechanical ventilation is unknown. We test the hypothesis that mechanical ventilation alters the responses of preterm airway epithelium to stimulation in culture. Respiratory epithelial cells from the trachea, left mainstem bronchi (LMSB), and distal bronchioles were harvested from unventilated preterm lambs, ventilated preterm lambs, and adult ewes. Epithelial cells were grown in culture or on air-liquid interface (ALI) and challenged with combinations of either media only, lipopolysaccharide (LPS; 10 ng/mL), bronchoalveolar fluid (BALF), or interleukin-13 (IL-13). Cell lysates were evaluated for mRNA changes in cytokine, cell type markers, Notch pathway, and acute phase markers. Mechanical ventilation altered preterm respiratory epithelium cell types. Preterm respiratory epithelial cells responded to LPS in culture with larger IL-8 induction than adults, and mechanical ventilation further increased cytokines IL-1ß and IL-8 mRNA induction at 2 h. IL-8 protein is detected in cell media after LPS stimulation. The addition of BALF from ventilated preterm animals increased IL-1ß mRNA to LPS (fivefold) in both preterm and adult cells and suppressed IL-8 mRNA (twofold) in adults. Preterm respiratory epithelial cells, when grown on ALI, responded to IL-13 with an increase in goblet cell mRNA. Preterm respiratory epithelial cells responded to LPS and IL-13 with responses similar to adults. Mechanical ventilation or exposure to BALF from mechanically ventilated animals alters the responses to LPS.NEW & NOTEWORTHY Preterm lamb respiratory epithelial cells can be extracted from the trachea and bronchi and frozen, and the preterm cells can respond in culture to stimulation with LPS or IL-13. Brief mechanical ventilation changes the distribution and cell type of preterm respiratory cells toward an adult phenotype, and mechanical ventilation alters the response to LPS in culture. Bronchoalveolar lavage fluid from preterm lambs receiving mechanical ventilation also alters unventilated preterm and adult responses to LPS.

Respiratory benefit in preterm lambs is progressively lost when the concentration of fetal plasma betamethasone is titrated below two nanograms per milliliter.

Antenatal steroid therapy is the standard of care for women at imminent risk of preterm delivery. Current dosing regimens use suprapharmacological doses to achieve extended fetal steroid exposures. We aimed to determine the lowest fetal plasma betamethasone concentration sufficient to achieve functional preterm lung maturation. Ewes with single fetuses underwent surgery to install a fetal jugular catheter. Adopting a stepwise design, ewes were randomized to either a saline-only group (negative control group; n = 9) or one of four betamethasone treatment groups. Each betamethasone group fetus received a fetal intravenous infusion to target a constant plasma betamethasone level of either 1) 2 ng/mL (2 ng/mL positive control group, n = 9); 2) 1 ng/mL, (1 ng/mL group, n = 10); 3) 0.5 ng/mL (0.5 ng/mL group, n = 10); or 4) 0.25 ng/mL (0.25 ng/mL group, n = 10). Fetuses were infused for 48 h, delivered, and ventilated. The positive control group, negative control group, and mid-point 0.5 ng/mL group animals were tested first. An interim analysis informed the final betamethasone group tested. Positive control group animals had large, statistically significant improvements in respiratory function. Based on an interim analysis, the 1.0 ng/mL group was studied in favor of the 0.25 ng/mL group. Treatment efficacy was progressively lost at plasma betamethasone concentrations lower than 2 ng/mL. We demonstrated that the acute respiratory benefit conveyed by antenatal steroid exposure in the fetal sheep is progressively lost when constant fetal plasma betamethasone concentrations are reduced below a targeted value of 2 ng/mL.NEW & NOTEWORTHY Lung maturation benefits in preterm lambs were progressively lost when fetal plasma betamethasone concentrations fell below 2 ng/mL. The effective floor threshold for a robust, lung-maturing exposure likely lies between 1 and 2 ng betamethasone per milliliter of plasma. Hypothalamic pituitary adrenal axis signaling and immunocyte populations remained materially disrupted at subtherapeutic steroid concentrations. These data demonstrate the potential to improve antenatal steroid therapy using reduced dose regimens informed by glucocorticoid pharmacokinetics and pharmacodynamics.

ChatGPT outscored human candidates in a virtual objective structured clinical examination in obstetrics and gynecology.

