Cell trajectory modeling identifies a primitive trophoblast state defined by BCAM enrichment.

In early placental development, progenitor cytotrophoblasts (CTB) differentiate along one of two cellular trajectories: the villous or extravillous pathways. CTB committed to the villous pathway fuse with neighboring CTB to form the outer multinucleated syncytiotrophoblast (SCT), whereas CTB committed to the extravillous pathway differentiate into invasive extravillous trophoblasts (EVT). Unfortunately, little is known about the processes controlling human CTB progenitor maintenance and differentiation. To address this, we established a single cell RNA sequencing (scRNA-seq) dataset from first trimester placentas to identify cell states important in trophoblast progenitor establishment, renewal and differentiation. Multiple distinct trophoblast states were identified, representing progenitor CTB, column CTB, SCT precursors and EVT. Lineage trajectory analysis identified a progenitor origin that was reproduced in human trophoblast stem cell organoids. Heightened expression of basal cell adhesion molecule (BCAM) defined this primitive state, where BCAM enrichment or gene silencing resulted in enhanced or diminished organoid growth, respectively. Together, this work describes at high-resolution trophoblast heterogeneity within the first trimester, resolves gene networks within human CTB progenitors and identifies BCAM as a primitive progenitor marker and possible regulator.

A Novel Anti-nucleocapsid Antibody Avidity Method for Identifying SARS-CoV-2 Reinfections.

Detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfections is challenging with current serology assays and is further complicated by the marked decrease in routine viral testing practices as viral transmission increased during Omicron. Here, we provide proof-of-principle that high-avidity anti-nucleocapsid (N) antibodies detects reinfections after a single infection with higher specificity (85%; 95% confidence interval [95% CI], 80%-90%) compared to anti-N antibody levels (72%; 95% CI, 66%-79%) in a vaccinated cohort. This method could be used to retroactively investigate the epidemiology and incremental long-term health consequences of SARS-CoV-2 reinfections.

Human complete NFAT1 deficiency causes a triad of joint contractures, osteochondromas, and B-cell malignancy.

The discovery of humans with monogenic disorders has a rich history of generating new insights into biology. Here we report the first human identified with complete deficiency of nuclear factor of activated T cells 1 (NFAT1). NFAT1, encoded by NFATC2, mediates calcium-calcineurin signals that drive cell activation, proliferation, and survival. The patient is homozygous for a damaging germline NFATC2 variant (c.2023_2026delTACC; p.Tyr675Thrfs∗18) and presented with joint contractures, osteochondromas, and recurrent B-cell lymphoma. Absence of NFAT1 protein in chondrocytes caused enrichment in prosurvival and inflammatory genes. Systematic single-cell-omic analyses in PBMCs revealed an environment that promotes lymphomagenesis with accumulation of naïve B cells (enriched for oncogenic signatures MYC and JAK1), exhausted CD4+ T cells, impaired T follicular helper cells, and aberrant CD8+ T cells. This work highlights the pleiotropic role of human NFAT1, will empower the diagnosis of additional patients with NFAT1 deficiency, and further defines the detrimental effects associated with long-term use of calcineurin inhibitors.

Total liquid ventilation in an ovine model of extreme prematurity: a randomized study.

BACKGROUND: This study aimed at comparing cardiorespiratory stability during total liquid ventilation (TLV)-prior to lung aeration-with conventional mechanical ventilation (CMV) in extremely preterm lambs during the first 6 h of life. METHODS: 23 lambs (11 females) were born by c-section at 118-120 days of gestational age (term = 147 days) to receive 6 h of TLV or CMV from birth. Lung samples were collected for RNA and histology analyses. RESULTS: The lambs under TLV had higher and more stable arterial oxygen saturation (p = 0.001) and cerebral tissue oxygenation (p = 0.02) than the lambs in the CMV group in the first 10 min of transition to extrauterine life. Although histological assessment of the lungs was similar between the groups, a significant upregulation of IL-1a, IL-6 and IL-8 RNA in the lungs was observed after TLV. CONCLUSIONS: Total liquid ventilation allowed for remarkably stable transition to extrauterine life in an extremely preterm lamb model. Refinement of our TLV prototype and ventilation algorithms is underway to address specific challenges in this population, such as minimizing tracheal deformation during the active expiration. IMPACT: Total liquid ventilation allows for remarkably stable transition to extrauterine life in an extremely preterm lamb model. Total liquid ventilation is systematically achievable over the first 6 h of life in the extremely premature lamb model. This study provides additional incentive to pursue further investigation of total liquid ventilation as a transition tool for the most extreme preterm neonates.

