The effect of comorbidities on the sFLT-1:PlGF ratio in preeclampsia.

Research indicates that soluble fms-like tyrosine kinase 1 (sFLT-1) and placental growth factor (PLGF) have diagnostic and prognostic significance for women with preeclampsia. However, sparse research has studied these biomarkers in women with preexisting comorbidities such as chronic hypertension, diabetes mellitus, systemic lupus erythematosus and chronic kidney disease. We undertook a prospective longitudinal cohort study to compare the sFLT-1: PlGF ratio between women with and without comorbidities who did and did not go on to develop preeclampsia. We found that women with comorbidities may develop preeclampsia with a milder elevation in sFLT-1: PlGF than do women without comorbidities. This has clinical and research implications.

Assessing the impact of gestational age of donors on the efficacy of amniotic epithelial cell-derived extracellular vesicles in experimental bronchopulmonary dysplasia.

BACKGROUND AND RATIONALE: Extracellular vesicles (EVs) are a potential cell-free regenerative medicine. Human amniotic epithelial cells (hAECs) are a viable source of cell therapy for diseases like bronchopulmonary dysplasia (BPD). However, little is known about the impact of gestational age of the donor on the quality of hAEC-derived EVs. AIMS: To determine the impact of gestational age on hAEC-derived EVs in experimental BPD. RESULTS: Term hAEC-derived EVs displayed a significantly higher density of surface epitopes (CD142 and CD133) and induced greater macrophage phagocytosis compared to preterm hAEC-EVs. However, T cell proliferation was more significantly suppressed by preterm hAEC-EVs. Using a model of experimental BPD, we observed that term but not preterm hAEC-EVs improved tissue-to-airspace ratio and septal crest density. While both term and preterm hAEC-EVs reduced the levels of inflammatory cytokines on postnatal day 7, the improvement in lung injury was associated with increased type II alveolar cells which was only observed in term hAEC-EV treatment group. Furthermore, only neonatal term hAEC-EVs reduced airway hyper-responsiveness, mitigated pulmonary hypertension and protected against right ventricular hypertrophy at 6 weeks of age. CONCLUSION: Term hAEC-EVs, but not preterm hAEC-EVs, have therapeutic efficacy in a mouse model of BPD-like lung injury. Therefore, the impact of donor criteria should be considered when applying perinatal cells-derived EV therapy for clinical use.

Silencing of Nrf genes in the human placenta as measured by SDS-PAGE and Western Blotting techniques.

Nuclear factor erythroid 2-related factor-2 (Nrf2), and the less well characterised proteins Nrf1 and Nrf3, are member of the cap 'n' collar family of transcription factors. Nrf proteins regulate the expression of endogenous antioxidant enzymes and have recently become the targets for various therapeutic treatments. Recently, Nrf proteins have been of particular interest as a target in placental-derived oxidative stress induced pregnancy disorders. Here, we report the presence of Nrf1, Nrf2 and Nrf3 proteins in both human primary trophoblast and human trophoblast choriocarcinoma cell line (BeWo). We also detail the steps taken to successfully silence all Nrf proteins in both human primary trophoblast cells and BeWo via detection of mRNA and protein using quantitative PCR, and SDS-PAGE and Western Blotting respectively.

Associations Between Maternal Immunisation and Reduced Rates of Preterm Birth and Stillbirth: A Population Based Retrospective Cohort Study.