BACKGROUND: Natural language processing is a form of artificial intelligence that allows human users to interface with a machine without using complex codes. The ability of natural language processing systems, such as ChatGPT, to successfully engage with healthcare systems requiring fluid reasoning, specialist data interpretation, and empathetic communication in an unfamiliar and evolving environment is poorly studied. This study investigated whether the ChatGPT interface could engage with and complete a mock objective structured clinical examination simulating assessment for membership of the Royal College of Obstetricians and Gynaecologists. OBJECTIVE: This study aimed to determine whether ChatGPT, without additional training, would achieve a score at least equivalent to that achieved by human candidates who sat for virtual objective structured clinical examinations in Singapore. STUDY DESIGN: This study was conducted in 2 phases. In the first phase, a total of 7 structured discussion questions were selected from 2 historical cohorts (cohorts A and B) of objective structured clinical examination questions. ChatGPT was examined using these questions and responses recorded in a script. Of note, 2 human candidates (acting as anonymizers) were examined on the same questions using videoconferencing, and their responses were transcribed verbatim into written scripts. The 3 sets of response scripts were mixed, and each set was allocated to 1 of 3 human actors. In the second phase, actors were used to presenting these scripts to examiners in response to the same examination questions. These responses were blind scored by 14 qualified examiners. ChatGPT scores were unblinded and compared with historical human candidate performance scores. RESULTS: The average score given to ChatGPT by 14 examiners was 77.2%. The average historical human score (n=26 candidates) was 73.7 %. ChatGPT demonstrated sizable performance improvements over the average human candidate in several subject domains. The median time taken for ChatGPT to complete each station was 2.54 minutes, well before the 10 minutes allowed. CONCLUSION: ChatGPT generated factually accurate and contextually relevant structured discussion answers to complex and evolving clinical questions based on unfamiliar settings within a very short period. ChatGPT outperformed human candidates in several knowledge areas. Not all examiners were able to discern between human and ChatGPT responses. Our data highlight the emergent ability of natural language processing models to demonstrate fluid reasoning in unfamiliar environments and successfully compete with human candidates that have undergone extensive specialist training.

Development of machine learning models to predict gestational diabetes risk in the first half of pregnancy.

BACKGROUND: Early prediction of Gestational Diabetes Mellitus (GDM) risk is of particular importance as it may enable more efficacious interventions and reduce cumulative injury to mother and fetus. The aim of this study is to develop machine learning (ML) models, for the early prediction of GDM using widely available variables, facilitating early intervention, and making possible to apply the prediction models in places where there is no access to more complex examinations. METHODS: The dataset used in this study includes registries from 1,611 pregnancies. Twelve different ML models and their hyperparameters were optimized to achieve early and high prediction performance of GDM. A data augmentation method was used in training to improve prediction results. Three methods were used to select the most relevant variables for GDM prediction. After training, the models ranked with the highest Area under the Receiver Operating Characteristic Curve (AUCROC), were assessed on the validation set. Models with the best results were assessed in the test set as a measure of generalization performance. RESULTS: Our method allows identifying many possible models for various levels of sensitivity and specificity. Four models achieved a high sensitivity of 0.82, a specificity in the range 0.72-0.74, accuracy between 0.73-0.75, and AUCROC of 0.81. These models required between 7 and 12 input variables. Another possible choice could be a model with sensitivity of 0.89 that requires just 5 variables reaching an accuracy of 0.65, a specificity of 0.62, and AUCROC of 0.82. CONCLUSIONS: The principal findings of our study are: Early prediction of GDM within early stages of pregnancy using regular examinations/exams; the development and optimization of twelve different ML models and their hyperparameters to achieve the highest prediction performance; a novel data augmentation method is proposed to allow reaching excellent GDM prediction results with various models.

Oxygen and steroids affect the regulatory role of natriuretic peptide receptor-C on surfactant secretion by type II cells.