Does Growth Velocity Affect Associations between Birth Weight and Neurodevelopment for Infants Born Very Preterm?

OBJECTIVE: To determine how neonatal growth velocity affects the association between birth weight and neurodevelopmental outcomes in infants born preterm. STUDY DESIGN: This study is a secondary analysis of the Maternal Omega-3 Supplementation to Reduce Bronchopulmonary Dysplasia in Very Preterm Infants (MOBYDIck) randomized multicenter trial conducted in breastfed infants born at <29 weeks of gestation, whose mothers were supplemented with docosahexaenoic acid or placebo during the neonatal period. Neurodevelopmental outcomes were assessed at 18-22 months of corrected age using the Bayley-III cognitive and language composite scores. The role of neonatal growth velocity was assessed with causal mediation and linear regression models. Subgroup analyses were stratified by birth weight z-score categories (<25th, ≥25th-≤75th, and >75th percentiles). RESULTS: Neurodevelopmental outcomes were available for 379 children (mean gestational age, 26.7 ± 1.5 weeks). Growth velocity partially mediated the relationships between birth weight and cognitive (ß = -1.1; 95% CI, -2.2 to -0.02; P = .05) and language scores (ß = -2.1; 95% CI, -3.3 to -0.8; P = .002). An increase by 1 g/kg/day in growth velocity was associated with an increase by 1.1 point in the cognitive score (95% CI, -0.03 to 2.1; P = .06) and 1.9 point in the language score (95% CI, 0.7 to 3.1; P = .001), after adjustment for birth weight z-score. For children with birth weight <25th percentile, a 1 g/kg/day increase in growth velocity was associated with an increase by 3.3 points in the cognitive score (95% CI, 0.5 to 6.0; P = .02) and 4.1 points in the language score (95% CI, 1.3 to 7.0; P = .004). CONCLUSIONS: Postnatal growth velocity mediated the relationship between birth weight and neurodevelopmental performance, with larger effects for children with lower birth weight. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT02371460.

Waning Immunity Against Respiratory Syncytial Virus During the Coronavirus Disease 2019 Pandemic.

Health jurisdictions have seen a near-disappearance of respiratory syncytial virus (RSV) during the first year of the coronavirus disease 2019 (COVID-19) pandemic. Over this corresponding period, we report a reduction in RSV antibody levels and live virus neutralization in sera from women of childbearing age and infants between May to June 2020 and February to June 2021, in British Columbia (BC), Canada. This supports that antibody immunity against RSV is relatively short-lived and that maintaining optimal antibody levels in infants requires repeated maternal viral exposure. Waning immunity may explain the interseasonal resurgence of RSV cases observed in BC and other countries.

A Higher Antibody Response Is Generated With a 6- to 7-Week (vs Standard) Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Vaccine Dosing Interval.

The optimal dosing interval for severe acute respiratory syndrome coronavirus 2 vaccines remains controversial. In this prospective study, we compared serology results of paramedics vaccinated with mRNA vaccines at the recommended short (17-28 days) vs long (42-49 days) interval. We found that a long dosing interval resulted in higher spike, receptor binding domain, and spike N terminal domain antibody concentrations.

Implementing genomics in the neonatal period: An assessment of parental decision making and anxiety.

As uptake of rapid genome sequencing (GS) in the neonatal period steadily increases, a clinical genetics service that is optimized to the needs of parents becomes increasingly important. We aimed to investigate factors that influence decision making about rapid GS by parents of infants admitted to neonatal intensive care units (NICU) and explore their experiences of decisional conflict and anxiety during this time. Parents of neonates suspected of having a genetic disorder and offered rapid GS in the NICU completed a questionnaire measuring experience with GS counseling, decisional conflict, and anxiety level. Our results demonstrate that despite a largely positive GS experience (70%; 21/30) among the survey respondents, 50.0% (14/28) experienced moderate to severe anxiety measured using the GAD-7 scale, and 34.6% (9/26) experienced decisional conflict measured using the SURE scale. We also showed that prematurity may be a modifier of anxiety in this group of parents and although not statistically significant, distance lived away from the hospital site could have practical significance. Open-ended responses to survey questions highlighted that feeling overwhelmed, the types of engagements parents had with healthcare providers, and the timing of information provision also influenced parental decision making in this setting. We suggest that the GAD-7 scale for generalized anxiety and SURE scale for decisional conflict could be incorporated by genetic counselors into routine care of parents of neonates who have been offered rapid GS to identify those who may need additional support (resources, information, or psychological). These tools may inform ways that communication between patients and providers can be improved and enhanced and clinical genetics services in the NICU can be optimized. We suggest that integrating genetic counselors into the NICU care team could increase access for this population and ensure delivery of optimized patient education and counseling.

