Research progress on m6A RNA methylation modification in human reproduction related diseases

m6A RNA methylation and its regulatory enzymes are ubiquitously present in follicles and early embryos across various developmental stages. Characterized by their dynamic, reversible, and highly responsive nature, these modifications exert post-transcriptional regulatory effects on key reproductive processes, including oocyte maturation, nuclear reprogramming in early embryos, lineage specification, embryo implantation, and pregnancy maintenance [ 61 , 62 ]. m6A RNA methylation plays pivotal roles in oocyte maturation by dynamically regulating maternal mRNA stability and degradation. The METTL3-METTL14 complex deposits m6A RNA methylation marks while ALKBH5/FTO mediate their removal, collectively maintaining precise RNA turnover. ALKBH5-mediated demethylation promotes selective mRNA decay, whereas METTL3 stabilizes crucial transcripts like Intersectin 2 (Itsn2) for proper meiotic spindle formation. FTO ensures chromosomal stability by regulating long interspersed element class 1 (LINE1) RNA. During maternal-to-zygotic transition, m6A RNA methylation marks orchestrate timely maternal mRNA clearance, with YTHDF2 mediating decay of methylated transcripts to enable embryonic development. For instance, m6A RNA methylation is preferentially deposited on maternal decay transcripts to facilitate their post-fertilization degradation, while also marking ZGA-related genes (such as Zscan4 ) to enhance their transcriptional activation. This m6A RNA methylation-demethylation balance governs meiotic progression and developmental competence [ 63 – 66 ]. In healthy individuals, m6A RNA methylation serves as a critical regulator of endometrial function by dynamically modulating gene expression, cellular activities, and hormonal responsiveness. The METTL3-METTL14 dimer plays a pivotal role in establishing endometrial receptivity, the specific timeframe when the endometrium becomes capable of embryo implantation. Notably, METTL3 promotes progesterone receptor (PGR) mRNA translation efficiency through YTHDF1, thereby maintaining proper progesterone signaling essential for endometrial preparation. Furthermore, m6A RNA methylation precisely coordinates the interplay between estrogen (E2) and progesterone (P4) signaling pathways. It governs key genes involved in epithelial morphogenesis and stromal proliferation, with METTL3-mediated m6A RNA methylation stabilizing critical transcripts like Aurora Kinase B (AURKB) to regulate cell cycle progression during endometrial remodeling. Importantly, m6A RNA methylation-dependent stabilization of PGR mRNA ensures sufficient progesterone receptor availability, which is fundamental for appropriate endometrial responsiveness and pregnancy maintenance [ 65 , 67 ]. Given their critical involvement in these mechanisms, m6A RNA methylation marks significantly influence female fertility and pregnancy outcomes, thereby serving as a fundamental determinant of reproductive success. Studies have shown that the occurrence of spontaneous abortion is related to aberrant m6A RNA methylation in trophoblast cells. It was found that compared with normal pregnant women, the expression levels of METTL3, FTO, IGF2BP1 and IGF2BP2 in placental villi tissues of spontaneous abortion women were lower, while the expression level of ALKBH5 was significantly increased [ 68 – 71 ]. Compared with normal early pregnant women, the m6A RNA methylation level in placental villi tissue of recurrent abortion patients was significantly decreased, and the expression level of ALKBH5 was increased. Knockdown of ALKBH5 promoted trophoblast invasion, while overexpression of ALKBH5 inhibited trophoblast invasion. It may be that overexpression of ALKBH5 can prolong the expression of cysteine-rich protein 61 (CYR61) in patients with spontaneous abortion. ALKBH5 suppressed trophoblast invasion by modulating the m6A-dependent stability of CYR61 mRNA. Mechanistically, ALKBH5 knockdown prolonged the half-life of CYR61 mRNA and upregulated its steady-state expression levels, suggesting a potential role for RNA epigenetic regulation in spontaneous abortion pathogenesis [ 69 ]. In addition, METTL3 was significantly down-regulated in trophoblast cells of patients with spontaneous abortion. By recognizing the m6A RNA methylation sequence of Zinc finger and BTB domain containing 4 (ZBTB4) mRNA by recognizing ZBTB4 m6A RNA motifs in the CDS, METTL3 increases the expression of ZBTB4 and enhances its stability, thereby inhibiting the invasion of trophoblast cells [ 70 ]. Future research will focus on the dynamic changes of m6A RNA modification in the maternal endometrium and embryonic tissues and its impact on embryonic development, the association between m6A RNA modification and placental function, such as trophoblast cell invasion, and therapeutic strategies targeting m6A RNA methylation regulatory factors, such as methyltransferase agonists or demethylase inhibitors, which may provide new intervention methods for spontaneous abortion. The