Artificial placenta support of extremely preterm ovine fetuses at the border of viability for up to 336 hours with maintenance of systemic circulation but reduced somatic and organ growth

Introduction: Artificial placenta therapy (APT) is an experimental life support system to improve outcomes for extremely preterm infants (EPI) less than 1,000 g by obviating the need for pulmonary gas exchange. There are presently no long-term survival data for EPI supported with APT. To address this, we aimed to maintain 95d-GA (GA; term-150d) sheep fetuses for up to 2 weeks using our APT system.Methods: Pregnant ewes (n = 6) carrying singleton fetuses underwent surgical delivery at 95d GA. Fetuses were adapted to APT and maintained for up to 2 weeks with constant monitoring of key physiological parameters and extensive time-course blood and urine sampling, and ultrasound assessments. Six age-matched in-utero fetuses served as controls. Data were tested for group differences with ANOVA.Results: Six APT Group fetuses (100%) were adapted to APT successfully. The mean BW at the initiation of APT was 656 ± 42 g. Mean survival was 250 ± 72 h (Max 336 h) with systemic circulation and key physiological parameters maintained mostly within normal ranges. APT fetuses had active movements and urine output constantly exceeded infusion volume over the experiment. At delivery, there were no differences in BW (with edema in three APT group animals), brain weight, or femur length between APT and in-utero Control animals. Organ weights and humerus lengths were significantly reduced in the APT group (p < 0.05). Albumin, IGF-1, and phosphorus were significantly decreased in the APT group (p < 0.05). No cases of positive blood culture were detected.Conclusion: We report the longest use of APT to maintain extremely preterm fetuses to date. Fetal systemic circulation was maintained without infection, but growth was abnormal. This achievement suggests a need to focus not only on cardiovascular stability and health but also on the optimization of fetal growth and organ development. This new challenge will need to be overcome prior to the clinical translation of this technology

Oral antenatal corticosteroids evaluated in fetal sheep

The use of antenatal corticosteroids (ACS) in low-resource environments is sporadic. Further, drug choice, dose, and route of ACS are not optimized. We report the pharmacokinetics and pharmacodynamics of oral dosing of ACS using a preterm sheep model. We measured pharmacokinetics of oral betamethasone-phosphate (Beta-P) and dexamethasone-phosphate (Dex-P) using catheterized pregnant sheep. We compared fetal lung maturation responses of oral Beta-P and Dex-P to the standard treatment with 2 doses of the i.m. mixture of Beta-P and betamethasone-acetate at 2, 5, and 7 days after initiation of ACS. Oral Dex-P had lower bioavailability than Beta-P, giving a lower maximum maternal and fetal concentration. A single oral dose of 0.33 mg/kg of Beta-P was equivalent to the standard clinical treatment assessed at 2 days; 2 doses of 0.16 mg/kg of oral Beta-P were equivalent to the standard clinical treatment at 7 days as assessed by lung mechanics and gas exchange after preterm delivery and ventilation. In contrast, oral Dex-P was ineffective because of its decreased bioavailability. Using a sheep model, we demonstrate the use of pharmacokinetics to develop oral dosing strategies for ACS. Oral dosing is feasible and may facilitate access to ACS in low-resource environments

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

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. 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. 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. 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. 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

A Reduction in Antenatal Steroid Dose Was Associated with Reduced Cardiac Dysfunction in a Sheep Model of Pregnancy

Despite widespread use, dosing regimens for antenatal corticosteroid (ACS) therapy are poorly unoptimized. ACS therapy exerts a programming effect on fetal development, which may be associated with an increased risk of cardiovascular disease. Having demonstrated that low-dose steroid therapy is an efficacious means of maturing the preterm lung, we hypothesized that a low-dose steroid exposure would exert fewer adverse functional and transcriptional changes on the fetal heart. We tested this hypothesis using low-dose steroid therapy (10 mg delivered to the ewe over 36 h via constant infusion) and compared cardiac effects with those of a higher dose treatment (30 mg delivered to the ewe over 24 h by intramuscular injection; simulating currently employed clinical ACS regimens). Fetal cardiac function was assessed by ultrasound on the day of ACS treatment initiation. Transcriptomic analyses were performed on fetal myocardial tissue. Relative to saline control, fetuses in the higher-dose clinical treatment group had significantly lower ratios between early diastolic ventricular filling and ventricular filling during atrial systole, and showed the differential expression of myocardial hypertrophy-associated transcripts including βMHC, GADD45γ, and PPARγ. The long-term implications of these changes remain unstudied. Irrespective, optimizing ACS dosing regimens to maximize respiratory benefit while minimizing adverse effects on key organ systems, such as the heart, offers a means of improving the acute and long-term outcomes associated with this important obstetric therapy

