Nitric Oxide Affects Heme Oxygenase-1, Hepcidin, and Transferrin Receptor Expression in the Placenta.

Nitric oxide (NO) is a gasotransmitter that avidly binds both free and heme-bound iron, forming relatively stable iron nitrosyl compounds (FeNOs). We have previously demonstrated that FeNOs are present in the human placenta and are elevated in preeclampsia and intrauterine growth restriction. The ability of NO to sequester iron raises the possibility of the NO-mediated disruption of iron homeostasis in the placenta. In this work, we tested whether exposure of placental syncytiotrophoblasts or villous tissue explants to sub-cytotoxic concentrations of NO would elicit the formation of FeNOs. Furthermore, we measured changes in the mRNA and protein expression levels of key iron regulatory genes in response to NO exposure. Ozone-based chemiluminescence was used to measure concentrations of NO and its metabolites. Our results showed a significant increase in FeNO levels in placental cells and explants treated with NO (p < 0.0001). The mRNA and protein levels of HO-1 were significantly increased in both cultured syncytiotrophoblasts and villous tissue explants (p < 0.01), and the mRNA levels of hepcidin and transferrin receptor were significantly increased in culture syncytiotrophoblasts and villous tissue explants, respectively, (p < 0.01), while no changes were seen in the expression levels of divalent metal transporter-1 or ferroportin. These results suggest a potential role for NO in iron homeostasis in the human placenta and could be relevant for disorders of pregnancy such as fetal growth restriction and preeclampsia.

In utero Treatment of Congenital High Airway Obstruction Syndrome via Fetal Laryngoscopy and EXIT Procedure.

INTRODUCTION: Congenital high airway obstruction syndrome (CHAOS) is a rare condition that can progress to fetal hydrops and demise in utero or at birth unless interventions are undertaken to alleviate the tracheal obstruction. While the ex-utero intrapartum treatment (EXIT) procedure for airway stabilization is technically feasible, abnormal pulmonary development as a result of the antenatal obstructive process may result in severe postnatal respiratory complications. CASE PRESENTATION: We describe a case of CHAOS with secondary hydrops treated in utero at 24 0/7 weeks' gestation by fetoscopic tracheal decompression via laser perforation of the airway obstruction. Interval imaging after the fetoscopic operation demonstrated resolution of the fetal hydrops. Tracheostomy for airway stabilization was performed at the time of the EXIT procedure near term (36 0/7 weeks). The patient underwent tracheal reconstruction and decannulation at 3 years of life. DISCUSSION/CONCLUSION: The primary goal of fetoscopic airway evaluation and intervention is not necessarily to perform definitive stabilization of the airway but rather to achieve sufficient decompression of the trachea to reverse fetal hydrops and salvage pulmonary development. In utero fetoscopic treatment may allow for prolongation of the pregnancy with delivery at or near term via EXIT procedure for definitive neonatal airway stabilization.

Dual demise following laser surgery for twin-twin transfusion syndrome: Analysis of 52 cases at a single fetal surgery center.

OBJECTIVE: To evaluate all individual cases of dual twin demise following laser surgery for twin-twin transfusion syndrome (TTTS). METHOD: This is an analysis of all monochorionic diamniotic twin gestations with TTTS complicated by dual demise following laser surgery from 2006 to 2019. Cases were reviewed by (1) a fetal surgeon researcher and (2) a panel of independent experienced maternal-fetal medicine specialists to code an etiology of demise for the donor and recipient, and to assess for possible preventability. RESULTS: Of 753 twins that underwent laser surgery for TTTS, 52 (6.9%) had postoperative dual demise. In this subgroup, gestational age at surgery was 19.5 (16.1-24.9) weeks, and 36 (69.2%) patients were Quintero stage III and IV. The most common etiology was the spectrum of disorders leading to preterm delivery, which included cervical insufficiency, preterm premature rupture of membranes, and preterm labor (44.2% and 48.1%, donor and recipient, respectively). Some degree of preventability was estimated for 23.1% of dual demises. CONCLUSIONS: The most common cause of dual demise post laser surgery for TTTS was preterm birth, reinforcing the need for studies regarding the etiology and prevention of post-fetoscopy prematurity. Nearly one-quarter of dual demise cases were deemed potentially preventable.

