{"paper_id":"52d9ee8f-a082-41d7-852e-8f784b1932d3","body_text":"Int. J. Biol. Sci. 2022, Vol. 18 \n \n \nhttps://www.ijbs.com \n3592 \nInternational Journal of Biological Sciences \n2022; 18(9): 3592-3604. doi: 10.7150/ijbs.69771 \nReview \nRole of m6A modification in female infertility and \nreproductive system diseases  \nJinyu Chen, Yiwei Fang, Ying Xu, Haotong Sun \nInstitute of Reproductive Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, P.R. China.  \n Corresponding author: Yiwei Fang, evayfang@163.com \n© The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). \nSee http://ivyspring.com/terms for full terms and conditions. \nReceived: 2021.12.06; Accepted: 2022.04.22; Published: 2022.05.16 \nAbstract \nGamete abnormalities and reproductive system tumors have become a dominant cause of infertility, \ntroubling people globally. In recent years, increasing evidence emerged and found that \nN6-methyladenosine (m6A) played a leading role in reproduction. The biological effects of m6A \nmodification are dynamically and reversibly regulated by methyltransferases (writers), WTAP, METTL3, \nMETTL14 and KIAA1429, demethylases (erasers), FTO and ALKBH5, and m6A binding proteins \n(readers), including YTH domain. In this review, we highlight the change of m6A modification in abnormal \noogenesis, female reproductive system diseases including reproductive system tumors, adenomyosis, \nendometriosis, premature ovarian failure and polycystic ovary syndrome. Moreover, we review some of \nthe mechanisms and the specific modified genes that have been identified. Especially, with the underlying \nmechanisms being uncovered, m6A and its protein machineries are expected to be the markers and \ntargets for the diagnosis and treatment of female reproductive dysfunction. \nKey words: RNA modification; N6-methyladenosine; Female reproductive diseases; Infertility; Reproductive system neoplasms \nIntroduction \nRecent studies have revealed that epigenetic \nmodification of diseases has emerged as an important \nregulator of a variety of physiological processes and \ndisease progression, attracting accumulating attention \nin bioscience research. Epigenetic processes, including \nDNA methylation, histone modifications, chromatin \nrearrangement, and RNA modifications, play crucial \nroles in the regulation of many physiological and \npathological processes, such as embryonic develop-\nment [1], nervous system development [2], and \ntumorigenesis [3]. Among them, RNA modification \ncomes into public view in recent years. There are \nnumerous types of RNA modifications, of which more \nthan 160 have been discovered up to now [4]. Studies \nhave widely reported certain types of RNA \nmodifications in eukaryotic mRNA, including m6A, \nN1-methyladenosine, and 5methylcytosine. m6A is \nthe most abundant internal modification of RNA in \nthe majority of eukaryotes. Since the pioneering \nresearch in the 1970s [5], with the identification of \nmore m6A-related enzymes, the important biological \nfunctions played by m6A modification have been \ngradually revealed around about half a century later. \nBesides, the rapid development of m6A detection \ntechnology pushes m6A research to a new height. \nCalled m6A iCLIP (miCLIP), an individual-nucleotide \nresolution UV crosslinking and immunoprecipitation \n(iCLIP)-based method was born in 2015, which allows \nthe transcriptome-wide mapping of individual m6A \nresidues at single-nucleotide resolution [6]. The \nadvance in miCLIP2 results in high-complexity \nmiCLIP2 libraries using less input material at less \neffort [7]. Encouragingly, single-base extension and \nlinkage qPCR amplification technology could greatly \nshorten the detection time of m6A level, and could \nuncover specific m6A modified genes and their \ntargets, paving the way for the possibility of m6A as a \ndiagnostic method. More recently, the technique of \nSELECT-m6A modified quantitative detection is \ngradually mature [8].This technological advance \nopens up the possibility of m6A being involved in the \nstudy and diagnosis of diseases. \n \nIvyspring  \nInternational Publisher \n\nInt. J. Biol. Sci. 2022, Vol. 18 \n \n \nhttps://www.ijbs.com \n3593 \nThe formation of m6A is a dynamic and \nreversible process [9], m6A “writers” with \nmethyltransferase activity are consisted of three \nindividual proteins: methyltransferase-like (METTL) \n-3, METTL14, Wilms-tumor associating protein \n(WTAP), Vir-like m6A methyltransferase-associated \n(VIRMA; also known as KIAA1429) [10]. The \nmethyltransferase complex catalyzed m6A modifi-\ncation by METTL3 and METTL14 and a regulatory \nsubunit WTAP. METTL3 was first shown to be m6A \nmethylation transferase, whose expression can direct \neffect the total methylation level of m6A, which has \neffects on mRNA stability, leading to dysregulated \ncellular functions [11]. METTL14 forms a stable \ncomplex with METTL3 and plays a key role in \nsubstrate recognition [12]. WTAP regulates \ntranscription and translation of niche factors by \ndepositing the m6A marks directly on transcripts \nencoding the niche factors or indirectly on \ntranscription [13]. Obesity-associated protein (FTO) \nand alkB homolog 5 (ALKBH5), m6A demethylases, \nare able to mediate that methylation reversal through \ngetting rid of the m6A modification [14]. Another \nprotein machineries functioned as m6A “readers,” \nincluding YTH domain family proteins (YTHDFs) and \nYTH domain-containing proteins 1-2 (YTHDCs), the \ninsulin-like growth factor 2 mRNA binding proteins \n(IGF2BPs) [15], heterogeneous nuclear ribonucleo-\nprotein A2B1 (HNRNPA2B1) [16], and eukaryotic \ninitiation factor 3 (eIF3) [17], which can recognize \nm6A modification to modulate mRNA fate[18]. For \nexample, YTHDF1 promotes the translation of m6A \nmodified mRNA, while YTHDF2 lowers mRNA \nstability, induces mRNA degradation, and mediates \nmRNA subcellular localization and selective splicing. \n(Figure 1) The other types of m6A protein \nmachineries have been