{"paper_id":"1ab9a78e-15a5-406e-9f59-043fd4753ecd","body_text":"Li et al. \nReproductive Biology and Endocrinology           (2023) 21:99  \nhttps://doi.org/10.1186/s12958-023-01151-0\nRESEARCH Open Access\n© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which \npermits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the \noriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or \nother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line \nto the material. 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The Creative Commons Public Domain Dedication waiver (http://creativecom-\nmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.\nReproductive Biology\nand Endocrinology\nMETTL3-regulated m6A modification impairs \nthe decidualization of endometrial stromal cells \nby regulating YTHDF2-mediated degradation \nof FOXO1 mRNA in endometriosis-related \ninfertility\nXiaoou Li1, Jie Jin1, Xuefeng Long1, Ruiwen Weng1, Wenqian Xiong1, Jiaxin Liang1, Junjun Liu1, Jingwen Sun1, \nXueqin Cai1, Ling Zhang1* and Yi Liu1* \nAbstract \nBackground Endometriosis-related infertility is a common worldwide reproductive health concern. Despite ongoing \nresearch, the causes of infertility remain unclear. Evidence suggests that epigenetic regulation is crucial in reproduc-\ntion. However, the role of N6-methyladenosine (m6A) modification of RNA in endometriosis-related infertility requires \nfurther investigation.\nMethods We examined the expression of m6A and methyltransferase-like 3 (METTL3) in endometrial samples taken \nfrom normal fertile women in the proliferative phase (the NP group) or the mid-secretory phase (the NS group) \nor from women with endometriosis-related infertility at the mid-secretory phase (the ES group). We treated primary \nendometrial stromal cells (ESCs) with medroxyprogesterone acetate and 8-Bromo-cyclic adenosine monophos-\nphate for in vitro decidualization and detected the expression of m6A, METTL3, and decidual markers. We analyzed \nthe expression of m6A, METTL3, and forkhead box O1 (FOXO1) in ESCs from normal fertile women (the ND group) \nor women with endometriosis-related infertility (the ED group). We also assessed the expression of m6A, METTL3, \nand decidual markers, as well as the embryo adhesion rate, upon METTL3 overexpression or knockdown. Additionally, \nwe investigated the role of METTL3 in embryo implantation in vivo by applying mice with endometriosis. Further-\nmore, we performed RNA stability assays, RNA immunoprecipitation (RIP), and methylated RIP assays to explore \nthe mechanisms underlying the regulation of FOXO1 by METTL3-mediated m6A.\nResults The expression of m6A and METTL3 was reduced only in the NS group; the NP and ES groups demon-\nstrated increased m6A and METTL3 levels. m6A and METTL3 levels decreased in ESCs with prolonged decidual \ntreatment. Compared to the ND group, m6A and METTL3 levels in the ED group increased after decidual treatment, \nwhereas the expression of FOXO1 decreased. METTL3 overexpression suppressed the expression of decidual markers \nand embryo implantation in vitro; METTL3 knockdown exhibited the opposite effect. Inhibition of METTL3 promoted \n*Correspondence:\nLing Zhang\nzhanglingxh@hust.edu.cn\nYi Liu\nliqun1994@hust.edu.cn\nFull list of author information is available at the end of the article\n\nPage 2 of 17Li et al. Reproductive Biology and Endocrinology           (2023) 21:99 \nembryo implantation in vivo. Furthermore, we observed that METTL3-mediated m6A regulated the degradation \nof FOXO1 mRNA through YTHDF2, a m6A binding protein.\nConclusions METTL3-regulated m6A promotes YTHDF2-mediated decay of FOXO1 mRNA, thereby affecting cellular \ndecidualization and embryo implantation. These findings provide novel insights into the development of therapies \nfor women with endometriosis-related infertility.\nKeywords Decidualization, Endometriosis, Infertility, N6-methyladenosine, Methyltransferase-like 3, Forkhead box O1\nBackground\nInfertility is a prevalent health issue affecting approxi -\nmately 8–12% of couples worldwide [1, 2]. Endometrio -\nsis, a condition characterized by the presence of tissue \nsimilar to the lining of the uterus outside the uterus, is \na multifactorial and systemic disease prevalent in 10% \nof women of reproductive age [3]. Approximately 50% \nof women with infertility suffer from endometriosis \n[4] and one third of women with endometriosis experi -\nence infertility [3]. Successful implantation of embryos \nin the uterus requires a well-functioning and synchro -\nnously developing endometrium, called the receptive \nendometrium, during the implantation window within \nthe mid-secretory phase [5]. However, women with \nendometriosis frequently experience endometrial disor -\nders [6] and lower rates of implantation and pregnancy \nthan those with tubal infertility after in  vitro fertiliza -\ntion (IVF) or intracytoplasmic sperm injection (ICSI) \ntreatments [7, 8]. Decidualization, the process by which \nendometrial fibroblast-like stromal cells transform into \nspecialized decidual cells, is crucial for establishing endo-\nmetrial receptivity, providing a nutritional and immuno -\nsuppressive environment for embryo implantation [7]. \nWomen with endometriosis-related infertility experience \nimpaired decidualization, which contributes to endo -\nmetrial defects [9–11]. Therefore, understanding the \nmechanisms involved in decidualization and endometrial \nreceptivity is essential for the detection and treatment of \nendometriosis-related infertility.\nN6-methyladenosine (m6A), one of the most preva -\nlent RNA modifications, has dynamic and reversible \nregulatory features. The catalysis of m6A modifica -\ntions is facilitated by a methyltransferase complex com -\nprising two subcomplexes, methyltransferase-like 3 \n(METTL3) and 14 (METTL14), along with other com -\nponents such as WT1 associated protein (WTAP), \nvir like m6A methyltransferase associated (VIRMA, \nalso known as KIAA1429) [12]. Conversely, fat mass \nand obesity-associated protein (FTO) [13] and alkB \nhomolog 5 (ALKBH5) [14] facilitate the reverse action. \nA balance between m6A methyltransferases and dem -\nethylases is involved in the dynamic regulation of m6A. \nThe recognition of m6A modifications is attributed to \nreader proteins, including YTH domain-containing \nproteins (YTH N6-methyladenosine RNA binding pro -\ntein C1/2 (YTHDC1/2) and F1/2/3 (YTHDF1/2/3)) \nand insulin-like growth factor 2 mRNA-binding pro -\ntein 1/2/3 (IGF2BP1/2/3) [15]. Several studies have \ndemonstrated the link between m6A modification and \ngametogenesis and fertility in both sexes [16, 17]. Addi -\ntionally, m6A modifications are associated with the \ndevelopment of endometrium-related diseases such \nas endometrial cancer [18] and endometriosis [19, \n20]. However, the precise role of m6A modifications \nin endometriosis-related infertility and endometrial \ndecidualization remains unclear.\nThe objective of this study was to elucidate the func -\ntional role of m6A modification in cellular deciduali -\nzation and to explore whether it is abnormal in women \nafflicted with endometriosis-related infertility. Addition -\nally, we attempted to uncover the effect of METTL3 and \nm6A expression on the decidualization of endometrial \nstromal cells and the potential mechanisms involved, \nthereby offering a novel therapeutic approach for improv-\ning endometriosis-associated infertility.