Exploring the role of m6A modification in the great obstetrical syndromes

Abstract Background N6-methyladenosine (m6A) is one of the predominant RNA epigenetic modifications that modify RNAs reversibly and dynamically by “writers” (methyltransferase), “erasers” (demethylase), and “readers.” Objective This review aimed to provide a comprehensive understanding of the complexity of m6A regulation in the great obstetrical syndromes to understand its pathogenesis and potential therapeutic targets. Methods The terms “placenta or trophoblast” and “m6A or N6-methyladenosine” were searched in PubMed databases (June 2023). Results In this review, we discuss the regulatory role of m6A in the great obstetrical syndromes such as preeclampsia (PE), spontaneous abortion (SA), hyperglycemia in pregnancy (HIP) and fetal growth to emphasize the clinical relevance of m6A dysregulation in pregnancy. We also describe mechanisms that potentially involve the participation of m6A methylation, such as proliferation, invasion, migration, apoptosis, autophagy, endoplasmic reticulum stress, macrophage polarization, and inflammation. Conclusion We summarize the recent research progress on the role of m6A modification in the great obstetrical syndromes and placental function and provide a brief perspective on its prospective applications.


Introduction
In the 1970s, N6-methyladenosine (m6A), one of the predominant internal RNA modifications in eukaryotes, was shown to modify RNAs dynamically and reversibly to support diverse biological processes.Approximately 0.1-0.4% of adenines exhibit m6A modification, with an average of 3-5 methylated sites in each mRNA in mammals [1].Methylation occurs at the sixth position of the nitrogen atom of adenosine at the posttranscriptional level.Currently, colorimetry [2], dot blotting [3], and liquid chromatography/mass spectrometry (LC/MS) [4] are mainly used to detect global m6A levels.Until m6A-specific methylated RNA immunoprecipitation sequencing (MeRIP-seq) was developed, the transcriptome-wide profile of m6A localization in RNA was described [5].It has been confirmed that the m6A modification usually occurs in the motif DRACH (D ¼ G/A/U, R ¼ G/A, H ¼ A/U/C).m6A is highly enriched in the 3 0 untranslated region (UTR) and near the stop codon, affecting the stability and function of RNA [6].
The placenta is an active interface between the mother and fetus that is essential for maternal-fetal nutrient and gas exchange, maintenance of healthy pregnancy, and fetal development."Great obstetrical syndromes" is a general term for several complications of pregnancy that conceivably lead to or the shortand long-term maternal and fetal risks and require close health monitoring and treatment [7].m6A provides a new perspective to explore the pathogenesis of placenta-related diseases.In this review, we will summarize m6A-related functional proteins and the current research progress in the great obstetrical syndromes.

RNA m6A methylation
m6A methylation is a reversible and dynamic modification of mRNA and other types of noncoding RNA (ncRNAs), including lncRNA, microRNA (miRNA), circ-RNA, ribosomal RNA (rRNA), transfer RNA (tRNA), and small nuclear RNA (snRNA).The installation and removal of m6A on RNAs is catalyzed by methyltransferases (writers) and demethylases (erasers), respectively.Altered methylation is recognized by readers, which exert a regulatory role in RNA stability, decay, splicing, translation and nuclear export [1].
Unlike mRNA, which can be translated into proteins, ncRNAs also show specialized functions in the modulation of gene expression at both the transcriptional and posttranscriptional levels.m6A modification could influence the generation and maturation of miRNAs, the splicing, generation, translation, subcellular trafficking (cytoplasmic export) and degradation of circ-RNAs, and RNA-protein interactions in lncRNAs [8][9][10][11][12].Interestingly, ncRNAs can also affect m6A modification by interacting with m6A regulatory proteins [13,14].

m6A Writers
The installation of m6A methylation is achieved by a highly conserved RNA methyltransferase complex.The RNA methyltransferase complex is mainly composed of methyltransferase-like 3 (METTL3), which plays a central role [2]; METTL14, which supports the METTL3 protein structure [15]; and Wilms' tumor 1-associated protein (WTAP), which regulates the recruitment of the complex to target mRNA [16].Knockdown of METTL3 led to a decrease in total m6A levels in trophoblasts [2].