Neonatal Outcomes and Maternal Characteristics in Monochorionic Diamniotic Twin Pregnancies: Uncomplicated versus Twin-to-Twin Transfusion Syndrome Survivors after Fetoscopic Laser Surgery.

Preterm birth remains a major complication of fetal laser surgery (FLS) due to twin-to-twin transfusion syndrome (TTTS). OBJECTIVES: We tested the hypothesis that neonatal outcomes in fetuses born at >24 weeks are worse in TTTS survivors compared to uncomplicated monochorionic diamniotic (MCDA) twins. METHODS: 196 patients with TTTS treated with laser therapy and 91 uncomplicated MCDA gestations were compared. Neonatal outcomes included respiratory distress syndrome (RDS), transient tachypnea of the newborn (TTN), bronchopulmonary dysplasia, intraventricular hemorrhage, necrotizing enterocolitis, and neonatal death. Risk factors assessed were TTTS, maternal age, maternal body mass index, race, premature prolonged rupture of membranes, stage of TTTS, parity, and gestational age (GA) at delivery. RESULTS: GA at delivery was lower in the TTTS group (31.0 ± 4.6 vs. 33.5 ± 2.4 weeks, p < 0.001). RDS and TTN occurred at higher rates in the TTTS than in the uncomplicated MCDA group. After multivariate logistic regression, the only factor significantly associated with the composite adverse outcome was GA at delivery (OR 0.61; 95% CI: 0.58-0.7). CONCLUSION: TTTS twins treated with FLS are deliver 2.5 weeks earlier than uncomplicated MCDA twins. Respiratory complications were significantly higher in TTTS twins and were mainly the consequence of the early GA at delivery.

A benchmarking study of SARS-CoV-2 whole-genome sequencing protocols using COVID-19 patient samples.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging new type of coronavirus that is responsible for the COVID-19 pandemic and the unprecedented global health emergency. Whole-genome sequencing (WGS) of SARS-CoV-2 plays a critical role in understanding the disease. Performance variation exists across SARS-CoV-2 viral WGS technologies, but there is currently no benchmarking study comparing different WGS sequencing protocols. We compared seven different SARS-CoV-2 WGS library protocols using RNA from patient nasopharyngeal swab samples under two storage conditions with low and high viral inputs. We found large differences in mappability and genome coverage, and variations in sensitivity, reproducibility, and precision of single-nucleotide variant calling across different protocols. For certain amplicon-based protocols, an appropriate primer trimming step is critical for accurate single-nucleotide variant calling. We ranked the performance of protocols based on six different metrics. Our findings offer guidance in choosing appropriate WGS protocols to characterize SARS-CoV-2 and its evolution.

Early gestational mesenchymal stem cell secretome attenuates experimental bronchopulmonary dysplasia in part via exosome-associated factor TSG-6.