\nMaterials and methods\nPatients and tissue collection\nThis study was approved by the local ethics committee of \nthe Union Hospital, Tongji Medical College, Huazhong \nUniversity of Science and Technology. Written informed \nconsent was obtained from patients before the collection \nof human tissues, in accordance with the guidelines of \nthe Declaration of Helsinki.\nNormal control endometrial samples were obtained \nfrom patients without endometriosis who visited our \nhospital with tubal infertility. These samples included \nproliferative (n = 21) and mid-secretory (n = 21) phase \nendometria collected through curettage. Additionally, \neutopic endometria in the mid-secretory phase (n = 14) \nwere obtained from patients with stage III and IV ovar -\nian endometriosis [21]. All the patients were premeno -\npausal and had regular menstrual cycles. Menstrual cycle \nphases were determined based on their menstrual history \nand endometrial histology confirmed by an independ -\nent pathologist. None of the patients received hormonal \ntreatment for at least three months prior.\n\nPage 3 of 17\nLi et al. Reproductive Biology and Endocrinology           (2023) 21:99 \n \nCell culture and in vitro decidualization\nPrimary endometrial stromal cells (ESCs) were isolated \nfrom the samples collected from the proliferative stage \nof the menstrual cycle by Pipelle biopsy from fertile, \nregularly cycling women and endometriosis-associated \ninfertile women under anesthesia as described previ -\nously [22]. The cells were routinely incubated in Dul -\nbecco’s Modified Eagle’s Medium (DMEM/F12) without \nphenol red, containing 10% activated carbon-adsorbed \nserum (BasalMedia, Shanghai, China). When perform -\ning in  vitro decidualization, cells were incubated in \nDMEM/F12 without phenol red containing 2% carbon-\nadsorbed serum with medroxyprogesterone acetate \n(MPA, 100 nM, MCE, HY-B0469S) and 8-Bromo-cyclic \nadenosine monophosphate (8-Br-cAMP , 0.5  mM, Sell -\neck, S7857) added to the cellular supernatant. After \n2–6 d, cellular decidualization was assessed by evaluat -\ning the expression of decidualization marker genes and \ncell morphology.\nHuman endometrial stromal cells (ThESCs) were pur -\nchased from the American Type Culture Collection \n(CRL-4003; ATCC) and cultured in the same medium \nand environment as ESCs. For METTL3 intervention \nassays, ThESCs were pretreated with a METTL3-over -\nexpressing vector (Dianjun, Shanghai, China), siRNAs of \nMETTL3 (Dianjun, Shanghai, China), or their own nega -\ntive control group for 24  h and then treated with MPA \nand 8-Br-cAMP for 4 d. The siRNA sequences are listed \nin Table S2.\nRNA m6A quantitative assays\nAn EpiQuik m6A RNA Methylation Quantification Kit \n(Epigentek, NY, USA) was used to quantify the m6A lev -\nels in total RNAs. First, the RNAs were added to strip \nwells containing an RNA high-binding solution. Accord -\ning to the instructions, capture and detection antibody \nsolutions were then added separately to the wells at \nappropriate dilutions. Finally, m6A levels were measured \ncolorimetrically by measuring the absorbance at 450 nm \nusing a microplate reader. Data were calculated using rel-\native quantification.\nRNA isolation and quantitative real‑time polymerase chain \nreaction (qRT‑PCR)\nTotal RNAs were extracted from cells using TRIzol \n(Vazyme, Nanjing, China) following the manufactur -\ner’s instructions. cDNA was synthesized and quanti -\nfied using a HiScript III 1st Strand cDNA Synthesis Kit \n(Vazyme, Nanjing, China). qRT-PCR was conducted with \n2 × TSINGKE® Master qPCR Mix (SYBR Green I with \nUDG; Tsingke, Beijing, China). All data were analyzed \nusing the  2−ΔΔCt  method. The primer sequences used are \npresented in Table S1.\nWestern blot analysis\nRadioimmunoprecipitation assay buffer (Beyotime) \nwas used to extract proteins, which were then quanti -\nfied using a BCA protein assay kit (Beyotime). Equal \namounts of protein (30  μg) were resolved using sodium \ndodecyl sulfate–polyacrylamide gel electrophoresis and \ntransferred onto polyvinyl difluoride membranes (Milli -\npore, MA, USA). After blocking with 5% skimmed milk \nin Tris-buffered saline containing 0.1% Tween-20 (TBST) \nfor 1  h, the membranes were incubated with primary \nantibodies against METTL3 (1:1000, Abcam, MA, USA), \nforkhead box O1 (FOXO1, 1:1000, CST, MA, USA), \nβ-actin (1:200000, Proteintech, Wuhan, China), and \nglyceraldehyde-3-phosphate dehydrogenase (GAPDH; \n1:200000, Proteintech, Wuhan, China) overnight at 4 °C. \nAfter washing, the membranes were incubated with anti-\nrabbit antibody (1:5000, Proteintech, Wuhan, China) at \n25 °C for 1 h. After washing, the membranes were visu -\nalized using a detection system, followed by incubation \nwith enhanced ECL detection reagent (Biology, Wuhan, \nChina). The gray values of the protein bands were ana -\nlyzed using Image J software.\nImmunohistochemical (IHC) staining\nAll the tissues were immediately placed in 4% buffered \nformalin for IHC staining. Paraffin embedding, section -\ning, and IHC staining were performed by Biosciences \nBiotechnology Co., Ltd. (Wuhan, China). IHC staining of \nparaffin sections was performed using primary antibod -\nies against METTL3 (1:400; Abcam, Cambridge, USA) \nand FOXO1 (1:1000; Cell Signaling Technology, Danvers, \nMA, USA). Finally, Image-Pro Plus software 6.0 was used \nto analyze the data.\nImmunofluorescence (IF) staining\nPrimary stromal cells were fixed with 4% paraform -\naldehyde for 30  min at 25  °C and then permeabilized \nwith phosphate buffer saline (PBS) containing 0.1% Tri -\ntonX-100 for 10  min at 25  °C. Non-specific sites were \nblocked with 1% bovine serum albumin in PBS for 1 h at \n37  °C. Endogenous proteins were stained with primary \nantibodies against METTL3 (1:400, Abcam, MA, USA) \nand FOXO1 (1:1000, CST, MA, USA) for 1  h at 25  °C. \nFluorescence-conjugated secondary antibodies (1:4000; \nProteintech) were used to visualize the signals. The nuclei \nwere stained with 4’ ,6-diamidino-2phenylindole dihy -\ndrochloride for 10 min. Finally, images were obtained by \nfluorescence confocal microscopy and processed using \nImage Pro Plus 6.0 software.\n\nPage 4 of 17Li et al. Reproductive Biology and Endocrinology           (2023) 21:99 \nEnzyme‑linked immunosorbent assay\nAfter in vitro cellular decidualization, cell culture super -\nnatants were harvested and centrifuged to remove cell \ndebris. A commercially available prolactin (PRL) enzyme-\nlinked immunosorbent assay kit (ruixinbio, Quanzhou, \nChina, RX106036H) and insulin-like growth factor-\nbinding protein 1 (IGFBP1) enzyme-linked immunosorb-\nent assay kit (ruixinbio, Quanzhou, China, RX104919H) \nwere used to detect PRL and IGFBP1 levels, respectively, \nin the collected supernatants. The samples were assayed \nin duplicate, and the concentrations were expressed as \nmIU/mL or ng/mL of the cell supernatant.\nRNA m6A dot blot assays\nTotal RNAs were spotted onto  N+ nylon membranes (GE \nHealthcare, MD, USA). After ultraviolet cross-linking, \nthe membranes were blocked with 5% fat-free milk in \nTBST for 1 h and then incubated with an anti-m6A anti -\nbody (1:1000, Proteintech, Wuhan, China) overnight at \n4 °C. After washing, the membranes were incubated with \nan anti-mouse antibody (1:5000, Proteintech, Wuhan, \nChina) for 1 h at 25 °C. After further washing, the mem -\nbranes were incubated with enhanced ECL detection \nreagent (Biology, Wuhan, China) and visualized using a \ndetection system. After washing, the membranes were \nstained with 0.2% methylene blue as a control.