Atrial natriuretic peptide (ANP) and its receptors natriuretic peptide receptor (NPR)-A and NPR-C are all highly expressed in alveolar epithelial type II cells (AEC2s) in the late-gestation ovine fetal lung and are dramatically decreased postnatally. However, of all the components, NPR-C stimulation inhibits ANP-mediated surfactant secretion. Since alveolar oxygen increases dramatically after birth, and steroids are administered to mothers antenatally to enhance surfactant lung maturity, we investigated the effects of O2 concentration and steroids on NPR-C-mediated surfactant secretion in AEC2s. NPR-C expression was highest at 5% O2 while being suppressed by 21% O2, in cultured mouse lung epithelial cells (MLE-15s) and/or human primary AEC2s. Surfactant protein-B (SP-B) was significantly elevated in media from both in vitro and ex vivo culture at 13% O2 versus 21% O2 in the presence of ANP or terbutaline (TER). Both ANP and C-ANP (an NPR-C agonist) attenuated TER-induced SP-B secretion; this effect was reversed by dexamethasone (DEX) pretreatment in AEC2s and by transfection with NPR-C siRNA in MLE-15 cells. DEX markedly reduced AEC2 NPR-C expression, and pregnant ewes treated with betamethasone showed reduced ANP in fetal sheep lung fluid. These data suggest that elevated O2 downregulates AEC2 NPR-C and that steroid-mediated NPR-C downregulation in neonatal lungs may provide a novel mechanism for their effect on perinatal surfactant production.

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.

Antenatal steroids (ANSs) are routinely administered to women judged to be at imminent risk of preterm delivery. Their principal benefit is precocious functional maturation of the preterm fetal lung. Current dosing regimens expose the mother and fetus to high steroid levels that may be unnecessary, increasing the potential risks of disruption to the maternal and fetal hypothalamic-pituitary-adrenal (HPA) axis and glucose regulation, alterations in placental function, and reduced fetal growth. Using a sheep model of pregnancy, we tested the hypothesis that direct fetal administration of an ultra-low dose course of betamethasone phosphate (∼0.33 mg) would be sufficient to elicit functional maturation of the fetal lung. A jugular catheter was installed in singleton ovine fetuses at 122-day gestation under general anesthesia. Animals were randomized to receive either: 1) fetal intravenous betamethasone phosphate to target fetal plasma betamethasone mean levels of 2 ng/mL for 26 h (fetal treatment group; n = 16); 2) fetal intravenous saline for 26 h and two maternal intramuscular injections of 0.25 mg/kg betamethasone phosphate + betamethasone acetate, simulating a standard clinical treatment (maternal treatment group; n = 12); or 3) fetal intravenous saline only for 26 h (negative control group; n = 10). Fetuses were delivered 48 h after surgery, ventilated for 30 min to allow the collection of lung function and physiological data, and euthanized. Quantitative PCR and Western blots were used to assess markers of lung maturation. The average total betamethasone phosphate dose for the fetal treatment group was 1% (0.3 mg) of the maternal treatment group (31-mg betamethasone phosphate + betamethasone acetate). At 30 min of ventilation, arterial [Formula: see text], pH, heart rate, and ventilation efficacy index (VEI) were significantly (P < 0.05) and equivalently improved in both the fetal treatment group and maternal treatment group, relative to the negative control group. Similarly, SP-A, SP-C, and AQ-5 mRNA expression was significantly higher in both the fetal treatment group and maternal treatment group, relative to negative control. Maternal steroid administration was not required to generate preterm fetal lung maturation in sheep. Using a low dose and targeting steroid treatments directly to the fetus has the potential to significantly reduce maternal exposures, while simultaneously reducing the potential risk of adverse outcomes associated with current clinical dosing regimens.

Continuous but not pulsed low-dose fetal betamethasone exposures extend the durability of antenatal steroid therapy.

Antenatal steroid (ANS) therapy is the standard care for women at imminent risk of preterm labor. Despite extensive and long-standing use, 40%-50% of babies exposed antenatally to steroids do not derive benefit; remaining undelivered 7 days or more after ANS treatment is associated with a lack of treatment benefit and increased risk of harm. We used a pregnant sheep model to evaluate the impact of continuous versus pulsed ANS treatments on fetal lung maturation at an extended, 8-day treatment to delivery interval. Continuous low-dose ANS treatments for more than 72 h in duration improved fetal lung maturation at 8 days after treatment initiation. If fetal ANS exposure was interrupted, the beneficial ANS effect was lost. Truncated treatments, including that simulating the current clinical treatment regimen, did not improve lung function. Variable fetal lung maturation was correlated to the amount of saturated phosphatidylcholine present in the lung fluid. These data demonstrate that 1) the durability of ANS therapy may be enhanced by employing an extended, low-dose treatment regimen by reducing total dose and 2) interrupting the continuity of fetal exposure by allowing it to fall below a minimal threshold was associated with comparably poor functional maturation of the preterm ovine lung.