Stillbirth and preterm birth (PTB) remain two of the most important, unresolved challenges in modern pregnancy care. Approximately 10% of all births are preterm with nearly one million children dying each year due to PTB. It remains the most common cause of death among children under five years of age. The numbers for stillbirth are no less shocking with 2.6 million babies stillborn each year. With minimal impact on the rate of these adverse birth outcomes over the past decade there is an urgent need to identify more effective interventions to tackle these problems. In this retrospective cohort study, we used whole-of-population data, to determine if maternal immunization during pregnancy against influenza and/or pertussis, is associated with a lower risk of PTB, delivering a small-for-gestational age (SGA) infant, developing preeclampsia or stillbirth. Women with a singleton pregnancy at 28 or more weeks' gestation delivering in Victoria, Australia from July 2015 to December 2018 were included in the analysis. Log-binomial regression was used to measure the relationship between vaccination during pregnancy against influenza and against pertussis, with preterm birth, SGA, preeclampsia and stillbirth. Variables included in the adjusted model were maternal age, body mass index, first or subsequent birth, maternal Indigenous status, socio-economic quintile, smoking, public or private maternity care and metropolitan or rural location of the hospital. Women who received influenza vaccine were 75% less likely to have a stillbirth (aRR 025; 95% CI 0.20, 0.31), and 31% less likely to birth <37 weeks (aRR 0.69; 95% CI 0.66, 0.72). Women who received pertussis vaccine were 77% less likely to have a stillbirth (aOR 0.23; 95% CI 0.18, 0.28) and 32% less likely to birth <37 weeks gestation (aRR 0.68; 95% CI 0.66, 0.71). Vaccination also reduced the odds of small for gestational age by 13% and reduced the odds of pre-eclampsia when restricted to primiparous women. This association was seen over four different influenza seasons and independent of the time of year suggesting that any protective effect on obstetric outcomes afforded by maternal vaccination may not be due to a pathogen-specific response but rather due to pathogen-agnostic immune-modulatory effects.

Impact of chemically defined culture media formulations on extracellular vesicle production by amniotic epithelial cells.

The therapeutic properties of cell derived extracellular vesicles (EVs) make them promising cell-free alternative to regenerative medicine. However, clinical translation of this technology relies on the ability to manufacture EVs in a scalable, reproducible, and cGMP-compliant manner. To generate EVs in sufficient quantity, a critical step is the selection and development of culture media, where differences in formulation may influence the EV manufacturing process. In this study, we used human amniotic epithelial cells (hAECs) as a model system to explore the effect of different formulations of chemically defined, commercially sourced media on EV production. Here, we determined that cell viability and proliferation rate are not reliable quality indicators for EV manufacturing. The levels of tetraspanins and epitope makers of EVs were significantly impacted by culture media formulations. Mass spectrometry-based proteomic profiling revealed proteome composition of hAEC-EVs and the influence of media formulations on composition of EV proteome. This study has revealed critical aspects including cell viability and proliferation rate, EV yield, and tetraspanins, surface epitopes and proteome composition of EVs influenced by media formulations, and further insight into standardised EV production culture media that should be considered in clinical-grade scalable EV manufacture for generation of therapeutic EVs.

A protocol for cell therapy infusion in neonates.

Cell therapies for neonatal morbidities are progressing to early phase clinical trials. However, protocols for intravenous (IV) delivery of cell therapies to infants have not been evaluated. It has been assumed the cell dose prescribed is the dose delivered. Early in our clinical trial of human amnion epithelial cells (hAECs), we observed cells settling in the syringe and IV tubing used to deliver the suspension. The effect on dose delivery was unknown. We aimed to quantify this observation and determine an optimal protocol for IV delivery of hAECs to extremely preterm infants. A standard pediatric infusion protocol was modeled in the laboratory. A syringe pump delivered the hAEC suspension over 60 minutes via a pediatric blood transfusion set (200-µm filter and 2.2 mL IV line). The infusion protocol was varied by agitation methods, IV-line volumes (0.2-2.2 mL), albumin concentrations (2% vs 4%), and syringe orientations (horizontal vs vertical) to assess whether these variables influenced the dose delivered. The influence of flow rate (3-15 mL/h) was assessed after other variables were optimized. The standard infusion protocol delivered 17.6% ± 9% of the intended hAEC dose. Increasing albumin concentration to 4%, positioning the syringe and IV line vertically, and decreasing IV-line volume to 0.6 mL delivered 99.7% ± 13% of the intended hAEC dose. Flow rate did not affect dose delivery. Cell therapy infusion protocols must be considered. We describe the refinement of a cell infusion protocol that delivers intended cell doses and could form the basis of future neonatal cell delivery protocols.

Potential treatment of keloid pathogenesis with follistatin 288 by blocking the activin molecular pathway.