\nIn vitro embryo implantation assays\nHTR-8/SVneo cells (from an immortalized cell line \nderived from first-trimester villous explants) were co-cul-\ntured with a confluent monolayer of ThESCs to simulate \nembryo attachment [23]. First, a single-cell suspension of \nHTR8 cells was placed in a low-adhesion 96-well plate. \nMulticellular spheroids of HTR8 cells were induced after \n72  h of culture and 70–100  μm diameter multicellular \nspheroids were sieved through filter sieves. Simultane -\nously, HTR8 spheroids were transferred onto a confluent \nmonolayer of ThESCs, which were treated accordingly \nin advance. After incubation at 37  °C for 12  h, cells \nwere washed with PBS to remove the unattached sphe -\nroids. The attached spheroids were counted under a light \nmicroscope, and the adhesion rate was expressed as a \npercentage of the total number of HTR8 spheroids added \nto the ThESC monolayer.\nAnimal experiments\nC57BL/6 mice (n = 57) were purchased from Hubei \nBeiente Biotechnology Co., Ltd. (Wuhan, China). Five \nfemale mice were subjected to a normal pregnancy assay. \nUterine tissues of 14 female donor mice were cut up and \ninjected into the abdominal cavity of 28 female recipi -\nent mice. After 21 d, 10 male C57 mice were mated with \nthe recipient mice, and the next day, when vaginal plugs \nwere observed, was regarded as day 1. Eight recipient \nmice were euthanized by cervical dislocation after deep \npentobarbital anesthesia on day 8, and the number of \nblastocysts in the uterus was counted. At the night of \nday 3, 10 μL of STM2457, a METTL3 inhibitor (Sellcek, \nS9870, 10  μM) and 10 μL dimethyl sulfoxide (DMSO) \nwere individually injected into the uterine horns of 20 \nrecipient mice. The mice were euthanized on day 8, and \nthe number of blastocysts in the uterus was counted. \nThe uterine tissues were collected and fixed in 4% (w/v) \nparaformaldehyde for histological and IHC analyses. All \nanimal experiments were approved by the Ethics Com -\nmittee of the Animal Center of the Tongji Medical Col -\nlege (approval number 3332).\nRNA immunoprecipitation PCR (RIP‑PCR)\nAn RNA immunoprecipitation kit (Bersinbio RIP Kit, \nGuangzhou, China) was used to perform the RIP assays. \nThe cells were UV-irradiated and lysed with lysis buffer \naccording to the manufacturer’s instructions. Immuno -\nprecipitation of endogenous factors was performed using \na primary antibody overnight at 4 °C. Protein A/G beads \nwere then added to capture the primary antibody. After \nwashing, proteinase K was added to the immunopre -\ncipitated complex to remove excess proteins. RNAs were \nextracted using TRIzol reagent and quantified by qRT-\nPCR using primers for FOXO1 . The data were normal -\nized to input or %IgG of input.\nMethylated RIP‑PCR (MeRIP‑PCR)\nA methylated RNA immunoprecipitation kit (Bersinbio \nRIP Kit, Guangzhou, China) was used to perform the \nMeRIP assays. RNAs were isolated from cells and frag -\nmented by ultrasonication for 1.5 min. An anti-m6A anti-\nbody was used for immunoprecipitation. Protein A/G \nbeads were then added to capture the anti-m6A antibody. \nAfter several washes, proteinase K was added to the \nimmunoprecipitated complex to remove excess proteins. \nFinally, RNAs were extracted using TRIzol reagent and \nquantified by qRT-PCR using primers for FOXO1 . The \ndata were normalized to the input or %IgG of input.\nRNA stability assays\nThESCs were pretreated with a METTL3-overexpressing \nvector (Dianjun, Shanghai, China) and its control group \n(or with the wild-type [ovM3-WT, Dianjun, Shanghai, \nChina], mutated METTL3-overexpressing vector [ovM3-\nMUT, D395A and W398A, Dianjun, Shanghai, China], \nand their control group) for 24  h and then treated with \nMPA and 8-Br-cAMP for 4 d. Next, actinomycin D (Act \nD, Selleck, S8964) was added to the culture media with \na final concentration of 2  μg/mL and cells were col -\nlected at 2, 4, and 6 h time points following actinomycin \n\nPage 5 of 17\nLi et al. Reproductive Biology and Endocrinology           (2023) 21:99 \n \nD addition. Finally, RNAs were extracted using TRIzol \nreagent and quantified by qRT-PCR using primers for \nFOXO1. The mutant of the METTL3-overexpressing vec-\ntor was constructed with disordered enzymatic activity, \nas described previously [24].\nStatistical analysis\nGraphPad Prism 7 was used for the statistical analyses. \nAll data were presented as the mean ± standard error of \nthe mean. All experiments were repeated in triplicate \nor quadruplicate. For data variables with a normal dis -\ntribution, a Student’s t test was used to analyze differ -\nences between the two groups and a one-way analysis of \nvariance was performed to analyze differences between \nmultiple groups. For non-normally distributed data, the \nMann–Whitney test was used for two groups, while the \nKruskal–Wallis test was used for multiple groups. Statis -\ntical significance was defined as P < 0.05.\nResults\nm6A and METTL3 are downregulated in mid‑secretory \nphase endometria from normal fertile women\nTo investigate the role of m6A modification in endome -\ntrial receptivity, we collected endometria from fertile \nwomen without endometriosis during both the prolifera -\ntive phase (the NP group) and the mid-secretory phase \n(the NS group) and measured m6A levels using a colori -\nmetric method. The results show that m6A levels in the \nNS group were downregulated compared to those in the \nNP group (Fig. 1a).\nAs previously stated, m6A is a dynamic RNA modifica -\ntion regulated by methyltransferases and demethylases. \nMETTL3 and METTL14, core components of the m6A \nmethyltransferase complex, are involved in the forma -\ntion of m6A [12], whereas the demethylases FTO [13] \nand ALKBH5 [14] are involved in demethylation. Fur -\nthermore, emerging studies have proposed that m6A \nreaders such as YTHDFs and IGF2BPs perform the real \nfunction of m6A by combining it with m6A tags [15]. \nTherefore, we measured the expression of these m6A-\nassociated genes in the NP and NS samples using qRT-\nPCR to determine the key molecules involved in m6A \ndownregulation in the NS group. The results show that \nMETTL3 and KIAA1429 were downregulated in the NS \ngroup than in the NP group; however, no difference in \nthe expression of METTL14, WTAP, FTO, and ALKBH5 \nwas observed between the NP and NS groups (Fig.  1b). \nIn addition, YTHDF3 expression was upregulated in the \nNS group compared to that in the NP group; however, \nno difference in YTHDF1 and YTHDF2 expression was \nobserved between the NP and NS groups (Fig.  1b). Nota-\nbly, the mRNA levels of all IGF2BPs were downregulated \nin the NS group compared with those in the NP group \n(Fig.  1b). These findings suggest an important role of \nm6A in endometrial receptivity.\nAs a catalytic factor in the methyltransferase complex, \nMETTL3 was chosen to further investigate the function \nof m6A in endometrial receptivity. Western blot analysis \nand IHC staining were performed for further confirma -\ntion. The results showed that METTL3 was downregu -\nlated in the NS group than in the NP group (Fig.  1c,d), \nsimilar to the qRT-PCR results. In addition, we observed \nthat METTL3 was expressed in the nuclei of epithelial \nand stromal cells and that METTL3 expression in both \nepithelial and stromal cells was downregulated in the NS \ngroup than in the NP group (Fig.  1d). Taken together, \nthese results suggest that the downregulation of m6A and \nMETTL3 in the mid-secretory phase may have a positive \neffect on the establishment of endometrial receptivity \nduring the implantation window.