BACKGROUND: Mesenchymal stem cells (MSCs) are promising tools for the treatment of human lung disease and other pathologies relevant to newborn medicine. Recent studies have established MSC exosomes (EXO), as one of the main therapeutic vectors of MSCs in mouse models of multifactorial chronic lung disease of preterm infants, bronchopulmonary dysplasia (BPD). However, the mechanisms underlying MSC-EXO therapeutic action are not completely understood. Using a neonatal mouse model of human BPD, we evaluated the therapeutic efficiency of early gestational age (GA) human umbilical cord (hUC)-derived MSC EXO fraction and its exosomal factor, tumor necrosis factor alpha-stimulated gene-6 (TSG-6). METHODS: Conditioned media (CM) and EXO fractions were isolated from 25 and 30 weeks GA hUC-MSC cultures grown in serum-free media (SFM) for 24 h. Newborn mice were exposed to hyperoxia (> 95% oxygen) and were given intraperitoneal injections of MSC-CM or MSC-CM EXO fractions at postnatal (PN) day 2 and PN4. They were then returned to room air until PN14 (in a mouse model of severe BPD). The treatment regime was followed with (rh)TSG-6, TSG-6-neutralizing antibody (NAb), TSG-6 (si)RNA-transfected MSC-CM EXO and their appropriate controls. Echocardiography was done at PN14 followed by harvesting of lung, heart and brain for assessment of pathology parameters. RESULTS: Systemic administration of CM or EXO in the neonatal BPD mouse model resulted in robust improvement in lung, cardiac and brain pathology. Hyperoxia-exposed BPD mice exhibited pulmonary inflammation accompanied by alveolar-capillary leakage, increased chord length, and alveolar simplification, which was ameliorated by MSC CM/EXO treatment. Pulmonary hypertension and right ventricular hypertrophy was also corrected. Cell death in brain was decreased and the hypomyelination reversed. Importantly, we detected TSG-6, an immunomodulatory glycoprotein, in EXO. Administration of TSG-6 attenuated BPD and its associated pathologies, in lung, heart and brain. Knockdown of TSG-6 by NAb or by siRNA in EXO abrogated the therapeutic effects of EXO, suggesting TSG-6 as an important therapeutic molecule. CONCLUSIONS: Preterm hUC-derived MSC-CM EXO alleviates hyperoxia-induced BPD and its associated pathologies, in part, via exosomal factor TSG-6. The work indicates early systemic intervention with TSG-6 as a robust option for cell-free therapy, particularly for treating BPD.

Biomimetic Culture Reactor for Whole-Lung Engineering.

Decellularized organs are now established as promising scaffolds for whole-organ regeneration. For this work to reach therapeutic practice, techniques and apparatus are necessary for doing human-scale clinically applicable organ cultures. We have designed and constructed a bioreactor system capable of accommodating whole human or porcine lungs, and we describe in this study relevant technical details, means of assembly and operation, and validation. The reactor has an artificial diaphragm that mimics the conditions found in the chest cavity in vivo, driving hydraulically regulated negative pressure ventilation and custom-built pulsatile perfusion apparatus capable of driving pressure-regulated or volume-regulated vascular flow. Both forms of mechanical actuation can be tuned to match specific physiologic profiles. The organ is sealed in an elastic artificial pleura that mounts to a support architecture. This pleura reduces the fluid volume required for organ culture, maintains the organ's position during mechanical conditioning, and creates a sterile barrier allowing disassembly and maintenance outside of a biosafety cabinet. The combination of fluid suspension, negative-pressure ventilation, and physiologic perfusion allows the described system to provide a biomimetic mechanical environment not found in existing technologies and especially suited to whole-organ regeneration. In this study, we explain the design and operation of this apparatus and present data validating intended functions.

Stem Cell Therapy in Neonatal Diseases.

Common complications in neonates occur in almost every organ system in the neonatal intensive care unit. While a number of them have short-term effects, a few of them also have long-term consequences. Among the latter are bronchopulmonary dysplasia and necrotizing enterocolitis in premature neonates, and hypoxic ischemic encephalopathy in borderline preterm and term neonates. While medical advances have improved our understanding of the pathogenesis, therapies to effectively prevent and/or significantly ameliorate the severity of these disorders, and to decrease their associated mortality and morbidity have not been found. One promising approach to make a potential impact in the outcomes of these neonatal conditions is the use stem cells, specifically, mesenchymal stem cells. The authors briefly review the potential role of stem cell therapy in the above-mentioned neonatal diseases. They focus primarily on human clinical trials.

Antenatal maternal hypoxic stress: adaptations in fetal lung Renin-Angiotensin system.

