Gestational diabetes mellitus (GDM) is a form of diabetes that is first diagnosed during pregnancy, with a worldwide prevalence of 9–21%. GDM frequently affects both short-term and long-term health in the mother and offspring, because of the diverse genetic background and epigenetic modifications that occur in response to nutritional and environmental factors (1, 2). Currently, the precise etiological mechanisms of GDM remain unclear; however, numerous studies have found that GDM is a multifactorial disease that involves genetic factors, lifestyle, and chronic inflammation. Insulin resistance (IR) and pancreatic β-cell dysfunction are regarded as essential for the pathogenesis of GDM (2, 3). Although the exact mechanisms remain to be clarified, chronic inflammation has been reported to participate in the development of IR and pancreatic β cell failure, which in turn leads to GDM (4). The placenta is a temporary organ formed during pregnancy, which serves as the only bridge connecting the mother and fetus, and has important endocrine function. Placenta-derived inflammatory cytokines, such as interleukin-1 beta (IL-1β), IL-6, IL-15, IL-10, IL-34, IL-38, and tumor necrosis factor alpha (TNF-α), can stimulate immune cells and aggravate immune and inflammatory responses, thereby exacerbating chronic inflammation and maternal IR and inducing β cell failure during pregnancy (5–11). Moreover, immune cells and inflammatory cytokines are important components of the placental microenvironment, which is essential for normal pregnancy (12, 13). Imbalanced immune infiltration in the placenta contributes to the pathogenesis and development of pregnancy-specific diseases, including GDM, and may affect GDM-related adverse pregnancy outcomes and clinical prognosis (13–15).

Chemical modifications of cellular RNAs can result in secondary structure modifications, splicing, degradation, or molecular stability, which are emerging layers of post-transcriptional gene regulation. More than 160 chemical modifications have been identified (16). N⁶- methyladenosine (m⁶A) RNA modification is the most prevalent type of RNA epigenetic processing (17, 18). m6A modification is mediated by its effector proteinsare in a dynamic and reversible pattern (17). m6A occurs mainly in the 3’- UTR and the vicinity of the termination codon mRNA, which is recogonized by “readers” (YTH domain family (YTHDF]1–3, and insulin-like growth factor 2 mRNA-binding proteins 1–3), catalyzed by methylases [methyltransferase-like (METTL)3/14, and Wilms’ tumour 1-associated protein], and removed by demethylases [fat-mass and obesity-associated protein (FTO), and alkylation repair homolog protein 5] (19). Recent evidence indicates that perturbations of m⁶A modifications dysregulate mRNA metabolism, including mRNA stability, mRNA splicing, RNA nucleation, RNA-protein teractions and mRNA translation, thereby contributing to various physiological and pathophysiological processes (20–22). Numerous m⁶A modifications have been shown to regulate adipogenesis, glucose metabolism, insulin resistance, and the related chronic immune response (17, 21, 23). This suggests that m⁶A modifications are implicated in the development of metabolic diseases, although the specific knowledge regarding GDM is still in its infancy.

In this study, we aimed to reveal the imbalanced immune infiltration in the placenta of patients with GDM, the differentially expressed m⁶A-related genes (DMRGs) involved, as well as the crosstalk between them, and also to develop a nomogram model for the diagnosis of GDM.

