Background

As a result of the growing interest in understanding how DNA and RNA modifications function, epigenomics has become a cutting-edge field [1]. Scientists have identified numerous layers of epigenetic modulation that are derived from the modification of DNA and proteins; however, RNA modification remains elusive [2]. Over 160 RNA chemical modifications have been discovered thus far [3]. The multitude of RNA modifications has added a new level of complexity to gene regulation. In eukaryotic mRNA, m6A RNA modification is the most abundant modification, which is defined as the methylation of adenosine 6 positions in mRNA and some noncoding RNA. N6-methyladenosine has been proven to be connected with various metabolic and physiologic processes, such as RNA transcript splicing, translation efficiency, nuclear export, stability, and decay [4]. m6A shares the same base pairing with unmodified adenosine, which prevents it from being detected by conventional sequencing or hybridization methods. A mystery surrounded the transcriptome distribution of m6A until a combination of m6A-specific methylated RNA immunoprecipitation and next-generation sequencing was devised. By employing these methods, scientists were able to determine that m6A residues were located in evolutionarily conserved regions within humans and mice. m6A residues were found to be contained primarily within internal mRNA sequences and appeared in the poly A tail, especially in the 3'UTR of mRNAs near the stop codon, and they always share a consensus motif of RRACH (R = A/G, H = A/C/U), and a terminal U is dominant part of the consensus [5,6,7,8].

The process of RNA methylation is controlled by methyltransferases, demethylases, and recognition factors. A methyltransferase and a demethylase catalyse the methylation of RNA dynamically, and the m6A function is determined by its recognition factor. Regulators may also be referred to as ''writers'', ''erasers'', and ''readers''. Although these regulators have been identified, the specific function of this process has not been well established. Cell types and environmental conditions may affect the expression level and biological function of m6A effectors. Furthermore, these factors can also be influenced by RNA species, abundances, secondary structures, intracellular location, translation state, and other heterogeneous factors. Taking these factors into account, it may be difficult to explain the exact function of m6A [9].

The normal physiological functions of the female reproductive system depend on the gonadal axis of the reproductive endocrine system, which regulates ovulation and the menstrual cycle. The maintenance of normal pregnancy depends on the establishment of endometrial receptivity, normal embryogenesis, and the formation of an immune microenvironment at the maternal-foetal interface. Several studies have shown that m6A modification is related to the regulation of physiological functions of the female reproductive system [10]. For example, by controlling the translation of Pgr mRNA through m6A modification, METTL3 is crucial for efficient P4 signaling during embryogenesis. m6A‑RNA immunoprecipitation‑qPCR showed that Pgr mRNA transcript was a target for METTL3-dependent m6A mRNA methylation [11]. In the process of ovarian aging, researchers found that the ovarian aging can be alleviated by up-regulating the FTO level in ovarian granulosa cells to reduce the m6A level of FOS-mRNA-3′UTR [12].However, the subtle mechanism of m6A modification involved in the physiological function of the female reproductive system remains to be elucidated.

Common disorders of the female reproductive system include benign diseases, such as inflammatory diseases of the reproductive organs, infertility, endometriosis, and polycystic ovarian syndrome; complications of pregnancy, such as miscarriage, preeclampsia, and gestational diabetes mellitus; and some malignant diseases, such as cervical cancer, endometrial cancer, and ovarian cancer. In recent years, it has been found that epigenetic abnormalities may be associated with the development of several female reproductive disorders. For example, epigenetic modifications such as DNA methylation, histone methylation and acetylation and long noncoding RNAs are involved in regulating follicular granulosa cell proliferation, endometrial receptivity and decidualization in early pregnancy, which affect ovulation, embryo implantation, and labour initiation [13]. Abnormalities in these epigenetic modifications may be associated with polycystic ovary syndrome, infertility due to repeated embryo implantation failure, and pregnancy complications. Some patients with endometriosis may be born with congenital epigenetic molecular abnormalities, and the recurrent bleeding and repair processes of endometriosis lesions may also trigger acquired epigenetic events [14]. In addition, some chemical toxicants, such as Bisphenol A (BPA), can affect ovarian function, embryonic development, and gamete quality during fertilization through epigenetic modifications such as CpG island methylation, histone modifications, and noncoding RNA production [15].

