Bulk RNA-sequencing, single-cell RNA-sequencing analysis, and experimental validation reveal iron metabolism-related genes CISD2 and CYP17A1 are potential diagnostic markers for recurrent pregnancy loss.

Yi-Bo He, Lu Han, Ju Fang, Qun Zhou, Cong Wang, Shi-Liang Chen, Zhe-Zhong Zhang, Yue Shang, Hua-Lei Cai, Jun-Yu Li, Yong-Lin Liu

Journal: Gene 2024;901():148168

PMID: 38244949

Full Text on Elsevier Science View on PubMed

Abstract

BACKGROUND

Recurrent pregnancy loss (RPL) is associated with variable causes. Its etiology remains unexplained in about half of the cases, with no effective treatment available. Individuals with RPL have an irregular iron metabolism. In the present study, we identified key genes impacting iron metabolism that could be used for diagnosing and treating RPL.

METHODS

We obtained gene expression profiles from the Gene Expression Omnibus (GEO) database. The Molecular Signatures Database was used to identify 14 gene sets related to iron metabolism, comprising 520 iron metabolism genes. Differential analysis and a weighted gene co-expression network analysis (WGCNA) of gene expression revealed two iron metabolism-related hub genes. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry were used on clinical samples to confirm our results. The receiver operating characteristic (ROC) analysis and immune infiltration analysis were conducted. In addition, we analyzed the distribution of genes and performed CellChat analysis by single-cell RNA sequencing.

RESULTS

The expression of two hub genes, namely, CDGSH iron sulfur domain 2 (CISD2)and Cytochrome P450 family 17 subfamily A member 1 (CYP17A1), were reduced in RPL, as verified by both qPCR and immunohistochemistry. The Gene Ontology (GO) analysis revealed the genes predominantly engaged in autophagy and iron metabolism. The area under the curve (AUC) demonstrated better diagnostic performance for RPL using CISD2 and CYP17A1. The single-cell transcriptomic analysis of RPL demonstrated that CISD2 is expressed in the majority of cell subpopulations, whereas CYP17A1 is not. The cell cycle analysis revealed highly active natural killer (NK) cells that displayed the highest communications with other cells, including the strongest interaction with macrophages through the migratory inhibitory factor (MIF) pathway.

CONCLUSIONS

Our study suggested that CISD2 and CYP17A1 genes are involved in abnormal iron metabolism, thereby contributing to RPL. These genes could be used as potential diagnostic and therapeutic markers for RPL.

Address: Department of Clinical Lab, The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Chinese Medicine), Hangzhou, Zhejiang Province, China.; Department of Gastroenterology, Guizhou Provincial People's Hospital, Guiyang, Guizhou Province, China.; Reproductive Center, Hainan Branch, Shanghai Children's Medical Center, School of Medicine, Shanghai Jiao Tong University, Sanya, Hainan Province, China.; Department of Emergency Obstetrics and Gynecology, Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou Province, China.; Obstetrics and Gynecology, First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Chinese Medicine), Hangzhou, Zhejiang Province, China.; Department of Clinical Lab, The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Chinese Medicine), Hangzhou, Zhejiang Province, China. Electronic address: [email protected].; Pharmacy Department, Hainan Branch, Shanghai Children's Medical Center, School of Medicine, Shanghai Jiao Tong University, Sanya, Hainan Province, China. Electronic address: [email protected].; Reproductive Center, The Second Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, Zhejiang Province, China. Electronic address: [email protected].

DOI: 10.1016/j.gene.2024.148168