Effect of Maternal Docosahexaenoic Acid Supplementation on Bronchopulmonary Dysplasia-Free Survival in Breastfed Preterm Infants: A Randomized Clinical Trial.

Importance: Maternal docosahexaenoic acid (DHA) supplementation may prevent bronchopulmonary dysplasia, but evidence remains inconclusive. Objective: To determine whether maternal DHA supplementation during the neonatal period improves bronchopulmonary dysplasia-free survival in breastfed infants born before 29 weeks of gestation. Design, Setting, and Participants: Superiority, placebo-controlled randomized clinical trial at 16 Canadian neonatal intensive care units (June 2015-April 2018 with last infant follow-up in July 2018). Lactating women who delivered before 29 weeks of gestation were enrolled within 72 hours of delivery. The trial intended to enroll 800 mothers, but was stopped earlier. Interventions: There were 232 mothers (273 infants) assigned to oral capsules providing 1.2 g/d of DHA from randomization to 36 weeks' postmenstrual age and 229 mothers (255 infants) assigned to placebo capsules. Main Outcomes and Measures: The primary outcome was bronchopulmonary dysplasia-free survival in infants at 36 weeks' postmenstrual age. There were 22 secondary outcomes, including mortality and bronchopulmonary dysplasia. Results: Enrollment was stopped early due to concern for harm based on interim data from this trial and from another trial that was published during the course of this study. Among 461 mothers and their 528 infants (mean gestational age, 26.6 weeks [SD, 1.6 weeks]; 253 [47.9%] females), 375 mothers (81.3%) and 523 infants (99.1%) completed the trial. Overall, 147 of 268 infants (54.9%) in the DHA group vs 157 of 255 infants (61.6%) in the placebo group survived without bronchopulmonary dysplasia (absolute difference, -5.0% [95% CI, -11.6% to 2.6%]; relative risk, 0.91 [95% CI, 0.80 to 1.04], P = .18). Mortality occurred in 6.0% of infants in the DHA group vs 10.2% of infants in the placebo group (absolute difference, -3.9% [95% CI, -6.8% to 1.4%]; relative risk, 0.61 [95% CI, 0.33 to 1.13], P = .12). Bronchopulmonary dysplasia occurred in 41.7% of surviving infants in the DHA group vs 31.4% in the placebo group (absolute difference, 11.5% [95% CI, 2.3% to 23.2%]; relative risk, 1.36 [95% CI, 1.07 to 1.73], P = .01). Of 22 prespecified secondary outcomes, 19 were not significantly different. Conclusions and Relevance: Among breastfed preterm infants born before 29 weeks of gestation, maternal docosahexaenoic acid supplementation during the neonatal period did not significantly improve bronchopulmonary dysplasia-free survival at 36 weeks' postmenstrual age compared with placebo. Study interpretation is limited by early trial termination. Trial Registration: ClinicalTrials.gov Identifier: NCT02371460.

RAPIDOMICS: rapid genome-wide sequencing in a neonatal intensive care unit-successes and challenges.