introduced in detail in a large \nnumber of reviews [19, 20]. \nStudies have reviewed the functions and roles of \nm6A protein machineries in diverse diseases, such as \nacute myeloid leukemia, glioblastoma, lung cancer, \nliver cancer [21], nonalcoholic fatty liver disease [22], \nazoospermia [23], heart failure [24]. m6A modification \nalso plays an important role in eukaryotes [25] and \ncell proliferation and differentiation [26]. Recently, \nstudies have revealed the role of m6A modification \nand its protein machineries in oogenesis and female \nreproductive tumors and other female reproductive \ndiseases. In oogenesis, the lack of YTHDF2 leads to \nthe failure of m6A modified mRNA degradation, \nwhich affects the quality of oocytes [27]. In \nreproductive tumors, METTL3 is upregulated in \novarian cancer (OC) [28]. \nThe incidence and prevalence of infertility and \nfemale reproductive system tumors increase steadily \nworldwide and have become a prevalent worldwide \nproblem in recent decades. Among them, abnormal \noogenesis in infertility accounted for a large \nproportion. So far, there is no better treatment for \ninfertility caused by abnormal gametes. For female \nreproductive system tumors, targeted drug therapy is \none of the important treatments, but there may be \nadverse consequences, as well as drug resistance. \nTherefore, the exploration for better treatment is \nurgent. Other diseases that severely impair female \nhealth and may cause infertility are endometriosis, \npremature ovarian failure, polycystic ovarian \nsyndrome (PCOS) and adenomyosis. Until now, the \npathogenesis of endometriosis and PCOS is still not \nwell understood, and there is no effective treatment. \nTherefore, greater insights into the mechanisms \nregulating spermatogenesis and male genital system \ntumors will help us found novel molecular targets to \ndevelop more effective treatment strategies for these \ndiseases.  \n \nTable 1. Roles of m6A protein machineries and biological mechanisms exerted in oogenesis.  \nType Regulator Role Mechanism Reference \nWriters METTL3 METTL3 loss caused failed mature gametes and impaired fertility Interrupted expression of genes important for sex hormone \nsynthesis and gonadotropin signaling pathway \n[45] \n METTL14 Reduced METTL14 enhanced the ability of meiosis maturation and \ndevelopment of porcine oocytes. \n/ [46] \n KIAA1429 KIAA1429-deficient germinal vesicle oocytes displayed abnormal \napoptosis and proliferation of granulosa cells \nThe alternative splicing of genes associated with oogenesis is \naffected. \n[47] \nReaders YTHDC1 YTHDC1 deficient oocytes are impeded at the primary follicular \nstage. \nA large number of alternative splicing deficiency in oocytes [48] \n YTHDC2 Adult female mice with YTHDC2 gene knockout were infertile YTHDC2 suppressed expression of the meiotic markers and \naffected the percent of FGCs at zygotene \n[50] \n YTHDF2/\n3 \nDouble mutation of YTHDF2 and YTHDF3 resulted in impaired \nfemale gonad development \nFailure of m6A modified mRNA degradation [52] \nErasers FTO The decrease of FTO mRNA and protein expression caused high \nrisk POI \n/ [53] \nFGCs: female germ cells; POI: premature ovarian insufficiency; \n\nInt. J. Biol. Sci. 2022, Vol. 18 \n \n \nhttps://www.ijbs.com \n3594 \n \nFigure 1. m6A modification is regulated by 3 components. m6A modification is added by “writers”, such as METTL3, METTL14, KIAA1429, WTAP. m6A could be \nreversibly removed by “erasers” (FTO and ALKBH5) or recognized by m6A binding proteins (“readers”, such as YTHDC1/2, YTHDF1/2/3 and IGF2BP1) to influence RNA \nsplicing and degradation. \n \nSo far, few reviews have addressed m6A in \nrelation to female reproductive health. Hence, we \nsummarize and focus on the role of m6A modification \nand its protein machineries in oogenesis and female \nreproductive system diseases including tumors and \nPCOS and so on. Moreover, we also review some of \nthe revealed mechanisms and specific genes modified \nby m6A, desiring to explore the possibility that some \nm6A target sites could be used to diagnose and treat \nreproductive disorders.  \nMechanisms of m6A Protein Machinery \nAs the most common and extensive base \nmodification method at the RNA level, m6A \nmethylation profoundly influences all aspects of \nmRNA-associated processes. m6A modification is \naffected by m6A protein machinery. So m6A protein \nmachinery can influence mRNA-associated process, \nincluding alternative splicing, nuclear export, \ntranslation, and stability.  \nm6A in mRNA splicing \nIn the term of alternative splicing, m6A \nmodification regulates gene expression by interfering \nwith this process. m6A methylation that directly \ninfluences splicing is usually located near exonic or \nintronic splice junctions, matching its function. \nMETTL3 dependent m6A modification has little effect \non alternative splicing. Instead, m6A-regulated \nsplicing is rapid and dynamic in changing \nenvironments and under pathological conditions. It \nonly occurs under specific circumstances, rather than \nfunctioning as a wide-ranging regulatory event that \npersists under normal physiological conditions [29]. \nBut another writer METTL16 rapidly induces the \nsplicing of the intron of MAT2A, encoding a SAM \nsynthetase, and maintains low levels of intracellular \nSAM [30]. \nm6A in mRNA nucleation \nAfter alternative spicing, mature mRNA enters \nthe cytoplasm from the nucleus for translation. m6A \nmodification is also involved in this process; in \nessence, this kind of regulation utilizes the formation \nof steric resistance to ultimately target translation. \nm6A reader YTHDC1 is involved in the process of \nmRNA nuclear export. The methylated mRNA is \nrecognized by the nuclear protein YTHDC1 and \ndelivered to the nuclear mRNA export receptor NXF1 \nvia interactions with the splicing factor and nuclear \nexport adaptor protein SRSF3 [31]. \nm6A in mRNA translation \nm6A modification can improve the translation \nefficiency