Recurrent miscarriage (RM) is defined as the occurrence of 2 or 3 consecutive abortions prior to the 20th week of pregnancy (Pei, et al., 2019). The prevalence of women with RM is approximately 2.5% in childbearing age women (Dimitriadis, et al., 2020). Currently, the known pathogenesis of RM includes genetic abnormalities, intrauterine structural abnormalities, antiphospholipid syndrome, thrombophilia, endocrine abnormalities, immune dysfunction, infection, and so on (Dimitriadis, et al., 2020). Due to the highly variable clinical presentation and the complexity of pathogenesis, there is a great challenge for the prediction, diagnosis, and treatment of RM.

At present, more and more scholars draw attention to pregnancy immune tolerance among the above causes (Li, et al., 2020). Immune cells including natural killer (NK) cells, T cells, macrophages, etc. all play crucial roles in maintaining maternal-fetal immune tolerance. As the most abundant immune cell subset in decidua, NK cells that encoded inhibitory killer cell immunoglobulin like receptor and supported embryonic growth were diminished in RM patients, while the ratio of another NK subset with cytotoxic and immune-active signature (CD56+CD16+ NK cells) was significantly increased (Wang, et al., 2021a). Increased cytotoxic T lymphocytes (CD3+CD8+/CD3+CD8+CD69+ cells) and the imbalance between pro-inflammatory Th17 cells and Treg cells in the peripheral blood were associated with RM (Krechetova, et al., 2020). The predominance of M2 towards M1 macrophages resulted in early miscarriage (Wang et al., 2011, Zhang et al., 2019b). Except for immune cells, cytokines and chemokines also played crucial role in the maintenance of pregnancy via regulating immune suppression, the invasion of trophoblast, and angiogenesis (Ali, et al., 2021).

Despite some mechanisms and pathogenesis were verified, unexplained RM still accounted for approximately 50% of the whole RM population (Dimitriadis, et al., 2020). In order to excavate the underlying mechanisms of RM, omics approaches, such as epigenomics, transcriptomics, metabolomics, proteomics, etc. have been applied. Genome-wide DNA methylation from placental villi was profiled in both RM patients and controls, which showed that hypomethylation of PRDM1 in RM patients leaded to trophoblast cell apoptosis and the inhibition of cell migration (Du, et al., 2020). Another study on DNA methylation in villi verified an increased methylation at AXL receptor tyrosine kinase and a decrease at defensin β 1 might contribute to or be a consequence of poor placental function in RM (Hanna, et al., 2013). Huang et al. performed RNA sequencing in decidual tissue, and elucidated that lnc-CES1-1 inhibited decidual cell migration by interacting with RNA-binding protein FUS and activating PPARγ in RM (Huang, et al., 2021). Another study using whole-transcriptome sequencing found out the important roles of focal adhesion, extracellular matrix-receptor interaction, and the PI3K-Akt signaling pathway in RM (Wang, et al., 2022). Metabolomics and proteomics were also used in the study of RM, suggesting low succinate accumulation in villi, and the interaction of angiotensinogen and embryonic development-related proteins in the decidua took a significant role in RM (Wang et al., 2021b, Xiong et al., 2021).

However, such attempts failed to address its pathogenesis from the networks of genes, RNAs, proteins and metabolites. As compared to studies of a single omics type, multi-omics offers the opportunity to understand the flow of information that underlies disease. Multi-omics has been widely used in tumor research. Chaudhary et al. provided a model that integrated multi-omics data (RNA sequencing, miRNA sequencing, and methylation data) from The Cancer Genome Atlas to explicitly predict hepatocellular carcinoma survival (Chaudhary, et al., 2018). Additionally, multi-omics analysis was applied to a cohort of 200 patient samples of chronic lymphocytic leukemia, profiled for somatic mutations, RNA expression, DNA methylation and ex vivo drug responses to effectively identify major dimensions of disease heterogeneity (Argelaguet, et al., 2018). Several integrated multi-omics analyses have also been applied in reproduction. Wu et al. integrated miRNA profile of villus and mRNA data of placenta, and the RM-related hub genes were enriched in immune responses, hormonal regulation, and metabolism (Wu, et al., 2021). The findings of Chen et al. via integrating mRNA, miRNA, and methylation data suggested the importance of osteoclast differentiation, leishmaniasis, NF-kappa B signaling pathway, Toll-like receptor signaling pathway, and tuberculosis in villus/placenta of women with RM (Chen, et al., 2019).

Nevertheless, the multi-omics analyses on RM were limited, and the above studies masked the contributions of different tissue types, as placenta consists of villus and decidua. Additionally, the development of RM is not only dependent on the damage of decidua or villus, but also associated with the dysfunction of feto-maternal immune cross-talk. Therefore, this study focused on biological functions of both villus and decidua, and restricted tissue types carefully from the very beginning. Multi-omics analysis was employed to figure out the gene expression patterns and hub genes of both villus and decidua in RM during early pregnancy. As maternal-fetal immune tolerance is crucial for the maintenance of pregnancy (Ander, et al., 2019), immune cell profiles and immunologic signatures of differentially expressed genes (DE-genes) were further conducted to elucidate the possible immune responses in RM, which might provide a novel perspective for the diagnosis and treatment of RM.