The complex challenge of antenatal steroid therapy nonresponsiveness.

Antenatal steroid therapy is standard care for women at imminent risk of preterm delivery. When deliveries occur within 7 days of treatment, antenatal steroid therapy reduces the risk of neonatal death and improves preterm outcomes by exerting diverse developmental effects on the fetal organs, in particular the preterm lung and cardiovascular system. There is, however, sizable variability in antenatal steroid treatment efficacy, and an important percentage of fetuses exposed to antenatal steroid therapy do not respond sufficiently to derive benefit. Respiratory distress syndrome, for example, is a central metric of clinical trials to assess antenatal steroid outcomes. In the present analysis, we addressed the concept of antenatal steroid nonresponsiveness, and defined a failed or suboptimal response to antenatal steroids as death or a diagnosis of respiratory distress syndrome following treatment. For deliveries at 24 to 35 weeks' gestation, the number needed to treat to prevent 1 case of respiratory distress syndrome was 19 (95% confidence interval, 14-28). Reflecting gestation-dependent risk, for deliveries at >34 weeks' gestation the number needed to treat was 55 (95% confidence interval, 30-304), whereas for elective surgical deliveries at term this number was 106 (95% confidence interval, 61-421). We reviewed data from clinical and animal studies investigating antenatal steroid therapy to highlight the significant incidence of antenatal steroid therapy nonresponsiveness (ie, residual mortality or respiratory distress syndrome after treatment), and the potential mechanisms underpinning this outcome variability. The origins of this variability may be related to both the manner in which the therapy is applied (ie, the treatment regimen itself) and factors specific to the individual (ie, genetic variation, stress, infection). The primary aims of this review were: (1) to emphasize to the obstetrical and neonatal communities the extent of antenatal steroid response variability and its potential impact; (2) to propose approaches by which antenatal steroid therapy may be better applied to improve overall benefit; and (3) to stimulate further research toward the empirical optimization of this important antenatal therapy.

Low-dose 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 of pregnancy.

BACKGROUND: The intramuscular administration of antenatal steroids to women at risk of preterm delivery achieves high maternal and fetal plasma steroid concentrations, which are associated with adverse effects and may reduce treatment efficacy. We have demonstrated that antenatal steroid efficacy is independent of peak maternofetal steroid levels once exposure is maintained above a low threshold. OBJECTIVE: This study aimed to test, using a sheep model of pregnancy, whether the low-dose antenatal steroid regimen proposed as part of the Antenatal Corticosteroids for Improving Outcomes in Preterm Newborns trial would achieve preterm lung maturation equivalent to that of the existing World Health Organization dexamethasone treatment regimen, but with reduced risk of adverse outcomes. STUDY DESIGN: Following ethical review and approval, date-mated ewes with single fetuses received intramuscular injections of either (1) four 6-mg maternal intramuscular injections of dexamethasone phosphate every 12 hours (n=22), (2) 4 2-mg maternal intramuscular injections of betamethasone phosphate every 12 hours (n=21), or (3) 4 2-mL maternal intramuscular injections of saline every 12 hours (n=16). Of note, 48 hours after first injection, (124±1 day), lambs were delivered, ventilated for 30 minutes, and euthanized for sampling. Arterial blood gas, respiratory, hematological, and biochemical data were analyzed for between-group differences with analysis of variance according to distribution and variance, with P<.05 taken as significant. RESULTS: After 30 minutes of ventilation, lambs from both steroid-treated groups had significant and equivalent improvements in lung function relative to saline control (P<.05). There was no significant difference in arterial blood pH, pO2, pCO2, lung compliance, ventilator efficiency index, or lung volume at necropsy with a static pressure of 40 cmH2O. The messenger RNA expression of surfactant protein (Sp)a, Spb, Spc, Spd, aquaporin (Aqp)1, Aqp5, and sodium channel epithelial 1 subunit beta (Scnn1b) was equivalent between both steroid groups. Maternal and fetal plasma neutrophil, glucose, and fetal plasma C-peptide levels were significantly elevated in the dexamethasone group, relative to the betamethasone group. Fetal plasma insulin-like growth factor 1 was significantly reduced in the dexamethasone group compared with the betamethasone group (P<0.05). Fetal adrenocorticotropic hormone (r=0.53), maternal glucose value (r=-0.52), and fetal glucose values (r=-0.42) were correlated with maternal weight in the betamethasone group (P<.05), whereas fetal pCO2 and pO2 were not correlated. There was no significant difference between male and female lamb outcomes in any groups for any of the items evaluated. CONCLUSION: This study reported that in preterm lambs, a low-dose treatment regimen of 8 mg betamethasone achieves lung maturation equivalent to that of a 24-mg dexamethasone-based regimen, but with smaller perturbations to the maternofetal hypothalamic-pituitary-adrenal axis. These data suggested that given steroid pharmacokinetic differences between sheep and humans, a betamethasone dose of 2 mg may remain above the minimum dose necessary for robust maturation of the preterm lung. Maternal weight-adjusted betamethasone doses might also be a key to reducing perturbations to the maternofetal hypothalamic-pituitary-adrenal axis.

