Publications

Abstract

Every year, 15 million babies are born preterm, putting them at increased risk of encephalopathy of prematurity (EoP). EoP is characterized by microglia-induced neuroinflammation, which can aggravate injury mechanisms leading to neuronal disorders, myelination delay, and subsequent functional consequences. While effective neuroprotective strategies in the preterm brain remain elusive, interventions such as skin-to-skin, developmental care, and music therapy have a positive impact on newborn brain development, potentially related to the oxytocinergic system. Endogenous oxytocin is recognized as a regulator of maternal-child social bonding, but its neuroprotective effect in the injured brain remains to be elucidated. Here, we investigated the effects of chemogenetic activation of oxytocinergic neurons on the neural correlates of EoP. Using a well-established mouse model of systemic interleukin-1β to induce EoP, we showed that neonatal chemogenetic activation of oxytocinergic neurons has anti-inflammatory effects in microglia, improving microstructural development of the corpus callosum and motor cortex, and rescuing typical social behavior. These neuroprotective effects were more pronounced in females, who showed a greater reduction in microgliosis and improved social behavior compared to males. This study provides a biological explanation for how developmental care and early interventions, linked to the oxytocinergic system, may induce neuroprotection in the developing brain.

Abstract

The newborn infant, particularly those born preterm, is vulnerable to brain injury resulting in lifelong neurodevelopmental sequalae. Conventional structural brain imaging correlates poorly with later individual neurodevelopmental trajectories. Therefore, assessing brain integrity with functional (particularly functional connectivity (FC)) neuroimaging, would be beneficial, as studies showed correlation between early FC assessment and later neurodevelopmental outcomes. However, these tools are absent of neonatal clinical settings, probably either due to lack of portability or restricted access to the deep structures. In this proof of concept (poc) work, we show that functional ultrasound imaging (fUS) has key characteristics for this challenge: including portability, sensitivity and spatiotemporal resolution. fUS can monitor fine grain brain activity in deep cerebral nuclei, detect changes in FC dynamics at different developmental stages, with capabilities for 3D imaging. Furthermore, we present a multimodal poc combining fUS with high-density diffuse optical tomography (HD-DOT). The results demonstrate correlation between fUS and HD-DOT signals in spatially overlapping areas of the brain. The complementary fields of view of fUS (in depth) and HD-DOT (shallow cortex) could enable for the first time cot-side whole brain assessment of FC. In the future, a system combining fUS and HD-DOT could be developed as a clinical tool to monitor the developing brain in high-risk infants.

Abstract

The preclinical stages of therapeutic agent development cost hundreds of millions of dollars, stymying innovation and slowing the development of products to improve human health. There is a striking unmet need for therapies that protect or repair the brain damage associated with preterm birth, i.e., delivery before 37 weeks of gestation. Of the more than 15 million babies born preterm every year, up to 60% will go on to develop a neurological disorder, with the earliest-born infants the most impacted. We have limited options with limited efficacy for preventing or treating these changes. Combining accurate knowledge of pathophysiology with high-throughput sequencing and computational biology approaches is a logical step towards an optimised screening pipeline. In this study, we conducted comprehensive testing of dose, timing, and route of administration, integrating multimodal data from preclinical models of brain injury common in preterm-born infants to validate the most effective therapeutic option for the cord-derived mesenchymal stem cell product (HuMSC). In this study, HuMSC serves as a working example, but the scoring system is therapy-agnostic. We developed a scoring protocol based on microglia transcriptome analyses and myelin protein expression to evaluate the efficacy of the HuMSC product in a rat model of inflammation-associated preterm infant brain injury. We identified the superiority of treatment delivered in the tertiary phase of injury over treatments in the acute or subacute stages, as well as the superiority of intranasal over intravenous delivery of HuMSCs. The optimal time, dose, and route of administration options for HuMSC were confirmed in a second model relevant to preterm infants, but with a different pathophysiology, namely germinal matrix haemorrhage. In conclusion, we have established a scoring protocol that expedites the collection of comprehensive dose, time and route of administration data critical for establishing large animal and clinical trials with the greatest chance of success

Abstract

The gut microbiome of preterm infants is highly vulnerable to perturbations. Members of the class Clostridia are among the first anaerobes colonizing the preterm gut, yet their ecological roles and antimicrobial resistance (AMR) properties remain poorly understood. We characterized 98 Clostridia isolates from fecal samples of preterm infants, spanning 17 species and 11 genera. Isolates were identified by MALDI-TOF and 16S rRNA sequencing, colonization levels were quantified, and antimicrobial susceptibility was assessed by disk diffusion and E-test. Resistance determinants were screened by PCR and sequenced. We focused on Clostridia that were present at low colonization levels (mean 5.3 log10 CFU g-1 of feces). While most isolates were susceptible to amoxicillin-clavulanic acid, imipenem, and metronidazole, resistance to tetracycline (12%), clindamycin (35%), and cefotaxime (35%) was observed. Distinct species-specific resistance included linezolid (Clostridium argentinense), chloramphenicol (Clostridium innocuum), and tigecycline (Paeniclostridium sordellii), and one Robinsonella peoriensis isolate displayed vancomycin resistance. The detection of tet and erm genes corresponded with phenotypic resistance, while β-lactamase activity was uncommon. Although colonizing at low levels, these findings highlight the ecological significance of rarely studied commensal Clostridia and their contribution to the neonatal resistome, acting as underappreciated reservoirs of AMR genes during a critical window of microbiome assembly.