through the binding of reader proteins to \nprotein factors required in the translation process, and \nm6A modifications located in different RNA regions \nexert effects by various modes of action. METTL3 \n\n\nInt. J. Biol. Sci. 2022, Vol. 18 \n \n \nhttps://www.ijbs.com \n3595 \npromotes translation by identifying 5′ UTR m6A and \n3′ UTR m6A [32]. Another model shows that METTL3 \nbinds to eIF3, which interacts with mRNA \ncap-associated proteins, resulting in the formation of \nan mRNA loop. However, direct METTL3 tethering \ncan promote translation only when bound to the 3′ \nUTR at a position near the stop codon [33]. \nYTHDF1/2/3 are all reported to enhance translation, \nbut the mechanism of YTHDF2 is still not clear \n[34-36]. Via interactions with the translation \nelongation factor eEF2, YTHDF1 mediates the CDS \nm6A-enhanced translation elongation of Snail mRNA, \nalthough a previous study indicated that it also binds \nto eIF3 in the 3′ UTR [34]. YTHDF3 significantly \npromotes the binding of eIF3a to m6A residues within \nthe 5′ UTR of YTHDF3 mRNA to enhance \ncap-independent translation in breast cancer brain \nmetastases [36]. \nm6A and mRNA stability \nm6A protein machinery is also essential for \nmaintaining the stability of mRNA. Through different \nmolecular mechanisms, m6A-containing transcripts \ncan mediate RNA decay, which is induced primarily \nby m6A readers. YTHDF2-bound m6A mRNAs are \ndegraded by at least two pathways. First, when a \nheat-responsive protein (HRSP)12-binding site and an \nRNase P/MRP (endoribonucleases)-directed cleavage \nsite exist upstream and downstream of the \nYTHDF2-binding site, respectively, HRSP12 functions \nas an adaptor to bridge YTHDF2 and RNase P or \nMRP, eliciting the rapid degradation of YTHDF2- \nbound RNAs by an endoribonucleolytic cleavage \npathway[37]. Second, via exosomes (3′-to-5′ \nexoribonuclease complex) and P bodies where the \ndecapping complex and 5′-to-3′ exoribonuclease \n(XRN1) are enriched, YTHDF2 directly recruits the \nCCR4/NOT deadenylase complex to trigger \ndeadenylation and subsequently initiates the \ndegradation of m6A-containing mRNA [38-40]. \nInterestingly, IGF2BPs get the opposite, they can \nmaintain the stability of mRNA. IGF2BPs stabilize \nmRNAs by binding to RNA stabilizers, such as HuR, \nmatrin 3 (MATR3), and poly(A)-binding protein \ncytoplasmic 1 (PABPC1) [15]. \nCurrently, studies on the regulatory mechanism \nof m6A protein machinery are not complete, and \nsubsequent studies need to study the specific \nmechanisms and find more RNA-binding proteins. \nOogenesis and female infertility  \nBeginning during fetal life, mammalian \noogenesis is completed after puberty [41]. In the \nembryonic ovary, the oogonia change abruptly from \nsuccessive mitotic divisions into meiosis and become \narrested at MPI (meiotic prophase I) stage. Enclosed \nby pregranulosa cells, the early oocytes form the \nprimordial follicles. After puberty, the oocytes resume \nmeiosis to finish the first meiotic division. The follicles \nkeep growing in size and putting on extra continuous \nlayers of granulosa cells around them, but only the \ndominant follicle is chosen to produce the mature egg \nfor ovulation. Then the eggs become arrested in \nmeiotic metaphase II (MII) until fertilization [42]. In \nthe last few years, studies have shown that the m6A \nmodifications are essential for oogenesis. The proof \ncomes from that there was significant enrichment of \ndifferentially expressed m6A methylated genes in \nseveral signaling pathways associated with \nsteroidogenesis, granulosa cell proliferation and \nfollicular development [43]. Recent studies have \nconfirmed that m6A protein machineries are also \ninvolved in ovulation, including METTL3, METTL14, \nYTHDC1, YTHDC 2, YTHDF1, YTHDF2, YTHDF3 \nand KIAA1429 (Table 1). \nThe loss of METTL3 leads to failed mature \ngametes and impaired fertility, possibly as a result of \nm6A downregulation and interrupted expression of \ngenes important for sex hormone synthesis and \ngonadotropin signaling pathway (e.g. npr, igf3, star, \n3βhsd, and cyp19a1a) [44]. Furthermore, the sex \nsteroids 11-ketone testosterone and estradiol have \nsignificant regulatory effects on germ cells to promote \ngametogenesis and gamete maturation [45]. The \nmRNA levels of METTL14 in L-ascorbic acid treated \nporcine oocytes were significantly reduced, which \nenhanced the ability of meiosis maturation and \ndevelopment of porcine oocytes [46]. This result \nsuggests that METTL14 may also play an important \nrole in ovulation. The newly discovered KIAA1429 is \na member of the family of m6A writers. \nKIAA1429-deficient germinal vesicle oocytes \ndisplayed abnormal apoptosis and proliferation of \ngranulosa cells, as well as abnormal chromatin \nconfiguration and RNA metabolism [47]. According \nto the above evidence, m6A writers plays an \nimportant role in oogenesis, but whether they can be \nused as a target for treating abnormal ovulation \nremains to be further studied. \nVarious studies have shown that ovulation \ncannot occur without YTH-domain including \nYTHDC1/2 and YTHDF1/2/3.