The regulation and potential roles of m6A modifications in early embryonic development and immune tolerance at the maternal-fetal interface.

Emerging studies have shown that the local tumor microenvironment (TME) required for tumor growth and survival plays important roles in tumor development and progression. TME is complex and contains not only cancer cells and stromal cells but also macrophages and distant recruited cells, such as infiltrating immune cells, characterized by hypoxia, immune escape, metabolic dysregulation and chronic inflammation ( 108 , 109 ). m6A has been widely investigated in regulating oncogenes or tumor suppressor genes in various cancers. Accumulating studies have recently reported a new role of m6A in the antitumour immune response. In addition to affecting classical immunotherapy, m6A also affects tumor-associated immune cell activation and infiltration and cytokine secretion in the tumor microenvironment, which play important roles in tumor initiation, progression, metastasis, and treatment response ( 110 , 111 ). The tumor immune microenvironment (TIME) generally consists of the infiltration of multiple immunosuppressive cells, especially MDSCs and Treg cells, and is often absent of antitumour immune cells ( 111 , 112 ). In recent years, emerging studies have deciphered the vital role of m6A modifications in the regulation of the local and systemic TIME, which mediate tumor progression and response to immunotherapy ( 113 ). The analysis of the m6A-related signatures from The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx) and the Gene Expression Omnibus (GEO) database revealed a significant relationship between the diverse m6A clusters and the TIME ( 114 , 115 ). In glioma, m6A signatures were associated with classification, including prognosis, grade, isocitrate dehydrogenase (IDH) status and 1p19q status. Patients in the high-risk group showed enhanced stroma and immune scores and a higher abundance of immune infiltration ( 116 – 119 ). Silencing ALKBH5 in glioblastoma multiforme (GBM) notably suppresses hypoxia-induced tumor-associated macrophage (TAM) recruitment and immunosuppression in allograft tumors by regulating CXCL8/IL-8 secretion ( 120 ). Reduced METTL3 in hepatocellular carcinoma (HCC) results in increased infiltration of DCs in the TIME, which leads to the overall upregulation of major histocompatibility complex (MHC) molecules, costimulatory molecules, and adhesion molecules and is closely related to the prognoses of HCC ( 121 ). In addition, overexpressed YTHDF1 in HCC was associated with low CD3+ and CD8+ T-cell infiltration ( 122 ). ALKBH5 regulated PD-L1 mRNA in a YTHDF2-dependent manner on monocytes/macrophages and infiltration of myeloid-derived suppressor-like cells in the TIME of intrahepatic cholangiocarcinoma (ICC) ( 123 ). Alkbh5 in melanoma and colorectal cancers (CRC) regulates the metabolism/cytokines and infiltration of immunosuppressive Treg cells and MDSCs, therefore enhancing PD-1 immunotherapy and GVAX vaccination therapy ( 124 , 125 ). In addition, Alkbh5 was related to the infiltration of monocytes in periodontitis, of which regulated m6A mediated the immune reaction of TNF-family-member receptors and cytokines, indicating the crucial roles of m6A in the diversity and complexity of the immune microenvironment of periodontitis ( 126 ). Additionally, Mettl3- or Mettl14-deficient tumors upregulated cytotoxic tumor-infiltrating CD8+ T cells and increased the production of IFN-γ, Cxcl9 and Cxcl10 in the TIME of CRC in vivo , thereby enhancing the response to anti-PD-1 treatment ( 127 ). METTL3, WTAP, IGF2BP3, YTHDF1, HNRNPA2B1 and HNRNPC were markedly increased in esophageal squamous cell carcinoma (ESCC) and positively related to the expression of PD-1, whose copy number dynamically affects the enrichment of tumor-infiltrating immune cells ( 128 ). Consensus clustering for 15 m6A regulators identified two molecular subtypes (clusters 1/2) in head and neck squamous cell carcinoma (HNSCC). Cluster 1 was enriched with G2 M checkpoint, mTORC1 signaling, and PI3K/AKT/mTOR signaling, while cluster 2 was associated with favorable prognosis, increased PD-L1, higher immune score and distinct immune cell infiltration ( 129 ). High-risk pancreatic adenocarcinoma (PAAD) contributed to the enhanced infiltration of M0 and M2 macrophages and decreased B cells, naïve T cells, CD8+ T cells and Treg cells ( 130 – 133 ). IGF2BPs, as functional downstream modulators of circNDUFB2, regulate the secretion of CXCL10, CXCL11, CCL5, and IFNβ in non-small cell lung cancer (NSCLC) ( 134 , 135 ). In addition, m6A-related genes in peripheral blood leukocytes are noninvasive biomarkers for NCCLC patients ( 136 ). Nucleophosmin 1 (NPM1) is a chameleon protein that shuttles between the nucleus and cytoplasm. NPM1 is overexpressed in lung adenocarcinoma (LUAD) and effectively distinguishes LUAD from normal samples. The expression level of NPM1 in LUAD is markedly related to tumor stage and prognosis. Multiple