Background Pregnant women are one of the most vulnerable groups that require additional precautions against SARS-CoV-2 infection. We hypothesized that SARS-CoV-2 virus infection has an impact on newborns from mothers infected in late stages of pregnancy and that this will be reflected in the urinary metabolomic fingerprint of newborns. Methods Term infants born to mothers with SARS-CoV2 at delivery and control term infants born to negative pregnant women during the same period in the same period were recruited and urine samples were collected in the first 48 - 72 hours after delivery. Untargeted metabolomic fingerprinting of urine samples was performed employing liquid chromatography-time-of-flight mass spectrometry Results A total of 49 term newborns were included in this study, 26 from mothers with acquired SARS-CoV-2 infection (GA= 40 weeks, IQR (38-40)) and 23 newborns from mothers without SARS-CoV-2 infection (GA= 40 weeks, IQR (39-40)). Sample analysis using two complementary methods allowed the annotation of 980 features. Differences in the urinary metabolomes between cases and controls were observed. Functional analysis indicated that metabolic pathways were altered, including pyrimidine metabolism, drug metabolism by cytochrome P450, a total of eight pathways related to amino acids metabolism and translation, and four pathways related to the metabolism of cofactors and vitamins. Also, Chemical Similarity Enrichment Analysis (ChemRICH) complemented the pathway enrichment analysis, indicating that several clusters of metabolites, such as dipeptides, fatty acids and chromans, are downregulated in urines samples from newborns with mothers affected by SARS-CoV-2. Conclusions This is the first study of the urinary metabolome of newborns from mothers with and without SARS-CoV-2 infection. Alterations on metabolic pathways and metabolite clusters confirm that neonates exhibited metabolic dysregulations when their mothers were infected late in pregnancy, although no intrauterine or transplacental transmission or clinical evidence was observed.

Method I ACQUITY BEH C18 (100 x 2.1 mm, 1.7 µm) column (Waters, Milford, MA, USA) running a stepwise gradient with solvent A (H2O with 0.1% v/v HCOOH) and solvent B (CH3CN, 0.1% v/v/ HCOOH) as mobile phase components as follows: 2% B were held for 0.5 min followed by a linear gradient to 25% B in 3 min and 98% B in 4.5 min; 98% B were maintained for 1.25 min followed by the return to initial conditions in 0.25 min, which were held for 2 min. The flow rate was set to 0.4 mL min-1, column and autosampler to 55 and 4 °C, respectively, and the injection volume was 3 µL. Method II a SynergiTM Hydro-RP 80Å LC C18 column (150 x 2 mm, 4 µm) (Phenomenex, Torrance, CA, USA) and a stepwise gradient were used as follows: 1% B was held for 2 min followed by a linear gradient from 1 to 80% B in 8 min and 98% B for 2 min before returning to initial conditions in 0.1 min and column equilibration with 1% B during 3 min. The flow rate was set to 0.4 mL min-1, column and autosampler to 55 and 4 °C, respectively, and the injection volume was 2 µL. Full scan MS detection was carried out in the ESI+ mode in the range between 50 and 1500 m/z at a scan frequency of 5 Hz using the following parameters: gas T, 200 °C; drying gas, 14 L min-1; nebulizer, 35 psi; sheath gas T, 350 °C; sheath gas flow, 11 L min-1. Reference standards were introduced into the source for automatic MS spectra recalibration during analysis via a reference sprayer valve using the m/z 149.02332 (background contaminant), 121.050873 (purine), and 922.009798 (HP-0921) as references.