m6A Erasers
Demethylation is mediated by alkylation repair homolog protein 5 (ALKBH5) [17] and fat-mass and obesityassociated protein (FTO) [18].Both of them belong to the AlkB family of nonheme Fe(II)/a-ketoglutarate (a-KG)-dependent dioxygenases, which catalyze a wide range of biological oxidations, including demethylation of m6A [12].Silencing of FTO or ALKBH5 both increased total m6A levels in RNA, and the demonstration of their demethylase activity proved that the posttranscriptional modification of mRNAs is reversible [17,18].

m6A Modification in the great obstetrical syndromes
Normal placental development is critical for sustaining intrauterine life.Trophoblasts proliferate and differentiate into villous and extravillous trophoblasts (EVTs) after implantation.Invasion and migration of trophoblasts mediate immunotolerance and remodeling of the uterine spiral artery at the maternal-fetal interface during embryo implantation [23].Under physiological conditions, trophoblasts maintain homeostasis by autophagy at a low basal level.Excessive autophagy can destroy normal cellular components and eventually lead to apoptosis [24].Dysfunctional trophoblast phenotypes and abnormal placentation can lead to adverse pregnancy-related complications.The molecular mechanisms of m6A modification governing placental formation and trophoblast cell lineage specification and differentiation are an emerging field of research.
We conducted a literature search using the PubMed database ranging from 2013 to 2023, and the keywords we searched mainly include "trophoblast or placenta" AND "m6A or N6-methyladenosine."This review included seven studies based only on the human placenta samples [3,, one study based on both human and rats placanta [29], one study based only on an in vitro trophoblast model [30], and one study based only on animal models [4].In addition, 18 studies were based on clinical specimens and in vitro model validation [2, 8-10, 13,14, 31-42], and three of them also conducted animal studies [40][41][42].Furthermore, four of these studies employed MeRIPseq [5,9,25,31].The m6A modification-related studies in various obstetrical syndromes is shown in Table 1, the regulation of placental function by m6A modification is shown in Figure 1, and the description in detail is as follows.PE: preeclampsia; loPE: late-onset preeclampsia; eoPE: early-onset preeclampsia; RSA: recurrent spontaneous abortion; SA: spontaneous abortion; GDM: gestational diabetes mellitus; IUGR: intrauterine growth restriction; OSO: oxidized soybean oil; LBW: low birth weight.