database analysis showed that NPM1 is negatively related to B cells and NK cells. Moreover, NPM1 expression was significantly correlated with one m6A modifier-related gene YTHDF2 and five glycolysis-related genes (ENO1, HK2, LDHA, LDHB and SLC2A1) ( 137 , 138 ). Four immune-related genes (IRGs), including CD274, CD8A, GZMA and PRF1, were screened and were consistent with the enrichment of CD8+ T cells and activated memory CD4+ T cells in the TIME of multiple cancers ( 132 ). In breast cancer, the three m6A clusters (writers, erasers and readers) are correlated with subsets of the infiltrating immune landscape, including activated CD8+ T cells, NK cells, activated DCs, macrophages and Treg cells. The low m6Ascore contributes to the increased mutation burden, immune activation and survival rates and is associated with an enhanced response to anti-PD-1/PD-L1 immunotherapy ( 139 – 141 ). In bladder cancer, 9 m6A-related lncRNAs were dramatically associated with overall survival outcomes of bladder cancer. The risk score of bladder cancer was correlated with the infiltration levels of multiple immune cells, including B cells, plasma cells, Tfh cells, Treg cells, resting NK cells, neutrophils, and M0, M1 and M2 macrophages, which indicated the important role of m6A-related lncRNAs in prognosis and shaping the tumor immune microenvironment ( 142 , 143 ), which was also found in papillary thyroid carcinoma (PTC) ( 114 ) and HCC ( 144 ). The above studies indicated that m6A methylation and m6A-related modifications play essential roles in the differentiation and function of immune cells and secretion of cytokines, therefore shaping the TIME and further regulating the response to immunotherapy ( Figure 7 ). The m6A modifications in the tumor immune microenvironment have been extensively studied ( Figure 8 ). In addition, emerging studies have reported that m6A modifications can regulate the functions of multiple immune cells and cytokine secretion and shape the TIME, thus participating in the progression of cancer. Currently, RNA modification has become a new direction for studying embryo development and maternal-fetal immune tolerance. m6A modifications in the endometrium and ovary have been reported to be related to multiple gynecological diseases, including gynecological cancers, adenomyosis, endometriosis, polycystic ovary syndrome and premature ovarian failure, which generally contribute to RPL ( 145 , 146 ). Researchers have proposed that receptivity at the maternal-fetal interface is more reminiscent of cancer immunology ( 35 , 147 ). Trophoblasts and tumor cells share many similarities, including invasion, angiogenesis, and immunosuppressive environments, both of which are supported by an abetting microenvironment. However, the immunosuppressive environment in tumors severely influences antitumour therapy, which is different from the decidual immune environment ( 147 ). Emerging studies of m6A in tumors have shown potential value and strengthened the functions of m6A in the decidual local immune microenvironment. The crosstalk between trophoblasts and decidual immune cells, including decidual NK cells, macrophages and T cells, determines the local immune microenvironment at the maternal-fetal interface, the imbalance of which may lead to adverse pregnancy outcomes, such as RPL/RIF and preeclampsia ( 148 ). Although little is known about m6A methylation in trophoblast and decidual immune cells, knowledge of m6A modifications in shaping multiple immune cells and the TIME would inspire us to explore the potential roles of m6A functions in maternal-fetal immune tolerance by targeting trophoblasts and decidual NK cells, T cells and macrophages as well as cytokine secretion to shape the local immune microenvironment, thus affecting placentation and immune tolerance at the maternal-fetal interface, the imbalance of which may affect placentation and disrupt the receptive microenvironment and further lead to adverse pregnancy, including RPL and RIF. However, trophoblast cells are more precisely regulated than tumor cells, and the regulatory mechanisms of m6A in trophoblast and decidual immune cells remains unknown and requires further investigation ( Figure 8 ). m6A modifies the tumor immune microenvironment and regulates tumor progression. m6A writers, erasers and readers regulate the tumor immune microenvironment in glioma, liver cancer, colorectal cancer, esophageal cancer, pancreatic cancer, lung cancer, breast cancer, and bladder cancer by controlling different immune cells and the PD-1/PD-L1 immune checkpoint mediating the efficacy of immune therapy. Insights into m6A modification in the tumor and decidual microenvironment. The m6A modifications in the tumor immune microenvironment have been extensively studied. However, the role of m6A modifications in decidual immune cells remains largely unknown. The same immune trajectory of decidual and tumor immune microenvironments encouraged us to study the mechanisms of m6A in decidual immune cells.