Antenatal maternal hypoxia (AMH) can lead to intrauterine growth restriction (IUGR), as well as idiopathic pulmonary hypertension of newborn and adult, the latter of which may be a consequence of alterations in the local pulmonary renin-angiotensin system (RAS). Little is known of these adaptations, however. Thus, we tested the hypothesis that antenatal maternal hypoxia is associated with alterations in gene and protein expression of the pulmonary renin-angiotensin system, which may play an important role in pulmonary disorders in the offspring. In FVB/NJ mice, we studied messenger RNA (mRNA) and protein expression, as well as promoter DNA methylation and microRNA (miRNA) levels in response to 48 hours hypoxia (10.5% O(2)) at 15.5 day post coitum (DPC). In response to AMH, the pulmonary mRNA levels of angiotensin-converting enzyme (ACE) 1.2, ACE-2, and angiotensin II type 1b (AT-1b) receptors were increased significantly, as compared to controls (N = 4). In response to antenatal hypoxia, pulmonary protein levels of renin and ACE-2 also were increased significantly, whereas ACE-1 protein expression was reduced. In fetal lungs, we also observed reduced expression of the miRNAs: mmu-mir -199b, -27b, -200b, and -468 that putatively increase the translation of renin, ACE-1, ACE-2, and AT-1 receptors, respectively. In response to AMH, promoter methylation of ACE was unchanged. We conclude that AMH leads to changes in expression of pulmonary RAS of fetal mice. The possible implications of these changes for the regulation of pulmonary vascular contractility in later life remain to be explored.

Brain renin-angiotensin system: fetal epigenetic programming by maternal protein restriction during pregnancy.

OBJECTIVE: Maternal protein malnutrition during pregnancy can lead to significant alterations in the systemic renin-angiotensin system (RAS) in the fetus. All components of the RAS are present in brain and may be altered in many disease states. Importantly, these disorders are reported to be of higher incidence in prenatally malnourished individuals. In the current study, we tested the hypothesis that antenatal maternal low protein diet (MLPD) leads to epigenetic changes and alterations in gene expression of brain RAS of the mouse fetus. METHODS: Mice dams were given control and 50% MLPD during second half of the gestation. We analyzed messenger RNA (mRNA), microRNA (miRNA), promoter DNA methylation, and protein expression of various RAS genes in the fetal offspring. RESULTS: As a consequence of 50% MLPD, fetal brains showed increased mRNA expression of angiotensinogen and angiotensin converting enzyme-1 (ACE-1), with a decrease in mRNA levels of angiotensin II type-2 (AT2) receptors. In contrast, while angiotensinogen protein expression was unaltered, the protein levels of ACE-1 and AT2 receptor genes were significantly reduced in the fetal brain from the MLPD dams. Our results also demonstrated hypomethylation of the CpG islands in the promoter regions of ACE-1 gene, and upregulation of the miRNAs, mmu-mir-27a and 27b, which regulate ACE-1 mRNA translation. Furthermore, our study showed reduced expression of the miRNA mmu-mir-330, which putatively regulates AT2 translation. CONCLUSION: For the developing fetal brain RAS, MLPD leads to significant alterations in the mRNA and protein expression, with changes in DNA methylation and miRNA, key regulators of hypertension in adults.

Gene expression in the placenta: maternal stress and epigenetic responses.

Successful placental development is crucial for optimal growth, development, maturation and survival of the embryo/fetus into adulthood. Numerous epidemiologic and experimental studies have demonstrated the profound influence of intrauterine environment on life, and the diseases to which one is subject as an adult. For the most part, these invidious influences, whether maternal hypoxia, protein or caloric deficiency or excess, and others, represent types of maternal stress. In the present review, we examine certain aspects of gene expression in the placenta as a consequence of maternal stressors. To examine these issues in a controlled manner, and in a species in which the genome has been sequenced, most of these reported studies have been performed in the mouse. Although each individual maternal stress is characterized by up- or down-regulation of specific genes in the placenta, functional analysis reveals some patterns of gene expression common to the several forms of stress. Of critical importance, these genes include those involved in DNA methylation and histone modification, cell cycle regulation, and related global pathways of great relevance to epigenesis and the developmental origins of adult health and disease.

Maternal protein deprivation: changes in systemic renin-angiotensin system of the mouse fetus.