proliferation, differentiation and apoptosis of granulosa cells are closely related to various developmental processes such as follicle selection and follicle atresia [ 72 , 73 ]. Abnormal m6A RNA methylation in granulosa cells led to apoptosis, which is associated with POI, PCOS and other diseases that impair ovarian function. POI refers to ovarian dysfunction in women before the age of 40. Previous research has clearly established that the etiology of POI is multifactorial, including genetic, chromosomal, autoimmune causes, environmental toxins, and chemicals [ 74 , 75 ]. Recent studies have found that the content of m6A RNA methylation in ovarian tissue of POI patients was significantly higher than that of normal women, indicating a certain correlation between m6A RNA methylation and POI. Aberrant m6A RNA methylation in granulosa cells affect apoptosis and proliferation and impair ovarian function, and further lead to POI [ 76 ]. These findings indicate that downregulated FTO expression at both the protein and mRNA levels may contribute to elevated m6A RNA methylation in POI, potentially exacerbating the risk of POI-related complications. Given these observations, m6A RNA hypermethylation warrants further investigation as a promising novel biomarker for POI [ 76 ]. The study demonstrated that cyclophosphamide (CTX), a widely used anticancer drug, induced m6A RNA methylation in a time- and dose-dependent manner. CTX upregulated the gene and protein expression of METTL3, METTL14, ZC3H13, and KIAA1429 in a concentration-dependent manner both in vitro and in vivo. However, prolonged CTX treatment suppressed their expression in a time-dependent manner. In contrast, CTX had no significant effect on the expression of the RNA methyltransferases WTAP and RBM15. This discrepancy may be attributed to the fact that WTAP and RBM15 function as regulatory components of the m6A RNA methylation complex, which requires METTL3 and METTL14 for their activity. Given the established association between CTX and reproductive impairment, including POI, its effects on m6A RNA regulatory enzymes in ovarian granulosa cells were further investigated. These findings suggest that CTX may impair ovarian function through dysregulation of m6A RNA methylation machinery [ 77 ]. In addition, the study found that the expression level of FTO protein and mRNA in ovarian tissue of POI patients was significantly lower than that of normal women. The decreased expression of FTO may be the reason for the increased m6A RNA methylation level in POI patients. FTO can delay the senescence of FTO-dependent ovarian granulosa cells by down-regulating the m6A RNA methylation, and reveal potential diagnostic and therapeutic targets for ovarian senescence and age-related reproductive diseases. Reactive oxygen species (ROS) can inhibit the expression of FTO, increase the m6A RNA methylation, and accelerate ovarian aging [ 78 ]. Therefore, reducing the accumulation of ROS may restore the expression of FTO and improve ovarian function. In addition, regulating the level of m6A RNA modification (such as using FTO activators or METTL16 inhibitors) may delay ovarian aging. The heterogeneity of ovarian aging (such as genetic and environmental factors) needs to be combined with multi-omics analysis, which cannot be ignored either. PCOS is the most common reproductive endocrine and metabolic disorder in women of childbearing age, and ovarian granulosa cell dysfunction is closely related to the incidence of PCOS. Research indicates that insulin resistance, cardiovascular diseases, abdominal obesity, psychological disorders, infertility, and cancer are all associated with PCOS, and these PCOS-related pathophysiological mechanisms are interrelated with each other [ 79 – 81 ]. Besides, the study found that m6A RNA methylation was significantly higher in luteinized granulosa cells in non-obese PCOS patients compared to normal women. The forkhead box O3 (FOXO3) gene plays key roles in cell apoptosis, metabolism and proliferation, and m6A RNA methylation is involved in regulating the stability of FOXO3 mRNA. In luteinized granular cells of non-obese PCOS patients after controlled ovarian hyperstimulation, m6A RNA methylation can induce up-regulation of FOXO3 mRNA expression, which may regulate FOXO3 mRNA transcription through YTHDF2-mediated RNA decay pathway. The above studies suggest that m6A RNA modification may serve as an important molecular bridge connecting metabolic abnormalities of PCOS and reproductive dysfunction [ 82 – 84 ]. The study also confirmed that FTO is associated with the risk of PCOS and insulin resistance. Overexpression of FTO can promote granulosa cell proliferation, inhibit cell apoptosis, and induce insulin resistance. The possible mechanism is that FTO promotes flotillin 2 (FLOT2) expression by reducing the m6A RNA methylation level of FLOT2 mRNA and increasing the stability of FLOT2 mRNA, thus inducing granulosa cell dysfunction. It is suggested that FTO/FLOT2 may be involved in the pathophysiological process of PCOS [ 85 ]. There are still many questions to be explored regarding the role of m6A RNA methylation in PCOS. Investigating the interplay between m6A RNA methylation and other epigenetic modifications, such as DNA methylation and histone modifications, may elucidate how these mechanisms collectively regulate the pathological processes of PCOS. And clinical translational research on how to transform the regulatory mechanism of m6A RNA methylation into precise diagnosis and treatment strategies for PCOS. EMs is a complex syndrome characterized by an estrogen-dependent chronic inflammatory process that primarily affects pelvic tissues, including the ovaries. This condition arises when endometrial tissue is shed and transported retrogradely into the lower abdominal cavity [ 86 ]. EMs is one of the important factors leading to infertility and miscarriage. Numerous theories, including coelomic metaplasia, implantation, and embryonic stem cell involvement, have been proposed to explain the pathophysiology of EMs. However, the precise etiology of the disease remains unknown [ 87 , 88 ]. Bioinformatic analysis revealed a decreased overall level of m6A RNA modification in ectopic endometrial tissues from EMs patients, along with an upregulation of FTO expression. By reducing the m6A RNA methylation level of GEF-H1 (a Rho guanine nucleotide exchange factor) mRNA, it promotes the migration and invasion of endometrial stromal cells and increases the risk of miscarriage. Inhibiting the activity of FTO or using its inhibitors (such as Rhein) can reduce the formation of ectopic lesions, providing a new target for the treatment of EMs-related miscarriage [ 89 ]. This overall hypomethylation state may be related to the abnormal estrogen and inflammatory microenvironment in EMs, as in vitro experiments have demonstrated that estrogen (E2) and inflammatory factors (IL-1β, LPS) can reduce the level of m6A RNA methylation in human endometrial stromal cells [ 89 ]. Moreover, the expression of lncRNA UBOX antisense RNA 1 (UBOX5-AS1) , which contains m6A RNA modification, was strongly and positively correlated with the expression of the demethylase ALKBH5 and autophagy levels. Functional analyses demonstrated that elevated ALKBH5 and UBOX5-AS1 expression enhanced autophagy, proliferation, and invasion in endometriosis cells in vitro. Furthermore, UBOX5-AS1 was found to mediate ALKBH5-dependent regulation of autophagy, proliferation, and invasive capacity. ALKBH5-induced autophagy was shown to promote cell proliferation, migration, and invasion. Mechanistically, ALKBH5 facilitates m6A RNA demethylation of UBOX5-AS1 , thereby upregulating its expression. Collectively, these findings suggest that the ALKBH5/UBOX5-AS1 axis may represent a promising therapeutic target for ovarian EMs in future clinical applications [ 90 , 91 ]. The METTL3/m6A RNA/microRNA-126 (miR-126) pathway plays an important role in the migration and invasion of endometrial stromal cells. That is, the down-regulation of METTL3 reduces the m6A RNA methylation level of pri-miR-126 and weakens the maturation of pri-miR-126 to miR-126. Thereby enhancing the migration and invasion of endometrial stromal cells [ 92 ]. Another study has shown that FTO/autophagy-related protein 5 (ATG5)/M2 recombinant pyruvate kinase isozymes M2. The key role of the pyruvate kinase M2 (PKM2) pathway in EMs is that the down-regulation of FTO reduces the expression of ATG5 in an m6A RNA methylation dependent manner and increases the expression of PKM2, thereby enhancing the glycolysis level of cells. It has been demonstrated that targeting the ATG5/PKM2 metabolic pathway through FTO can inhibit the glycolysis, proliferation and metastasis of endometriotic stromal cells [ 93 ]. In addition, studies have reported that compared with normal endometrium and ectopic endometrium, most m6A RNA methylation modified regulatory factors (METTL3, YTHDF2, YTHDF3, HNRNPA2B1, HNRNPC, and FTO) in ectopic samples are significantly downregulated. HNRNPA2B1 and HNRNPC may play regulatory roles in immune pathways and modulate immune cell infiltration in EMs. Dysregulation within the gene transcription factor network associated with EMs pathogenesis could potentially alter the expression patterns of HNRNPA2B1 and HNRNPC. These molecular alterations may serve as promising diagnostic biomarkers for EMs detection and clinical management [ 94 ]. In conclusion, m6A RNA methylation in ectopic and eutopic endometrial tissues of EMs patients are significantly lower than those in normal tissues, suggesting that the abnormal activation of m6A RNA methylation demethylases (such as FTO, ALKBH5) may be one of the disease characteristics. This also provides new strategies for targeted therapy. For example, the combination of m6A RNA methylation regulators and hormone therapy (such as progesterone) may overcome hormone resistance problems. Furthermore, in terms of prognosis assessment, the high expression of FTO or ALKBH5 may be associated with the risk of postoperative recurrence and can be used as monitoring indicators.