The function of epigenetic modification in the female reproductive system has received increasing attention over the last few years. Despite this, research on m6A modification and the female reproductive system is still in its infancy. A comprehensive review of recent research advances in m6A modification and its roles in pathogenesis, diagnosis, and molecular targeted therapies for gynaecological reproductive disease is presented in this paper. We discuss the potential directions of m6A modification for future research in obstetrical and gynaecological disorders, aiming to clarify the role played by RNA methylation in female reproductive system diseases and malignant tumours. In doing so, we can gain a novel perspective on these diseases.

The mechanism and regulation of m6A RNA methylation

m6A writers

An N6-adenosine methyltransferase complex (MTC) is required for the installation of this modification (Fig. 1). The MTC comprises the METTL3 catalytic subunit, as well as the METTL14, WTAP, VIRMA, RBM15, and ZC3H13 accessory subunits. A 70 kDa protein named METTL3 was the first known m6A writer [3, 16]. The METTL3 subunit is a key component of the catalytic process, facilitating the reception of methyl groups from the SAM moiety by RNA adenine. METTL14, a molecular homologue of METTL3, was discovered by researchers as a new methyltransferase [17]. When METTL14 and METTL3 combine, a stable heterodimer is formed. It is believed that this complex mediates m6A deposition on nuclear RNA in mammals. A structural requirement for the activation of METTL3 is METTL14. METTL14 acts as an essential component to facilitate RNA recognition and binding. Although METTL14 is not directly involved in catalysis, it stabilizes the conformation of METTL3 to enhance catalytic activity [18, 19]. By decreasing METTL14 expression, methylated RNA binding and the splicing-related protein DGCR8, which plays an important role in maturing RNA, were suppressed [20].

Fig. 1
figure 1

Detailed mechanism of m6A readers, writers, and erasers. N6-methyladenosine is methylated by the methyltransferase complex, which contains METTL3, METTL14, WTAP, KIAA1429, and RBM15/15B. The m6A erasers FTO and ALKBH5 can reversibly remove m6A. The reader is responsible for performing the biological functions of m6A, such as RNA translation, decay, splicing, and translation

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RNA-binding domains in RBM15 and RBM15B enable WTAP–METTL3 to bind to specific mRNAs and determine the methylated m6A consensus sequences. As part of the m6A methylation complex, RBM15/15B binds to XIST, and X-chromosome genes can be silenced by this transcription factor [21]. Researchers have identified that WTAP contains no obvious catalytic domains, but it initiates the localization of METTL3-METTL14 in the nuclear speckle and facilitates catalysis. Both WTAP and METTL3 regulate transcription and RNA processing genes by regulating their expression and alternative splicing. Morpholino-mediated knockdown of WTAP or METTL3 in zebrafish embryos resulted in defects in tissue differentiation and an increase in apoptosis [23]. In HeLa cells, KIAA1429 mediates the preferred deposition of m6A in the 3'UTR near the stop codon and contributes to APA [24]. Furthermore, its homologue, Virilizer, is essential for the alternative splicing of the sex determination factor in Drosophila [25].

Due to METTL16’s role in regulating SAM homeostasis, it has been linked to several modifications in the epitranscriptome of m6A [26]. Scientists have investigated whether METTL16 methylates MAT2A (encoding SAM synthetase) and U6 snRNA [82]. Table 1 summarizes the physiological functions of m6A RNA methylation in the female reproductive system.

Table 1 The physiological function of m6A RNA methylation in female reproductive system
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m6A RNA methylation in pregnancy and female reproductive disorder

Preeclampsia and gestational diabetes mellitus

Research has found that placental trophoblasts of preeclampsia patients had significantly higher levels of m6A, METTL3, and HNRNPC1/C2 [83]. A recent study demonstrated that METTL3 promotes m6A RNA methylation in the placenta of preeclamptic patients, resulting in a change from pre-miRNA 497-5p/145-5p to mature miRNA 497-5p/145-5p [84]. In preeclampsia samples, the levels of several heat shock proteins were elevated, and the peak of m6A was primarily located in the CDS region near the 3'UTR. Researchers have discovered that the m6A readers IGF2BPs can enhance gene expression by stabilizing mRNA when bound to mRNA [140,141,142]. Although research on these chemical modifications is not as intensive as that on m6A, the role of these chemical modifications in female reproductive disorders deserves attention and has great therapeutic potential.