[48-52]. The knockout \nof YTHDC1 leads to extensive selective polyadenyla-\ntion in oocytes, changes the length of 3’ Untranslated \nRegion (3'-UTR), and eventually causes a lot of \nalternative splicing deficiency in oocytes, which \nhinders the development of oocytes and leads to the \nlack of secondary follicles or antral follicles in ovaries \n[48]. \n\nInt. J. Biol. Sci. 2022, Vol. 18 \n \n \nhttps://www.ijbs.com \n3596 \nTable 2. Roles of m6A protein machineries and biological mechanisms exerted in female reproductive system tumor. \nCancers Regulator Role in cancer  Mechanism Functional classification Reference \nOvarian \ncancer \nMETTL3 Oncogene Through upregulating the receptor tyrosine kinase AXL \ntranslation and epithelial to mesenchymal transition. \nPromoting OC growth and invasion  [28] \n METTL3 Oncogene Through AKT pathway Functioning in the progression of human OC cells [57] \n METTL3 Oncogene Via modulating the aberrant m6A RNA methylation on \ngenes including EIF3C, AXL, CSF-1 \nIndicating poor malignancy and survival of \nendometrioid epithelial OC \n[58] \n YTHDF1 Oncogene Through m6A-YTHDF1-mediated TRIM29 pathway  Indicating a poor prognosis in the cisplatin-resistant \nOC cells \n[59] \n YTHDF1 Oncogene Enhancing EIF3C translation by binding to m6A-modified \nEIF3C mRNA \nIndicating poor prognosis [60] \n YTHDF2 Oncogene FBW7 can suppress OC development by targeting YTHDF2 Promoting proliferation and migration of OC [62] \n YTHDF2 Oncogene miR-145 can repress the proliferation and migration of OC \nby suppress YTHDF2 \nPromoting proliferation and migration of OC [61] \n YTHDF3 Oncogene / Increasing the pathological grade of OC [63] \n FTO Tumor \nSuppressor \nBy blocking cAMP signaling FTO inhibited the self-renewal of ovarian CSC and \nsuppressed tumorigenesis in vivo \n[64] \n ALKBH5 Oncogene Through NF-κB pathway. Participating in the tumorigenesis of OC [65] \nCervical \ncancer \nMETTL3 Oncogene Through enhancing Warburg effect Promoting the proliferation and invasion of CC cells [72] \n METTL3 Oncogene Through enhancing the m6A modification of PDK4 Resulting in the growth progression of CC [73] \n METTL3 Oncogene By repressing the activity of miR-193b, which can regulate \nthe expression of CCND1 positively \nPromoting CC aggressiveness [74] \n FTO Oncogene Through interacting with E2F1 and Myc mRNAs Promoting CC cells proliferation and migration. [75] \n FTO Oncogene Through regulating expression of β-catenin Enhancing the chemo-radiotherapy resistance both \nin vitro and in vivo \n[76] \n YTHDF1 Oncogene Through regulating RANBP2 translation Indicating poor prognosis [77] \nEndometrial \ncancer \nMETTL3 Oncogene Through AKT pathway Promoting the proliferation and tumorigenicity of \nEC \n[82] \n IGF2BP1 Oncogene Stabilizing PEG10 mRNA in an m6A-dependent manner Indicating poor prognosis [92] \n WTAP Oncogene Via CAV-1/NF-κB axis Promoting EC progression.  [83] \n FTO Oncogene Through activating Wnt signaling pathway Promoting EC metastasis [86] \n ALKBH5 Oncogene Through enhancing IGF1R mRNA stability and promoting \nIGF1R translation \nPromoting the proliferation and tumorigenicity of \nEC \n[87] \n YTHDF2 Tumor \nSuppressor \nVia downregulating the expression of IRS1 methylated with \nm6A \nInhibiting the tumorigenicity of EC [89] \nCSC: cancer stem cell; OC: ovarian cancer; CC: cervical cancer; EC: endometrial cancer; IRS1: Insulin Receptor Substrate 1; PDK4: pyruvate dehydrogenase kinase 4; PEG10: \npaternally expressed gene 10 \n \nInterestingly, a recent study confirms that the \nadult female mice with YTHDC2 gene knockout were \ninfertile due to the lack of developing follicles, and the \nfetal female germ cells could not carry out normal \nearly pregnancy [50]. m6A may regulate female \ngermline stem cells self-renewal through m6A \nbinding protein YTHDF1 [51]. The lack of YTHDF2 \nleads to the failure of m6A modified mRNA \ndegradation, which affects the quality of oocytes [27]. \nMoreover, double mutation of YTHDF2 and YTHDF3 \nresulted in impaired female gonad development [52], \nconsistent with previous works proposing m6A and \nits protein machineries as regulators of gametogenesis \n[49]. At present, the specific mechanism of the YTH \nfamily in ovulation is still unclear, and more studies \nare needed to find the specific genes regulated by the \nYTH-domain.  \nMore interestingly, up-regulation of m6A is a \nhigh-risk factor of premature ovarian insufficiency \n(POI). Concretely, in patients with POI and mice \nmodel, the levels of m6A modified mRNA was \nsignificantly higher than that in the control group, \nwhile the expression of FTO was the opposite. \nHowever, the specific mechanism of m6A in POI still \nremains unclear up to now [53].  \nThe role of m6A in ovulation has only been \npreliminarily explained, and there is still a lot of gaps, \nfor example, the exact role of how m6A modification \ninfluences oogenesis at different developmental \nstages remains largely unknown, especially in \nhumans, owing to inaccessibility of the early human \ngerm line in vivo.  \nm6A modification in female reproductive \nsystem neoplasms \nm6A has been shown to play an important role \nin many physiological processes and various cancers. \nEpithelial transcription of tumor cells promotes \ncarcinogenesis by up-regulating or down-regulating \nthe expressions of m6A “writer”, “reader”, and \n“eraser”. Same is true in female reproductive system \nneoplasms, including OC, CC and EC (Table 2). We \nanalyzed the expression of the m6A protein machi-\nneries in cervical cancer and endometrial cancer using \ndatabases such as The Cancer Genome Atlas (TCGA) \ndataset and Genotype Tissue Expression (GTEx) \ndataset. Compared to normal tissue, bioinformatics \nanalysis of multiple m6A protein machineries in \ncervical and endometrial cancer revealed that the \nexpression of multiple m6A protein machineries \nvaried in cancer tissues (Figure 2A-B). We found that \nthe expression of m6A writers including METTL3 and \nMETTL14 were down-expressed in EC, the same as \n\nInt. J. Biol. Sci. 2022, Vol. 18 \n \n \nhttps://www.ijbs.com \n3597 \nreported in the literature. In CC, YTHDF1/2 were \noverexpressed. To some extent, this validates and \ncomplements the changes in m6A protein machineries \nreviewed in our literature. More importantly, the \nmechanism of m6A modification exerted in these \ntumors are also reviewed (Figure 3). \n \n \nFigure 2. Expression heatmap of m6A released genes in A) cervical cancer, and B) endometrial cancer. On behalf of the heatmap of m6A related genes in cancers. \nThe left part of the figure represents normal tissue, and right represents cancer tissue. The color of the grid in the heatmap represents the relative expression of the gene. All \ntumor tissue data were obtained from TCGA database and that for normal tissue came from the