\nm6A and METTL3 are upregulated \nin the mid‑secretory phase of endometria from women \nwith endometriosis‑related infertility\nWomen with endometriosis often develop infertility, \nwhich is closely associated with defective endometrial \nreceptivity [9–11]. To investigate the role of m6A and \nMETTL3 in patients with endometriosis-related infer -\ntility, we collected mid-secretory endometrial samples \nfrom women with endometriosis-related infertility (the \nES group). As expected, m6A levels in the ES group were \nupregulated compared to those in the NS group (Fig.  1e). \nIn addition, METTL3 expression, as determined by west -\nern blot analysis and IHC staining, was upregulated in \nthe ES group than in the NS group, consistent with m6A \nupregulation (Fig.  1f,g). Furthermore, we observed that \nMETTL3 was upregulated in epithelial and stromal cells \nin the ES group than in the NS group (Fig.  1g). Taken \ntogether, these results suggest that the upregulation of \nMETTL3 and m6A probably accounts for the impaired \nendometrial receptivity in women with endometriosis-\nrelated infertility.\nReduction of m6A and METTL3 contributes \nto the decidualization of primary endometrial stromal cells\nDecidualization of endometrial stromal cells is a crucial \nstep in establishing endometrial receptivity [25]. Previous \nresults indicate the potential role of m6A and METTL3 \nin establishing endometrial receptivity; therefore, we \nexplored the function of METTL3 in the decidual pro -\ncess of stromal cells. First, we established in vitro decidu -\nalization of ESCs using MPA and 8-Br-cAMP treatment \nand examined the expression levels of decidual mark -\ners at different time points. The results showed that \nthe mRNA and protein levels of PRL and IGFBP1 were \nincreased over time, with the highest expression on day \n\nPage 6 of 17Li et al. Reproductive Biology and Endocrinology           (2023) 21:99 \n6 (Fig.  2a-d). In addition, the mRNA and protein levels \nof FOXO1, which regulates the transcription of PRL and \nIGFBP1, increased over time (Fig.  2e,f). Furthermore, IF \nstaining showed that the FOXO1 expression increased \non day 6 (Fig.  2g, Supplementary Figure S1a). Addition -\nally, compared to ESCs on day 0, cellular morphology \non day 6 was more rounded (Fig.  2h). These results sug -\ngest successful establishment of in  vitro decidualiza -\ntion. Next, we measured METTL3 and m6A levels after \ndecidual treatment and found that METTL3 expression \ndecreased over time, with the lowest expression observed \non day 6 (Fig. 2i). IF staining also showed lower METTL3 \nFig. 1 Expression of N6-methyladenosine (m6A) and methyltransferase-like 3 (METTL3) in different endometrial samples. m6A and METTL3 levels \nwere downregulated in the mid-secretory endometria of normal fertile women (the NS group) without endometriosis, whereas it was upregulated \nduring the same phase in the endometria of women with endometriosis-related infertility (the ES group). a m6A levels in the endometria of normal \nfertile women in the proliferative phase (the NP group) (n = 21) and the NS group (n = 21). b mRNA levels of m6A-associated genes in the tissues \ndetected by qRT-PCR. c Protein levels of METTL3 in sampled tissues detected by western blot analysis. d Protein levels of METTL3 in sampled tissues \ndetected by immunohistochemical (IHC) staining (scale bar = 100 μm or scale bar = 50 μm). e m6A levels in the NS (n = 14) and ES (n = 14) groups. \nf Protein levels of METTL3 in sampled tissues detected by western blot analysis. g Protein levels of METTL3 in sampled tissues detected by IHC \nstaining (scale bar = 100 μm or scale bar = 50 μm). All experiments were repeated in triplicate or quadruplicate. The blots in this figure are cropped \n(please refer to supplementary files for details). Data with error bars are presented to indicate the mean ± standard error of the mean (SEM) values. \n*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001\n\nPage 7 of 17\nLi et al. Reproductive Biology and Endocrinology           (2023) 21:99 \n \nFig. 2 METTL3 expression was downregulated during the decidualization of primary endometrial stromal cells (ESCs) in vitro. These cells were \ntreated with medroxyprogesterone acetate (MPA) and 8-Bromo-cyclic adenosine monophosphate (8-Br-cAMP) for the decidual treatment for 0, \n2, 4, and 6 d. The mRNA (a, b) and protein (c, d) levels of prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1) were detected \nusing qRT-PCR and western blot analysis, respectively. e, f mRNA and protein levels of forkhead box O1 (FOXO1) were detected by qRT-PCR \nand western blot analysis, respectively. g Immunofluorescence (IF) staining was performed to detect FOXO1 levels (scale bar = 100 μm). h IF staining \nwas performed to detect morphological changes in cells after decidual treatment (scale bar = 50 μm). i, j Protein levels of METTL3 were detected \nby western blot analysis and IF staining (scale bar = 100 μm). k Dot blot assays were performed to detect m6A changes in cells after decidual \ntreatment. Methylene blue staining was used as a control. l Protein levels of METTL3 in ESCs derived from normal (ND) and endometriosis (ED) \ngroups, both of which received decidual treatments. All experiments were repeated in triplicate or quadruplicate. The blots in this figure are \ncropped (please refer to supplementary files for details). Data with error bars are presented as the mean ± SEM values. **P < 0.01, ***P < 0.001, \n****P < 0.0001\n\nPage 8 of 17Li et al. Reproductive Biology and Endocrinology           (2023) 21:99 \nexpression on day 6 than on day 0 (Fig. 2j, Supplementary \nFigure S1b). In addition, the level of m6A on day 6 was \nlower than that on day 0 (Fig.  2k). These results suggest \nthat decreased m6A and METTL3 levels contribute to \nthe decidualization of endometrial stromal cells.\nWe then extracted ESCs derived from women with \nendometriosis-related infertility and performed the \nsame in  vitro decidualization assay for six days (the ED \ngroup). Compared with those of normal ESCs treated \nwith in  vitro decidualization (the ND group), m6A and \nMETTL3 levels in the ED group were significantly \nincreased (Fig.  2l, Supplementary Figure S1c), while the \ndecidual marker FOXO1 was decreased (Supplementary \nFigure S1d). Taken together, these results suggest that the \nreduction in METTL3 and m6A levels contributes to the \ndecidual process of endometrial stromal cells. Therefore, \nwhen m6A and METTL3 levels are increased in stromal \ncells derived from women with endometriosis-related \ninfertility, cellular decidualization is disrupted, resulting \nin defective endometrial receptivity.\nMETTL3 is involved in regulating the decidualization \nof endometrial stromal cells and embryo implantation\nTo further confirm the correlation between METTL3/\nm6A and cellular decidualization, we transfected a \nMETTL3-overexpressing vector or siRNAs of METTL3 \ninto ThESCs and then performed in  vitro decidualiza -\ntion. METTL3 was effectively overexpressed and silenced \nat the mRNA and protein levels (Fig.  3a-d). As expected, \nMETTL3 overexpression increased m6A levels com -\npared to the negative control (Fig.  3e), whereas METTL3 \nknockdown decreased m6A levels (Fig.  3f), suggesting \nthat METTL3 regulates m6A levels in decidualizing stro -\nmal cells.