m5C is a conserved and common RNA modification. It is primarily found in eukaryotic tRNAs and rRNAs [143]. In an animal experiment, deletion of the m5C methyltransferase Nsun5 resulted in suppression of ovarian function and embryonic development arrest in mice [144]. In several bioinformatics studies on ovarian and endometrial cancers, m5C-related genes were closely associated with tumour chemotherapy sensitivity and overall survival [145,146,147]. In addition, abnormal m5C modification may be involved in TRDMT1-mediated granulosa cell death, which in turn causes premature ovarian failure [148]. Another study in Drosophila confirmed that YPS promotes the proliferation and differentiation of germinal stem cells in the Drosophila ovary by binding RNA containing m5C modifications [149]. However, the role of m5C in human ovarian function remains to be further investigated.

m1A and m7G modifications are also present in eukaryotic mRNAs [150]. m1A modifications may produce their biological effects by enhancing RNA‒protein interactions or by altering RNA secondary structures [151]. m7G modification is usually catalysed by METTL1 and located at the 5’ caps and internal positions of eukaryotic mRNA [152]. Several studies on endometrial cancer have found that m1A- and m7G-related lncRNAs and miRNAs can be used to construct tumour-related prognostic models and are associated with different immune infiltration phenotypes and drug susceptibility in endometrial cancer [153,154,155,156]. Pseudouridine is the most abundant modified nucleotide in RNA [157]. In a study on ovarian cancer, a pseudouridine synthase, PUS7, was considered a potential diagnostic marker for ovarian cancer [158].

m6A RNA methylation sheds light on the diagnosis and treatment of several diseases

The TNM stage of gastric cancer is associated with METTL14 expression. According to an overall survival analysis, overall survival rates were higher for patients with higher levels of METTL14 expression. Based on multivariate analysis, METTL14 expression levels were associated with improved outcomes [159]. Non-small cell lung carcinoma cells undergo drug resistance and metastasis via diverse pathways when METTL3 increases m6A modification of both YAP and lncRNA MALAT1 [160]. FTO promotes cell proliferation of acute myeloid leukaemia in an m6A-dependent manner. Two inhibitors of FTO have been successfully developed by Huang and colleagues. These inhibitors, FB23 and FB23-2, bind directly to FTO and inhibit its m6A demethylase activity. The inhibitors display significantly improved inhibitory activity on FTO demethylation of m6A-RNA in vitro. Using these inhibitors, researchers inhibited the proliferation of a panel of AML cell lines and primary AML leukaemia stem cells in patient-derived xenotransplantation mice. Therefore, FTO and its analogues may be effective molecular targets for inhibiting leukaemogenesis in leukaemia stem cells [161,162,163]. Previous studies also indicated that YTHDF1 was strongly associated with a poor prognosis for ovarian cancer, breast cancer, and other female reproductive disorders [132, 164, 165]. The low expression of KIAA1429, an m6A methyltransferase, significantly increased dendritic cell infiltration. Researchers have shown that KIAA1429 expression affects immune checkpoint blockade therapeutic efficacy and boosts intratumoral antitumour immunity. Patients with low KIAA1429 expression experienced survival benefits from anti-PD-L1 immunotherapy [166,167,168].

It has been demonstrated that m6A regulators are associated with the prognosis of cervical cancer patients. m6A methylation regulators may be key regulators of PD-L1 expression and immune cell infiltration and may have an important impact on the TIME of cervical cancer [169]. In recent years, the role of m6A modification in PARP resistance in ovarian cancer has attracted the interest of several scientists. In BRCA-mutated ovarian cancer cells, m6A modification of FZD10 mRNA promoted PARP resistance through upregulation of the Wnt/β-catenin pathway [170]. Another study on olaparib resistance in ovarian cancer cells showed that m6A modifications of the olaparib pharmacogene were increased in resistant ovarian cancer cells [171]. These m6A modification sites could be used as potential pharmacoepitranscriptomics markers of drugs. Studies have confirmed that ALKBH5 expression is upregulated in cisplatin-resistant ovarian cancer and that overexpression of the ALKBH5-HOXA10 loop activates the JAK2/STAT3 signalling pathway, leading to chemoresistance in ovarian cancer [172]. In future studies, targeted blockade against the ALKBH5-HOXA10 loop may reverse chemoresistance in ovarian cancer cells. Based on the above studies, it is clear that m6A regulators are closely linked to clinical treatment and diagnosis.