Variability in the efficacy of a standardized antenatal steroid treatment was independent of maternal or fetal plasma drug levels: evidence from a sheep model of pregnancy

Administration of antenatal steroids is standard of care for women assessed to be at imminent risk of preterm delivery. There is a marked variation in antenatal steroid dosing strategy, selection for treatment criteria, and agent choice worldwide. This, combined with very limited optimization of antenatal steroid use per se, means that treatment efficacy is highly variable, and the rate of respiratory distress syndrome is decreased to perhaps as low as 40%. In some cases, antenatal steroid use is associated with limited benefit and potential harm. We hypothesized that individual differences in maternofetal steroid exposure would contribute to observed variability in antenatal steroid treatment efficacy. Using a chronically catheterized sheep model of pregnancy, we aimed to explore the relationship between maternofetal steroid exposure and antenatal steroid treatment efficacy as determined by functional lung maturation in preterm lambs undergoing ventilation. Ewes carrying a single fetus underwent surgery to catheterize a fetal and maternal jugular vein at 119 days’ gestation. Animals recovered for 24 hours before being randomized to either (1) a single maternal intramuscular injection of 2 mL saline (negative control group, n=10) or (2) a single maternal intramuscular injection of 0.25 mg/kg betamethasone phosphate plus acetate (antenatal steroid group, n=20). Serial maternal and fetal plasma samples were collected from each animal after 48 hours before fetuses were delivered and ventilated for 30 minutes. Total and free plasma betamethasone concentration was measured by mass spectrometry. Fetal lung tissue was collected for analysis using quantitative polymerase chain reaction. One animal from the control group and one animal from the antenatal steroid group did not complete their treatment protocol and were removed from analyses. Animals in the antenatal steroid group were divided into a responder subgroup (n=12/19) and a nonresponder subgroup (n=7/19) using a cutoff of partial pressure of arterial CO2 at 30-minute ventilation within 2 standard deviations of the mean value from saline-treated negative control group animals. Although antenatal steroid improved fetal lung maturation in the undivided antenatal steroid group and in the responder subgroup both physiologically (blood gas– and ventilation-related data) and biochemically (messenger ribonucleic acid expression related to fetal lung maturation), these values did not improve relative to saline-treated control group animals in the antenatal steroid nonresponder subgroup. No differences in betamethasone distribution, clearance, or protein binding were identified between the antenatal steroid responder and nonresponder subgroups. This study correlated individual maternofetal steroid exposures with preterm lung maturation as determined by pulmonary ventilation. Herein, approximately 40% of preterm lambs exposed to antenatal steroids had lung maturation that was not significantly different to saline-treated control group animals. These nonresponsive animals received maternal and fetal betamethasone exposures identical to animals that had a significant improvement in functional lung maturation. These data suggest that the efficacy of antenatal steroid therapy is not solely determined by maternofetal drug levels and that individual fetal or maternal factors may play a role in determining treatment outcomes in response to glucocorticoid signaling

The duration of fetal antenatal steroid exposure determines the durability of preterm ovine lung maturation