Betamethasone phosphate reduces the efficacy of antenatal steroid therapy and is associated with lower birthweights when administered to pregnant sheep in combination with betamethasone acetate.

BACKGROUND: Antenatal corticosteroid therapy is a standard of care for women at imminent risk of preterm labor. However, the optimal (maximum benefit and minimal risk of side effects) antenatal corticosteroid dosing strategy remains unclear. Although conveying overall benefit when given to the right patient at the right time, antenatal corticosteroid treatment efficacy is highly variable and is not risk-free. Building on earlier findings, we hypothesized that when administered in combination with slow-release betamethasone acetate, betamethasone phosphate and the high maternal-fetal betamethasone concentrations it generates are redundant for fetal lung maturation. OBJECTIVE: Using an established sheep model of prematurity and postnatal ventilation of the preterm lamb, we aimed to compare the pharmacodynamic effects of low-dosage treatment with betamethasone acetate only against a standard dosage of betamethasone phosphate and betamethasone acetate as recommended by the American College of Obstetricians and Gynecologists for women at risk of imminent preterm delivery between 24 0/7 and 35 6/7 weeks' gestation. STUDY DESIGN: Ewes carrying a single fetus at 122±1 days' gestation (term=150 days) were randomized to receive either (1) maternal intramuscular injections of sterile saline (the saline negative control group, n=12), (2) 2 maternal intramuscular injections of 0.25 mg/kg betamethasone phosphate+betamethasone acetate administered at 24-hour dosing intervals (the betamethasone phosphate+betamethasone acetate group, n=12); or (3) 2 maternal intramuscular injections of 0.125 mg/kg betamethasone acetate administered at 24-hour dosing intervals (the betamethasone acetate group, n=11). The fetuses were surgically delivered 48 hours after treatment initiation and ventilated for 30 minutes to determine functional lung maturation. The fetuses were euthanized after ventilation, and the lungs were collected for analysis using quantitative polymerase chain reaction and Western blot assays. Fetal plasma adrenocorticotropic hormone levels were measured in the cord blood samples taken at delivery. RESULTS: Preterm lambs were defined as either antenatal corticosteroid treatment responders or nonresponders using an arbitrary cutoff, being a PaCO2 level at 30 minutes of ventilation being more extreme than 2 standard deviations from the mean value of the normally distributed saline control group values. Compared with the animals in the saline control group, the animals in the antenatal corticosteroid treatment groups showed significantly improved lung physiological responses (blood gas and ventilation data) and had a biochemical signature (messenger RNA and surfactant protein assays) consistent with functional maturation. However, the betamethasone acetate group had a significantly higher treatment response rate than the betamethasone phosphate+betamethasone acetate group. These physiological results were strongly correlated to the amount of surfactant protein A. Birthweight was lower in the betamethasone phosphate+betamethasone acetate group and the fetal hypothalamic-pituitary-adrenal axis was suppressed to a greater extent in the betamethasone phosphate+betamethasone acetate group. CONCLUSION: Low-dosage antenatal corticosteroid therapy solely employing betamethasone acetate was sufficient for fetal lung maturation. The elevated maternal-fetal betamethasone concentrations associated with the coadministration of betamethasone phosphate did not in addition improve lung maturation but were associated with greater fetal hypothalamic-pituitary-adrenal axis suppression, a lower antenatal corticosteroid treatment response rate, and lower birthweight-outcomes not desirable in a clinical setting. These data warranted a clinical investigation of sustained low-dosage antenatal corticosteroid treatments that avoid high maternal-fetal betamethasone exposures.