Conclusion

The actions of m6A modification have gradually become apparent in recent years as high-throughput sequencing technologies and highly specific antibodies against m6A have been developed. In the field of m6A RNA methylation, great progress has been made in revealing potential mechanisms of the onset and progression of female reproductive system disorders. Differentially expressed m6A regulator genes are found in a large number of gynaecological cells compared to normal cells and serve as triggers in gynaecological disease progression. Additionally, imbalanced RNA m6A has also been identified in these diseases. To clarify the molecular mechanisms underlying certain refractory obstetrics and gynaecology diseases, such as endometriosis and preeclampsia, abnormal RNA m6A modification is an important research direction.

Various female reproductive cancers are known to be affected by m6A modification and its regulators. Crystal structure data, for example, have provided insight into cancer-associated mutations in METTL14. Mutations in METTL14 that cause an inefficient RNA-binding domain are found in endometrial cancers, and the mutated form of the enzyme shows partially reduced activity. It has also been found that m6A mRNA methylation modulates AKT activity, which is necessary for endometrial cancer proliferation and tumorigenicity [126]. By binding to a locus of the MYC gene, IGF2BP2 enhances the proliferation, metastasis, and aerobic glycolysis of cervical cancer cells [173]. These findings indicate that the role of m6A regulators in female reproductive organs is different based on cell type, therapeutic issues, and pathological conditions.

Clinical diagnostics and therapeutics can also target m6A regulators in disorders of the female reproductive system. The oncogenic mechanism of mRNA m6A modification in gynaecologic malignant tumorigenesis may become a direction for future research. m6A regulators and their upstream and downstream signalling pathways may provide certain methods to elucidate the pathophysiological mechanisms of gynaecologic malignant tumours and establish prognostic models. Modification of pharmacogene mRNAs may alter their pharmacokinetics and pharmacodynamics, thus affecting drug efficacy. In future studies, not only do the pathophysiological mechanisms of female reproductive system diseases in which m6A is involved deserve attention but also the m6A modifications occurring in the pharmacodynamic gene targets corresponding to drugs need to be considered.

For the successful design of tissue- or cell-specific RNA methylation agonists or inhibitors, more multicentre and large-scale studies are needed. Many essential issues must be addressed in a detailed manner. A very limited number of cell and animal models are used in all cited papers, and the results are hence subject to variation. First, although METTL4, FTO, and ALKBH5 are commonly known and well-studied m6A regulatory factors, the role of less studied m6A regulatory factors, such as RBM15/15B and ZC3H13, may only be the tip of the iceberg. Further research is needed on the interactions between RNA modification regulators and their downstream targets in the female reproductive system. Second, the reproductive-endocrine system plays a pivotal role in the progression of gynaecological diseases. Research currently focuses on the influence of m6A on cellular behaviour. The interplay between the hormonal milieu and endocrine system related to m6A modification remains a mystery and should be addressed in the future. Finally, the majority of published studies have concentrated only on the molecular mechanism of m6A modification. A greater focus should be placed on the diagnostic and therapeutic properties of m6A. According to a large body of evidence, m6A modifications are related to clinical phenotypes and prognoses. m6A is involved in the common pathological features shared by benign and malignant diseases of the female reproductive system, and different m6A phenotypes in these diseases may be involved in the transformation between benign and malignant diseases and between different pathological types in the same disease.

Compared with other reviews in this field, our article provides a more comprehensive overview of the relationship between m6A and the physiology and pathology of the female reproductive system. Whether it is germ cell genesis, embryonic development or benign and malignant diseases of the female reproductive system, we searched for appropriate literature to address them. In particular, we briefly outlined other RNA modifications. The connection between these RNA modifications and m6A modifications may become a new direction in future studies. In addition, we look forward to the involvement of m6A modifications in the diagnosis and treatment of female reproductive system diseases. m6A modifications deserve to be noticed for their clinical applications in female reproductive system diseases.

Undoubtedly, the pathogenesis of female reproductive system diseases is quite complex, and changes in the levels of m6A regulators dynamically regulate the level of m6A modifications in the female reproductive system, altering the abundance and function of related mRNAs and proteins and ultimately determining the onset of disease. However, the application of m6A regulators to clinical diagnosis and treatment requires improving the sensitivity and specificity of the relevant biomarkers. RNA m6A modification can likely be used to classify clinical phenotypes of several female reproductive disorders and to predict the mutual transformation of these disorders.