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.

CONTEXT: Intrauterine inflammation, a representative stressor for the fetus, has been shown to alter the hypothalamus-pituitary-adrenal (HPA) axis reactivity in preterm fetuses and increase postnatal cortisol production. However, the mechanism of this alteration has not yet been elucidated. OBJECTIVE: We aimed to clarify the effects of endotoxin-induced intrauterine inflammation on the HPA axis of periviable sheep fetuses. METHODS: Fetal sheep (0.63 term) were divided into 2 groups: (1) the endotoxin group, in which the endotoxin was injected into the amniotic fluid; and (2) the control group, in which the saline solution was injected instead. A corticotropin-releasing hormone (CRH) challenge test was performed on the third day after injection to evaluate the cortisol-producing capacity of each group. Gene expression levels in the fetal adrenal glands of each group were analyzed by RNA-seq. RESULTS: The cortisol levels were significantly higher in the endotoxin group than in the control group after CRH challenge (P = .02). There were no significant differences in the responsiveness of adrenocorticotropin and cortisone between the 2 groups. Gene expression levels of the following enzymes involved in cortisol synthesis were significantly elevated in the endotoxin group: cytochrome P450 family (CYP) 11 subfamily A member 1 (log2FC 1.75), CYP 17 subfamily A member 1 (log2FC 3.41), 3β-hydroxysteroid dehydrogenase type I (log2FC 1.13), steroidogenic acute regulatory protein (log2FC 1.09), and CYP 21 (log2FC 0.89). CONCLUSION: Periviable fetuses exposed to inflammation in utero have altered the responsiveness of the HPA axis with increased expression of enzymes involved in cortisol synthesis in the adrenal gland.

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.

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.

Recent studies, using magnetic resonance imaging (MRI) to assess white matter injury in preterm brains, increasingly recognize punctate white matter lesions (PWML) as the primary lesion type. There are some papers showing the relationship between the size and number of PWML and the prognosis of infants. However, the histopathological features are still unknown. In this study, we experimentally induced periventricular leukomalacia (PVL) in a sheep fetus model, aiming to find whether MRI can visualize necrotic foci (small incipient lesions of PVL) as PWML. Three antenatal insults were employed to induce PVL in preterm fetuses at gestational day 101-117: (i) hypoxia under intrauterine inflammation, (ii) restriction of artificial placental blood flow, and (iii) restriction of artificial placental blood flow after exposure to intrauterine inflammation. MRI was performed 3-5 days after the insults, and standard histological studies of the PVL validated its findings. Of the 89 necrotic foci detected in histological samples from nine fetuses with PVL, 78 were visualized as PWML. Four of the lesions detected as abnormal findings on MRI could not be histologically detected as corresponding abnormal findings. The diagnostic sensitivity and positive predictive values of histologic focal necrosis visualized as PWML were 0.92 and 0.95, respectively. The four lesions were excluded from these analyses. These data suggest that MRI can visualize PVL necrotic foci as PWML 3-5 days after the injury induction. PWML can spontaneously become obscure with time after birth, so their accurate diagnosis in the acute phase can prevent overlooking mild PVL.