\nNext, we investigated the effect of changes in METTL3 \nexpression on cellular decidualization. The results \nshowed that METTL3 overexpression decreased the \nmRNA and protein levels of PRL and IGFBP1 (Fig.  3g,h, \nSupplementary Figure S1e,f), whereas METTL3 knock -\ndown increased the mRNA and protein levels of PRL \nand IGFBP1 (Fig.  3i,j, Supplementary Figure S1g,h). In \naddition, FOXO1 expression decreased at the mRNA \nand protein levels when METTL3 was overexpressed \n(Fig.  3k,l), whereas FOXO1 expression increased when \nMETTL3 was silenced (Fig.  3m,n). These results suggest \nthat METTL3 is involved in regulating the decidualiza -\ntion of stromal cells.\nWe then investigated whether alterations in METTL3 \nexpression in stromal cells affect embryo implantation \nusing in  vitro embryo implantation assays. The results \nshowed that METTL3 overexpression in ThESCs reduced \nthe adhesion rate of HTR8 spheroids from the HTR-8/\nSVneo trophoblast cell line, whereas METTL3 knock -\ndown increased this rate (Fig.  3o). We further exam -\nined whether changes in METTL3 expression affect \nembryo implantation by regulating FOXO1 expression. \nThe results showed that FOXO1  overexpression rescued \nthe reduced FOXO1 levels induced by METTL3 over -\nexpression (Fig.  3p); however, treatment with a FOXO1 \ninhibitor reduced the increased FOXO1 levels induced by \nMETTL3 knockdown (Fig.  3q). Additionally, the overex -\npression of FOXO1  restored the adhesion rate of HTR8 \nspheroids reduced by METTL3 overexpression; however, \ntreatment with a FOXO1 inhibitor reduced the adhesion \nrate increased by METTL3 knockdown (Fig.  3r). Over -\nall, we demonstrate that METTL3 is involved in cellu -\nlar decidualization by modulating FOXO1 expression, \nthereby affecting embryo implantation in vitro.\nFor further confirmation, mouse models of endome -\ntriosis were established for in-depth in vivo investigation, \nas shown in Fig.  4a. Figure  4b shows the ectopic lesions \nof model mice with endometriosis. After mating, the day \nthe virginal plugs were observed was regarded as day 1 \n(D1), and the mice were euthanized on day 8 (D8). The \nresult showed that the number of blastocysts implanted \nin normal pregnant mice was significantly higher than \nFig. 3 METTL3 was involved in regulating cellular decidualization and embryo implantation. a, b Overexpression and knockdown (ovM3, siM3) \nefficacy of METTL3 at the mRNA level in human endometrial stromal cells (ThESCs). c, d Overexpression and knockdown efficacy of METTL3 \nat the protein level in ThESCs. e, f Dot blot assays were used to detect m6A changes upon METTL3 overexpression or knockdown in ThESCs. \nMethylene blue staining was performed as a control. g-j Protein levels of PRL and IGFBP1 upon METTL3 overexpression or knockdown in ThESCs. \nk, l mRNA and protein levels of FOXO1 upon METTL3 overexpression in ThESCs. m, n mRNA and protein levels of FOXO1 upon METTL3 knockdown \nin ThESCs. o Cell percentage of HTR-8/SVneo trophoblast cell line spheroids attached to ThESCs following METTL3 overexpression or knockdown \nwere evaluated (scale bar = 200 μm). p Protein level of FOXO1 reduced by METTL3 overexpression was rescued by FOXO1 overexpression. q \nProtein level of FOXO1 increased by METTL3 knockdown was decreased by a FOXO1 inhibitor. r Cell percentage of HTR-8/SVneo spheroids \nattached to ThESCs transfected with the negative control and METTL3-overexpressing vector were evaluated in the absence or presence \nof the FOXO1-overexpressing vector. The cell percentage of HTR-8/SVneo spheroids attached to ThESCs transfected with the negative control \nand siRNA of METTL3 were evaluated in the absence or presence of a FOXO1 inhibitor (scale bar = 200 μm). ThESCs were treated with METTL3 \nintervention for 24 h and then subjected to decidual treatment with MPA + 8-br-cAMP for 4 d. All experiments were repeated in triplicate \nor quadruplicate. The blots in this figure are cropped (please refer to supplementary files for details). Data with error bars are presented to indicate \nthe mean ± SEM values. *P < 0.05, **P < 0.01, ***P < 0.001\n(See figure on next page.)\n\nPage 9 of 17\nLi et al. Reproductive Biology and Endocrinology           (2023) 21:99 \n \nthat implanted in mice with endometriosis (Fig.  4c). \nNext, we injected an METTL3 inhibitor (STM2457) and \ncontrol solvent (DMSO) individually into the horn of \nthe uterus on opposite sides of the mice with endome -\ntriosis. The number of blastocysts implanted on the side \ninjected with STM2457 was higher than that implanted \non the side injected with DMSO (Fig.  4d). In addition, \nIHC staining showed that FOXO1 expression in stromal \ncells on the STM2457-injected side was higher than that \non the DMSO-injected side (Fig.  4e); however, no dif -\nference in FOXO1 expression was observed in epithelial \ncells (Supplementary Figure S2). Taken together, these \nfindings demonstrate that increased METTL3 expres -\nsion impairs the decidualization of endometrial stromal \nFig. 3 (See legend on previous page.)\n\nPage 10 of 17Li et al. Reproductive Biology and Endocrinology           (2023) 21:99 \nFig. 4 Changes in METTL3 expression were involved in embryo implantation by regulating cellular decidualization in vivo. a Flowchart of animal \nexperiments. b Mouse models of endometriosis were established and the ectopic lesions are shown. c Number of blastocysts in normal \npregnant mice (n = 5) and mice with endometriosis (n = 8) was counted (the left panel, scale bar = 1 cm). d Either the METTL3 inhibitor (STM2457) \nor the control solvent dimethyl sulfoxide (DMSO) were injected into the horn of the uterus on either side of the mice with endometriosis (n = 10). \nThe number of blastocysts per side of the uterus was counted (the left panel, scale bar = 1 cm). e Protein levels of FOXO1 on the DMSO-injected \nand STM2457-injected sides of the mouse uterus were detected by IHC staining on day 8 (scale bar = 100 μm or scale bar = 50 μm). All experiments \nwere repeated in triplicate or quadruplicate. Data with error bars are presented to indicate the mean ± SEM values. ***P < 0.001, ****P < 0.0001\n\nPage 11 of 17\nLi et al. Reproductive Biology and Endocrinology           (2023) 21:99 \n \ncells and thus affects embryo implantation, which might \nlargely contribute to endometriosis-related infertility.\nMETTL3‑mediated m6A promotes the degradation \nof FOXO1 mRNA in a YTHDF2‑dependent manner\nWe then investigated the potential mechanisms by which \nMETTL3 regulates FOXO1 expression during the cel -\nlular decidualization process. Various studies have dem -\nonstrated that m6A is crucial in several RNA processes, \nincluding RNA transcription, splicing, stability, and \ntranslation [26–29], among which RNA stability and \ntranslation are the most widely studied. Based on previ -\nous results, we hypothesized that METTL3 regulates the \nstability of FOXO1  mRNA in an m6A-dependent man -\nner, thus affecting cellular decidualization. To test this \nhypothesis, METTL3-overexpressing cells with decidual \ntreatment were treated with the transcriptional inhibi -\ntor Act D to determine the stability of FOXO1  mRNA. \nThe results showed a lower stability of FOXO1  mRNA \nin METTL3-overexpressing cells (Fig.  5a), suggesting \nthat METTL3 overexpression promotes the decay of \nFOXO1 mRNA during the decidual process. We then \nperformed RNA immunoprecipitation on ThESCs using \nan anti-METTL3 antibody. The results showed that \nMETTL3 coprecipitated with FOXO1  (Fig.  5b). MeRIP-\nPCR using an anti-m6A antibody was then performed to \ntest whether METTL3 regulates FOXO1 expression in \nan m6A-dependent manner. We observed that the level \nof FOXO1 modified by m6A was elevated in METTL3-\noverexpressing cells (Fig.  5c). These results reveal that \nMETTL3-mediated m6A promotes FOXO1 mRNA decay \nduring cellular decidualization.