Abstract

Objective: Early-onset neonatal sepsis (EONS) due to ascending infection is a potentially preventable complication of preterm premature rupture of membranes (PPROM). Our objective was to determine whether the analysis of bacteria from vaginal swab samples is predictive of the risk of EONS in PPROM.

Study design: In a prospective 3-center observational cohort, patients with PPROM were enrolled between 22 and 36 weeks’ gestation (WG) + 6 days. Vaginal swab samples at delivery were analyzed using two different approaches, classical bacterial cultures and shotgun metagenomic sequencing analysis. A metagenomics score was constructed combining the characterization of the vaginal microbiome and the presence of pathogens and the optimal cut-off to predict EONS was tested on a receiver operating curve.

Results: 563 PPROM cases were enrolled, with 646 liveborn neonates. PPROM occurred < 32 WG in 41.9% and deliveries were < 34 WG in 41.0%. The incidence of EONS was 29/646 (4.5%). When considering all central and peripheral microbiological samples available for 26 neonates, the main pathogens isolated were Escherichia coli in 14 cases (53.8 %), other gram-negatives in 5 (19.2%), strict anaerobes in 3 (11.5%); there was a single case (3.8%) each with Group B Streptococcus (GBS), Streptococcus anginosus, Staphylococcus aureus and Ureaplasma urealyticum. We studied the prediction of EONS among 272 mothers and their 310 neonates (20 EONS, 6.4%) with both culture and metagenomic data available. A culture positive for a major or intermediate pathogen in the vaginal sample at delivery had a sensitivity of 80.0 % (95% CI=56.3-94.3) and a specificity of 37.9% (95% CI=32.3-43.8), adjusted odds ratio (aOR) of 1.6 (95 % CI [0.5-5.0]) to predict EONS. The presence of E. coli was associated with an EONS risk of 10.6% vs 4.9%, in the absence of E. coli (p=0.07). The metagenomics score was highly associated with EONS, with an area under the receiver operating curve of 0.75 (95% CI, 0.61-0.90). At the optimal cutoff value, sensitivity was 70% (95% CI, 64-95%), specificity was 85% (95% CI, 81-89%). A metagenomics score greater than 40 was associated with a significantly increased risk of EONS with an aOR of 8.9 (95 % CI [3.5; 22.3]) in multivariate analysis adjusted for latency period and gestational age, p<0.001.

Conclusion: In PPROM, conventional microbial culture of maternal vaginal samples was associated with EONS, but its predictive values remain insufficient to guide perinatal care. Metagenomic microbial signatures improved predictive values. This opens the perspective for a rapid point-of-care test.

Abstract

[This corrects the article DOI: 10.3389/fped.2023.1198016.]

Abstract

Microfluidics mixing is the current lab-scale method used for producing mRNA-loaded lipid nanoparticles (mRNA-LNPs) thanks to reproducibility and robustness of microfluidic mixing. Despite these advantages, the production of small LNP volumes is associated with significant material waste. Given the high cost of synthetic mRNA, this waste can be a major limitation, particularly for early-stage screening of formulations. This study proposes alternative methods for mRNA-LNP formulation aiming to improve their stability for both formulation and mRNA screening, while reducing material waste on a research scale. Specifically, we investigated post-encapsulation of mRNA into pre-formed vesicles (PFVs) obtained by microfluidic mixing. These PFVs were complexed with mRNA by: (1) a microfluidic or (2) a manual pipetting method. The resulting mRNA-LNPs produced using these two post-encapsulation methods exhibit similar physicochemical properties and morphologies to those obtained by conventional microfluidic protocol. These mRNA-LNPs were assessed on in vitro and in vivo expression. mRNA-LNPs prepared by our alternative methods showed a similar transfection level compared to the conventional formulation taken as a control. The suitability of post-encapsulation methods to other lipids, mRNAs and microfluidic systems was also confirmed. This work offers robust, simple and economic alternative methods for preparing small volumes of mRNA-LNPs. The versatility of post-encapsulation methods allows to screen mRNA formulations in a wide range of laboratories. These methods could be applied to encapsulate tailored doses of mRNA and various mRNA constructs to achieve an optimal and personalized therapy.