m6A RNA methylation-associated enzymes can be classified into three types [ 10 , 11 ]. Methyltransferases, namely “writers”, include methyltransferase-like protein 3 (METTL3), METTL14, METTL16, Wilms’ tumour 1-associating protein (WTAP), vir-like m6A methyltransferase-associated protein (VIRMA, also known as KIAA1429), zinc finger CCCH-type containing 13 (ZC3H13), RNA-binding motif protein 15 (RBM15) and RBM15B [ 12 ]. m6A RNA writers catalyze the methylation of RNA at the N6 position of adenine, targeting the amino group on the nucleobase. The writers complex adds methyl groups to specific adenosine residues within the RRACH motif of mRNA and non-coding RNAs (R represents A or G; H represents A, C or U). Initially characterized as a S-adenosyl methionine (SAM)-binding protein, METTL3 establishes a stable heterodimer with METTL14, despite both proteins possessing methyltransferase domains [ 10 , 11 ]. Besides, Methyltransferases can be broadly classified into two categories: “bona fide” methyltransferases, which directly methylate RNA, and “scaffolding” proteins, which facilitate complex formation and substrate specificity but do not methylate. Among these, METTL3, METTL14, and METTL16 are bona fide methyltransferases, whereas WTAP, VIRMA, ZC3H13, RBM15, and RBM15B function as scaffolding proteins that stabilize and organize the methyltransferase machinery [ 13 – 15 ]. Demethylases, also referred to as “erasers”, composed of ALKB homolog 5 (ALKBH5) and fat mass and obesity-associated protein (FTO), which can mediate methylation reversal by removing m6A RNA methylation. The discovery of FTO confirms that m6A RNA methylation modification is a dynamic and reversible process [ 6 ]. The binding proteins, also known as “readers”, are composed of YTH domain family proteins (YTHDF1-3), YTH domain containing proteins (YTHDC1 and YTHDC2), heterogeneous nuclear ribonucleoproteins (such as hnRNPA2B1, hnRNPC, and hnRNPG), eukaryotic translation initiation factor 3 (eIF3), proline rich coiled-coil 2 A (PRRC2A), and insulin-like growth factor 2 binding proteins (IGF2BP1-3). The binding proteins can recognize m6A RNA methylation and regulate mRNA function [ 16 – 21 ]. Upon methylation of adenosine, a diverse array of m6A-binding proteins is recruited, which subsequently regulate the fate of target RNAs by modulating processes such as splicing, stability, translation, nuclear export, and secondary structure formation [ 22 – 27 ]. For example, m6A-modified RNAs undergo accelerated deadenylation, a process mediated by the CCR4-NOT deadenylase complex. The CCR4–NOT complex is a nine-subunit complex containing two deadenylase subunits, CAF1 (or its paralogue POP2) and CCR4A (or its paralogue CCR4B). This regulation occurs through YTHDF2 recruitment of the CCR4-NOT complex via direct interaction between the N-terminal domain of YTHDF2 and the superfamily homology (SH) domain of the CNOT1 subunit [ 28 ]. YTHDC1 facilitates exon inclusion in target mRNAs by recruiting the pre-mRNA splicing factor serine/arginine rich splicing factor 3 (SRSF3) while competitively inhibiting serine/arginine rich splicing factor 10 (SRSF10) mRNA binding. Specifically, YTHDC1 modulates mRNA splicing by orchestrating the recruitment and spatial positioning of pre-mRNA splicing factors, thereby regulating their accessibility to binding motifs on target transcripts [ 29 ]. Notably, whereas METTL3 and METTL14 methylate a broad range of transcripts, METTL16 requires a specific secondary structure for the methylation of its RNA targets [ 30 – 32 ]. As a single subunit methyltransferase, METTL16 primarily methylates targets including the U6 small nuclear RNA (U6 snRNA) and the methionine adenosyltransferase 2 A (MAT2A) mRNA hairpins, while it is also known to bind to the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) long non-coding RNA (lncRNA) [ 33 , 34 ]. To summarize, m6A RNA methylation is dynamically reversible and relies on a series of enzymes. Multiple binding proteins work together to regulate the complex and important physiological functions of m6A RNA methylation, while abnormalities in a certain regulatory factor are closely related to human reproductive diseases (Fig.  1 ). Fig. 1 m6A RNA methylation genes involved in the human reproduction-related diseases m6A RNA methylation genes involved in the human reproduction-related diseases