Genetic disorders are one of the leading causes of infant mortality and are frequent in neonatal intensive care units (NICUs). Rapid genome-wide sequencing (GWS; whole genome or exome sequencing (ES)), due to its diagnostic capabilities and immediate impacts on medical management, is becoming an appealing testing option in the NICU setting. RAPIDOMICS was a trio-based rapid ES pilot study of 25 babies with suspected genetic disorders in the BC Women's Hospital NICU. ES and bioinformatic analysis were performed after careful patient ascertainment. Trio analysis was performed using an in-house pipeline reporting variants in known disease-causing genes. Variants interpreted by the research team as definitely or possibly causal of the infant's phenotype were Sanger validated in a clinical laboratory. The average time to preliminary diagnosis was 7.2 days. Sanger validation was pursued in 15 patients for 13 autosomal dominant and 2 autosomal recessive disorders, with an overall diagnostic rate (partial or complete) of 60%.Conclusion: In total, 72% of patients enrolled had a genomic diagnosis achieved through ES, multi-gene panel testing or chromosomal microarray analysis. Among these, there was an 83% rate of significant and immediate impact on medical decision-making directly related to new knowledge of the diagnosis. Health service implementation challenges and successes are discussed. What is Known: • Rapid genome-wide sequencing in the neonatal intensive care setting has a greater diagnostic hit rate and impact on medical management than conventional genetic testing. However, the impact of consultation with genetics and patient ascertainment requires further investigation. What is New: • This study demonstrates the importance of genetic consultation and careful patient selection prior to pursuing exome sequencing (ES). • In total, 15/25 (60%) patients achieved a diagnosis through ES and 18/25 (72%) through ES, multi-gene panel testing or chromosomal microarray analysis with 83% of those having immediate effects on medical management.

A Time-Based Analysis of Inflammation in Infants at Risk of Bronchopulmonary Dysplasia.

OBJECTIVE: To precisely delineate the timing and contribution of inflammation to bronchopulmonary dysplasia (BPD) in preterm infants during the neonatal period. STUDY DESIGN: Longitudinal study of blood inflammatory biomarkers (interleukin [IL]-6, IL-8, and granulocyte colony-stimulating factor) measured between birth and 42 days of age, at high temporal (daily) resolution, in infants born at or below 30 weeks of gestation. Cytokine predictors of BPD at 36 weeks postmenstrual age were adjusted for infant-specific and time-dependent factors, using hierarchical mixed effects regressions models. RESULTS: A total of 1518 data points were obtained in 62 infants (mean gestational age of 27 weeks). Infants who developed BPD later on presented increased inflammation after birth compared with infants without BPD. Inflammation was sustained, with gradual attenuation over 2 weeks (IL-8: OR: 6.5 [95% CI: 1.8-24]; granulocyte colony-stimulating factor: 3.3 [1.5-7.6]) and was higher in boys and in infants of lower birth weight. This inflammation preceded the clinical increased requirement in supplemental oxygen characteristic of BPD, and preceded the peak occurrence of neonatal sepsis or necrotizing enterocolitis. CONCLUSIONS: Systemic inflammation occurs early in the neonatal period and precedes clinical symptoms in infants with BPD. These data provide a discrete vulnerability window period, supporting a role for targeted intensive care interventions during the early phase of BPD.

Glucose control and autonomic response during acute stress in youth with type 1 diabetes: A pilot study.

BACKGROUND: Type 1 diabetes (T1D) is a chronic source of metabolic and neuropsychological stress, which may eventually lead to autonomic neuropathy and other complications related to micro- and macro-vasculopathies. We aimed to investigate the relationship between T1D chronic stress and autonomic response to acute stress testing that was expected being affected by chronic stress. METHODS: Twenty youths with confirmed diagnosis of T1D were assessed. Chronic stress assessment included hemoglobin A1c (HbA1c) ≥7.5%, psychological stress assessed by perceived stress scale (PSS), hypoglycemic events, and proinflammatory cytokines. The acute stress testing used standardized stress video games. Autonomic response to acute stress was assessed by the amplitude and direction of changes in heart rate variability. Analyses determined correlations between changes in parasympathetic nervous system during stress testing and chronic diabetes stressors. RESULTS: A strong correlation was found between the amplitude of high frequency (HF) changes and HbA1c values (ρ = 0.74, P < .001). Youths with HbA1c ≥7.5% showed a larger amplitude of HF changes during acute stress (49% vs 16%, P < .001) and a higher PSS score (22.5 vs 19.0, P = .003), compared to those with HbA1c <7.5%. Additionally, among youths with HbA1c ≥7.5%, those with positive changes in HF had a lower level of IL-8 than those with negative changes (5.40 vs 7.85 pg/mL, P = .009). CONCLUSIONS: Study findings support the need for better understanding the health effects of stress-related autonomic dysfunction in youth with T1D.

A functional ATG16L1 (T300A) variant is associated with necrotizing enterocolitis in premature infants.