Most mammalian genomes undergo RNA transcription, and many RNA transcripts can never be translated into proteins ( 1 ), which may lead to functional defects. RNA is not only an essential intermediate in the flux from DNA to proteins but also a regulatory molecule for fundamental cellular processes, the dysfunction of which contributes to important pathological processes ( 2 ). The coding and noncoding transcriptomes are widely and dynamically regulated by chemical modification, which adds new modifications of complexity and functionality to the emerging roles of RNAs in physiological and pathological conditions ( 3 ). Covalent modifications sense the changing environment directly and rapidly without changing the DNA and RNA sequences ( 4 ). In contrast to the epigenetic modifications on DNA and histones that work at the transcriptional level, RNA methylation has a notable effect on gene regulation at the posttranscriptional level ( 5 ). RNA modifications affect transcripts by altering the charge, base-pair potential, secondary structure and RNA–protein interactions, which, in turn, regulates gene expression via RNA processing, localization, translation and degradation ( 3 ). N6-methyladenosine (m6A) is the most abundant internal mRNA modification. Identifying of the proteins that mediate m6A modifications has elucidated the roles of mRNA modifications in nearly every aspect of the mRNA life cycle, as well as various cellular, developmental, and disease processes ( 3 ). In mammals, approximately 0.1%- 0.6% of adenines undergo m6A modification, with an average of 3-5 methylated sites in each mRNA. Remarkably, m6A modifications can be deposited onto transcripts in tissue- and cell-type-specific- manners ( 6 ). The m6A modifications commonly occur in yeast, plants, flies, bacteria, humans and other mammals, which implicates its multiple functions in RNA, including precursor mRNA (pre-mRNA) splicing, mRNA translation, stability, structure, export and decay, implying an association with several cellular processes, such as cell differentiation and reprogramming, further contributing to various human diseases (reviewed in ( 7 )). For m6A detection, methods, including analytical chemistry, high-throughput sequencing, m6A-CLIP and miCLIP, have emerged to determine the specific methylation sites and the modification fractions at these sites to promote biological studies of RNA modifications ( 3 , 8 – 11 ), which provided the available ways to study the roles of m6A modification proteins in physical and pathological processes. Similar to DNA methylation, the deposition and removal of mRNA methylation also depend on a multiunit methyltransferase complex that was initially documented in 1994 ( 7 ). mRNA methylation is governed by three types of proteins, namely, methyltransferases as “writers”, demethylases as “erasers”, and specific m6A-binding proteins (YTHDF1-3) as “readers” ( 12 ). The deposition of m6A methylation is catalyzed by multicomponent methyltransferases, mainly methyltransferase-like 3 (METTL3), METTL14, Wilms’ tumor 1-associated protein (WTAP), RNA-binding motif proteins 15 (RBM15s), virlike m6A methyltransferase associated (VIRMA/KIAA1429), zinc finger CCCH-Type containing 13 (ZC3H13) and METTL16 ( 13 – 19 ). METTL3 is the most important component of the m6A methyltransferase complex (MTC) and highly conserved in eukaryotes from yeast to humans ( 20 ); METTL3 forms a stable heterodimer core complex regulated by WTAP and catalyzes the transfer of meth1 groups ( 13 , 21 ). Similar to WTAP, RBM15s have no catalytic function but bind to METTL3 and WTAP to guide these two proteins to specific RNA sites for m6A modification. KIAA1429-mediated m6A methylation of mRNAs takes place near the 3’-UTR and the stop codon ( 22 ). m6A modification occurs when METTL3 and METTL14 are recruited into the nucleus ( 14 ). Only METTL3 has methyltransferase activity in the MTC. METTL16 encodes S-adenosylmethionine (SAM) synthase and is expressed in most cells. However, METTL16-mediated m6A sites were located in introns or intron–exon boundaries, which is different from the common m6A sites in UTRs ( 19 ). The m6A demethylases as “erasers” make the m6A methylation dynamic and reversible, which could be passively removed from the transcriptome via degradation of the modified RNA