Given its widespread regulatory functions in both male and female reproductive system, m6A RNA methylation holds significant promise as a biomarker for early diagnosis, a therapeutic target for reproductive disorders, and a prognostic indicator for clinical outcomes (Table  2 ). However, the field remains in its infancy, hindered by technical and ethical hurdles that currently limit research largely to animal models. Addressing these gaps necessitates a shift toward human tissue-based analyses and innovative in vitro human models, like those employing embryonic stem cell-derived germ cells, to decipher disease mechanisms, identify biomarkers, and forge targeted therapeutic strategies. Table 2 Roles of m6A RNA methylation in human reproduction-related diseases Disease Effect and regulatory mechanism Refs. Asthenozoospermia The expression of METTL3 and METTL14 were significantly higher. Increased m6A RNA methylation content is a risk factor [ 45 , 46 ] Azoospermia two missense variations of FTO have harmful effects on the function of FTO. The overall m6A RNA methylation levels of METTL3, ALKBH5, and YTHDF3 were obviously dysregulated [ 8 , 50 , 51 ] URPL Impairing RNA stability and translation, ultimately disrupting key signaling pathways essential for ZGA [ 60 ] Poor-quality Semen ALKBH5 and FTO gene mutations were strongly associated with changes in semen quality. m6A RNA methylation level decreased, reducing testosterone synthesis and secretion [ 50 , 54 , 55 ] Spontaneous Abortion Knockdown of Alkbh5 promoted trophoblast invasion. METTL3 increases the expression of ZBTB4 and enhances its stability [ 69 , 70 ] POI Aberrant m6A RNA methylation level in granulosa cells affect apoptosis and proliferation and impair ovarian function. CTX can significantly inhibit the expression of demethylase FTO. ROS can inhibit the expression of FTO [ 76 – 78 ] PCOS m6A RNA methylation is involved in regulating the stability of FOXO3 mRNA. FTO promotes FLOT2 expression by reducing the m6A RNA methylation level of FLOT2 mRNA [ 82 , 85 ] EMs Reduced m6A methylation promotes the migration and invasion of endometrial stromal cells. Targeting the ATG5/PKM2 metabolic axis via FTO suppression effectively inhibits glycolysis, proliferation, and metastasis in endometriotic stromal cells [ 89 , 93 ] Roles of m6A RNA methylation in human reproduction-related diseases Several m6A RNA methylation inhibitors such as STC-15 and Bisantrene are currently under clinical trials, for instance, in tumor therapy. Although targeting m6A RNA methylation holds promise for developing highly specific therapeutics, concerns remain regarding potential off-target effects from germline editing of regulators like ALKBH5. Furthermore, the impacts of established m6A inhibitors (e.g., 3-deazaadenosine) on primordial germ cells are poorly understood. To address these challenges, the establishment of humanized organotypic toxicity assessment platforms is necessary. A comprehensive, multi-scale research approach that integrates molecular, cellular, and organismal levels is thus imperative to fully decipher the molecular pathogenesis of reproductive disorders and facilitate their translation into effective clinical strategies for disease prevention, early diagnosis, and therapy.

The incidence of infertility has increased steadily in recent years, which has become an urgent problem to cope with the challenge of population aging in the world. Reproductive diseases are the important reasons for the decrease of fertility. Epigenetic modification plays important roles in the regulation of various physiological and pathological processes, among which m6A RNA methylation is the most popular RNA modification in eukaryotes, including mammals, plants, yeast, fruit flies and other eukaryotic, and viral RNAs [ 1 – 4 ]. The m6A RNA methylation relies on a series of enzymes, including methyltransferases, demethylases, and the reader proteins [ 5 , 6 ]. As a dynamic and reversible methylation modification, dysregulated m6A RNA methylation has been shown to be linked to disorders of human male infertility, including asthenozoospermia, azoospermia, and abnormal semen [ 7 , 8 ]. In addition, m6A RNA methylation is also critical for female reproductive health, and its aberration can lead to infertility such as polycystic ovary syndrome (PCOS), premature ovarian insufficiency (POI), spontaneous abortion, and endometriosis (EMs) [ 9 ]. In this review, the mechanism of m6A RNA methylation is briefly reviewed, with emphasis on the research progress in human reproduction related diseases, providing a new direction for disease prevention, diagnosis, and treatment.