BACKGROUND: Intraamniotic inflammation is associated with up to 40% of preterm births, most notably in deliveries occurring prior to 32 weeks' gestation. Despite this, there are few treatment options allowing the prevention of preterm birth and associated fetal injury. Recent studies have shown that the small, non-competitive allosteric interleukin (IL)-1 receptor inhibitor, rytvela, may be of use in resolving inflammation associated with preterm birth (PTB) and fetal injury. We aimed to use an extremely preterm sheep model of chorioamnionitis to investigate the anti-inflammatory efficacy of rytvela in response to established intra-amniotic (IA) lipopolysaccharide (LPS) exposure. We hypothesized that rytvela would reduce LPS-induced IA inflammation in amniotic fluid (AF) and fetal tissues. METHODS: Sheep with a single fetus at 95 days gestation (estimated fetal weight 1.0 kg) had surgery to place fetal jugular and IA catheters. Animals were recovered for 48 hours before being randomized to either: i) IA administration of 2 ml saline 24 hours before 2 ml IA and 2 ml fetal intravenous (IV) administration of saline (Saline Group, n = 7); ii) IA administration of 10 mg LPS in 2 ml saline 24 hours before 2 ml IA and 2 ml fetal IV saline (LPS Group, n = 10); 3) IA administration of 10 mg LPS in 2 ml saline 24 hours before 0.3 mg/fetal kg IA and 1 mg/fetal kg fetal IV rytvela in 2 ml saline, respectively (LPS + rytvela Group, n = 7). Serial AF samples were collected for 120 h. Inflammatory responses were characterized by quantitative polymerase chain reaction (qPCR), histology, fluorescent immunohistochemistry, enzyme-linked inmmunosorbent assay (ELISA), fluorescent western blotting and blood chemistry analysis. RESULTS: LPS-treated animals had endotoxin and AF monocyte chemoattractant protein (MCP)-1 concentrations that were significantly higher at 24 hours (immediately prior to rytvela administration) relative to values from Saline Group animals. Following rytvela administration, the average MCP-1 concentrations in the AF were significantly lower in the LPS + rytvela Group relative to in the LPS Group. In delivery samples, the expression of IL-1β in fetal skin was significantly lower in the LPS + rytvela Group compared to the LPS Group. CONCLUSION: A single dose of rytvela was associated with partial, modest inhibition in the expression of a panel of cytokines/chemokines in fetal tissues undergoing an active inflammatory response.

BACKGROUND: 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. OBJECTIVE: 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. STUDY DESIGN: 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. RESULTS: 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. CONCLUSION: 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.

BACKGROUND: Ex vivo uterine environment therapy is an experimental intensive care strategy for extremely preterm infants born between 21 and 24 weeks of gestation. Gas exchange is performed by membranous oxygenators connected by catheters to the umbilical vessels. The fetus is submerged in a bath of synthetic amniotic fluid. The lungs remain fluid filled, and pulmonary respiration does not occur. Intrauterine inflammation is strongly associated with extremely preterm birth and fetal injury. At present, there are no data that we are aware of to show that artificial placenta-based systems can be used to support extremely preterm fetuses compromised by exposure to intrauterine inflammation. OBJECTIVE: To evaluate the ability of our ex vivo uterine environment therapy platform to support extremely preterm ovine fetuses (95-day gestational age; approximately equivalent to 24 weeks of human gestation) exposed to intrauterine inflammation for a period of 120 hours, the following primary endpoints were chosen: (1) maintenance of key physiological variables within normal ranges, (2) absence of infection and inflammation, (3) absence of brain injury, and (4) gross fetal growth and cardiovascular function matching that of age-matched in utero controls. STUDY DESIGN: Ten ewes with singleton pregnancies were each given a single intraamniotic injection of 10-mg Escherichia coli lipopolysaccharides under ultrasound guidance 48 hours before undergoing surgical delivery for adaptation to ex vivo uterine environment therapy at 95-day gestation (term=150 days). Fetuses were adapted to ex vivo uterine environment therapy and maintained for 120 hours with constant monitoring of key vital parameters (ex vivo uterine environment group) before being killed at 100-day equivalent gestational age. Umbilical artery blood samples were regularly collected to assess blood gas data, differential counts, biochemical parameters, inflammatory markers, and microbial load to exclude infection. Ultrasound was conducted at 48 hours after intraamniotic lipopolysaccharides (before surgery) to confirm fetal viability and at the conclusion of the experiments (before euthanasia) to evaluate cardiac function. Brain injury was evaluated by gross anatomic and histopathologic investigations. Eight singleton pregnant control animals were similarly exposed to intraamniotic lipopolysaccharides at 93-day gestation and were killed at 100-day gestation to allow comparative postmortem analyses (control group). Biobanked samples from age-matched saline-treated animals served as an additional comparison group. Successful instillation of lipopolysaccharides into the amniotic fluid exposure was confirmed by amniotic fluid analysis at the time of administration and by analyzing cytokine levels in fetal plasma and amniotic fluid. Data were tested for mean differences using analysis of variance. RESULTS: Six of 8 lipopolysaccharide control group (75%) and 8 of 10 ex vivo uterine environment group fetuses (80%) successfully completed their protocols. Six of 8 ex vivo uterine environment group fetuses required dexamethasone phosphate treatment to manage profound refractory hypotension. Weight and crown-rump length were reduced in ex vivo uterine environment group fetuses at euthanasia than those in lipopolysaccharide control group fetuses (P<.05). There were no biologically significant differences in cardiac ultrasound measurement, differential leukocyte counts (P>.05), plasma tumor necrosis factor α, monocyte chemoattractant protein-1 concentrations (P>.05), or liver function tests between groups. Daily blood cultures were negative for aerobic and anaerobic growth in all ex vivo uterine environment group animals. No cases of intraventricular hemorrhage were observed. White matter injury was identified in 3 of 6 lipopolysaccharide control group fetuses and 3 of 8 vivo uterine environment group fetuses. CONCLUSION: We report the use of an artificial placenta-based system to support extremely preterm lambs compromised by exposure to intrauterine inflammation. Our data highlight key challenges (refractory hypotension, growth restriction, and white matter injury) to be overcome in the development and use of artificial placenta technology for extremely preterm infants. As such challenges seem largely absent from studies based on healthy pregnancies, additional experiments of this nature using clinically relevant model systems are essential for further development of this technology and its eventual clinical application.

