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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.

Background Antenatal steroids (ANS) are routinely administered to women at risk of preterm birth to accelerate fetal lung maturation. Despite extensive clinical use, dosing and route of ANS administration remain unoptimized beyond intramuscular (IM) injection. We aimed to undertake a proof-of-principle assessment of transdermal ANS administration for accelerated fetal lung maturation. Methods We formulated and tested a transdermal ANS (betamethasone) patch in ex vivo studies. To confirm in vivo transdermal distribution of the ANS patch, we undertook a 24-h pharmacokinetic study in non-pregnant cynomolgus macaques (NHP). To assess the efficacy of transdermal betamethasone on fetal lung maturation, we used a preterm sheep model of pregnancy comparing postnatal ventilation outcomes. Date mated ewes received either of the following: (i) IM normal saline (Saline Control, n  = 13); (ii) IM ANS (betamethasone) delivery 2 or 8 days post-treatment (2-Day IM ANS, n  = 14; 8-day IM ANS Group, n  = 6); (iii) IM saline + 2 × transdermal ANS (Betamethasone) patches delivery 2 days post-treatment (2-Day ANS Patch, n  = 10); or (iv) IM saline + 2 × transdermal ANS (betamethasone) patches replaced with 2 × new patches after 48 h, delivery 8 days post-treatment (8-Day ANS Patch, n  = 11). Lambs were delivered at 123 ± 1 dGA (term 150 days), then ventilated for 30 min to assess lung maturation status. Arterial blood gas, delivery, and ventilation data were analyzed ( p  < 0.05 significant). Results Transdermal administration of ANS in NHPs achieved rapid plasma accumulation, with a plasma half-life of 8.9 h, similar to that achieved with IM dosing in clinical practice. In preterm sheep, all 2- and 8-day IM and transdermal ANS patch groups had clinically important and statistically significant improvements in ventilation parameters (umbilical PaCO 2 , pH, tidal volume) compared to saline control. Conclusions We demonstrate, for the first time, the potential for using transdermal ANS for preterm fetal lung maturation. Transdermal ANS administration eliminates the need for injections, pain, and infection risk. Moreover, transdermal ANS delivery demonstrates advantages in dose control, continuity of exposure, and potential to remove drug exposure should the risk of preterm birth resolve. Further development of this technology may improve preterm outcomes through delivery of a low dose, constant fetal steroid exposure.

Background A lack of tests to assess fetal development impacts decision making around antenatal steroid use in women at risk of preterm birth. We analyzed the expression of 21 cfRNA targets related to human fetal lung maturation. Discovery studies were performed using maternal and fetal sheep plasma, with results compared to fetal lung mRNA expression. These findings were then validated in first, second, and third trimester human maternal plasma samples. Methods Discovery studies utilized a preterm sheep model of pregnancy. Date mated ewes received saline (control n  = 6), or antenatal steroids (dexamethasone n  = 12) (betamethasone n  = 11) prior to delivery and ventilation. We analyzed the expression of 21 human cfRNA targets related to lung maturation in maternal and fetal sheep plasma and compared this to mRNA expression in fetal lung tissue. Findings were first validated in a separate cohort of sheep exposed to betamethasone ( n  = 8), intraamniotic LPS endotoxin for lung maturation ( n  = 6), or untreated term animals ( n  = 6). Findings were further validated in maternal plasma from a human cohort of uncomplicated term pregnancies ( n  = 10). Delivery and ventilation data were analyzed with ANOVA, Tukey HSD, and Dunnett T3 tests. A Random Forest algorithm identified genes that separated mature from immature fetal lung subgroups and determined AUC values for maternal and fetal cell-free RNA (cfRNA) feature sets to predict fetal lung maturation. Results We demonstrate that the analysis of 21 human cfRNA targets in maternal plasma is highly predictive of fetal lung maturation status across antenatal steroid induced (Dexamethasone AUC = 0.93; Betamethasone AUC = 1) and physiological (AUC = 1) lung development models. Maternal plasma cfRNA expression in the dexamethasone antenatal steroid group closely resembled direct fetal lung tissue mRNA expression. These findings were then validated in human maternal plasma samples (1st vs. 3rd trimester AUC = 0.96; 2nd vs. 3rd trimester AUC = 1). Conclusions Further development of this technology may provide a rapid, minimally invasive, and cost-effective clinical tool to optimize patient selection for initial and repeat courses of antenatal steroids, along with insights into the molecular mechanisms underlying fetal lung development. Graphical Abstract Graphical abstract illustrating the potential future application of maternal plasma cell-free RNA analysis. Created in https://BioRender.com .