BACKGROUND: Antenatal corticosteroids (ACS) are the standard of care for maturing the fetal lung and improving outcomes for preterm infants. Antenatal corticosteroid dosing remains nonoptimized, and there is little understanding of how different treatment-to-delivery intervals may affect treatment efficacy. The durability of a lung maturational response is important because the majority of women treated with antenatal corticosteroids do not deliver within the widely accepted 1- to 7-day window of treatment efficacy. OBJECTIVE: We used a sheep model to test the duration of fetal exposures for efficacy at delivery intervals from 1 to 10 days. MATERIALS AND METHODS: For infusion studies, ewes with single fetuses were randomized to receive an intravenous bolus and maintenance infusion of betamethasone phosphate to target 1-4 ng/mL fetal plasma betamethasone for 36 hours, with delivery at 2, 4,or 7 days posttreatment or sterile saline solution as control. Animals receiving the clinical treatment were randomised to receive either a single injection of 0.25 mg/kg with a 1: 1 mixture of betamethasone phosphate thorn betamethasone acetate with delivery at either 1 or 7 days posttreatment, or 2 treatments of 0.25 mg/kg betamethasone phosphate thorn betamethasone acetate spaced at 24 hours (giving similar to 48 hours of fetal steroid exposure) with delivery at 2, 5, 7, or 10 days posttreatment. Negative control animals were treated with saline solution. All lambs were delivered at 121 +/- 3 days gestational age and ventilated for 30 minutes to assess lung function. RESULTS: Preterm lambs delivered at 1 or 2 days post-antenatal corticosteroid treatment had significant improvements in lung maturation for both intravenous and single-dose intramuscular treatments. After 2 days, the efficacy of 36-hour betamethasone phosphate infusions was lost. The single dose of 1:1 betamethasone phosphate thorn betamethasone acetate also was ineffective at 7 days. In contrast, animals treated with 2 doses had significant improvements in lung maturation at 2, 5, and 7 days, with treatment efficacy reduced by 10 days. CONCLUSION: In preterm lambs, the durability of antenatal corticosteroids treatment depends on the duration of fetal exposure and is independent of the intravenous or intramuscular maternal route of administration. For acute 24- to 48-hour posttreatment deliveries, a 24-hour fetal antenatal corticosteroids exposure was sufficient for lung maturation. A fetal exposure duration of at least 48 hours was necessary to maintain long-term treatment durability. A single-dose ACS treatment should be sufficient for women delivering within <48 hours of antenatal corticosteroids treatment

Upregulation of hepatic nuclear receptors in extremely preterm ovine fetuses undergoing artificial placenta therapy

Extremely preterm infants have low Nuclear Receptor (NR) expression in their developing hepatobiliary systems, as they rely on the placenta and maternal liver for compensation. NRs play a crucial role in detoxification and the elimination of both endogenous and xenobiotic substances by regulating key genes encoding specific proteins. In this study, we utilized an Artificial Placenta Therapy (APT) platform to examine the liver tissue expression of NRs of extremely preterm ovine fetuses. This fetal model, resembling a “knockout placenta,” lacks placental and maternal support, while maintaining a healthy extrauterine survival. Six ovine fetuses at 95 ± 1 d gestational age (GA; term = ∼150 d)/∼600 g delivery weight were maintained on an APT platform for a period of 120 h (APT Group). Six age-matched, in utero control fetuses were delivered at 99–100 d GA (Control Group). Fetal liver tissue samples and blood samples were collected at delivery from both groups and assessed mRNA expression of NRs and target transporters involved in the hepatobiliary transport system using quantitative PCR. Data were tested for group differences with ANOVA (p  mRNA expression of NRs was identified in both the placenta and the extremely preterm ovine fetal liver. The expression of HNF4α, LRH1, LXR, ESR1, PXR, CAR, and PPARα/γ were significantly elevated in the liver of the APT Group compared to the Control Group. Moreover, target transporters NTCP, OATP1B3, BSEP, and MRP4 were upregulated, whereas MRP2 and MRP3 were unchanged. Although there was no evidence of liver necrosis or apoptotic changes histologically, there was an impact in the fetal liver of the ATP group at the tissue level with a significant increase in TNFα mRNA, a cytokine involved in liver inflammation, and blood elevation of transaminases. A number of NRs in the fetal liver were significantly upregulated after loss of placental-maternal support. However, the expression of target transporter genes appeared to be insufficient to compensate role of the placenta and maternal liver and avoid fetal liver damage, potentially due to insufficient excretion of organic anions.</p