Assessment of synthetic red cell therapy for extremely preterm ovine fetuses maintained on an artificial placenta life-support platform.

BACKGROUND: Artificial placenta therapy (APT) is an experimental care strategy for extremely preterm infants born at 21-24 weeks' gestation. In our previous studies, blood taken from the maternal ewe was used as the basis of priming solutions for the artificial placenta circuit. However, the use of maternal blood as a priming solution is accompanied by several challenges. We explored the use of synthetic red cells (hemoglobin vesicles; HbV) as the basis of a priming solution for APT used to manage extremely early preterm ovine fetuses. METHODS: Six ewes with singleton pregnancies at 95 d gestation (term = 150 d) were adapted to APT and maintained with constant monitoring of key vital parameters. The target maintenance period was 72 h in duration. A synthetic red cell solution consisting of HbV, sheep albumin and electrolytes was used as priming solutions for the APT circuit. Fetuses were evaluated on gross appearance, physiological parameters and bleeding after euthanasia. RESULTS: Two out of six APT fetuses were successfully maintained for the targeted 72 h experimental period with controllable anemia (>10 g/dl) and methemoglobinemia (<10%) using an infusion of blood transfusion and nitroglycerin delivered >1 h after APT commencement, a sufficient period of time to cross-match blood products and screen for viral agents of concern. CONCLUSIONS: Extremely preterm sheep fetuses were maintained for a period of up to 72 h using APT in combination with circuit priming using a synthetic red cell (HbV) preparation. Although significant further refinements are required, these findings demonstrated the potential clinical utility of synthetic blood products in the eventual clinical translation of artificial placenta technology to support extremely preterm infants.

Mesenchymal Stem Cells-Induced Trophoblast Invasion Is Reduced in Patients with a Previous History of Preeclampsia.

Endometrial stromal cells play an important role in reproductive success, especially in implantation and placentation. Although Mesenchymal stem cells (MSCs) have been studied to assess decidualization disorders in preeclampsia (PE), their role during trophoblast invasion remains unclear. This study aims to determine: (i) whether MSCs isolated from menstrual fluid (MenSCs) from nulliparous, multiparous, and women with a previous history of preeclampsia exhibited different patterns of proliferation and migration and (ii) whether reproductive history (i.e., prior pregnancy or prior history of PE) was able to produce changes in MenSCs, thus altering trophoblast invasion capacity. MenSCs were collected from nulliparous and multiparous women without a history of PE and from non-pregnant women with a history of PE. Proliferation and migration assays were performed on MenSCs with sulforhodamine B and transwell assays, respectively. Trophoblast invasion was analyzed by culturing HTR-8/SVneo trophospheres on a matrigel overlying MenSCs for 72 h at 5% O2, simulating a 3D implantation model. A previous history of pregnancy or PE did not impact the proliferative capacity or migratory behavior of MenSCs. Following exposure to physiological endometrial conditions, MenSCs demonstrated upregulated expression of IGFBP-1 and LIF mRNA, decidualization and window of implantation markers, respectively. The mRNA expression of VIM, NANOG, and SOX2 was upregulated upon trophosphere formation. Relative to co-culture with multiparous MenSCs, co-culture with PE-MenSCs was associated with reduced trophoblast invasion. The findings of this study suggest a potential role for communication between maternal MenSCs and invading trophoblast cells during the implantation process that could be implicated in the etiology of PE.

Glucocorticoids regulate mitochondrial fatty acid oxidation in fetal cardiomyocytes.