GDM is a common complication of pregnancy, adversely affecting both the mother and fetus (1, 2). The etiology of GDM, which involves genetic background and epigenetic modifications, remains unclear. Chronic low-grade inflammation during pregnancy can contribute to the pathogenesis of GDM by exacerbating maternal IR and inducing β cell failure (4). As an endocrine organ, the placenta derives inflammatory cytokines that stimulate immune cells and aggravates the immune/inflammatory response (12, 13). Moreover, the disturbance of immune cell infiltration in the placenta is attributed to pregnancy-specific diseases, including GDM, as well as GDM-related adverse outcomes (13–15). In this study, we found that the infiltration of monocytes was higher in GDM placentas than in control samples, while the infiltration level of macrophages (M1 and M2) in GDM placentas was lower than that in the controls. Monocyte infiltration has been shown to be crucial during inflammation. As important mediators of the innate immunity, monocytes circulate in the bloodstream and pass into tissues during the steady state and in increased quantities during inflammation (27). GDM is considered as a low-degree inflammation, and elevated levels of monocytes in the peripheral blood of patients with GDM have been previously reported (28). Based on the expression of superficial CD14 and CD16 in flow cytometry, monocytes can be divided into three subsets: classical (CD14++CD16-), intermediate (CD14+CD16++) and non-classical (CD14+CD16+) (29). Angelo et al. (28) observed increased percentage of classical monocytes, decreased frequency of intermediate monocytes in the peripheral blood of patients with GDM compared to controls. By contraries, an increase in the intermediate subset and a decreased frequency of classical monocytes were detected in healthy pregnancy compared to non-pregnant women (30). Considering that the variation in levels of monocyte subsets may contribute to the development of inflammation in GDM, it is essential to develop new studies on this topic to validate these findings. During gestation, bone marrow-derived monocytes can migrate from the bloodstream to the uterus and differentiate into decidua-specific macrophages upon exposure to this local microenvironment (31–34). A proportion of tissue-resident macrophages is constantly replaced by blood monocytes, and the mechanisms behind these differential renewal patterns are not fully understood and may be controlled by the tissue specific microenvironment (27, 35, 36). Inflammatory stimuli often depleted macrophages and induce monocyte recruitment; these monocytes might potentially contribute to tissue-resident macrophages upon the resolution of inflammation (27). Therefore, the decrease in macrophages and increased monocytes may be due to the inflammation during GDM. Decidual macrophages are highly plastic (37). It is generally accepted that macrophages are mainly the M1 (pro-inflammatory) phenotype during the pre-implantation period, and change to M2 (anti-inflammatory) phenotype following trophoblast attachment and invasion; macrophages seem to revert to M1 phenotype at the time of delivery (37–39). Inappropriate macrophage polarization may cause adverse pregnancy outcomes (30, 37). There are controversies regarding the use of placental macrophages in describing GDM. An imbalance of M2 to M1 macrophages has been observed in the placentas of diabetic patients and rats (40), as well as in placentas of GDM patients (41). Opposing conclusions have been reported in other studies, in which macrophages maintain the M2 phenotype in GDM compared to controls (42–44). In the present study, we also observed higher propotion of M2 than M1 phenotype in GDM compared to controls ( Supplementary Figure 1 ).

m⁶A methylation plays a vital role in glucose/lipid metabolism as well as its related chronic inflammatory processes (23, 45–47). FTO moduates glucose metabolism via regulating forkhead box protein O1 and activating transcription factor 4 of m6A modification (46, 48). FTO also regulates adipogenesis by controlling cell cycle progression in an YTHDF2 dependent mechanism (45). METTL3 regulates lipid metabolism via mediating JAK1 mRNA stability an m6A-YTHDF2 dependent manner (47), and regulating NF-κB and MAPK via meditating m6A modification of TNF receptor associated factor 6 (49). Due to the dynamic and reversible nature, m6A methylation can be reversed by environmental stressors, including changes in nutrition. High-fat diet affecs METTL3 and FTO mRNA expression, and fasting state leads to the reduced FTO mRNA expression and increases m6A levels (50, 51). It remains unknown whether m⁶A modifications play a role in GDM. Exploration of the crosstalk between m6A modification and GDM may provide a potential strategy for the diagnosis, prognosis and treatment. We obtained m⁶A-related genes from the RMBase and RMvar databases and identified DMRGs based on the GSE70493 dataset. Enrichment analysis was conducted to determine the biological functions of the DMRGs. Notably, several pathways, such as type 1 diabetes mellitus and autoimmune thyroid disease, were closely correlated with the development and mal outcome of GDM. Recent studies have revealed that a small but significant population of patients with GDM develop postpartum T1DM (52, 53). Emerging evidence suggests that perturbations of the thyroid hormone signaling pathway and antibodies are associated with GDM development and adverse outcomes (54, 55). In terms of the GO biological process category, the DMRGs were closely related to inflammatory- and immune-related biological processes. Therefore, we suggest that, in addition to chronic inflammation, the immune response may also contribute to the pathophysiology of GDM.