Keloids are benign tumours caused by abnormal wound healing driven by increased expression of cytokines, including activin A. This study compared effects of activins on normal and keloid-derived human dermal fibroblasts and investigated a novel treatment for keloids using follistatin. Normal skin and keloid tissue samples from 11 patients were used to develop primary fibroblast cultures, which were compared in terms of their histology and relevant gene (qRT-PCR and RNAseq) and protein (ELISA) expression. Activin A (INHBA) and connective tissue growth factor (CTGF) gene expression were significantly upregulated in keloid fibroblasts, as was activin A protein expression in cell lysates and culture medium. Activator protein 1 inhibitor (SR11302) significantly decreased INHBA and CTGF expression in keloid fibroblasts and a single treatment of follistatin over 5 days significantly inhibited activin and various matrix-related genes in keloid fibroblasts when compared to controls. Follistatin, by binding activin A, suppressed CTGF expression suggesting a novel therapeutic role in managing keloids and perhaps other fibrotic diseases.

Window of opportunity for human amnion epithelial stem cells to attenuate astrogliosis after umbilical cord occlusion in preterm fetal sheep.

There is increasing evidence that administration of many types of stem cells, including human amnion epithelial cells (hAECs), can reduce hypoxic-ischemic injury, including in the perinatal brain. However, the therapeutic window for single dose treatment is not known. We compared the effects of early and delayed intracerebroventricular administration of hAECs in fetal sheep at 0.7 gestation on brain injury induced by 25 minutes of complete umbilical cord occlusion (UCO) or sham occlusion. Fetuses received either 1 × 106 hAECs or vehicle alone, as an infusion over 1 hour, either 2 or 24 hours after UCO. Fetuses were killed for brain histology at 7 days post-UCO. hAEC infusion at both 2 and 24 hours had dramatic anti-inflammatory and anti-gliotic effects, including significantly attenuating the increase in microglia after UCO in the white and gray matter and the number of astrocytes in the white matter. Both protocols partially improved myelination, but had no effect on total or immature/mature numbers of oligodendrocytes. Neuronal survival in the hippocampus was increased by hAEC infusion at either 2 or 24 hours, whereas only hAECs at 24 hours were associated with improved neuronal survival in the striatum and thalamus. Neither protocol improved recovery of electroencephalographic (EEG) power. These data suggest that a single infusion of hAECs is anti-inflammatory, anti-gliotic, and neuroprotective in preterm fetal sheep when given up to 24 hours after hypoxia-ischemia, but was associated with limited white matter protection after 7 days recovery and no improvement in the recovery of EEG power.

Prematurity negatively affects regenerative properties of human amniotic epithelial cells in the context of lung repair.

There is a growing appreciation of the role of lung stem/progenitor cells in the development and perpetuation of chronic lung disease including idiopathic pulmonary fibrosis. Human amniotic epithelial cells (hAECs) were previously shown to improve lung architecture in bleomycin-induced lung injury, with the further suggestion that hAECs obtained from term pregnancies possessed superior anti-fibrotic properties compared with their preterm counterparts. In the present study, we aimed to elucidate the differential effects of hAECs from term and preterm pregnancies on lung stem/progenitor cells involved in the repair. Here we showed that term hAECs were better able to activate bronchioalveolar stem cells (BASCs) and type 2 alveolar epithelial cells (AT2s) compared with preterm hAECs following bleomycin challenge. Further, we observed that term hAECs restored TGIF1 and TGFß2 expression levels, while increasing c-MYC expression despite an absence of significant changes to Wnt/ß-catenin signaling. In vitro, term hAECs increased the average size and numbers of BASC and AT2 colonies. The gene expression levels of Wnt ligands were higher in term hAECs, and the expression levels of BMP4, CCND1 and CDC42 were only increased in the BASC and AT2 organoids co-cultured with hAECs from term pregnancies but not preterm pregnancies. In conclusion, term hAECs were more efficient at activating the BASC niche compared with preterm hAECs. The impact of gestational age and/or complications leading to preterm delivery should be considered when applying hAECs and other gestational tissue-derived stem and stem-like cells therapeutically.