GTEx database. All the above analysis methods and R package were implemented \nby R version 4.0.3 and software packages ggplot2 and pheatmap. \n \nFigure 3. The momentous biological pathways of m6A exerted in female reproductive system tumors. Female reproductive system tumors including OC, CC and \nEC. \n\n\nInt. J. Biol. Sci. 2022, Vol. 18 \n \n \nhttps://www.ijbs.com \n3598 \nOvarian cancer \nOC is the leading cause of death in women \ndiagnosed with gynecological cancers. In general, it is \nalso the fifth most frequent cause of death in women \n[54]. Most OC patients are diagnosed at an advanced \nstage, so the selection or invention of an efficient \ndiagnosis and screening method has become an \neffective measure for early detection of OC patients. \nThe standard line of care treatment includes surgery \nand platinum-based chemotherapy. Moreover, \ncurrent treatments for OC are associated with high \nrecurrence rates and poor prognosis in some patients. \nDiscovering efficient and safe diagnosis and treatment \nof OC has become a valuable research topic. In recent \nyears, immune-checkpoint inhibitors (ICIs) have \nemerged in cancer therapy, but they do not seem to be \nideal in OC. This may be related to the inhibitory \neffect of tumor microenvironment (TME) in the \ntreatment of OC. The absence of an immune response \nin OC may reflect the inefficiency or absence of \nantigen presentation and adaptive immune response \ninitiation. However, recent research into the tumor \nmicroenvironment seems to provide insight for a \nbreakthrough on that. Recent study shows METTL3 \nplays an important role in TME. METTL3 depletion in \nmacrophages reshaped the TME by increasing M1- \nand M2-like tumor-associated macrophages (TAMs) \nand regulatory T (Treg) cell infiltration in vivo, \nresulting in tumor growth, metastasis, and drug \nresistance. Mechanistically, knockout of METTL3 in \nmacrophages inhibits the YTHDF1-mediated SPRED2 \ntranslation to upregulate ERK expression to activate \nNF-κB and STAT3 signaling [55]. Whether this effect \nalso exists in ovarian cancer, as well as other \nneoplasm of reproductive system, is worth further \nexploration. Also, Luo et al.[56] found m6A affected \nthe process of antigen presentation in the immune \nsystem and played an important role in TME cell \ninfiltration in OC. \nSome m6A protein machineries and methylated \ngene loci have been found in OC, which may be used \nas therapeutic targets or prognostic markers in the \nfuture. However, m6A protein machinery is worrying \nas a treatment target because an m6A protein \nmachinery can regulate the metabolic process of \nmultiple gene transcription products, which leads to \nits lower specificity and more adverse reactions. \nFurther study can be focused on exploring the specific \nrole of m6A modification in TME. And then the \ntreatment of OC may be promoted to a new height. \nMETTL3 was frequently upregulated in OC and \nthat a high level of METTL3 was significantly \nassociated with higher tumor grade. Hua et al. [28] \nfound that stable overexpression of METTL3 in vitro \nsignificantly increased cellular proliferation, focus \nformation, motility, invasion, and tumor formation in \nnude mice. However, silencing METTL3 expression in \ncell lines with short hairpin RNA effectively inhibited \nits oncogenic function. Mechanism analysis shows \nthat METTL3 promotes ovarian carcinoma growth \nand invasion through upregulating the receptor \ntyrosine kinase AXL translation and epithelial to \nmesenchymal transition [28]. In METTL3 knockdown \nOC cells, apoptosis rates increased, which may have \nbeen mediated by activating the mitochondrial apop-\ntosis pathway. METTL3 knockdown downregulated \nthe phosphorylation levels of AKT and the expression \nof the downstream effector Cyclin D1. These results \nsuggested that METTL3 may serve an oncogenic \nfunction in the progression of human OC cells \npartially through the AKT signaling pathway [57]. \nMETTL3 knockdown reduced m6A enrichment of the \ngenes associated with OC including EIF3C, AXL, \nCSF-1, FZD10 in vitro. And the high expressed \nMETTL3 indicated poor malignancy and survival of \nendometrioid epithelial OC via modulating the \naberrant m6A RNA methylation [58].  \nYTH-domain affects the development of OC \ndirectly or indirectly through m6A modification. \nYTHDF1 can promote OC by enhancing the \nexpression of m6A modified mRNA of some specific \ngenes. And genes identified include TRIM29 and \nEIF3C [59, 60]. \nLi et al. [61] demonstrated that YTHDF2 \npromoted proliferation and migration, inhibited \napoptosis, and reduced global mRNA m6A levels of \nepithelial OC (EOC) cell lines. YTHDF2 has been \nidentified as a novel substrate for the enzyme FBW7 \nwhich is markedly down-regulated in OC tissues and \nis negatively correlated with the prognosis. FBW7 \ncounteracts the tumor-promoting effect of YTHDF2 \nby inducing proteasomal degradation of the latter in \nOC [62]. Additionally, EOC can be negatively \nregulated by miR-145, resulting in cell proliferation \ninhibition [61]. Similarly, the expression of YTHDF3 \nwas positively correlated with OC malignancy, but \nthis was only based on the validation of \nbioinformatics [63].  \nFTO inhibited the self-renewal of ovarian cancer \nstem cell (CSC) and suppressed tumorigenesis in vivo. \nIntegrative RNA-sequencing and m6A mapping \nanalysis revealed significant transcriptomic changes \nassociated with FTO overexpression and m6A loss \ninvolving stem cell signaling, RNA transcription, and \nmRNA splicing pathways. By reducing m6A levels at \nthe 3‘-UTR and the mRNA stability of two \nphosphodiesterase genes (PDE1C and PDE4B), FTO \naugmented second messenger 3‘,5’-cyclic adenosine \nmonophosphate signaling and suppressed stemness \n\nInt. J. Biol. Sci. 2022, Vol. 18 \n \n \nhttps://www.ijbs.com \n3599 \nfeatures of OC cells [64]. However, ALKBH5 got the \nopposite result. NANOG, one dispensable gene in cell \nproliferation, whose expression was up-regulated by \nALKBH5, was involved in the tumorigenesis of OC \n[65]. \nBioinformatics indicate that m6A protein \nmachineries are associated with the prognosis of OC \npatients [66]. Regression models identified that \nprognosis is associated with HNRNPA2B1, \nKIAA1429, and WTAP [67]. However, m6A protein \nmachineries are still not used to determine the \nprognosis of OC patients clinically, maybe the \naccuracy still needs to be improved. \nFrom above all, METTL3, YTHDF1/2/3, \nALKBH5 play positive role in the occurrence and \ndevelopment of OC, while FTO is a tumor suppressor. \n(Table 2) More studies of m6A modification in OC \nother reproductive tumors lay a foundation for us to \nhave a clearer understanding of the pathogenesis of \nOC, which may be conducive to better prevention and \ntreatment. But it is also important to know that there \nare many categories of OC including epithelial \ncarcinoma of the ovary, malignant germ cell tumor of \novary and malignant sex cord-stromal tumors. It still \nneeds to be considered whether the effect of m6A \nmodification is consistent across different categories \nof OC. As the most malignant tumor in OC, whether \nm6A modification is participated in hyaline cell \ncarcinoma of ovary (one kind of epithelial carcinoma \nof the ovary) is still not clear. All of these can be \ncontained in the future study. \nCervical cancer \nCervical cancer (CC) is the most common \ngynecological tumor worldwide. Persistent infection \nof high-risk HPV-induced chronic inflammation is \nconsidered to be an important risk factor for CC. TME \nalso plays an important role in the progress of the \ntumorigenesis, development, and prognosis of CC \n[68]. CC has higher tumor mutation burden (TMB) \nlevel and inflammatory gene expression, suggesting \nthat there may be a continuous functional suppression \nof the immune response, better response to ICIs. On \nthis basis, they respond better to PD-1/PD-L1 or \nCTLA-4 inhibitors. However, the treatment will be \nless effective because of immune avoidance or \nimmunosuppressive signaling pathways. These \nmechanisms include adaptive immune response, loss \nof tumor antigen expression, insensitivity to \nantibiotics, and imbalance of metabolites and \ncytokines [69], which may affect the therapeutic \neffectiveness of ICIs. Recent study shows m6A \nmodification participates in the expression of PD-L1 \nindirectly. METTL14 can induce the expression of \nseven in absentia homolog 2 (Siah2), which has been \ninvolved in tumorigenesis and cancer progression. \nSiah2 knockdown inhibited T cells expansion and \ncytotoxicity by sustaining tumor cell PD-L1 \nexpression. Analysis of specimens from patients \nreceiving anti-PD1 immunotherapy suggested that \ntumors with low Siah2 levels were more sensitive to \nanti-PD1 immunotherapy [70]. Whether this is \neffective in CC still remains to be studied. \nMETTL3 can promote the proliferation and \ninvasion of CC cells [71]. So far, several studies have \nexplored the specific functions of METTL3 in CC. \nWang et al. [72] found METTL3 was significantly \nupregulated in CC tissue and cells, which was closely \ncorrelated with the lymph node metastasis and poor \nprognosis of CC patients. Mechanistically, METTL3 \ntargeted the 3’-UTR of hexokinase 2 (HK2) mRNA, \nagain recruited YTHDF1 to enhance HK2 stability, \npromoting Warburg effect of CC [72]. Interestingly, \nthe m6A modification of pyruvate dehydrogenase \nkinase 4 is mediated by METTL3.Via binding with \nYTHDF1 and IGF2BP3, the translation of m6A \nmodified PDK4 mRNA is enhanced, promoting the \nglycolysis of cancer cells, resulting in the growth and \nprogression of CC [73]. A recent study reveals a \ndifferent mechanism, METTL3 can promote CC \naggressiveness by repressing the activity of miR-193b, \nwhich can regulate the expression of “CCND1” \npositively [74].  \nFTO was frequently overexpressed in human CC \ntissues and highly correlated with CC progression. \nFTO serves as an oncogenic regulator for CC cells \nproliferation and migration. Mechanistically, FTO \ndirectly interacted with E2F1 and Myc mRNAs and \ninhibition FTO dramatically impaired these two \nimportant oncogenes translation, thus suppressed CC \ncells proliferation and migration [75]. However, \ndifferent researchers hold different opinions on the \nrelationship between FTO and CC. Zhou et al. [76] \ndiscovered FTO enhances the chemo-radiotherapy \nresistance both in vitro and in vivo through regulating \nexpression of β-catenin by reducing m6A levels in its \nmRNA transcripts.  \nIn CC, YTHDF1 was overexpressed, and it was \nclosely associated with poor prognosis [77]. YTHDF1 \nregulated RANBP2 translation in an m6A-dependent \nmanner, which potentiated the growth, migration and \ninvasion of CC cells.  \nCurrently, m6A protein machineries found in \ncervical cancer including METTL3, YTHDF1 and FTO \nall play a role in promoting the occurrence of cancer \n(Table 2), and whether other machineries are \ninvolved in CC can be explored in the future.  \nEndometrial cancer \nEndometrial carcinoma (EC) is the most frequent \n\nInt. J. Biol. Sci. 2022, Vol. 18 \n \n \nhttps://www.ijbs.com \n3600 \ngynecological malignancy in developed countries and \nrequires a relatively invasive diagnostic evaluation \nand operative therapy as the primary therapeutic \napproach [78]. It has been confirmed that significant \nchanges in the endometrial cancer immune \nmicroenvironment, such as the number of CD8+ T \ncells decreases [79]. In addition, Garzetti et al. [80] \nsuggested that locally advanced stage I and II ECs had \nsignificantly lower mean values of NK cell activity \ncompared with healthy controls, which means that \nNK cells are less able to kill tumor cells. Figuring out \nhow to enhance the immune response to EC by \nregulating tumor immunosuppressive microenviron-\nment has become the focus of future EC \nimmunotherapy research. Dong et al. [81] found that \nmacrophage-specific knockout of an