The late gestational rise in glucocorticoids contributes to the structural and functional maturation of the perinatal heart. Here, we hypothesized that glucocorticoid action contributes to the metabolic switch in perinatal cardiomyocytes from carbohydrate to fatty acid oxidation. In primary mouse fetal cardiomyocytes, dexamethasone treatment induced expression of genes involved in fatty acid oxidation and increased mitochondrial oxidation of palmitate, dependent upon a glucocorticoid receptor (GR). Dexamethasone did not, however, induce mitophagy or alter the morphology of the mitochondrial network. In vivo, in neonatal mice, dexamethasone treatment induced cardiac expression of fatty acid oxidation genes. However, dexamethasone treatment of pregnant C57Bl/6 mice at embryonic day (E)13.5 or E16.5 failed to induce fatty acid oxidation genes in fetal hearts assessed 24 h later. Instead, at E17.5, fatty acid oxidation genes were downregulated by dexamethasone, as was GR itself. PGC-1α, required for glucocorticoid-induced maturation of primary mouse fetal cardiomyocytes in vitro, was also downregulated in fetal hearts at E17.5, 24 h after dexamethasone administration. Similarly, following a course of antenatal corticosteroids in a translational sheep model of preterm birth, both GR and PGC-1α were downregulated in heart. These data suggest that endogenous glucocorticoids support the perinatal switch to fatty acid oxidation in cardiomyocytes through changes in gene expression rather than gross changes in mitochondrial volume or mitochondrial turnover. Moreover, our data suggest that treatment with exogenous glucocorticoids may interfere with normal fetal heart maturation, possibly by downregulating GR. This has implications for clinical use of antenatal corticosteroids when preterm birth is considered a possibility. KEY POINTS: Glucocorticoids are steroid hormones that play a vital role in late pregnancy in maturing fetal organs, including the heart. In fetal cardiomyocytes in culture, glucocorticoids promote mitochondrial fatty acid oxidation, suggesting they facilitate the perinatal switch from carbohydrates to fatty acids as the predominant energy substrate. Administration of a synthetic glucocorticoid in late pregnancy in mice downregulates the glucocorticoid receptor and interferes with the normal increase in genes involved in fatty acid metabolism in the heart. In a sheep model of preterm birth, antenatal corticosteroids (synthetic glucocorticoid) downregulates the glucocorticoid receptor and the gene encoding PGC-1α, a master regulator of energy metabolism. These experiments suggest that administration of antenatal corticosteroids in anticipation of preterm delivery may interfere with fetal heart maturation by downregulating the ability to respond to glucocorticoids.

Antenatal corticosteroids: a reappraisal of the drug formulation and dose.

We review the history of antenatal corticosteroid therapy (ACS) and present recent experimental data to demonstrate that this, one of the pillars of perinatal care, has been inadequately evaluated to minimize fetal exposure to these powerful medications. There have been concerns since 1972 that fetal exposures to ACS convey risk. However, this developmental modulator, with its multiple widespread biologic effects, has not been evaluated for drug choice, dose, or duration of treatment, despite over 30 randomized trials. The treatment used in the United States is two intramuscular doses of a mixture of 6 mg betamethasone phosphate (Beta P) and 6 mg betamethasone acetate (Beta Ac). To optimize outcomes with ACS, the goal should be to minimize fetal drug exposure. We have determined that the minimum exposure needed for fetal lung maturation in sheep, monkeys, and humans (based on published cord blood corticosteroid concentrations) is about 1 ng/ml for a 48-h continuous exposure, far lower than the concentration reached by the current dosing. Because the slowly released Beta Ac results in prolonged fetal exposure, a drug containing Beta Ac is not ideal for ACS use. IMPACT: Using sheep and monkey models, we have defined the minimum corticosteroid exposure for a fetal lung maturation. These results should generate new clinical trials of antenatal corticosteroids (ACS) at much lower fetal exposures to ACS, possibly given orally, with fewer risks for the fetus.

Budesonide with surfactant decreases systemic responses in mechanically ventilated preterm lambs exposed to fetal intra-amniotic lipopolysaccharide.

BACKGROUND: Chorioamnionitis is associated with increased rates of bronchopulmonary dysplasia (BPD) in ventilated preterm infants. Budesonide when added to surfactant decreased lung and systemic inflammation from mechanical ventilation in preterm lambs and decreased the rates and severity of BPD in preterm infants. We hypothesized that the addition of budesonide to surfactant will decrease the injury from mechanical ventilation in preterm lambs exposed to intra-amniotic (IA) lipopolysaccharide (LPS). METHODS: Lambs at 126 ± 1 day GA received LPS 10 mg IA 48 h prior to injurious mechanical ventilation. After 15 min, lambs received either surfactant mixed with: (1) saline or (2) Budesonide 0.25 mg/kg, then ventilated with normal tidal volumes for 4 h. Injury markers in the lung, liver, and brain were compared. RESULTS: Compared with surfactant alone, the addition of budesonide improved blood pressures, dynamic compliance, and ventilation, while decreasing mRNA for pro-inflammatory cytokines in the lung, liver, and multiple areas of the brain. LPS caused neuronal activation and structural changes in the brain that were not altered by budesonide. Budesonide was not retained within the lung beyond 4 h. CONCLUSIONS: In preterm lambs exposed to IA LPS, the addition of budesonide to surfactant improved physiology and markers of lung and systemic inflammation. IMPACT: The addition of budesonide to surfactant decreases the lung and systemic responses to injurious mechanical ventilation preterm lambs exposed to fetal LPS. Budesonide was present in the plasma by 15 min and the majority of the budesonide is no longer in the lung at 4 h of ventilation. IA LPS and mechanical ventilation caused structural changes in the brain that were not altered by short-term exposure to budesonide. The budesonide dose of 0.25 mg/kg being used clinically seems likely to decrease lung inflammation in preterm infants with chorioamnionitis.