Cell therapy for the preterm infant: promise and practicalities.

Recent decades have seen the rapid progress of neonatal intensive care, and the survival rates of the most preterm infants are improving. This improvement is associated with changing patterns of morbidity and new phenotypes of bronchopulmonary dysplasia and preterm brain injury are recognised. Inflammation and immaturity are known contributors to their pathogenesis. However, a new phenomenon, the exhaustion of progenitor cells is emerging as an important factor. Current therapeutic approaches do not adequately address these new mechanisms of injury. Cell therapy, that is the use of stem and stem-like cells, with its potential to both repair and prevent injury, offers a new approach to these challenging conditions. This review will examine the rationale for cell therapy in the extremely preterm infant, the preclinical and early clinical evidence to support its use in bronchopulmonary dysplasia and preterm brain injury. Finally, it will address the challenges in translating cell therapy from the laboratory to early clinical trials.

Hydroxychloroquine Mitigates the Production of 8-Isoprostane and Improves Vascular Dysfunction: Implications for Treating Preeclampsia.

In preeclampsia, widespread maternal endothelial dysfunction is often secondary to excessive generation of placental-derived anti-angiogenic factors, including soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng), along with proinflammatory cytokines such as tumour necrosis factor-α (TNF-α) and activin A, understanding of which offers potential opportunities for the development of novel therapies. The antimalarial hydroxychloroquine is an anti-inflammatory drug improving endothelial homeostasis in lupus. It has not been explored as to whether it can improve placental and endothelial function in preeclampsia. In this in vitro study, term placental explants were used to assess the effects of hydroxychloroquine on placental production of sFlt-1, sEng, TNF-α, activin A, and 8-isoprostane after exposure to hypoxic injury or oxidative stress. Similarly, human umbilical vein endothelial cells (HUVECs) were used to assess the effects of hydroxychloroquine on in vitro markers of endothelial dysfunction. Hydroxychloroquine had no effect on the release of sFlt-1, sEng, TNF-α, activin A, or 8-isoprostane from placental explants exposed to hypoxic injury or oxidative stress. However, hydroxychloroquine mitigated TNF-α-induced HUVEC production of 8-isoprostane and Nicotinanamide adenine dinucleotide phosphate (NADPH) oxidase expression. Hydroxychloroquine also mitigated TNF-α and preeclamptic serum-induced HUVEC monolayer permeability and rescued the loss of zona occludens protein zona occludens 1 (ZO-1). Although hydroxychloroquine had no apparent effects on trophoblast function, it may be a useful endothelial protectant in women presenting with preeclampsia.

Brain inflammation and injury at 48 h is not altered by human amnion epithelial cells in ventilated preterm lambs.

BACKGROUND: Mechanical ventilation of preterm neonates is associated with neuroinflammation and an increased risk of adverse neurological outcomes. Human amnion epithelial cells (hAECs) have anti-inflammatory and regenerative properties. We aimed to determine if intravenous administration of hAECs to preterm lambs would reduce neuroinflammation and injury at 2 days of age. METHODS: Preterm lambs were delivered by cesarean section at 128-130 days' gestation (term is ~147 days) and either ventilated for 48 h or humanely killed at birth. Lambs received 3 mL surfactant (Curosurf) via endotracheal tube prior to delivery (either with or without 90 × 106 hAECs) and 3 mL intravenous phosphate-buffered saline (with or without 90 × 106 hAECs, consistent with intratracheal treatment) after birth. RESULTS: Ventilation increased microglial activation, total oligodendrocyte cell number, cell proliferation and blood-brain barrier permeability (P < 0.05, PBS + ventilation and hAEC + ventilation vs. control), but did not affect numbers of immature and mature oligodendrocytes. Ventilation reduced astrocyte and neuron survival (P < 0.05, PBS + ventilation and hAEC + ventilation vs. control). hAEC administration did not alter markers of neuroinflammation or injury within the white or gray matter. CONCLUSIONS: Mechanical ventilation for 48 h upregulated markers of neuroinflammation and injury in preterm lambs. Administration of hAECs did not affect markers of neuroinflammation or injury. IMPACT: Mechanical ventilation of preterm lambs for 48 h, in a manner consistent with contemporary neonatal intensive care, causes neuroinflammation, neuronal loss and pathological changes in oligodendrocyte and astrocyte survival consistent with evolving neonatal brain injury.Intravenous administration of hAECs immediately after birth did not affect neonatal cardiorespiratory function and markers of neuroinflammation or injury.Reassuringly, our findings in a translational large animal model demonstrate that intravenous hAEC administration to the preterm neonate is safe.Considering that hAECs are being used in phase 1 trials for the treatment of BPD in preterm infants, with future trials planned for neonatal neuroprotection, we believe these observations are highly relevant.

