Clinical serum biochemistry is used to assess maternal and fetal health; however, it involves complex physiological adaptations during fetal development, c

Background/objectives: Maternal hypothyroidism during pregnancy, including overt and subclinical hypothyroidism, is a common endocrine disorder complicating pregnancy and is associated with increased risks of adverse maternal and fetal outcomes. However, its effects on neonatal metabolism remain inadequately understood. This study aimed to assess the association between hypothyroidism in pregnancy and neonatal amino acid and acylcarnitine levels. Methods: This retrospective case–control study included 1,987 pregnant women with overt hypothyroidism in pregnancy, 2,023 with subclinical hypothyroidism in pregnancy, and 2,043 euthyroid pregnant women from Fujian Provincial Maternity and Children’s Hospital between 2016 and 2024. Their newborns all underwent newborn screening for 11 amino acids and 31 acylcarnitines using tandem mass spectrometry. Results: After adjusting for potential confounding factors including gestational age at delivery, birth weight, and neonatal sex, newborns born to mothers with hypothyroidism in pregnancy (overt and subclinical) showed significant differences in the levels of 9 amino acids compared with those born to mothers with normal thyroid function. These amino acids are involved in the arginine-proline metabolism, glycine metabolism, and the urea cycle pathway. Amino acid levels also differed between the two subtypes. In addition, the levels of short-chain, medium-chain, and long-chain acylcarnitines in newborns from the hypothyroidism groups were significantly higher than those in the control group, with several acylcarnitine indicators (C16, C16:1, C16OH, C18:1OH, and C6DC) exhibiting characteristic differences between the two subtypes. The levels of thyroid-stimulating hormone (TSH) in newborns from both hypothyroidism groups were significantly higher than those in the control group, whereas no statistically significant difference in TSH levels was observed between the overt and subclinical hypothyroidism groups. Conclusion: Our findings revealed a significant association between hypothyroidism in pregnancy (overt and subclinical) and neonatal amino acid and acylcarnitine levels, which were mainly involved in branched-chain amino acid, arginine, proline, and glycine metabolism, as well as the urea cycle and fatty acid oxidation pathways. These results underscore the importance of identifying maternal conditions that correlate with neonatal metabolites, providing a reference for exploring strategies to optimize neonatal nutrition and improving long-term health outcomes.