Phased tracking (PT) is a high-precision ultrasonic technology that enables measurements of pulse pressure (PP). The aim of this study was to verify the accuracy of estimated PP using PT. Estimated PPs were compared with measured PPs in three sheep fetuses that were connected to an artificial placenta system. Similarly, estimated and measured PPs of 30 human neonates were compared. PP was calculated using the Water-Hammer equation (PP = ρ × PWV (pulse wave velocity) × ΔU). PWV was estimated by measuring the transit times of pulse waves at two sites along the aorta using the PT method, and ΔU was obtained by subtracting end-diastolic velocity from peak systolic velocity. The correlation between the estimated and measured PPs of the sheep fetuses was strong (r = 0.95, p ˂ 0.01), as was the case with the human neonates (r = 0.88, p ˂ 0.05). It can be concluded from the results of this study that PT may be a non-invasive alternative method used to predict PP.

BACKGROUND: Arginine vasopressin (AVP) infusion has been shown to be a useful strategy for the management of systemic perfusion failure in premature infants. Our objective was to determine the characteristics of the blood flow redistribution induced by AVP infusion in premature fetal sheep. METHODS: Nine sheep fetuses at 99 to 113 days of gestation were continuously infused with AVP. Measurement of blood flow to individual fetal organs was performed using a colored microsphere technique, with measurements performed at 30 min before and 90 min after the initiation of AVP infusions. RESULTS: The AVP infusion significantly increased blood flow to the medulla oblongata (P < 0.05), and significantly decreased flow to the adrenal glands (from 492.0 ± 239.6 to 364.9 ± 143.3 mL/min/100 g, P < 0.05) and heart (from 592.6 ± 184.5 to 435.6 ± 137.4 mL/min/100 g, P < 0.05). The infusion significantly increased the vascular resistance in adrenal glands, kidneys, ileum, colon, heart, and cerebellum. In the brain, except for the cerebellum, no significant increase in resistance was identified. CONCLUSIONS: There was no significant response to AVP infusion in cerebral blood flow in mid-gestation fetal sheep. Our observations suggest that, under AVP stimulation, the blood flow to the adrenal glands and myocardium might be decreased due to an increase in vascular resistance.

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 + betamethasone acetate with delivery at either 1 or 7 days posttreatment, or 2 treatments of 0.25 mg/kg betamethasone phosphate + betamethasone acetate spaced at 24 hours (giving ∼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 + 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.