BACKGROUND: The genetic basis of dysfunctional immune responses in necrotizing enterocolitis (NEC) remains unknown. We hypothesized that variants in nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs) and autophagy (ATG) genes modulate vulnerability to NEC. METHODS: We genotyped a multi-center cohort of premature infants with and without NEC for NOD1, NOD2, ATG16L1, CARD8, and NLRP3 variants. Chi-square tests and logistic regression were used for statistical analysis. RESULTS: In our primary cohort (n = 1,015), 86 (8.5%) infants developed NEC. The A allele of the ATG16L1 (Thr300Ala) variant was associated with increased NEC (AA vs. AG vs. GG; 11.3 vs. 8.4 vs. 4.8%, P = 0.009). In regression models for NEC that adjusted for epidemiological confounders, GA (P = 0.033) and the AA genotype (P = 0.038) of ATG16L1 variant were associated with NEC. The association between the A allele of the ATG16L1 variant and NEC remained significant among Caucasian infants (P = 0.02). In a replication cohort (n = 259), NEC rates were highest among infants with the AA genotype but did not reach statistical significance. CONCLUSION: We report a novel association between a hypomorphic variant in an autophagy gene (ATG16L1) and NEC in premature infants. Our data suggest that decreased autophagy arising from genetic variants may confer protection against NEC.

Impaired NLRP3 inflammasome activity during fetal development regulates IL-1ß production in human monocytes.

Interleukin-1ß (IL-1ß) production is impaired in cord blood monocytes. However, the mechanism underlying this developmental attenuation remains unclear. Here, we analyzed the extent of variability within the Toll-like receptor (TLR)/NLRP3 inflammasome pathways in human neonates. We show that immature low CD14 expressing/CD16(pos) monocytes predominate before 33 weeks of gestation, and that these cells lack production of the pro-IL-1ß precursor protein upon LPS stimulation. In contrast, high levels of pro-IL-1ß are produced within high CD14 expressing monocytes, although these cells are unable to secrete mature IL-1ß. The lack of secreted IL-1ß in these monocytes parallels a reduction of NLRP3 induction following TLR stimulation resulting in a lack of caspase-1 activity before 29 weeks of gestation, whereas expression of the apoptosis-associated speck-like protein containing a CARD and function of the P2×7 receptor are preserved. Our analyses also reveal a strong inhibitory effect of placental infection on LPS/ATP-induced caspase-1 activity in cord blood monocytes. Lastly, secretion of IL-1ß in preterm neonates is restored to adult levels during the neonatal period, indicating rapid maturation of these responses after birth. Collectively, our data highlight important developmental mechanisms regulating IL-1ß responses early in gestation, in part due to a downregulation of TLR-mediated NLRP3 expression. Such mechanisms may serve to limit potentially damaging inflammatory responses in a developing fetus.

Attenuation of respiratory syncytial virus-induced and RIG-I-dependent type I IFN responses in human neonates and very young children.

Newborn infants, including those born at term without congenital disorders, are at high risk of severe disease from respiratory syncytial virus (RSV) infection. Indeed, our current local surveillance data demonstrate that approximately half of children hospitalized with RSV were ≤3 mo old, and 74% were born at term. Informed by this clinical epidemiology, we investigated antiviral innate immune responses in early life, with the goal of identifying immunological factors underlying the susceptibility of infants and young children to severe viral lower respiratory tract infections. We compared RSV-induced innate cytokine production in blood mononuclear cells from neonates, young children aged 12-59 mo, and healthy adults. RSV-induced IFN-α production was primarily mediated by plasmacytoid dendritic cells (pDCs), and was significantly lower in term infants and young children < 5 y of age than in adults (p < 0.01). RSV-induced IFN-α production in human pDCs proceeded independently of endosomal TLRs, and human pDCs from healthy adult donors produced IFN-α in a retinoic acid-inducible gene I protein (RIG-I)-dependent manner. Of interest, young age and premature birth were independently associated with attenuated RIG-I-dependent IFN-α responses (p < 0.01). In contrast to IFN-α production, proinflammatory IL-6 responses to RSV were mediated by monocytes, appeared less dependent on RIG-I, and were significantly impaired only among preterm infants, not in term infants and young children. Our results suggest that human pDCs are less functional in early life, which may contribute to the increased susceptibility of infants and young children to severe RSV disease.