m6A \nmethyltransferase METTL14 drives CD8+ T cell \ndifferentiation along a dysfunctional trajectory, \nimpairing CD8+ T cells to eliminate tumors, which \nwas found in colorectal cancer. But it also provides \ninsights into EC. It shows that m6A modification may \nparticipate in tumor immunosuppressive microenvi-\nronment of EC. Future studies could also explore \nwhether m6A modifications play a role in the \nEC-induced immunosuppressive microenvironment, \nso as to provide more possibilities for immuno-\ntherapy. Up to now, research on M6A modifications \nand EC relationships has made preliminary progress, \nbut there are many phenomena still difficult to \nexplain. \nThe downregulation of METTL3 enhance the \nproliferation and tumorigenesis of EC through AKT \npathway. That resulted in changes in the expression \nlevels of PHLPP2 and mTORC2. PHLPP2, a \nphosphatase regulating AKT phosphorylation, and \nmTORC2, a kinase that phosphorylates AKT [82]. \nReductions in m6A methylation lead to decreased \nexpression of the negative AKT regulator PHLPP2 \nand increased expression of the positive AKT \nregulator mTORC2 and the activity of AKT pathway, \npromoting abnormal cell proliferation. [82].WTAP, \none of the most important enzymes catalyzing \ngeneration of m6 A on mRNA could methylate 3'-UTR \nof CAV-1 and downregulate CAV-1 expression to \nactivate NF-κB signaling pathway in EC, which \npromoted EC progression [83].  \nThe expression of KIAA1429 observed in EC was \nsignificantly decreased, leading to the reduction of \nm6A levels [84]. KIAA1429 gene expression is \nassociated with cellular nucleic metabolism. It was \ndiscovered that KIAA1429 contributed to liver cancer \nprogression through N6-methyladenosine-dependent \npost-transcriptional modification of GATA binding \nprotein 3, which is a highly conserved, essential \ntranscription factor expressed in a number of tissues \n[85]. But in EC the mechanism is still not explicit. \nm6A “erasers” including FTO and ALKBH5 can \npromote EC through enhancing the mRNA stability \nand protein expression of some important genes. FTO \ncan decrease HOXB13 mRNA decay and increase \nHOXB13 protein expression, promoting Wnt \nsignaling pathway activation and the expression of \ndownstream proteins, leading to tumor metastasis \nand invasion [86]. ALKBH5 demethylated target \ntranscripts IGF1R and enhanced IGF1R mRNA \nstability, consequently promoting IGF1R translation \nand activating IGF1R signaling pathway, eventually \nenhancing proliferation and invasion of EC [87]. \nYTHDC1 knockdown promoted the proliferation \nand invasion of EC cells [88]. But YTHDF2 was \nidentified to inhibit the proliferation and invasion of \nEC cell lines. Mechanistically, the m6A reader \nYTHDF2 bind the methylation sites of target \ntranscripts Insulin Receptor Substrate 1 (IRS1) and \npromoted IRS1 mRNA degradation, consequently \ninhibiting the expression of IRS1 and inhibiting \nIRS1/AKT signaling pathway, finally inhibit the \ntumorigenicity of EC [89]. IRS1 plays a key role in \ncancer cell proliferation and mediates the resistance to \nanticancer drugs [90]. IGF2BP1 expression increased \nin EC, and high expression of this protein correlated \nwith poor prognosis [91]. IGF2BP1 overexpression can \npromote cell proliferation and regulate the tumor cell \ncycle and cancer progression, both in vivo and in \nvitro. Mechanistically, IGF2BP1 can recognize m6A \nsites in the 3’-UTR of Paternally Expressed Gene 10 \n(PEG10) mRNA and recruits polyadenylate-binding \nprotein 1 (PABPC1) to enhance PEG10 mRNA \nstability, which consequently promotes PEG10 \nprotein expression. Additionally, it would appear that \na large number of PEG10 proteins bind p16 and p18 \ngene promoter sequences, thereby repressing \nexpression and accelerating the cell cycle [92]. \nAnother study showed that IGF2BP1 was enriched in \nmicroRNAs in cancer pathway, contributing to the \nprogression of EC [93]. \nMETTL3, IGF2BP1, WTAP, FTO, ALKBH5 are \ninvolved in the occurrence and development of EC. \nHowever, as a tumor suppressor in EC, YTHDF2 can \ninhibit the tumorigenicity of EC (Table 2). \nOther female reproductive system \ndiseases and m6A \nm6A modification is not well studied in other \nfemale reproductive system diseases including \nadenomyosis, endometriosis, polycystic ovary \nsyndrome and premature ovarian failure. The future \nresearch should focus on further exploring whether \nm6A modification plays roles in the occurrence and \ndevelopment of these diseases. And then the \n\nInt. J. Biol. Sci. 2022, Vol. 18 \n \n \nhttps://www.ijbs.com \n3601 \nresearchers can further explore the specific target of \nm6A modification. \nAdenomyosis \nAs a common uterine disease, adenomyosis is \ncharacterized by abnormal findings of endometrial \nepithelial cells and stromal fibroblasts in the \nmyometrium, where they cause proliferation and \nhypertrophy of surrounding smooth muscle cells[94]. \nAt present, the pathological mechanism of \nadenomyosis is not very clear, which makes it difficult \nto find a good diagnosis and treatment. Zhai et al. [95] \nfound that m6A protein machineries contributed to \nthe pathogenesis of adenomyosis. Bioinformatics \nanalysis showed that METTL3, ZC3H13, FTO, and \nYTHDC1 were significantly reduced in patients with \nadenomyosis, which caused decreased m6A levels. \nPossible target genes are cadherin 3(CDH3), sodium \nchannelβ-subunit 4 (SCN4B), and placenta-specific \nprotein 8 (PLAC8), which are involved in cell \nadhesion, muscle contraction and immune response \nin the myometrium of adenomyosis patients were also \nvalidated [95]. Their findings undoubtedly provide \nnew ideas for the diagnosis and treatment of \nadenomyosis, but it is worth noting that the above \nexperiments have not been verified in animal models, \nand more research is still needed to find effective drug \ntherapeutic targets. \nEndometriosis \nEndometriosis patients have lower levels of m6A \nin the endometrium than normal