BACKGROUND: Ex vivo uterine environment therapy is an experimental life support platform designed to reduce the risk of morbidity and mortality for extremely preterm infants born at the border of viability (21-24 weeks' gestation). To spare the functionally immature lung, this platform performs gas exchange via a membranous oxygenator connected to the umbilical vessels, and the fetus is submerged in a protective bath of artificial amniotic fluid. We and others have demonstrated the feasibility of extended survival with ex vivo uterine environment therapy therapy in late preterm fetuses; however, there is presently no evidence to show that the use of such a platform can support extremely preterm fetuses, the eventual translational target for therapy of this nature. OBJECTIVE: The objective of the study was to use our ex vivo uterine environment therapy platform to support the healthy maintenance of 600-700 g/95 days gestational age (equivalent to 24 weeks of human gestation) sheep fetuses. Primary outcome measures were as follows: (1) maintenance of key physiological variables; (2) absence of infection; (3) absence of brain injury; and (4) growth and cardiovascular function patterns matching that of noninstrumented, age-matched in utero controls. STUDY DESIGN: Singleton fetuses from 8 ewes underwent surgical delivery at 95 days' gestation (term, 150 days). Fetuses were adapted to ex vivo uterine environment therapy and maintained for 120 hours with real-time monitoring of key physiological variables. Umbilical artery blood samples were regularly collected to assess blood gas data, differential counts, inflammation, and microbial load to exclude infection. Brain injury was evaluated by gross anatomical and histopathological approaches after euthanasia. Nine pregnant control animals were euthanized at 100 days' gestation to allow comparative postmortem analyses. Data were tested for mean differences with an analysis of variance. RESULTS: Seven of 8 ex vivo uterine environment group fetuses (87.5%) completed 120 hours of therapy with key parameters maintained in a normal physiological range. There were no significant intergroup differences (P > .05) in final weight, crown-rump length, and body weight-normalized lung and brain weights at euthanasia compared with controls. There were no biologically significant differences in hematological parameters (total or differential leucocyte counts and plasma concentration of tumor necrosis factor-α and monocyte chemoattractant protein 1) (P > .05). Daily blood cultures were negative for aerobic and anaerobic growth in all ex vivo uterine environment animals. There was no difference in airspace consolidation between control and ex vivo uterine environment animals, and there was no increase in the number of lung cells staining positive for the T-cell marker CD3. There were no increases in interleukin-1, interleukin-6, interleukin-8, tumor necrosis factor-α, and monocyte chemoattractant protein 1 mRNA expression in lung tissues compared with the control group. No cases of intraventricular hemorrhage were observed, and white matter injury was identified in only 1 ex vivo uterine environment fetus. CONCLUSION: For several decades, there has been little improvement in outcomes of extremely preterm infants born at the border of viability. In the present study, we report the use of artificial placenta technology to support, for the first time, extremely preterm ovine fetuses (equivalent to 24 weeks of human gestation) in a stable, growth-normal state for 120 hours. With additional refinement, the data generated by this study may inform a treatment option to improve outcomes for extremely preterm infants.

BACKGROUND: Antenatal corticosteroids are among the most important and widely used interventions to improve outcomes for preterm infants. Antenatal corticosteroid dosing regimens remain unoptimized and without maternal weight-adjusted dosing. We, and others, have hypothesized that, once a low concentration of maternofetal steroid exposure is achieved and maintained, the duration of the steroid exposure determines treatment efficacy. Using a sheep model of pregnancy, we tested the relationship among steroid dose, duration of exposure, and treatment efficacy. OBJECTIVE: The study was conducted to investigate the relative importance of duration and magnitude of fetal corticosteroid exposure to mature the preterm fetal ovine lung. STUDY DESIGN: Ewes with single fetuses at 120 days gestation received an intravenous bolus (loading dose) followed by a maintenance infusion of betamethasone phosphate to target 12-hour fetal plasma betamethasone concentrations of (1) 20 ng/mL, (2) 10 ng/mL, or (3) 2 ng/mL. In a subsequent experiment, fetal plasma betamethasone concentrations were targeted at 2 ng/mL for 26 hours. Negative control animals received sterile saline solution. Positive control animals received 2 intramuscular injections of 0.25 mg/kg Celestone Chronodose (betamethasone phosphate + betamethasone acetate) spaced at 24 hours. Preterm lambs were delivered surgically and ventilated 48 hours after treatment commenced. Maternal and fetal plasma betamethasone concentrations were confirmed by mass spectrometry in a parallel study of chronically catheterized, corticosteroid-treated ewes and fetuses. RESULTS: The loading and maintenance doses were achieved and maintained the desired fetal plasma betamethasone concentrations of approximately 20, 10, and 2 ng/mL for 12 hours. Compared with the 12-hour infusion-treated animals, lambs from the positive control (2 intramuscular doses of 0.25 mg/kg Celestone Chronodose) group had the greatest functional lung maturation (compliance, gas exchange, arterial pH) and molecular evidence of maturation (glucocorticoid receptor signaling activation), despite having maximum fetal plasma betamethasone concentrations 2.5 times lower than animals in the 20 ng/mL betamethasone infusion group. Lambs from the 12-hour 2-ng/mL betamethasone infusion group had little functional lung maturation. In contrast, lambs from the 26-hour 2-ng/mL betamethasone infusion group had functional lung maturation equivalent to lambs from the positive control group. CONCLUSION: In preterm lambs that were exposed to antenatal corticosteroids, high maternofetal plasma betamethasone concentrations did not correlate with improved lung maturation. The largest and most consistent improvements in lung maturation were in animals that were exposed to either the clinical course of Celestone Chronodose or a low-dose betamethasone phosphate infusion to achieve a fetal plasma betamethasone concentration of approximately 2 ng/mL for 26 hours. The duration of low-concentration maternofetal steroid exposure, not total dose or peak drug exposure, is a key determinant for antenatal corticosteroids efficacy. These findings underscore the need to develop an optimized steroid dosing regimen that may improve both the efficacy and safety of antenatal corticosteroids therapy.