Background Antenatal steroid (ANS) therapy is given to women at risk of preterm delivery to accelerate fetal lung maturation. However, the benefit of ANS therapy is variable and how maternal and fetal factors contribute to this observed variability is unknown. We aimed to test the degree of concordance in preterm lung function, and correlate this with genomic, transcriptomic, and pharmacokinetic variables in preterm dizygotic twin ovine fetuses. Methods Thirty-one date-mated ewes carrying twin fetuses at 123 ± 1 days’ gestation received maternal intramuscular injections of either (i) 1 × 0.25 mg/kg betamethasone phosphate and acetate (CS1, n  = 11 twin pairs) or (ii) 2 × 0.25 mg/kg betamethasone phosphate and acetate, 24 h apart (CS2, n  = 10 twin pairs) or (iii) 2 × saline, 24 h apart (negative control, n  = 10 twin pairs). Fetuses were surgically delivered 24 h after their final treatment and ventilated for 30 min. Results ANS-exposed female fetuses had lower arterial partial pressure of carbon dioxide (PaCO 2 ) values than male fetuses (76.5 ± 38.0 vs. 97.2 ± 42.5 mmHg), although the observed difference was not statistically significant ( p  = 0.1). Only 52% of ANS-treated twins were concordant for lung maturation responses. There was no difference in fetal lung tissue or plasma steroid concentrations within or between twin pairs. Genomic analysis identified 13 single-nucleotide polymorphisms (SNPs) statistically associated with ANS-responsiveness, including in the proto-oncogene MET and the transcription activator STAT1. Conclusions Twin fetal responses and ANS tissue levels were comparable with those from singleton fetuses in earlier studies. Twin ovine fetuses thus benefit from ANS in a similar manner to singleton fetuses, and a larger dose of betamethasone is not required. Assuming no difference in input from the placental or maternal compartments, fetal lung responses to ANS therapy in dizygotic twin preterm lambs are dependent on the fetus itself. These data suggest a potential heritable role in determining ANS responsiveness.

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. 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. 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-1[beta] in fetal skin was significantly lower in the LPS + rytvela Group compared to the LPS Group. 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.

BackgroundThe 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.MethodsWe 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.ResultsOral 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.ConclusionUsing 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.

Abstract 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.

Background Labial fusion is defined as adhesions of the labia minora or majora. Labial fusion may cause urinary retention. Surgical treatment based on an accurate anatomic assessment may be needed, but the usefulness of endoscopic examination for this disease has not been reported. Case presentation A 76-year-old Japanese woman undergoing chemoradiation treatment for esophageal cancer was referred to our department for evaluation of high accumulation in the vagina on a positron emission tomography scan. On physical examination, her labia were noted to be extensively fused with a pinhole opening at the midline. Endoscopic examination revealed that her vagina was filled with urine and there were no abnormalities in her urethral meatus and cervix. The adhesions were separated under anesthesia and there has been no recurrence during follow-up. Conclusions We present a case of a postmenopausal patient with labial fusion who underwent successful surgical management. An endoscopic examination enabled us to determine the precise anatomic position and adopt a safe surgical procedure.

Intrauterine inflammation is a significant cause of early preterm birth and fetal injury. There is a lack of effective interventions for intrauterine inflammation. This study aimed to determine whether direct fetal treatment with IL-1 receptor antagonists (IL-1RA), specifically anakinra (competitive IL-1RA) or rytvela (allosteric IL-1RA), could reduce intrauterine inflammation caused by intraamniotic injection of E. coli lipopolysaccharides (LPS) in a sheep model of pregnancy. We hypothesized the fetal intramuscular administration of IL1-RA therapy would comprehensively resolve intrauterine inflammation caused by LPS in the pregnant sheep model. Date-mated Merino ewes carrying single fetuses were randomized into four groups: LPS Group (10 mg intraamniotic LPS injection followed by saline), RYTVELA Group (10 mg LPS injection followed by 5 mg rytvela), ANAKINRA Group (LPS injection followed by 100 mg anakinra), and SALINE Group (saline injection followed by saline). All LPS-exposed fetuses had elevated bilirubin levels, leukopenia, and increased inflammatory mediators IL-1β, IL-8, tumour necrosis factor alpha (TNFα), and monocyte chemoattractant protein 1 (MCP-1) in amniotic fluid and lung tissue. Both anakinra and rytvela treatments reduced immunocyte infiltration in chorioamniotic membranes and lungs, and microglial staining, and increased the oligodendrocyte staining, but did not significantly resolve overall inflammation compared to the SALINE Group. In conclusion, fetal intramuscular administration of anakinra and rytvela did not effectively resolve intrauterine inflammation but showed potential in reducing tissue invasion and brain injury markers. These findings suggest that modest inflammation reduction may protect against brain injury and preterm birth, though no additional benefit was observed compared to intraamniotic IL-1RA treatment.