Preeclampsia (PE)
PE, defined as the development of hypertension and proteinuria after 20 weeks of gestation, is one of the main pregnancy complications contributing to preterm birth, perinatal death, maternal mortality, and intrauterine growth retardation (IUGR).Early-onset PE (eoPE, with delivery at <34 weeks of gestation) and late-onset PE (loPE, with delivery at 34 þ 0 weeks of gestation) are two types of PE [45].Abnormal proliferation, invasion, migration, and dysfunctional syncytialization of human placental trophoblasts and a severe endoplasmic reticulum (ER) stress state are involved in the pathogenesis of PE [46].In most studies, m6A levels were increased in PE placentas, and the expression of METTL3 and METTL14, the main methyltransferases, was upregulated in PE placentas [2,5,32,43].METTL3 affected the expression of methylated transmembrane BAX inhibitor motif containing 6 (TMBIM6) by regulating its mRNA stability via YTHDF2 in HTR-8/SVneo cells, which regulated ER stress [43].METTL14 overexpression inhibited proliferation and invasion and induced light chain 3 (LC3)-dependent autophagy in HTR-8/SVneo cells by epigenetically elevating forkhead box O3a (FOXO3a) [32].METTL14 overexpression also augmented circPAPPA2 m6A methylation but decreased its reader IGF2BP3, leading to a decline in circPAPPA2 levels and inhibition of TEV1 cell invasion [9].Knockdown of METTL3 in rats could reduce the adverse effects of PE, including reductions in fetal weight, placental weight, and renal pathological changes [43].These outcomes suggest that METTL3 is a potential target for the treatment of PE.
However, a few studies have proposed that the global m6A levels are significantly reduced in the eoPE placenta [33], and RNA methyltransferases, including METTL3 and WTAP, are downregulated in the PE placenta [33,34].Knockdown of METTL3 and WTAP significantly inhibited the invasion and migration of HTR-8/SVneo cells through m6A modification of myosin light chain kinase (MYLK) [34] and high mobility group nucleosomal binding domain 3 (HMGN3) [33], respectively.It has also been proven that the expression of ALKBH5 was upregulated in PE placenta and HTR- 8/SVneo cells treated with hypoxia/reoxygenation (H/R) [32,40].The abnormal expression of METTL3 may be involved in the occurrence of PE by regulating the m6A modification levels of different factors, thereby affecting trophoblast invasion and ER stress, respectively.Given the complexity of METTL3 functionality, further investigations remain to be performed.Guo et al. further demonstrated that inhibition of ALKBH5 promotes histone lysine demethylase 3B (KDM3B)-mediated activated leukocyte cell adhesion molecule (ALCAM) promoter demethylation by facilitating peroxisome proliferator-activated receptor gamma (PPARG) mRNA m6A modification and further activates the Wnt/b-catenin pathway, which promotes HTR-8/SVneo cell proliferation and migration and alleviates PE-like features in pregnant mice [40].Therefore, ALKBH5 could also be a candidate target for the treatment of PE.
In addition, abnormal expression of m6A readers was also observed in the PE placenta.The expression of IGF2BP2/3 was downregulated in the placenta of PE patients, and the mRNA level of IGF2BP1, a protective factor of PE, was also decreased in the blood plasma of pregnant women with PE [13,14,35].miR-423-5p and miR-181a-5p were increased in both the plasma and placenta of patients with severe PE, and both suppressed the invasion and migration of HTR-8/SVneo cells by directly inhibiting IGF2BP1 and IGF2BP2 through a conserved binding site in the 3 0 -UTR, respectively [13,14].In addition, IGF2BP2 regulates the RNA stability of Linc01116, which is related to poor uterine spiral artery remodeling via m6A methylation in HTR8/SVneo cells [8].IGF2BP3 significantly inhibits the invasion and migration capacities of HTR8/SVneo cells and EVTs from the first-trimester human placental villi and decreases the mRNA level of IGF2 in HTR8/SVneo cells [35].The findings of IGF2BPs function and its crosstalk with ncRNAs expand our understanding of the role of m6A in trophoblast dysfunction and PE development.