Chorioamnionitis induces changes in ovine pulmonary endogenous epithelial stem/progenitor cells in utero.

BACKGROUND: Chorioamnionitis, an intrauterine infection of the placenta and fetal membranes, is a common risk factor for adverse pulmonary outcomes in premature infants including BPD, which is characterized by an arrest in alveolar development. As endogenous epithelial stem/progenitor cells are crucial for organogenesis and tissue repair, we examined whether intrauterine inflammation negatively affects these essential progenitor pools. METHODS: In an ovine chorioamnionitis model, fetuses were intra-amniotically exposed to LPS, 2d or 7d (acute inflammation) before preterm delivery at 125d of gestation, or to intra-amniotic Ureaplasma parvum for 42d (chronic inflammation). Lung function, pulmonary endogenous epithelial stem/progenitor pools, and downstream functional markers were studied. RESULTS: Lung function was improved in the 7d LPS and 42d Ureaplasma groups. However, intrauterine inflammation caused a loss of P63+ basal cells in proximal airways and reduced SOX-9 expression and TTF-1+ Club cells in distal airways. Attenuated type-2 cell numbers were associated with lower proliferation and reduced type-1 cell marker Aqp5 expression, indicative for impaired progenitor function. Chronic Ureaplasma infection only affected distal airways, whereas acute inflammation affected stem/progenitor populations throughout the lungs. CONCLUSIONS: Acute and chronic prenatal inflammation improve lung function at the expense of stem/progenitor alterations that potentially disrupt normal lung development, thereby predisposing to adverse postnatal outcomes. IMPACT: In this study, prenatal inflammation improved lung function at the expense of stem/progenitor alterations that potentially disrupt normal lung development, thereby predisposing to adverse postnatal outcomes. Importantly, we demonstrate that these essential alterations can already be initiated before birth. So far, stem/progenitor dysfunction has only been shown postnatally. This study indicates that clinical protocols to target the consequences of perinatal inflammatory stress for the immature lungs should be initiated as early as possible and ideally in utero. Within this context, our data suggest that interventions, which promote function or repair of endogenous stem cells in the lungs, hold great promise.

Intestinal Goblet Cell Loss during Chorioamnionitis in Fetal Lambs: Mechanistic Insights and Postnatal Implications.

Chorioamnionitis, an important cause of preterm birth, is linked to necrotizing enterocolitis (NEC). NEC is characterized by a disrupted mucus barrier, goblet cell loss, and endoplasmic reticulum (ER) stress of the intestinal epithelium. These findings prompted us to investigate the mechanisms underlying goblet cell alterations over time in an ovine chorioamnionitis model. Fetal lambs were intra-amniotically (IA) exposed to lipopolysaccharides (LPS) for 5, 12, or 24 h, or 2, 4, 8, or 15 d before premature delivery at 125 d gestational age (GA). Gut inflammation, the number, distribution, and differentiation of goblet cells, ER stress, and apoptosis were measured. We found a biphasic reduction in goblet cell numbers 24 h-2 d after, and 15 d after IA LPS exposure. The second decrease of goblet cell numbers was preceded by intestinal inflammation, apoptosis, and crypt ER stress, and increased SAM-pointed domain-containing ETS transcription factor (SPDEF)-positive cell counts. Our combined findings indicated that ER stress drives apoptosis of maturating goblet cells during chorioamnionitis, ultimately reducing goblet cell numbers. As similar changes have been described in patients suffering from NEC, these findings are considered to be clinically important for understanding the predecessors of NEC, and targeting ER stress in this context is interesting for future therapeutics.