or active demethylation by the m6A demethylases fat mass and obesity-associated (FTO) or a-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5), both belonging to the AlkB family of dioxygenases known to demethylate N-methylated nucleic acids ( 23 , 24 ). FTO was the first demethylase discovered in 2011 ( 23 ). The ALKBH5 catalytic domain can demethylate m6A-containing single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) ( 25 ). The discovery of reader proteins has made great progress in elucidating the impact of m6A methylation in mammalian cells. Different ‘readers’ with different cellular localizations influence almost all aspects of RNA metabolism, such as YT521-B homology domain family proteins (YTHDFs), insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs), eukaryotic translation initiation factor 3 (EIF3) and heterogeneous nuclear ribonucleoproteins (HNRNPs) ( 26 – 29 ). Proteins with YTH domains located in the cytoplasm (YTHDF1, YTHDF2 and YTHDF3) and nuclei (YTHDC1 and YTHDC2) directly recognize m6A marks ( 27 , 30 ), which promote the degradation and translation of m6A-modified RNA. In contrast to YTHDFs, IGF2BPs promote the stability and storage of their target mRNAs in a m6A-dependent manner under normal stress conditions and therefore affect gene expression output ( 28 ). EIF3 directly binds a single 5’ UTR m6A and recruits the 43S complex to initiate translation in a cap-dependent manner ( 26 ). Numerous studies have shown that m6A modifications play essential roles in multiple biological and pathological processes: hematopoietic development, central nervous and system development, the adaptive and innate immune system, carcinogenesis and the tumor microenvironment, as well as gametogenesis and early embryo development, the dysfunction of which generally results in various diseases by abnormal m6A modifications of the target genes ( 31 ) ( Figure 1 ). Currently, with the development of assisted reproductive technology (ART), which helps to exclude abnormalities in the embryo, there remains the occurrence of recurrent pregnancy loss (RPL) in child-bearing women ( 32 ). Embryo implantation into the endometrium successfully relies on strict and coordinated regulation of trophoblasts derived from the fetus and decidual stromal cells and immune cells derived from the maternal sides ( 33 ). Crosstalk could be regulated by molecules associated with implantation, including hormones, signaling molecules, transcription factors and cytokines ( 34 ). However, the occurrence of RPL remains high, and more efforts should be made to discover the mystery of embryo implantation. Emerging studies emphasize the essential roles of m6A modifications in embryo implantation, while their roles in trophoblasts and immune tolerance at the maternal-fetal interface are worthy of further investigation. Overview of the reversible m6A RNA modifications and related functions. m6A modifications are regulated by m6A writers (METTL3, METTL14, RBM15s and WATP) and readers (FTO and ALKBH5). m6A methylation is recognized by different readers (YTHDC1-2, YTHDF1-3 and EIF3) by regulating RNA splicing, exportation, translation, degradation, stabilization and storage. Successful blastocyst implantation was first established by normal embryo development after fertilization. Its further development relies on the dynamically coordinated balance between the fetal-derived invading trophoblasts and the receptive maternal decidua ( 35 ). Once implantation is initiated, the trophoblasts, originating from the outer layer of the blastocyst, will differentiate into invasive extravillous trophoblasts (EVTs) to attach and invade the maternal decidua and therefore promote placentation ( 36 ), making the first contact with the maternal immune system. The placenta not only mediates the hormonal, nutritional and oxygen support to the fetus but also plays an essential immunoregulatory role at the maternal-fetal interface ( 37 ). Blastocysts that take paternal antigens as semiallogeneic to the maternal side can be recognized and accepted by the immunosuppressive maternal immune system ( 38 ). After normal implantation, the implantation sites are infiltrated with diverse immune cells, which are mainly characterized by an anti-inflammatory Th2-type immune microenvironment ( 39 ). Previously, embryo