We tested the hypothesis that maternal protein deprivation during gestation results in changes in expression of the systemic renin-angiotensin system in fetal mice. Fetal weight was decreased significantly as a consequence of 50% maternal protein deprivation during second half of gestation. In fetal liver, angiotensinogen protein expression was reduced significantly despite a significant increase in messenger RNA (mRNA). In fetal kidneys, both mRNA and protein levels of renin were increased significantly. In the lungs, we observed a decrease in both angiotensin-converting enzyme I and II mRNA expression, whereas protein expression of both isoforms was increased significantly. The fetal heart showed significant increases in expression of angiotensin II type 1 (AT-1) and type 2 (AT-2) receptors mRNA. Protein expression of AT-1 receptors increased, while that of AT-2 receptors decreased. We conclude that maternal low-protein diet during gestation leads to significant changes in expression of the systemic renin-angiotensin system in fetal mice and may be important in the genesis of hypertension in the adult.

Gene expression patterns in the hypoxic murine placenta: a role in epigenesis?

Hypoxia has been identified as a major stress or in placental and fetal development. To test the hypothesis that hypoxic stress responses are associated with gene expression changes, the authors measured gene expression in the mouse placenta in response to 48 hours of hypoxia. Embryonic day 15.5 pregnant mice were exposed to 48 hours of hypoxia (10.5% O(2)), after which the Affymetrix Mouse 430A_2.0 array was used to measure gene expression changes in the placenta. The authors observed 171 probe sets, corresponding to 163 genes, that were regulated by hypoxia (P < .01). Ninety genes were upregulated, and 73 were downregulated. The authors functionally annotated the regulated genes and examined overrepresented functional categories. Among the upregulated and downregulated genes, several overrepresented functional categories were observed. Upregulated genes included those involved in metabolism, oxygen transport, proteolysis, cell death, metabolism of reactive oxygen species, and DNA methylation. Genes involved in transcription, cell cycle regulation, and cell structure were downregulated. Microarray analysis has allowed the description of the genetic responses to hypoxia in the mouse placenta. The observation that hypoxia upregulates reactive oxygen species metabolism, in conjunction with DNA methylation enzymes, suggests that hypoxia may contribute to long-term epigenetic changes in stressed fetal tissues and organs.

Pre- and postjunctional alpha(2)-adrenergic receptors in fetal and adult ovine cerebral arteries.

In ovine cerebral arteries, adrenergic-mediated vasoconstrictor responses differ significantly with developmental age. We tested the hypothesis that, in part, these differences are a consequence of altered alpha(2)-adrenergic receptor (alpha(2)-AR) density and/or affinity. In fetal (approximately 140 days) and adult sheep, we measured alpha(2)-AR density and affinity with the antagonist [(3)H]idazoxan in main branch cerebral arteries and other vessels. We also quantified contractile responses in middle cerebral artery (MCA) to norepinephrine (NE) or phenylephrine in the presence of the alpha(2)-AR antagonists yohimbine and idazoxan and contractile responses to the alpha(2)-AR agonists clonidine and UK-14304. In fetal and adult cerebral artery homogenates, alpha(2)-AR density was 201 +/- 18 and 52 +/- 6 fmol/mg protein, respectively (P < 0.01); however, antagonist affinity values did not differ. In fetal, but not adult, MCA, 10(-7) M yohimbine significantly decreased the pD(2) for NE-induced tension in the presence of 3 x 10(-5) M cocaine, 10(-5) M deoxycorticosterone, and 10(-6) M tetrodotoxin. In fetal, but not adult, MCA, UK-14304 induced a significant decrease in pD(2) for the phenylephrine dose-response relation. In addition, stimulation-evoked fractional NE release was significantly greater in fetal than in adult cerebral arteries. In the presence of 10(-6) M idazoxan to block alpha(2)-AR-mediated inhibition of prejunctional NE release, the fractional NE release was significantly increased in both age groups. We conclude that in fetal and adult ovine cerebral arteries, alpha(2)-AR appear to be chiefly prejunctional. Nonetheless, the fetal cerebral arteries appear to have a significant component of postjunctional alpha(2)-AR.