Mechanical ventilation causes lung injury and systemic inflammatory responses in preterm sheep and is associated with bronchopulmonary dysplasia (BPD) in preterm infants. Budesonide added to surfactant decreased BPD by 20% in infants. We wanted to determine the effects of budesonide and surfactant on injury from high tidal volume (VT) ventilation in preterm lambs. Ewes at 125 ± 1 days gestational age had fetal surgery to expose fetal head and chest with placental circulation intact. Lambs were randomized to 1) mechanical ventilation with escalating VT to target 15 ml/kg by 15 min or 2) continuous positive airway pressure (CPAP) of 5 cmH2O. After the 15-min intervention, lambs were given surfactant 100 mg/kg with saline, budesonide 0.25 mg/kg, or budesonide 1 mg/kg. The fetuses were returned to the uterus for 24 h and then delivered and ventilated for 30 min to assess lung function. Budesonide levels were low in lung and plasma. CPAP groups had improved oxygenation, ventilation, and decreased injury markers compared with fetal VT lambs. Budesonide improved ventilation in CPAP lambs. Budesonide decreased lung weights and lung liquid and increased lung compliance and surfactant protein mRNA. Budesonide decreased proinflammatory and acute-phase responses in lung. Airway thickness increased in animals not receiving budesonide. Systemically, budesonide decreased monocyte chemoattractant protein-1 mRNA and preserved glycogen in liver. Results with 0.25 and 1 mg/kg budesonide were similar. We concluded that budesonide with surfactant matured the preterm lung and decreased the liver responses but did not improve lung function after high VT injury in fetal sheep.

BACKGROUND: Antenatal steroids are standard of care for women who are at risk of preterm delivery; however, antenatal steroid dosing and formulation have not been evaluated adequately. The standard clinical 2-dose treatment with betamethasone-acetate+betamethasone-phosphate is more effective than 2 doses of betamethasone-phosphate for the induction of lung maturation in preterm fetal sheep. We hypothesized that the slowly released betamethasone-acetate component induces similar lung maturation to betamethasone-phosphate+betamethasone-acetate with decreased dose and fetal exposure. OBJECTIVE: The purpose of this study was to investigate pharmacokinetics and fetal lung maturation of antenatal betamethasone-acetate in preterm fetal sheep. STUDY DESIGN: Groups of 10 singleton-pregnant ewes received 1 or 2 intramuscular doses 24 hours apart of 0.25 mg/kg/dose of betamethasone-phosphate+betamethasone-acetate (the standard of care dose) or 1 intramuscular dose of 0.5 mg/kg, 0.25 mg/kg, or 0.125 mg/kg of betamethasone-acetate. Fetuses were delivered 48 hours after the first injection at 122 days of gestation (80% of term) and ventilated for 30 minutes, with ventilator settings, compliance, vital signs, and blood gas measurements recorded every 10 minutes. After ventilation, we measured static lung pressure-volume curves and sampled the lungs for messenger RNA measurements. Other groups of pregnant ewes and fetuses were catheterized and treated with intramuscular injections of betamethasone-phosphate 0.125 mg/kg, betamethasone-acetate 0.125 mg/kg, or betamethasone-acetate 0.5 mg/kg. Maternal and fetal betamethasone concentrations in plasma were measured for 24 hours. RESULTS: All betamethasone-treated groups had increased messenger RNA expression of surfactant proteins A, B, and C, ATP-binding cassette subfamily A member 3, and aquaporin-5 compared with control animals. Treatment with 1 dose of intramuscular betamethasone-acetate 0.125mg/kg improved dynamic and static lung compliance, gas exchange, and ventilation efficiency similarly to the standard treatment of 2 doses of 0.25 m/kg of betamethasone-acetate+betamethasone-phosphate. Betamethasone-acetate 0.125 mg/kg resulted in lower maternal and fetal peak plasma concentrations and decreased fetal exposure to betamethasone compared with betamethasone-phosphate 0.125 mg/kg. CONCLUSION: A single dose of betamethasone-acetate results in similar fetal lung maturation as the 2-dose clinical formulation of betamethasone-phosphate+betamethasone-acetate with decreased fetal exposure to betamethasone. A lower dose of betamethasone-acetate may be an effective alternative to induce fetal lung maturation with less risk to the fetus.