Abstract

Clostridium butyricum exhibits a dual role, acting not only as a probiotic but also as an opportunistic pathogen associated with neonatal necrotizing enterocolitis (NEC) and infant botulism. We aimed to establish high-resolution genotyping frameworks to improve molecular surveillance and outbreak investigations. We analyzed 297 C. butyricum genomes, including 200 isolates from preterm neonates across 13 French neonatal intensive care units over a 20-year period and 97 publicly available genomes. A core-genome multilocus sequence typing (cgMLST) scheme was developed using chewBBACA, defining 2,621 loci, and applied to genomes with ≥95% locus presence. Core-genome single-nucleotide polymorphism (cgSNP) analysis was performed for complementary resolution. Phylogenetic cgMLST classified isolates into nine major clades. Some clinical strains displayed clonal relationships, whereas others were geographically and temporally unrelated. All botulinum neurotoxin type E-producing strains were grouped within a single clade. NEC-associated isolates showed geographic and temporal clustering, but no clade was uniquely linked to NEC. cgSNP analysis identified 11 clusters with overall discriminatory power similar to cgMLST while providing finer resolution for NEC-related strains. We propose robust cgMLST and cgSNP schemes for C. butyricum, enabling high-resolution genotyping and supporting epidemiological surveillance and outbreak investigation of this emerging opportunistic pathogen in neonatal settings.

Importance: Clostridium butyricum has been identified in fecal samples from both asymptomatic neonates and cases of necrotizing enterocolitis (NEC). Using a large collection of strains from different origins and spatiotemporal contexts, we developed and established a cgMLST scheme for the molecular typing of C. butyricum. Our results show that most C. butyricum strains cluster independently of origin and spatiotemporal context factors. However, specific cgMLST clades of C. butyricum were found for plant and botulinum neurotoxin type E strains. Clonal strains were also identified. No specific cgMLST clade was found to be genetically associated with NEC. cgSNP showed higher discriminatory power compared to cgMLST. Importantly, cgSNP provided better discriminatory power for strain relatedness with respect to strains isolated from NEC patients.

Abstract

Background: Bronchopulmonary dysplasia (BPD) is the most common morbidity of very preterm (VPT) infants born <32 weeks’ gestation with life-long consequences. Studies document wide variation between regions and units in BPD prevalence.

Research question: Which unit-level factors contribute to the variation in BPD prevalence among very preterm infants between European neonatal units?

Study design and methods: Analyses were conducted using the prospective population-based EPICE cohort in 19 regions in 11 European countries. We compared prevalence of moderate/severe BPD among VPT infants without severe congenital anomalies in neonatal units with ≥40 annual VPT admissions (83 units, 5,285 infants). Unit prevalence was adjusted for individual risk factors using standardised morbidity rates. Spearman correlation and multilevel logistic regression were used to assess associations of BPD with unit-level variables: unit mortality rates, first week oxygen saturation targets, proportion of infants ventilated within the first 24 hours, unit practice of postnatal corticosteroid use for hypotension or BPD prevention and unit volume.

Results: Unadjusted BPD prevalence ranged from 2%-47% (median:13%) between units and was 8%-42% (median:17%) after adjustment and standardisation. Oxygen saturation targets, proportion of initial mechanical ventilation and postnatal corticosteroid use partly explained the between-unit variability (proportional change of variance: 25%, 5%, 17% respectively), leaving 53% unexplained. Risk-adjusted in-hospital mortality (range 8%-21%) and patient volume were not correlated with BPD prevalence.

Interpretation: Large variability in BPD prevalence exists between European units, which was only partially explained by patient characteristics. Our findings suggest that improving respiratory management for VPT infants could be beneficial for reducing BPD prevalence. The association of unit postnatal corticosteroid use practice with BPD requires further investigation.

Abstract

Background: Pregnancy complications are known to be risk factors for the onset of depression and anxiety symptoms. This study assessed associations between pregnancy complications, including concurrent complications, and symptoms of anxiety and depression among pregnant women living in France.

Methods: A cross-sectional study was carried out among 492 pregnant women. Sociodemographic and obstetric characteristics were collected using an online questionnaire. Depression and anxiety symptoms were evaluated using the Edinburgh Postnatal Depression Scale and the Spielberger State-Trait Anxiety Inventory, respectively. Multivariate logistic regressions were employed to identify associations between mental health outcomes and pregnancy complications.

Results: While 37% of women declared no pregnancy complications, 9.76% declared two or more complications, and 63% of participants had at least one complication. Among these latter, 68.9% had a high risk of depression, 83.9% elevated state anxiety, and 77.4% elevated trait anxiety. State anxiety scores were significantly higher in women who felt they did not receive adequate social support from their partner, family, and friends and who reported dissatisfaction with medical care. Adjusting for confounders, we identified that women with complications had higher odds of experiencing higher state anxiety scores (adjusted OR: 2.94; 95% CI: 1.40-6.10). Positive associations were also observed between gestational diabetes mellitus and increased likelihood of reporting depressive symptoms (adjusted OR: 1.99; CI:1. 20-3.29) and high state anxiety scores (OR: 3.31; CI: 1.22-9.01).

Conclusion: We found a high prevalence of depression and anxiety among pregnant women with complications. Gestational diabetes mellitus was positively associated with antenatal depression and high state anxiety levels. These findings suggest that women with complications have a higher risk of developing depressive and anxious symptoms. Screening for and treating physical and mental health problems in women experiencing pregnancy complications and poor mental health symptoms are crucial to safeguard the well-being of the mother and the fetus.