Measuring Sulforaphane and Its Metabolites in Human Plasma: A High Throughput Method.

(1) Background: There is increasing understanding of the potential health benefits of cruciferous vegetables. In particular sulforaphane (SFN), found in broccoli, and its metabolites sulforaphane-glutathione (SFN-GSH), sulforaphane-cysteine (SFN-Cys), sulforaphane cysteine-glycine (SFN-CG) and sulforaphane-N-acetyl-cysteine (SFN-NAC) have potent antioxidant effects that may offer therapeutic value. Clinical investigation of sulforaphane as a therapeutic antioxidant requires a sensitive and high throughput process for quantification of sulforaphane and metabolites; (2) Methods: We collected plasma samples from healthy human volunteers before and for eight hours after consumption of a commercial broccoli extract supplement rich in sulforaphane. A rapid and sensitive method for quantification of sulforaphane and its metabolites in human plasma using Liquid Chromatography-Mass Spectrometry (LC-MS) has been developed; (3) Results: The LC-MS analytical method was validated at concentrations ranging between 3.9 nM and 1000 nM for SFN-GSH, SFN-CG, SFN-Cys and SFN-NAC and between 7.8 nM and 1000 nM in human plasma for SFN. The method displayed good accuracy (1.85%-14.8% bias) and reproducibility (below 9.53 %RSD) including low concentrations 3.9 nM and 7.8 nM. Four SFN metabolites quantitation was achieved using external standard calibration and in SFN quantitation, SFN-d8 internal standardization was used. The reported method can accurately quantify sulforaphane and its metabolites at low concentrations in plasma; (4) Conclusions: We have established a time- and cost-efficient method of measuring sulforaphane and its metabolites in human plasma suitable for high throughput application to clinical trials.

The Effects of Early-Onset Pre-Eclampsia on Placental Creatine Metabolism in the Third Trimester.

Creatine is a metabolite important for cellular energy homeostasis as it provides spatio-temporal adenosine triphosphate (ATP) buffering for cells with fluctuating energy demands. Here, we examined whether placental creatine metabolism was altered in cases of early-onset pre-eclampsia (PE), a condition known to cause placental metabolic dysfunction. We studied third trimester human placentae collected between 27-40 weeks' gestation from women with early-onset PE (n = 20) and gestation-matched normotensive control pregnancies (n = 20). Placental total creatine and creatine precursor guanidinoacetate (GAA) content were measured. mRNA expression of the creatine synthesizing enzymes arginine:glycine aminotransferase (GATM) and guanidinoacetate methyltransferase (GAMT), the creatine transporter (SLC6A8), and the creatine kinases (mitochondrial CKMT1A & cytosolic BBCK) was assessed. Placental protein levels of arginine:glycine aminotransferase (AGAT), GAMT, CKMT1A and BBCK were also determined. Key findings; total creatine content of PE placentae was 38% higher than controls (p < 0.01). mRNA expression of GATM (p < 0.001), GAMT (p < 0.001), SLC6A8 (p = 0.021) and BBCK (p < 0.001) was also elevated in PE placentae. No differences in GAA content, nor protein levels of AGAT, GAMT, BBCK or CKMT1A were observed between cohorts. Advancing gestation and birth weight were associated with a down-regulation in placental GATM mRNA expression, and a reduction in GAA content, in control placentae. These relationships were absent in PE cases. Our results suggest PE placentae may have an ongoing reliance on the creatine kinase circuit for maintenance of cellular energetics with increased total creatine content and transcriptional changes to creatine synthesizing enzymes and the creatine transporter. Understanding the functional consequences of these changes warrants further investigation.