Preterm twins have a higher morbidity rate of patent ductus arteriosus (PDA) than do singletons. However, the effect of multiple births on maturation of the ductus arteriosus (DA) has not been reported. Because intimal thickening (IT) is required for DA anatomical closure, we examined IT development in the DA of preterm twins and singletons. Sheep DA tissues obtained from preterm fetuses were subjected to elastica van Gieson staining to evaluate IT. The total IT score in each DA was the sum of the IT scores obtained from six evenly divided parts of the DA, which was positively correlated with gestational ages in singletons. Total IT scores were smaller in preterm twins than in singletons, although no difference in gestational age, birth weight, or gender ratio was observed. These data suggest that IT development of the DA is attenuated in sheep preterm twins, which may affect the higher morbidity of PDA.

BACKGROUND: Extremely preterm infants born at the border of viability (22-24 weeks' gestation) have high rates of death and lasting disability. Ex vivo uterine environment therapy is an experimental neonatal intensive care strategy that provides gas exchange using parallel membranous oxygenators connected to the umbilical vessels, sparing the extremely preterm cardiopulmonary system from ventilation-derived injury. OBJECTIVE: In this study, we aimed to refine our ex vivo uterine environment therapy platform to eliminate fetal infection and inflammation, while simultaneously extending the duration of hemodynamically stable ex vivo uterine environment therapy to 1 week. STUDY DESIGN: Merino-cross ewes with timed, singleton pregnancies were surgically delivered at 112-115 days of gestation (term is ∼150 days) and adapted to ex vivo uterine environment therapy (treatment group; n = 6). Physiological variables were continuously monitored; humerus and femur length, ductus arteriosus directional flow, and patency were estimated with ultrasound; serial blood samples were collected for hematology and microbiology studies; weight was recorded at the end of the experiment. Control group animals (n = 7) were euthanized at 122 days of gestation and analyzed accordingly. Bacteremia was defined by positive blood culture. Infection and fetal inflammation was assessed with white blood cell counts (including differential leukocyte counts), plasma and lung proinflammatory cytokine measurements, and lung histopathology. RESULTS: Five of 6 fetuses in the treatment group completed the 1-week study period with key physiological parameters, blood counts remaining within normal ranges, and no bacteremia detected. There were no significant differences (P > .05) in arterial blood oxygen content or lactate levels between ex vivo uterine environment therapy and control groups at delivery. There was no significant difference (P > .05) in birthweight between control and ex vivo uterine environment groups. In the ex vivo uterine environment group, we observed growth of fetal humerus (P < .05) and femur (P < .001) over the course of the 7-day experimental period. There was no difference in airway or airspace morphology or consolidation between control and ex vivo uterine environment animals, and there was no increase in the number of lung cells staining positive for T-cell marker CD3+. CONCLUSION: Five preterm lambs were maintained in a physiologically stable condition for 1 week with significant growth and without clinically significant bacteremia or systemic inflammation. Although substantial further refinement is required, a life support platform based around ex vivo uterine environment therapy may provide an avenue to improve outcomes for extremely preterm infants.