endometrium, and \nthis reduction is due to lower levels of METTL3. Li et \nal. [96] found that METTL3 knockdown promotes \nmigration and invasion of human endometrial \nstromal cells (HESCs), while METTL3 overexpression \nhas the opposite effect, suggesting that METTL3 \nknockdown may promote the development of \nendometriosis by promoting cell migration and \ninvasion. In addition, they found the pathway that \nmediated this reaction. Specifically, suppressive \nMETTL3 enhances cell migration and invasion by \nattenuating DGCR8-mediated maturation of \npri-miR126 in an m6A-dependent manner, thus \ncontributing to endometriosis development. Bioinfor-\nmatics analysis also supports this finding, METTL3, \nYTHDF2, YTHDF3, HNRNPA2B1, HNRNPC and \nFTO are found decreased in ectopic endometrium. \nHNRNPA2B1 and HNRNPC may be associated with \nimmune response and can be used as useful \nbiomarkers in the diagnosis of endometriosis [97]. \nCurrently, there are few studies on the role of m6A \nprotein machineries in endometriosis, but existing \nstudies have shown that they play a significant role in \nendometriosis, and some new drugs for the treatment \nof endometriosis can be developed by targeting m6A \nprotein machineries. \nPolycystic ovary syndrome \nThe pathophysiological feature of polycystic \novary syndrome (PCOS) is granulosa cells (GCs) \ndysfunction. A recent study found that m6A levels \nwere elevated in luteinized granulosa cells in PCOS \npatients. It is found that FOXO3 mRNA lacked m6A \nmodification in luteinized granulosa cells from PCOS \npatients. Selective knockout of m6A methyltrans-\nferase or demethylase altered FOXO3 expression in \nluteinized GCs in the control group, but not in PCOS \npatients. These results suggest that m6A-mediated \nFOXO3 transcription is absent in luteinized GCs in \nPCOS patients. Forkhead Box O3 (FOXO3) plays \nimportant roles in diverse cellular processes including \napoptosis, metabolism, cell proliferation and cell \nsurvival[98]. This study sheds light on the potential \nmechanism of PCOS. \nPremature Ovarian Failure \nAs one of the most commonly used alkylated \nanticancer drugs, Cyclophosphamide (CTX) is \nassociated with premature ovarian failure. Huang et \nal. [99] found that CTX may affect ovarian function by \naffecting m6A levels. They found CTX increased m6A \nlevels in a time and concentration- dependent \nmanner. Except for RBM15 and WTAP, the expression \nlevel of RNA methyltransferase in CTX treatment \ngroup was significantly higher than that in control \ngroup in a time-dependent and concentration-depen-\ndent manner. CTX significantly inhibited the \nexpression of RNA demethylase FTO in a time- \ndependent and concentration-dependent manner, but \ndid not significantly inhibit ALKBH5 [99]. Although \nthey performed in vivo and in vitro studies, they did \nnot identify the specific mechanism by which CTX \naffects m6A protein machineries or the specific target \nof premature ovarian failure. \nPerspective \nDespite the researches of m6A in reproduction \nhave make dramatic roles in recent years, a large \nnumber of challenges still exist. Firstly, most of the \nconclusions are derived from bioinformatics analysis \nor in vitro experiments, in vivo experiments are rarely \ninvolved. Secondly, it is possible some enzymes that \nmodify m6A have not been identified. Thirdly, the \nmechanisms of m6A protein machineries in some \ninfertility diseases are still unclear, more efforts are \nneeded to explore the specific mechanism of m6A in \nthe various pathways that regulate gene expression. \nFourthly, the specific sites of m6A modification are \nrarely reported, which greatly limits clinical \n\nInt. J. Biol. Sci. 2022, Vol. 18 \n \n \nhttps://www.ijbs.com \n3602 \ntransformation. Fifthly, studies showed that \nregulation of m6A level and its protein machineries \nmay be potential therapeutic targets for some \nreproduction diseases, but lack of the specific \napplications in clinical practice with a large sample \nsize, and the safety, effectiveness, even the \ncorresponding side effects are largely unknown. \nSixthly, it is uncertain whether m6A modification \nplays the same role in different tumor subtypes. All of \nthese issues should be addressed. \nConclusions \nRNA modification, especially m6A modification, \nhas become a hot topic in recent years. m6A is \nextremely important for mRNA metabolism at \ndifferent stage, from processing in the nucleus to \ntranslation and decay in the cytoplasm. In this review, \nwe summarized that m6A modification and its \nregulators played a key role in the occurrence and \ndevelopment of oogenesis and female reproductive \nsystem diseases. The change of m6A protein \nmachineries contributed to the proliferation and \naggressiveness of tumors. With the introduction of \nm6A detection technology into large-scale commercial \nuse, the m6A level and its protein machineries have \nbecome more possible for the diagnosis of oogenesis \nand female reproductive system diseases. In addition, \nwe reviewed the regulatory mechanisms and target \ngenes that have been discovered so far, providing \nprospects for the study of related drugs and \ntreatments. \nAbbreviations \nOC: ovarian cancer; CC: cervical cancer; EC: \nendometrial cancer; WTAP: Wilms-tumor associating \nprotein; VIRMA/ KIAA1429: Vir-like m6A \nmethyltransferase-associated; FTO: \nObesity-associated protein; ALKBH5: alkB homolog 5; \nPOI: premature ovarian insufficiency; CSC: cancer \nstem cell; EOC: epithelial OC; HK2: hexokinase 2; \nIRS1: Insulin Receptor Substrate 1; PEG10: Paternally \nExpressed Gene 10; PABPC1: polyadenylate-binding \nprotein 1; AF: angiogenic factor. \nAcknowledgements \nThis work was funded by National Key R&D \nProgram of China (Grant numbers: 2018YFC1004300, \n2018YFC1004304), National Natural Foundation of \nChina (Grant numbers: 81701539). \nAuthor Contributions \nY.W.F., and J.Y.C. designed the work. 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