Spontaneous abortion (SA)
Spontaneous abortion (SA) is one of the most common complications of pregnancy and may be associated with inadequate trophoblast cell invasion.Recurrent spontaneous abortion (RSA), also called recurrent miscarriage (RM), is defined as the failure of two or more clinically recognized pregnancies before 20-24 weeks of gestation and includes embryonic and fetal losses [47].However, the current research findings on m6A modification of SA/RSA have not reached complete unity.
Qiu et al. [3] suggested that FTO expression was significantly downregulated in trophoblasts of SA patients with aberrant m6A accumulation, which was also correlated with oxidative stress.The expression of genes involved in immunotolerance, immune cell infiltration and angiogenesis at the maternal-fetal interface, including human leukocyte antigen (HLA)-G, vascular endothelial growth factor receptor (VEGFR), and matrix metalloproteinase-2 (MMP-2) bound to YTHDF2 and FTO, is also decreased in patients with SA [3].Moreover, Qin et al. proposed that eukaryotic initiation factor (eIF) 5 A could regulate METTL14 expression via interaction with its promoter, which influences the viability, proliferation, and migration of HTR8 cells; the eIF5A/METTL14 pathway was also downregulated in the villi of SA [30].METTL3/METTL14-catalyzed m6A RNA methylation on lncRNA HZ01(lnc-HZ01) enhanced its RNA stability, and lnc-HZ01 inhibited the proliferation and invasion of HTR-8/SVneo and Swan 71 cells via the MAX dimerization protein 1 (MXD1)/eIF4E pathway, which might be part of the mechanism of organic pollutant-related abortion [41,42].These findings indicate a tight crosstalk among these m6A modulators.
The effect of ALKBH5 on trophoblast function in RSA remains controversial.Zheng et al. reported that ALKBH5 was expressed at low levels in the EVT of RSA [37].Knockdown of ALKBH5 in the mouse placenta suppressed the weight of the fetus and placentas and decreased the area of the placental labyrinth, which significantly led to fetal abortion in vivo [37].ALKBH5 activated the transforming growth factor-b (TGF-b) signaling pathway by promoting the expression and phosphorylation of small mothers against decapentaplegic (SMAD)1/5 by erasing their m6A modification to induce the expression of matrix metalloproteinase (MMP)-9 and integrin subunit alpha 1 (ITGA1), which can consequently enhance the migration and invasion of HTR8/SVneo cells [37].However, Li et al. revealed that global mRNA m6A methylation was significantly decreased and the expression of ALKBH5 was increased in villous tissue from patients with RSA [36].Inhibition of ALKBH5 also significantly promoted the migration and invasive ability of HTR-8/SVneo cells and human villous explants via cysteine-rich angiogenic inducer 61 (CYR61) m6A modification [36].Therefore, these investigations demonstrated the functional significance of ALKBH5 in RSA placenta, as well as in PE placenta.In addition, METTL3 and IGF2BP3 have also been found to be downregulated in the placenta of RSA [31,38].METTL3-mediated m6A modification of the coding sequences (CDS) of zinc finger and BTB domain containing 4 (ZBTB4) regulates its RNA stability and expression, which further affects trophoblast invasion [31].IGF2BP3 is involved in the regulation of inflammatory pathways in trophoblasts, such as the Hippo pathway, interleukin (IL)-4 and 13 inflammatory, tumor necrosis factor (TNF)-a, and nucleotide oligomerization domain (NOD)-like receptor (NLR) signaling pathways in trophocytes [38].Knockdown of IGF2BP3 in HTR8/SVneo cells decreased the expression of IL-10 by activating the nuclear factor jB (NF-jB) pathway, which further promoted M1 macrophage polarization.An imbalance in the ratio of M2/M1 macrophages ultimately induces abortion [38].However, the specific regulatory mechanisms remain to be explored.

Hyperglycemia in pregnancy (HIP)
HIP manifests as gestational diabetes mellitus (GDM) and diabetes mellitus in pregnancy (DIP), also known as pregestational diabetes mellitus (PGDM), and is defined as the existence of type 1 (T1DM) or type 2 diabetes mellitus (T2DM) [48].Approximately 16.7% of live-birth pregnancies are affected by HIP, and HIP is associated with a significantly increased risk of shortand long-term maternal and fetal complications [49].Growing evidence has shown that m6A and regulators are critical for the pathogenesis of HIP.METTL14 was downregulated and m6A levels in total RNA were lower in GDM placentas.The m6A RNA profile revealed that m6A levels in both the 3 0 -UTR and CDS near the stop codons of placental mRNAs were strongly decreased in the GDM group, and the m6A levels of insulin receptor (INSR) and insulin receptor substrate 1 (IRS1), GDM-related genes, were also significantly reduced in GDM, which might be involved in GDM development [25].Linc00667, which may notably contribute to the development of GDM, has been shown to directly bind to YTHDF3 in HTR8/SVneo cells [11].The gene polymorphisms of IGF2BP2 and FTO might be associated with the risk of GDM or might affect some of the clinical parameters for newborns in some populations [26].However, Franzago et al. [27] found no association between placental FTO DNA methylation and GDM.RNA m6A methylation in the placenta with maternal obesity, a condition closely related to diabetes, was also decreased along with reduced gene expression of WTAP [44].However, the specific mRNA targets of m6A modification and their specific mechanisms in the HIP placenta are still not unclear, and further investigations remain to be done.