Ex vivo uterine environment (EVE) therapy is an experimental neonatal intensive care strategy wherein gas exchange is performed by membranous oxygenators attached to the umbilical vessels. Our aim was to assess the ability of a newly refined EVE system to maintain key physiological parameters in preterm lambs within optimal ranges for 48 h. EVE group; n = 6: Preterm lambs were delivered under general anesthesia at 115 ± 2 days of gestational age. Animals were submerged in a bath of artificial amniotic fluid on EVE therapy for 48 h. Physiological parameters were monitored in real-time over the length of the experiment. Control group; n = 11: Ewes carrying a single fetus (115 ± 2 days of gestational age) underwent recovery surgery to allow placement of a fetal carotid artery catheter. Fetuses received an infusion of sterile saline only. After euthanasia, EVE and Control group fetuses underwent necroscopy to perform static pressure-volume curves and for sampling of lung and cord blood plasma for molecular analyses. Five out of six fetuses in the EVE group completed the study period with key physiological variables remaining within their respective reference ranges for the duration of the 48 h study. Bacteremia was identified in four out of five EVE fetuses, and was associated with a systemic inflammatory response. Using our refined EVE therapy platform, preterm lambs were maintained in a stable physiological condition for 48 h. These findings represent a significant advance over earlier work with this system; however, the identification of bacteremia and a fetal inflammatory response suggests that further refinement to the EVE therapy platform is required.

BACKGROUND: Antenatal steroids are standard of care for cases of anticipated preterm labor to improve neonatal outcomes. However, steroids are potent drugs, and their use in pregnancy remains largely unoptimized. OBJECTIVE: The objective of the study was to measure the maternofetal pharmacokinetics of constant, low-dose intravenous betamethasone phosphate infusions and correlate these data with the transcriptional effect exerted by subclinical betamethasone exposures on the ovine fetal lung. STUDY DESIGN: Thirty-two ewes carrying a single fetus had surgery to catheterize fetal and maternal jugular veins at 116 days of gestation (term, 150 days). Animals were recovered for 2 days and then were randomized to receive 2 sequential maternal intravenous infusions of either (n = 4/group) of the following: 1) saline, 0.125, 0.04, or 0.0125 mg/kg betamethasone phosphate over 3 hours; or 2) saline, 0.25, 0.08, or 0.025 mg/kg betamethasone phosphate over 12 hours. Each infusion was separated by 2 days. Fetal lung tissue was collected for analysis using quantitative polymerase chain reaction and an ovine-specific microarray. Plasma betamethasone levels from time-course catheter samples were determined by mass spectrometry. Data were assessed for distribution, variance, and tested by an analysis of variance. RESULTS: Betamethasone was detectable (>1 ng/mL) in fetal plasma only in animals randomized to 0.125 mg/kg 3 hour or 0.250 mg/kg 12 hour infusions. Fetal betamethasone half-lives were 1.7-2.8 times greater than maternal values. At maximum concentration, fetal plasma betamethasone levels were approximately 10% of maternal levels. Compared with saline control, all animals, other than those receiving 0.0125 mg/kg 3 hour betamethasone phosphate infusions, had evidence of dose-dependent glucocorticoid transcriptional responses in the fetal lung. CONCLUSION: Constant maternal betamethasone infusions delivering substantially lower fetal and maternal betamethasone maximal concentrations than those achieved with current clinical treatment protocols were associated with dose-dependent changes in glucocorticoid-response markers in the fetal lung. Further studies to determine the minimally efficacious dose of steroids for improving outcomes in preterm infants should be viewed as a priority.

Surgical ligation for patent ductus arteriosus (PDA) in extremely low birth weight infants (ELBWIs) has been shown a possible association with neurodevelopmental impairment (NDI) because of its invasiveness. However, we have undergone surgical ligation for ELBWIs immediately after cyclooxygenase inhibitor failed to close a hemodynamically significant PDA (hsPDA) to maintain proper systemic circulation. We aimed to determine the effect of surgical ligation for hsPDA on NDI in ELBWIs. In enrolled 71 ELBWIs, the clinical parameters, including the developmental quotient (DQ), were collected and compared among three groups that were divided by closure mode: spontaneous closure (n = 11), cyclooxygenase inhibitor therapy (n = 37) and surgical ligation (n = 23). No significant differences in DQ at the age of 36 months among the three groups were found: Median (interquartile range): 92.0 (31.0), 89.0 (22.0) and 92.0 (24.5), respectively. In a comparison between groups of DQ < 70 (n = 15) and DQ ≥ 70 (n = 56), a significant difference was found in the parameters related to prematurity (p < 0.05 for each): gestational age [23.9 (1.70) vs. 25.4 (2.50) weeks], birth weight [595 (183) vs. 714 (192) g], Apgar score < 5 (1 min) (67% vs. 36%), and laser photocoagulation for retinopathy of prematurity (73% vs. 43%), but there was no significant association with hsPDA. Therefore, we propose that surgical ligation for hsPDA in ELBWIs should be immediately carried out for preventing future neurodevelopmental deterioration if the cyclooxygenase inhibitor failed to close hsPDA.