quality was shown to contribute to adverse pregnancy complications. ART tools make the selection of high-quality embryos available; however, implantation rates and successful pregnancy with ART are still relatively low. This indicated that uterine receptivity might also play a crucial role in the establishment of normal pregnancy ( 40 ). Diverse mechanisms, including cytokine/chemokine and hormonal signaling as well as DNA modifications, contribute to the pathology of pregnancy complications ( 40 ), which is attributed to the abnormal gene expression of cytokines/chemokines and abnormal signaling in a specific time and space during placentation and further fetal development. Early mammalian embryos are capable of strong pluripotent stemness, which could be reprogrammed by epigenetic modifications. m6A modification is highly conserved from yeast to mammals and can regulate gene expression output, determine stem cell fate and cell differentiation, and further shape the local microenvironment. Recent studies have shown that m6A modifications are associated with animal reproductive processes, including gametogenesis, maternal-zygote transition (MZT) and early embryonic development ( 41 – 45 ). In addition, m6A modifications also play an essential roles in fine-tuning the immune response, including innate and adaptive immune responses and immune system development ( 8 ). Here, we reviewed the potential roles and novel insights of m6A methylation in embryo development and immune tolerance during early pregnancy during maternal-fetal tolerance.

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Numerous studies have elucidated the crucial roles of m6A- and m6A-associated regulators in embryogenesis during implantation. Additionally, other authors uncovered their essential roles in the differentiation and function of innate and adaptive immune cells as well as in shaping the local and systemic immune microenvironment. Indeed, m6A- and m6A-associated- regulators target cytokine secretion and multiple immune cells in the development and metastasis of various cancers by mediating the response to immunotherapy. Interestingly, the process of blastocyst implantation was similar to the metastasis of tumors; currently, the occurrence of RIF and RPL still disturbs child-bearing age women with unknown pathologies ( 35 , 149 ). It is essential to determine the potential unknown factors causing RPL and RIF to provide clear targets for reproductive physicians. Although it is relatively straightforward to diagnose RPL, the progress of predicting and preventing RPL has been hampered by a lack of a standardized definition, uncertainties around the pathogenesis and highly variable clinical presentation. Moreover, the effectiveness of many medical interventions is controversial due to the available treatments targeting the putative risk factors for RPL ( 150 ). Therefore, it is urgent to explore the underlying pathologies that lead to RPL. Dysfunction of trophoblasts, stromal cells and decidual immune cells contributes to RPL and RIF. The m6A- and m6A-related- regulators participate in embryogenesis and shape the local microenvironment, which draws inspiration from studies in tumors and provides novel insight for investigating the potential pathologies causing RPL and RIF. Although some studies have reported the role of m6A- and m6A-related- regulators in the function of trophoblasts and are therefore correlated with pathological pregnancies, the relationship between m6A-and m6A-related- regulators and maternal decidual cells and the local immune microenvironment at the maternal-fetal interface is still unknown and needs further exploration, which would fill the gap in m6A in the shaping microenvironment at the maternal-fetal interface.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

We thank Kahinho P Muyayalo and Zhibing Zhang for the language editing. The figures in this paper were created with BioRender.com .

This work is supported by the Women and Children’s Hospital of Hubei Province (2021SFYY002).

HL: conceptualization, investigation, data curation, writing – original draft, visualization, funding acquisition. JZ: data curation, writing – review & editing. AL: conceptualization, writing – review & editing. All authors have read and approved the final manuscript.