The present study is focused on the determination of low-volatile chemosignals excreted or secreted by mouse pups in their early days of life involved in maternal care induction in mice adult females. Untargeted metab-olomics was employed to differentiate between samples collected with swabs from facial and anogenital area from neonatal mouse pups receiving maternal care (first two weeks of life) and the elder mouse pups in the weaning period (4th week old). The sample extracts were analysed by ultra-high pressure liquid chromatography (UHPLC) coupled to ion mobility separation (IMS) in combination with high resolution mass spectrometry (HRMS). After data processing with Progenesis QI and multivariate statistical analysis, five markers present in the first two weeks of mouse pups life and putatively involved in materno-filial chemical communication were tentatively identified: arginine, urocanic acid, erythro-sphingosine (d17:1), sphingosine (d18:1) and sphinga-nine. The four-dimensional data and the tools associated to the additional structural descriptor obtained by IMS separation were of great help in the compound identification. The results demonstrated the great potential of UHPLC-IMS-HRMS based untargeted metabolomics to identity putative pheromones in mammals.

Motherhood is a critical period modulating behavioural changes to favour survival in mammals. In mice, olfaction is a key driver of social behaviours, and adult neurogenesis in the olfactory bulb is modulated at this stage, contributing to pup recognition. The primary olfactory cortex, known as the piriform cortex, contains a special population of neurons of embryonic origin which continues expressing immature markers such as doublecortin (DCX) in young and adult mice, and present a long period of protracted maturation and integration into the circuit as the animal ages. While these immature neurons are considered a potential source of plasticity, their precise role and whether motherhood affects their maturation rate remains unclear. To investigate this question, we analysed the expression of DCX in prepubescent vs. young adults, in virgin vs. pregnant and in pup-sensitized virgins vs. lactating female mice. We found that the density of DCX cells sharply decreased in the piriform cortex with age, previously demonstrated as a proxy of their integration as glutamatergic neurons by contrast, pregnancy and lactation failed to significantly alter the density of these cells. To further analyse how motherhood could affect DCX-ir cells, we co-labelled DCX cells with NeuN, an archetypical marker of mature neurons. We did not find significant differences in the percentage of double-labelled cells nor in features related to maturation like number of neurites or main diameter in lactating dams as compared to pup-sensitized virgin females. Our results suggest that the first pregnancy does not significantly affect the differentiation of immature neurons of the piriform cortex.

Background: Esophageal atresia (EA) is a complex congenital anomaly frequently associated with additional malformations and genetic conditions. Despite advances in prenatal imaging and genomic technologies, establishing an etiologic diagnosis and performing accurate risk stratification remain challenging due to marked clinical and genetic heterogeneity. Methods: We conducted a retrospective cohort study of neonates diagnosed with EA and admitted to a level IIIc neonatal intensive care unit between 2005 and 2024. Prenatal findings, associated anomalies, genetic testing results, mortality, and neurodevelopmental outcomes beyond 12 months were analyzed. Results: A total of 105 neonates were included, of whom 10.5% were diagnosed prenatally. Isolated EA was identified in 55.2% of patients, whereas 44.8% had associated anomalies, most commonly congenital cardiac defects. Clinically relevant genetic findings were identified in 10.5% of the total cohort (23.4% of complex EA cases). These findings reflect a clinically selected subgroup and should not be interpreted as diagnostic yields applicable to unselected populations or as a comparison between testing modalities. Overall mortality was 11.4%. Lower birth weight showed the strongest association with mortality in univariable analyses; however, no independent predictors were inferred due to the limited number of events. All deceased patients had complex malformative conditions and/or extreme prematurity. Among children with follow-up beyond 12 months, 88.5% demonstrated age-appropriate neurodevelopment. Conclusions: EA is characterized by substantial etiologic and phenotypic heterogeneity. Prenatal detection remains challenging, although advances in fetal imaging may improve diagnostic accuracy. A phenotype-guided approach integrating clinical evaluation and genetic testing may support etiologic diagnosis, recurrence counseling, and follow-up planning in selected patients. However, because testing was indication-driven and evolved over time, the reported diagnostic yields should not be generalized to unselected EA populations or interpreted as comparative performance across testing modalities.

Programs for Undiagnosed Rare Diseases (URD) with anonymized data sharing are contributing to the earlier genetic diagnosis of patients and to the identification and characterization of novel genetic disorders. Recently, de novo pathogenic variants in two non-coding spliceosomal small nuclear RNAs (snRNAs), RNU4-2 and RNU2-2 , key regulators of gene expression during neurodevelopment, have been linked to neurodevelopmental disorders (NDD). With the aim to identify individuals with causal genetic variants in known and novel snRNA genes, we analyzed WGS and clinical data of 1,708 probands with undiagnosed NDD enrolled in the Spanish URD Programs. Selected individuals underwent in-depth re-phenotyping including facial mask analyses (Face2Gene), and blood-DNA methylation profiling was obtained (Infinium methylation Epicv2 microarrays). We identified 38 patients (2.22%) with de novo pathogenic variants in snRNA genes, 28 in RNU4-2 (1.64%), 6 in RNU2-2 (0.35%), and 4 in RNU5B-1 (0,23%). A clinically recognizable NDD (ReNU syndrome) is caused by RNU4-2 variants, with dysmorphic features making a disease-associated facial mask, RNU2-2 associates an early onset epileptic encephalopathy, and RNU5-B1 a less defined NDD with overlapping features. A DNA methylomic episignature was also identified for RNU4-2 , which does not overlap with the other conditions. We have also defined autosomal recessive inheritance in 8 additional patients (0.47%) with NDD and biallelic variants in three snRNA genes (3 in RNU4-2 , 3 in RNU2-2 , and 2 in RNU4-1 ). Our study has facilitated the quick diagnosis, management, and genetic counselling of 2.69% previously undiagnosed patients with NDD, further reinforcing the value and utility of the URD Programs. GRAPHICAL

Motherhood is a critical period modulating behavioural changes to favour survival in mammals. In mice, olfaction is a key driver of social behaviours, and adult neurogenesis in the olfactory bulb is modulated at this stage, contributing to pup recognition. However, whether motherhood would also promote changes in the population of immature neurons of the piriform cortex is unknown. To investigate this question, we analysed the expression of doublecortin (DCX), a marker of immature neurons, in prepubescent vs young adults, in virgin vs pregnant and in pup-sensitized virgins vs lactating female mice. We found that the density of DCX cells sharply decreased in the piriform cortex with age, but pregnancy and lactation failed to significantly alter the density of these cells. To further analyse how motherhood could affect DCX-ir cells, we co-labelled DCX cells with NeuN, an archetypical marker of mature neurons. We did not find significant differences in the percentage of double-labelled cells nor in features related to maturation like number of neurites or main diameter in lactating dams as compared to pup-sensitized virgin females. Our results suggest that the first pregnancy does not significantly affect the differentiation of immature neurons of the piriform cortex.