An artificial placenta (AP) is an arterio-venous extracorporeal life support system that is connected to the fetal circulation via the umbilical vasculature. Previously, we published an article describing a pumpless AP system with a small priming volume. We subsequently developed a parallelized system, hypothesizing that the reduced circuit resistance conveyed by this modification would enable healthy fetal survival time to be prolonged. We conducted experiments using a premature lamb model to test this hypothesis. As a result, the fetal survival period was significantly prolonged (60.4 ± 3.8 vs. 18.2 ± 3.2 h, P < 0.01), and circuit resistance and minimal blood lactate levels were significantly lower in the parallel circuit group, compared with our previous single circuit group. Fetal physiological parameters remained stable until the conclusion of the experiments. In summary, parallelization of the AP system was associated with reduced circuit resistance and lactate levels and allowed preterm lamb fetuses to survive for a significantly longer period when compared with previous studies.

Using uterine explants from Per1::Luc rats and in situ hybridization, we recently reported that the circadian property of the molecular clock in the uterus and placenta is stably maintained from non-pregnancy, right through to the end stage of pregnancy under regular light-dark (LD) cycles. Despite long-lasting increases in progesterone during gestation and an increase in estrogen before delivery, the uterus keeps a stable Per1::Luc rhythm throughout the pregnancy. The study suggests the importance of stable circadian environments for fetuses to achieve sound physiology and intrauterine development. This idea is also supported by epidemiological and animal studies, in which pregnant females exposed to repeated shifting of the LD cycles have increased rates of reproductive abnormalities and adverse pregnancy outcomes. Leading from this, we introduced artificial circadian environments with controlled lighting conditions to human preterm infants by developing and utilizing a specific light filter which takes advantage of the unique characteristics of infants' developing visual photoreceptors. In spite of growing evidence of the physiological benefits of nighttime exposure to darkness for infant development, many Japanese Neonatal Intensive Care Units (NICUs) still prefer to maintain constant light in preparation for any possible emergencies concerning infants in incubators. To protect infants from the negative effects of constant light on their development in the NICU, we have developed a new device similar to a magic mirror, by which preterm infants can be shielded from exposure to their visible wavelengths of light even in the constant light conditions of the NICU while simultaneously allowing medical care staff to visually monitor preterm infants adequately. The device leads to significantly increased infant activity during daytime than during night time and better weight gains.

Maternal circadian information has been reported to play an important role in fetal physiology and development. Hormones and nutrition have been mainly investigated as circadian cues from mother to fetus. However, the influences of circadian properties of the pregnant reproductive organs on fetuses have not been fully investigated. To gain an insight on the circadian functions of the reproductive organs, we examined molecular clocks in the pregnant rat uterus and placenta. By using a Period1-luciferase (Per1-luc) rat, whose tissues express luciferase corresponding to activation of Period1, a "key clock gene", we examined the uterus clock during non-pregnancy, on embryonic day 12 (E12), and on E22 (the end of pregnancy) in a light-dark (LD) cycle and constant darkness (DD). By in situ hybridization we further explored Per1 mRNA rhythms in the placenta on E12 and E22. The uterus in vitro showed clear circadian Per1-luc rhythms both in and out of pregnancy, having peaks at around the time corresponding to dusk in LD. Likewise, in DD, the uterus in vitro had the same Per1-luc rhythms. The decidua in LD showed circadian Per1 mRNA rhythms, peaking during night 6 h after dusk, while the decidua in DD showed the same Per1 mRNA rhythms only on E22. In contrast, the labyrinth showed no circadian Per1 mRNA rhythms in LD or DD during pregnancy. These results suggest that the uterus and decidua, a maternally-originated tissue of the placenta, but not the labyrinth, a fetus-originated tissue of the placenta, can provide the fetus with circadian information.