\nFor further verification, we constructed a mutant \n(D395A and W398A, ovM3-MUT) of the METTL3-\noverexpressing vector with disordered enzymatic activity, \nas described previously [24]. We found that the mutant \ncould elevate the expression of METTL3 similar to the \nwild-type METTL3-overexpressing (ovM3-WT) vector \n(Fig.  5d) but failed to elevate the m6A level of FOXO1  \nmRNA in ThESCs compared with transfection with the \novM3-WT vector (Fig.  5e). In addition, the mRNA and \nprotein levels of FOXO1 were decreased in ThESCs \ntransfected with the ovM3-WT vector, but not in cells \ntransfected with the ovM3-MUT vector (Fig.  5f,g). In \naddition, the decay rate of FOXO1 mRNA accelerated \nrapidly in ThESCs transfected with the ovM3-WT vec -\ntor but not in cells transfected with the ovM3-MUT vec -\ntor in the transcription inhibition assay (Fig.  5h). These \nresults indicate that METTL3-mediated m6A promotes \nthe degradation of FOXO1 mRNA.\nAccumulating evidence suggests that YTHDF2 plays \nan important role in regulating RNA stability [28, 30]. \nThus, RIP-PCR using an anti-YTHDF2 antibody was \nperformed to test whether YTHDF2 is involved in the \nMETTL3-mediated regulation of FOXO1  mRNA degra -\ndation. The mRNA level of FOXO1  bound to YTHDF2 \nincreased in ThESCs transfected with the ovM3-WT vec-\ntor but not in cells transfected with the ovM3-MUT vec -\ntor (Fig. 5i), which is consistent with our previous results \n(Fig.  5e), suggesting the involvement of YTHDF2 in the \nMETTL3-mediated regulation of FOXO1  mRNA degra -\ndation. Taken together, we conclude that METTL3-medi-\nated m6A promotes the degradation of FOXO1 mRNA in \na YTHDF2-dependent manner (Fig. 5j).\nDiscussion\nThe findings of the present study indicate that \nMETTL3-mediated m6A modification impairs the \ndecidualization of endometrial stromal cells by pro -\nmoting YTHDF2-dependent degradation of FOXO1  \nmRNA, thus affecting embryo implantation and \nresulting in endometriosis-related infertility to a large \nextent. The main findings were as follows: (a) m6A \nand METTL3 levels in mid-secretory phase endo -\nmetria were decreased compared to those in prolif -\nerative phase endometria in normal fertile women \nFig. 5 METTL3-mediated m6A regulated the degradation of FOXO1 mRNA in a YTHDF2-dependent manner. a The curve and statistical analysis \nof the FOXO1 mRNA decay slope in the negative or METTL3-overexpressing ThESCs after transcriptional inhibition. b RNA immunoprecipitation-PCR \n(RIP-PCR) assays showing an enrichment of FOXO1 bound to METTL3 in ThESCs. c Methylated RNA immunoprecipitation-PCR (MeRIP-PCR) assays \nshowing an enrichment of FOXO1 with m6A in METTL3-overexpressing ThESCs. d Protein levels of METTL3 in the wild METTL3-overexpressing \n(ovM3-WT) and mutated METTL3-overexpressing (ovM3-MUT) ThESCs were analyzed by western blotting. e Methylation of FOXO1 mRNA \nin the ovM3-WT and ovM3-MUT ThESCs were analyzed by MeRIP-PCR. f mRNA levels of FOXO1 in the ovM3-WT and ovM3-MUT ThESCs were \nanalyzed by qRT-PCR. g Protein levels of FOXO1 in the ovM3-WT and ovM3-MUT ThESCs were analyzed by western blotting. h Curve and statistical \nanalysis of the FOXO1 mRNA decay slope in the ovM3-WT and ovM3-MUT ThESCs after transcriptional inhibition. i Enrichment of FOXO1 mRNA \nbound to YTHDF2 in the ovM3-WT and ovM3-MUT ThESCs were analyzed by RIP-PCR. j Model of a pattern of METTL3-mediated m6A in regulating \nthe decidualization of endometrial stromal cells: METTL3 increases the m6A level of FOXO1 mRNA, thus promoting the binding of YTHDF2 \nand enhancing the degradation of FOXO1 mRNA, contributing to the defective decidualization of endometrial stromal cells in endometriosis. All \nexperiments were repeated in triplicate or quadruplicate. The blots in this figure are cropped (please refer to supplementary files for details). Data \nwith error bars are presented to indicate the mean ± SEM values. ***P < 0.001, ****P < 0.0001\n(See figure on next page.)\n\nPage 12 of 17Li et al. Reproductive Biology and Endocrinology           (2023) 21:99 \nwithout endometriosis, while both m6A and METTL3 \nlevels were elevated in the mid-secretory phase endo -\nmetria of women with endometriosis-related infer -\ntility; (b) METTL3 expression gradually decreased \nduring cellular decidualization, contrary to the expres -\nsion of decidual markers, and METTL3 expression \nin decidual-treated ESCs derived from women with \nendometriosis-related infertility was higher than that in \nESCs derived from normal fertile women; (c) increased \nMETTL3 levels impaired the decidualization of ESCs \nby regulating the expression of FOXO1, thus affect -\ning embryo implantation; and (d) METTL3-mediated \nm6A promoted the degradation of FOXO1  mRNA in a \nYTHDF2-dependent manner.\nFig. 5 (See legend on previous page.)\n\nPage 13 of 17\nLi et al. Reproductive Biology and Endocrinology           (2023) 21:99 \n \nIt is widely recognized that m6A plays a crucial role \nin various biological and cellular processes [26–29]. \nThrough the regulation of methyltransferases (writ -\ners), demethylases (erasers), and m6A-binding proteins \n(readers), m6A is dynamically and reversibly involved \nin these processes [15]. Recently, a growing body of evi -\ndence has demonstrated a connection between m6A and \nendometriosis. Down-regulated METTL3, heterogene -\nous nuclear ribonucleoprotein C (HNRNPC), and A2/\nB1 (HNRNPA2B1) have been linked to the development \nof endometriosis [19, 20]. Our prior research demon -\nstrated that the suppression of METTL3 promoted the \nmigration and invasion of endometrial stromal cells in \nendometriosis through the METTL3/m6A/miR126 axis \n[20]. Subsequent studies have provided similar results, \nindicating that reduced METTL3 stimulates the pro -\nliferation, invasion, and migration of endometrial stro -\nmal cells through m6A-mediated differential expression \nof downstream target genes, thus contributing to the \ndevelopment of endometriosis [31–33]. Additionally, \ntwo recent bioinformatics studies have identified several \nm6A regulators associated with endometriosis [34, 35]. \nOne study reported that METTL3 and YTHDF2 were \nidentified as potential diagnostic targets for endometri -\nosis, suggesting the importance of the METTL3-m6A-\nmRNA/long non-coding RNA (lncRNA)-YTHDF2 axis \nin the development of endometriosis [34]. Our present \nstudy also supports the importance of this axis. Spe -\ncifically, we have demonstrated the involvement of the \nMETTL3-m6A-FOXO1-YTHDF2 axis in the decidu -\nalization of endometrial stromal cells. Another study \nhas also identified three different m6A regulators (FTO, \nHNRNPC, and HNRNPA2B1) between the endome -\ntriosis and non-endometriosis groups [35]. Based on \nthe analysis of these three candidate genes, the study \nidentified three molecular subtypes, among which clus -\nterB was found to be highly linked to endometriosis, \nwith high levels of T helper 17 cells, neutrophil infiltra -\ntion, and overexpression of pyroptosis-related genes [35]. \nAlthough numerous studies have emphasized the sig -\nnificance of m6A and its regulators in the pathogenesis \nof endometriosis, our understanding of m6A-associated \nmolecular and cellular events in cellular decidualization \nand endometriosis-related infertility is still limited. Our \npresent study demonstrated that m6A and METTL3 \nwere upregulated in the mid-secretory phase of endome -\ntria from women with endometriosis-related infertility \ncompared to those in fertile women without endome -\ntriosis, and that the upregulation of METTL3 impaired \nthe decidualization of endometrial stromal cells, thus \ncontributing to defective uterine receptivity and poor \nembryo implantation. While our previous studies as well \nas those of others have shown that decreased METTL3 \nis involved in the pathogenesis of endometriosis [20, \n31–33]. Taken all together, METTL3 may have different \nfunctions in the pathogenesis and cellular decidualization \nof endometriosis.