Wenqi Lv ^1,2,3 Han Xie ¹ Shengyu Wu ^1,2 Jiaqi Dong ^1,2 Yuanhui Jia ^1,4 Hao Ying ^1,5*

• ¹ Shanghai First Maternity and Infant Hospital, Shanghai, China
• ² Department of Clinical Laboratory, Shanghai Tongji Hospital, Tongji University School of Medicine, Shanghai, China
• ³ Shanghai Key Laboratory of Maternal Fetal Medicine, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai, China
• ⁴ Other, Shanghai, China
• ⁵ Department of Obstetrics and Gynecology, Shanghai First Maternity and Infant Hospital, School of Medicine, Tongji University, Shanghai, China

Background Spontaneous preterm birth (sPTB) is a global disease that is a leading cause of death in neonates and children younger than 5 years of age. However, the etiology of sPTB remains poorly understood. Recent evidence has shown a strong association between metabolic disorders and sPTB. To determine the metabolic alterations in sPTB patients, we used various bioinformatics methods to analyze the abnormal changes in metabolic pathways in the preterm placenta via existing datasets. Methods In this study, we integrated two datasets (GSE203507 and GSE174415) from the NCBI GEO database for the following analysis. We utilized the “Deseq2” R package and WGCNA for differentially expressed genes (DEG) analysis; the identified DEGs were subsequently compared with metabolism-related genes. To identify the altered metabolism-related pathways and hub genes in sPTB patients, we performed multiple functional enrichment analysis and applied three machine learning algorithms, LASSO, SVM-RFE, and RF, with the hub genes that were verified by immunohistochemistry. Additionally, we conducted single-sample gene set enrichment analysis to assess immune infiltration in the placenta. Results We identified 228 sPTB-related DEGs that were enriched in pathways such as arachidonic acid and glutathione metabolism. A total of 3 metabolism-related hub genes, namely, ANPEP, CKMT1B, and PLA2G4A, were identified and validated in external datasets and experiments. A nomogram model was developed and evaluated with 3 hub genes; the model could reliably distinguish sPTB patients and term labor patients with an area under the curve (AUC) > 0.75 for both the training and validation sets. Immune infiltration analysis revealed immune dysregulation in sPTB patients. Conclusion Three potential hub genes that influence the occurrence of sPTB through shadow participation in placental metabolism were identified; these results provide a new perspective for the development and targeting of treatments for sPTB.