As stated above, monocyte infiltration is aberrant in the placentas of patients with GDM. We obtained DMRGs related to monocyte infiltration, of which seven DMRGs (CD81, CFH, FABP5, GBP1, GNG11, IL1RL1, and SLAMF6) were selected through LASSO regression analysis to construct a nomogram. FABP5 belongs to the calycin superfamily and fatty-acid binding protein family, and serves as a gatekeeper for mitochondrial integrity to modulate regulatory T cells (Treg) and subdue immune responses (56). It has been reported that increased intra-tumoral FABP5 contributes to CD8+ T-cell infiltration and is linked to overall and recurrence-free survival, indicating that FABP5 could be an immunometabolic marker in hepatocellular carcinoma (57). Moreover, FABP5 has been observed to be enriched in classical monocytes of heart failure patients, suggesting that FABP5 contributes to monocyte activation (58). CD81 is a tetraspanin that participates in adaptive immunity and host-virus interactions (59, 60). As an inhibitor of the alternative complement pathway, CFH protects self-surfaces from immune attacks, thereby engaging in host-virus interactions and innate immunity (61–63). GBP1 is involved in macrophage apoptosis and pyroptosis (64). Interleukin-33 (IL-33) is the only known ligand of IL1RL1, and IL1RL1/IL-33 signaling participates in various inflammatory diseases (65). SLAMF6 is expressed in a variety of immune cells and may be involved in crosstalk between different microenvironments (66–68). Finally, GNG11, a member of the guanine nucleotide-binding protein family, is involved in various transmembrane signaling systems (69, 70). The nomogram showed a robust performance in distinguishing GDM patients from normal controls in the training set (GSE70493), with an AUC of 83%. GDM diagnosis is usually confirmed by a 75 g-oral glucose tolerance test during the second trimester. GSE92772, which is based on blood samples extracted during 24–28 weeks of gestation, was selected to externally validate the diagnostic capacity of the nomogram. The nomogram model exhibited a high diagnostic value with an AUC value of 85.9%, although it lacked the expression matrix of CFH. Therefore, our findings suggest that this m⁶A-related signature, correlated with monocyte infiltration, can be regarded as a novel biomarker and potential therapeutic target for GDM.

This study had a few limitations. A comprehensive analysis of the placenta and peripheral blood is warranted to verify the mRNA expression, protein expression and m⁶A-modification status of CD81, CFH, FABP5, GBP1, GNG11, IL1RL1, and SLAMF6. The diagnostic ability of the nomogram model may require further validation using a larger sample size. For subsequent research, more clinical parameters regarding valuable prognosis risk characteristics should be incorporated to verify the predictive ability of the nomogram.

In this study, we analyzed the immune landscape and DMRGs in the placentas of patients with GDM. Some DMRGs were strongly associated with monocyte infiltration, which was higher in GDM placentas than in the control group. Based on seven selected DMRGs linked to monocyte infiltration in GDM placentas, we developed and validated a highly accurate nomogram for recognizing GDM.

The raw data of GSE70493 and GSE92772 were obtained from public databases (https://www.ncbi.nlm.nih.gov/geo/). The processed data are available from the corresponding author upon reasonable request.

RD: Conceptualization, Data curation, Formal analysis, Software, Writing—original draft. YW Conceptualization, Supervision, Validation, Writing—review and editing. LL: Funding acquisition, Methodology. All authors contributed to the article and approved the submitted version.

The study was supported by the Clinical Research Project of Liaoning Diabetes Medical Nutrition Prevention Society [grant number LNSTNBYXYYFZXH-RS01A].

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.

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