Fetal growth
m6A deposition in different locations may result in specific regulatory functions.Taniguchi et al. [39] proposed that m6A modification near the stop codon and at the 5 0 -UTR in placental mRNA may play important roles in fetal growth and disease.The m6A levels at the 5 0 -UTR in mRNAs of small for gestational age (SGA) placenta were increased and the m6A levels in the vicinity of stop codons were decreased in large for gestational age (LGA) placenta.By performing m6A-circRNA epitranscriptomic microarray analysis and MeRIP-qPCR assays, circMPP1 showed high m6A modification levels in both LGA and IUGR samples, indicating a possible correlation between circMPP1 and placental dysfunction [10].circMPP1 promotes placental inflammation and dysfunction by activating the NF-jB and signal transducer and activator of transcription (STAT)3 pathways.YTHDC1 can suppress the expression of circMPP1 via m6A modification in JEG-3 cells and inhibit the growth and development of neonatal rats stimulated by circMPP1 knockdown [10].As a potential marker of fetoplacental development, whether circMPP1 could be a new therapeutic target remains to be elucidated.
FTO is highly expressed in the placenta and is associated with increased placental weight, fetal weight and fetal length [28].A 2013 study revealed that reduced FTO gene expression in both rat and human placenta was linked with IUGR [29].The expression of FTO was also decreased in the low birth weight (LBW) placentas of piglets, and m6A levels were increased [4].However, placental FTO expression was not altered in macrosomia in human or pregestational maternal high-fat diet (HFD)-induced overgrowth in rat fetuses.Maternal food restriction reduces FTO expression in the placenta of rats, and maternal HFD reduces FTO expression in the female placentas of mice [29].In the Chinese population, the FTO promoter methylation level at a specific CpG site is negatively associated with birth weight, which might influence fetal intrauterine weight gain by reducing adipocyte lipolytic activity [28].FTO could be involved in the cellular sensing of amino acids through the mechanistic target of the rapamycin (mTOR)C1 pathway, which might regulate fetal growth [50].These studies strongly suggest that FTO-mediated demethylation of m6A may be a vital target for epigenetic transcription, which might be instrumental in the regulation of fetal growth and development, and the exact mechanism still needs to be explored.

Potential clinical application of m6A modification
Some studies have demonstrated that the expression levels of m6A-related miRNAs such as miR-423-5p and miR-181a-5p, and the m6A reader IGF2BP1 were changed in the blood plasma of pregnant women with PE [13,14], indicating that changes in m6A-modified target ncRNAs and m6A modification-related proteins in circulation could serve as biomarkers for the promising noninvasive predictive diagnosis of specific diseases as early as early pregnancy.In addition, based on bioinformatics analysis, the m6A-related module could be used for the diagnosis of specific diseases and regarded as a key target of disease mechanism research [11].The aforementioned studies have explored the various m6A-related mechanisms of some obstetrical syndromes from multiple perspectives and these findings have enriched the pathogenesis of these diseases and indicated a way to develop effective treatment strategies.To date, the therapeutic effects of METTL3 and ALKBH5 knockdown have been observed in PE rats [40,43].Moreover, m6A-associated ncRNAs, as a potential target, can also provide theoretical guidance for clinical treatment.However, the specific mechanism of noncoding RNAs for use in targeted therapy needs to be further confirmed.
In summary, the analysis of m6A modifications could provide multiple potential biotargets or biomarkers for diagnosis and treatment against these trophoblast-related adverse pregnancy outcomes, and more specific mechanisms remain to be further confirmed.

Conclusion
Although m6A modification is currently reported to be associated with the proliferation, invasion, migration, and apoptosis of trophoblastic cells, further studies are needed to elucidate the role of m6A enzymes and the modification of the biological functions of trophoblasts, which further provide a wide range of RNA epigenetic regulatory patterns in both physical and pathological pregnancies.

Table 1 .
Summary of current reported m6A studies in the great obstetrical syndromes.