\nDecidualization is a fundamental process in which \nendometrial fibroblast-like stromal cells undergo differ -\nentiation to form specialized decidual cells [25]. This pro-\ncess plays an important role in establishing endometrial \nreceptivity, embryo implantation, and placental develop -\nment, as it provides a vital nutritional and immunosup -\npressive substrate [25]. Unlike in most other mammals, \ndecidualization in humans is triggered by an increase \nin progesterone and local cAMP secretion during the \npostovulatory phase of each menstrual cycle [25]. The \ntranscription factor FOXO1 is activated by an increase \nin progesterone and cAMP levels in endometrial stro -\nmal cells, leading to cell cycle arrest and differentiation \nof stromal cells into decidual cells. These decidual cells \nare responsible for encasing and safeguarding the fetal/\nplacental unit during gestation and offer specific endo -\ncrine and immune functions [36]. Therefore, FOXO1 \nis regarded as a vital marker for investigating cellular \ndecidualization.\nIn this study, we investigated the effect of m6A on \nendometrial receptivity during normal physiological \nmenstrual cycles and whether it is associated with defec -\ntive endometrial receptivity in women with endome -\ntriosis-related infertility. To achieve this, we collected \nproliferative and mid-secretory endometria from normal \nfertile women and mid-secretory endometria from infer -\ntile women with endometriosis. Our results showed that \nm6A was expressed at a lower level in the mid-secretory \nphase of the normal endometrium than in the prolifera -\ntive phase, suggesting that the downregulation of m6A \nmay be associated with endometrial receptivity. We \ndetected a series of m6A-related genes and found that \nMETTL3, KIAA1429, and IGF2BPs were downregu -\nlated during the implantation window, whereas YTHDF3 \nwas upregulated. Since METTL3 plays a core role in the \nm6A methyltransferase complex [12], we chose it for our \nin-depth study. Next, we detected the levels of m6A and \nMETTL3 in the mid-secretory phase of the endometria \nof infertile women with endometriosis and found that \nboth were expressed at higher levels in the mid-secretory \nendometria of these women than in those from nor -\nmal fertile women. These results suggest that increased \nm6A/METTL3 expression is associated with defective \nendometrial receptivity in women with endometriosis. \nAdditionally, we treated normal primary stromal cells \nwith MPA and 8-Br-cAMP for in  vitro decidualization \nand found that METTL3 expression decreased further \nwith prolonged treatment time and was accompanied \nby reduced m6A levels. Various studies have shown that \n\nPage 14 of 17Li et al. Reproductive Biology and Endocrinology           (2023) 21:99 \npatients with endometriosis-related infertility have poor \ndecidualization and reduced endometrial receptivity in \nthe eutopic endometrium [9–11, 37–39]. Therefore, we \ntreated two groups of primary stromal cells, one derived \nfrom women with endometriosis-infertility and the \nother from normal fertile women without endometrio -\nsis. We found that m6A and METTL3 levels were higher \nin eutopic stromal cells than in normal stromal cells, \nwhereas FOXO1 levels were lower. These findings indi -\ncate that METTL3 negatively affects the cellular decidual \nprocess, contributing to defective endometrial recep -\ntivity in women with endometriosis. Furthermore, we \ndemonstrated that METTL3 overexpression suppressed \nthe expression of decidualization markers, including \nFOXO1, PRL, and IGFBP1, and embryo implantation, \nwhereas METTL3 knockdown had the opposite effect. \nUsing RIP-PCR and MeRIP-PCR, we demonstrated that \nMETTL3-mediated m6A promoted the decay of FOXO1  \nmRNA in a YTHDF2-dependent manner. In summary, \nwe revealed that METTL3-mediated m6A disrupts the \ncellular decidual process by promoting the YTHDF2-\ndependent decay of FOXO1 mRNA, thereby contributing \nto defective endometrial receptivity in infertile women \nwith endometriosis.\nAdditionally, the expression of IGF2BPs was notably \nlower in the mid-secretory phase endometria of normal \nfertile women than in the proliferative phase endometria. \nIGF2BP family members act as readers of m6A modifi -\ncation and are responsible for recognizing and binding \nthe m6A signature, ultimately carrying out a variety of \ndownstream functions. Previous studies have observed \nlower methylation ratios of IGF2BP2 in the endometrium \non day 15 of gestation than on day 5, indicating that \nIGF2BP2 expression may be vital for embryo implanta -\ntion [40]. IGF2BP3 controls embryonic development via \nalternative splicing of diverse genes [41]. During embry -\nonic development in mice, IGF2BP1 and IGF2BP3 are \nprimarily expressed in the snout, viscera, forebrain, hind-\nbrain, branchial arch, skin, and tail vertebrae of mice [42, \n43], whereas IGF2BP3 gradually degrades during the \nlate embryogenesis stages, and IGF2BP1 remains in the \nkidney, intestine, and liver [44]. Conversely, IGF2BP2 is \ncontinuously expressed in many adult tissues [45, 46]. \nThese findings reveal the essential roles of IGF2BPs in \nregulating embryonic development and suggest that their \naberrant expression may play a role in tissue and organ \ndysplasia. Taken together, we propose that IGF2BPs \nmay perform their functions in establishing endometrial \nreceptivity, though more research is needed to provide \nfurther insights.\nA recent study revealed the negative role of METTL3 \nin embryo implantation, which corroborates our view -\npoint [47]. The study indicated a significant increase \nin global m6A methylation and METTL3 expression \nin the endometrial tissues of women with recurrent \nimplantation failure compared to that in controls [47]. \nOverexpression of METTL3 in Ishikawa cells led to a \ndecrease in the ratio of BeWo spheroid attachment and \nhindered the expression of homeobox A10 (HOXA10) \nand its downstream targets, whereas overexpression \nof HOXA10 in Ishikawa cells effectively restored the \nimpact of METTL3 overexpression on embryo attach -\nment in vitro [47]. Mechanistically, METTL3-mediated \nm6A contributed to the decay of HOXA10, thereby \nshortening its half-life [47]. Our study focused on the \nfunction of METTL3 in the decidualization of endo -\nmetrial stromal cells and revealed a negative effect of \nMETTL3 on the cellular decidual process in endome -\ntriosis, resulting in poor endometrial receptivity and \ndisrupted embryo implantation. Interestingly, we found \nthat the conclusions of two recent articles differed from \nour findings. Both of these studies [48, 49] used Mettl3-\nablated mice and proposed a positive role for METTL3 \nin uterine receptivity and embryo implantation, as well \nas its crucial importance in the transmission of proges -\nterone signals. Therefore, further studies are required \nto determine the effects of m6A/METTL3 on cellular \ndecidualization, endometrial receptivity, and embryo \nimplantation.