Two-year outcomes of infants enrolled in the first-in-human study of amnion cells for bronchopulmonary dysplasia.

We previously reported on the immediate safety and neonatal outcomes of six premature infants with severe bronchopulmonary dysplasia (BPD) who were administered human amnion epithelial cells (hAECs). One infant died in the neonatal period due to unrelated causes. In this study, we aimed to assess the long-term safety and follow-up outcomes of the five surviving infants until 2 years corrected age (CA). hAECs were administered intravenously at a dose of 1 × 106 cells per kilogram after 36 weeks postconceptional age in infants with established BPD. Study follow-up consisted of assessment of any adverse events, growth, and respiratory, cardiac, and neurodevelopmental outcomes over four time points (6, 12, 18, and 24 months CA). Investigations included chest x-rays, cranial and abdominal ultrasounds, and echocardiograms at regular intervals as well as a magnetic resonance imaging (MRI) brain at 2 years CA. All five infants were alive at 2 years CA. Median time to wean off oxygen was 24 (10-36) months. Two infants had pulmonary hypertension, which resolved by 2 years of age. Four infants were rehospitalized briefly for viral or bacterial infections during the 2 years. MRI brain findings included normal (n = 1), and mild to moderate white matter loss (n = 2). Neurodisabilities diagnosed included hemiplegic cerebral palsy (n = 1), global developmental delay (n = 3), and severe hearing loss (n = 3). No evidence of tumor formation was noted on physical examinations or on any imaging. There were no long-term adverse events observed that could be attributed to hAEC administration. We observed long-term effects of extreme prematurity and severe BPD in the cohort.

Prolong: a double-blind randomised placebo-controlled trial of broccoli sprout extract in women with early onset preeclampsia. A clinical trial protocol.

INTRODUCTION: Preeclampsia is a leading cause of maternal and perinatal morbidity and mortality. There is a need for adjuvant, targeted therapies to improve outcomes. Broccoli sprout extract, rich in the antioxidant sulforaphane, reduces oxidative stress and placental secretion of the antiangiogenic factors that contribute to vascular dysfunction in preeclampsia. We propose a phase III trial investigating broccoli sprout extract. We will assess broccoli sprout extract in women with early onset (<34 weeks) preeclampsia, investigating (1) the interval between enrolment and delivery (days), (2) biomarkers of placental and endothelial function and (3) maternal and fetal outcomes. METHODS: A double-blind, placebo-controlled randomised trial will be conducted at Monash Health, Melbourne, Australia. One hundred and eighty women (45 each arm of each stratum) with early onset preeclampsia (defined as per Society for Obstetric Medicine of Australia and New Zealand guidelines) will be recruited. Consenting women will be randomised to receive an oral dose of either broccoli sprout extract (24 mg of activated sulforaphane) or identical placebo, twice daily until delivery. Maternal blood will be collected antenatally for measurement of biomarkers of preeclampsia, including soluble fms-like tyrosine kinase 1 (sFlt-1), placental growth factor (PlGF), soluble endoglin (sEng) and activin A, as well as circulating sulforaphane metabolites. Maternal and perinatal outcomes will be monitored throughout. All clinical care decisions, including the timing of delivery, will be made by the treating team, blinded to treatment allocation. Participation in this trial will not affect routine care. At delivery, maternal and cord blood and placentae will be collected to measure sulforaphane metabolites and sFlt-1, PlGF, sEng and activin A. ETHICS AND DISSEMINATION: Approval to conduct the trial has been granted by Monash Health Human Research and Ethics Committee (RES-18-0000-109A). Deidentified data will be published in peer-reviewed journals and presented at learnt society conferences, both nationally and internationally. This study has not yet commenced and is pre-results.Trial registration numberACTRN12618000216213.

Human amnion cells for the prevention of bronchopulmonary dysplasia: a protocol for a phase I dose escalation study.