\nThe uterine epithelium plays an important role \nin embryonic implantation. The uterine epithelium \nincludes the luminal epithelium (LE) and glandular \nepithelium (GE), which extends from the LE to the \nstromal layer. The LE is the first maternal contact for \nan implanted embryo and serves as a transient gate -\nway for embryo implantation and subsequent embryo \ndevelopment in the uterus [50]. During the implanta -\ntion window, blastocyst attachment to the LE results in \ncellular and ultrastructural changes, including gradual \nloss of uterine epithelial cell polarity and formation \nof microprotrusions on the epithelial apical surface, \nwhich are called pinopodes or uterodomes [51, 52]. \nEfficient removal of the epithelial barrier by hatched \nblastocysts is a crucial step in embryo implantation \n[52]. Recent studies have shown that m6A plays an \nimportant role in epithelial development and differ -\nentiation. Mettl3 deletion in the epidermis and oral \nepithelium results in broad developmental defects, \nincluding significant failure of hair morphogenesis, \npremature interfollicular differentiation, and loss of \nfiliform papillae in the tongue [53]. The deletion of \nMettl14 in the murine epidermis impairs the m6A-\ndependent association between the long non-coding \nRNA plasmacytoma variant translocation 1 (Pvt1) \nand MYC, which is critical for the promotion of epi -\ndermal stemness and wound-healing capabilities [54]. \n\nPage 15 of 17\nLi et al. Reproductive Biology and Endocrinology           (2023) 21:99 \n \nThese significant phenotypic abnormalities demon -\nstrate the critical role of m6A in epithelial homeosta -\nsis. Increased METTL3-mediated m6A promotes the \nupregulation of p63 and K14, the downregulation of \nK10, and cell proliferation in cutaneous squamous cell \ncarcinoma [55]. Increased m6A mediated by METTL3/\nMETTL14 promotes tumorigenesis and tumor metas -\ntasis by regulating the stability and translation of onco -\ngenic mRNA in several epithelial cancers [55– 58]. \nThese findings suggest a vital role for m6A in regulat -\ning the development, differentiation, and biological \nfunction of epithelial cells, which provides a direc -\ntion for exploring endometrial receptivity and embryo \nimplantation from the perspective of the association \nbetween m6A and the uterine epithelium.\nConclusion\nOur research reveals that m6A modification regulated \nby METTL3 impairs the decidual process of endome -\ntrial stromal cells by promoting YTHDF2-mediated \ndegradation of FOXO1  mRNA. This ultimately leads to \npoor uterine receptivity and embryo implantation fail -\nure. Our novel findings shed new light on the potential \nof METTL3 as a therapeutic target to enhance cellular \ndecidual function in patients with endometriosis-related \ninfertility and ultimately increase the probability of suc -\ncessful embryo implantation.\nAbbreviations\nm6A  N6-methyladenosine\nMETTL3  Methyltransferase-like 3\nWTAP  WT1 associated protein\nVIRMA  Vir like m6A methyltransferase associated\nESCs  Primary endometrial stromal cells\nDMEM/F12  Dulbecco’s Modified Eagle’s Medium\nMPA  Medroxyprogesterone acetate\n8-Br-cAMP  8-Bromo-cyclic adenosine monophosphate\nThESCs  Human endometrial stromal cells\nqRT-PCR  Quantitative real-time polymerase chain reactioN\nFOXO1  Forkhead box O1\nIHC  Immunohistochemical\nIF  Immunofluorescence\nPRL  Prolactin\nIGFBP1  Insulin-like growth factor-binding protein 1\nDMSO  Dimethyl sulfoxide\nRIP-PCR  RNA immunoprecipitation PCR\nMeRIP-PCR  Methylated RIP-PCR\novM3-WT  Wild-type METTL3-overexpressing vector\novM3-MUT  Mutated METTL3-overexpressing vector\nAct D  Actinomycin D\nHNRNPC  Heterogeneous nuclear ribonucleoprotein C\nHNRNPA2B1  Heterogeneous nuclear ribonucleoprotein A2B1\nlncRNA  Long non-coding RNA\nHOXA10  Homeobox A10\nLE  Luminal epithelium\nGE  Glandular epithelium\nPvt1  Plasmacytoma variant translocation 1\nSupplementary Information\nThe online version contains supplementary material available at https:// doi. \norg/ 10. 1186/ s12958- 023- 01151-0.\nAdditional file 1: Table S1. The sequences of Primers. Table S2. The \nsequences of siRNAs. Figure S1. (a) Statistical analysis panel of FOXO1 \nlevels in ESCs treated with the decidual treatment for 0 or 6 days. (b) Sta-\ntistical analysis panel of METTL3 levels in ESCs treated with the decidual \ntreatment for 0 or 6 days. (c) Dot blot assays were performed to detect \nm6A changes in ESCs derived from normal (ND) and endometriosis (ED) \ngroups, both of which received decidual treatments. Methylene blue \nstaining was used as a control. (d) Protein levels of FOXO1 in ESCs derived \nfrom ND and ED groups, both of which received decidual treatments. \n(e-h) mRNA levels of PRL and IGFBP1 upon METTL3 overexpression or \nknockdown in ThESCs. All experiments were repeated in triplicate or \nquadruplicate. Data with error bars are presented to indicate the mean \n± SEM values. *P < 0.05, **P < 0.01, ***P < 0.001. Figure S2. Statistical \nanalysis panel of the FOXO1 levels in the epithelial cells on the DMSO-\ninjected and STM2457-injected sides of the mouse uterus by IHC staining. \nAll experiments were repeated in triplicate or quadruplicate. Data with \nerror bars are presented to indicate the mean ± SEM values. *P < 0.05, **P \n< 0.01, ***P < 0.001.\nAdditional file 2. \nAcknowledgements\nThe authors thank all the patients who agreed to participate in this study.\nAuthors’ contributions\nConceptualization, X.L., L.Z. and Y.L.; Data curation, X.L.; Formal analysis, X.L.; \nFunding acquisition, W.X. and Y.L.; Investigation, X.L., J.S., J.L. and X.C.; Method-\nology, X.L. and J.J.; Project administration, X.L.; Resources, X.L.; Software, X.L.; \nSupervision, X.L.; Validation, X.L., X.L. (Xuefeng Long), R.W. and J.L. (Junjun Liu); \nVisualization, X.L.; Writing – original draft, X.L.; Writing – review & editing, X.L., \nL.Z. and Y.L. All authors reviewed the manuscript.\nFunding\nThis research was funded by National Natural Science Foundation of China, \n(grant numbers 81974242, 82071722, and U20A20349).\nAvailability of data and materials\nThe data that support the findings of this study are available from the cor-\nresponding author upon reasonable request.\nDeclarations\nEthics approval and consent to participate\nThe study was conducted in accordance with the Declaration of Helsinki \nand approved by the local ethics committee of the Union Hospital, \nTongji Medical College, Huazhong University of Science and Technology \n(approval number 0268). The animal study protocol was approved by \nthe Ethics Committee of the Animal Center of the Tongji Medical College \n(approval number 3332).\nConsent for publication\nNot applicable.\nCompeting interests\nThe authors declare no competing interests.\nAuthor details\n1 Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical \nCollege, Huazhong University of Science and Technology, Wuhan 430022, \nHubei, China. \nReceived: 15 August 2023   Accepted: 13 October 2023\n\n\nPage 16 of 17Li et al. Reproductive Biology and Endocrinology           (2023) 21:99 \nReferences\n 1. Ombelet W, Cooke I, Dyer S, Serour G, Devroey P . Infertility and the provi-\nsion of infertility medical services in developing countries. Hum Reprod \nUpdate. 2008;14:605–21.\n 2. Boivin J, Bunting L, Collins JA, Nygren KG. International estimates \nof infertility prevalence and treatment-seeking: potential need and \ndemand for infertility medical care (vol 22, pg 1507, 2007). 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