INTRODUCTION: Bronchopulmonary dysplasia (BPD), an important sequela of preterm birth, is associated with long-term abnormalities of lung function and adverse neurodevelopmental outcomes. Inflammation, inhibition of secondary septation and vascular maldevelopment play key roles in the pathogenesis of BPD. Human amnion epithelial cells (hAECs), stem-like cells, derived from placental tissues are able to modulate the inflammatory milieu and, in preclinical studies of BPD-like injury, restore lung architecture and function. Allogeneic hAECs may present a new preventative and reparative therapy for BPD. METHODS AND ANALYSIS: In this two centre, phase I cell dose escalation study we will evaluate the safety of intravenous hAEC infusions in preterm infants at high risk of severe BPD. Twenty-four infants born at less than 29 weeks' gestation will each receive intravenous hAECs beginning day 14 of life. We will escalate the dose of cells contained in a single intravenous hAEC infusion in increments from 2 million cells/kg to 10 million cells/kg. Further dose escalation will be achieved with repeat infusions given at 5 day intervals to a maximum total dose of 30 million cells/kg (three infusions). Safety is the primary outcome. Infants will be followed-up until 2 years corrected age. Additional outcome measures include a description of infants' cytokine profile following hAEC infusion, respiratory outcomes including BPD and pulmonary hypertension and other neonatal morbidities including neurodevelopmental assessment at 2 years. ETHICS AND DISSEMINATION: This study was approved on the June12th, 2018 by the Human Research Ethics Committee of Monash Health and Monash University. Recruitment commenced in August 2018 and is expected to take 18 months. Accordingly, follow-up will be completed mid-2022. The findings of this study will be disseminated via peer-reviewed journals and at conferences. PROTOCOL VERSION: 5, 21 May 2018. TRIAL REGISTRATION NUMBER: ACTRN12618000920291; Pre-results.

Sharing the pain: lessons from missed opportunities for healthcare improvement from patient complaints and litigation in the Australian health system.

Learning from medical errors to prevent their recurrence is an important component of any healthcare system's quality and safety improvement functions. Traditionally, this been achieved principally from review of adverse clinical outcomes. The opportunity to learn systematically and in a system manner from patient complaints and litigation has been less well harnessed. Herein we describe the pathways and processes for both patient complaints and medicolegal claims in Victoria, and Australia more broadly, and assess the potential for these to be used for system improvement. We conclude that both patient complaints and medicolegal claims could afford the potential to additionally inform and direct safety and quality improvement. At present neither patient complaints nor medicolegal claims are used systematically to improve patient safety. We identify how this may be done, particularly through sharing findings across agencies.

Delayed intranasal infusion of human amnion epithelial cells improves white matter maturation after asphyxia in preterm fetal sheep.

Perinatal hypoxic-ischemic (HI) brain injury remains highly associated with neurodevelopmental disability after preterm birth. There is increasing evidence that disability is linked with impaired white matter maturation, but there is no specific treatment. In this study, we evaluated whether, in preterm fetal sheep, delayed intranasal infusion of human amnion epithelial cells (hAECs) given 1, 3 and 10 days after severe HI, induced by umbilical cord occlusion for 25 min, can restore white matter maturation or reduce delayed cell loss. After 21 days recovery, asphyxia was associated with reduced electroencephalographic (EEG) maturation, brain weight and cortical area, impaired maturation of oligodendrocytes (OLs), no significant loss of total OLs but a marked reduction in immature/mature OLs and reduced myelination. Intranasal infusion of hAECs was associated with improved brain weight and restoration of immature/mature OLs and fractional area of myelin basic protein, with reduced microglia and astrogliosis. Cortical EEG frequency distribution was partially improved, with reduced loss of cortical area, and attenuated cleaved-caspase-3 expression and microgliosis. Neuronal survival in deep grey matter nuclei was improved, with reduced microglia, astrogliosis and cleaved-caspase-3-positive apoptosis. These findings suggest that delayed intranasal hAEC administration has potential to alleviate chronic dysmaturation after perinatal HI.