Increased METTL3-mediated m6A methylation inhibits embryo implantation by repressing HOXA10 expression in recurrent implantation failure

Background Recurrent implantation failure (RIF) is a major limitation of assisted reproductive technology, which is associated with impaired endometrial receptivity. Although N6-methyladenosine (m6A) has been demonstrated to be involved in various biological processes, its potential role in the endometrium of women with RIF has been poorly studied. Methods Global m6A levels and major m6A methyltransferases/demethylases mRNA levels in mid-secretory endometrium from normal and RIF women were examined by colorimetric m6A quantification strategy and quantitative real-time PCR, respectively. The effects of METTL3-mediated m6A modification on embryo attachment were evaluated by an vitro model of a confluent monolayer of Ishikawa cells co-cultured with BeWo spheroids, and the expression levels of homeo box A10 (HOXA10, a well-characterized marker of endometrial receptivity) and its downstream targets were evaluated by quantitative real-time PCR and Western blotting in METTL3-overexpressing Ishikawa cells. The molecular mechanism for METTL3 regulating HOXA10 expression was determined by methylated RNA immunoprecipitation assay and transcription inhibition assay. Results Global m6A methylation and METTL3 expression were significantly increased in the endometrial tissues from women with RIF compared with the controls. Overexpression of METTL3 in Ishikawa cells significantly decreased the ration of BeWo spheroid attachment, and inhibited HOXA10 expression with downstream decreased β3-integrin and increased empty spiracles homeobox 2 expression. METTL3 catalyzed the m6A methylation of HOXA10 mRNA and contributed to its decay with shortened half-life. Enforced expression of HOXA10 in Ishikawa cells effectively rescued the impairment of METTL3 on the embryo attachment in vitro. Conclusion Increased METTL3-mediated m6A modification represents an adverse impact on embryo implantation by inhibiting HOXA10 expression, contributing to the pathogenesis of RIF.


Background
Embryo implantation is an important and complex process in the establishment of pregnancy in mammals, which requires the simultaneous development of highquality embryos and endometrial receptivity [1][2][3]. Endometrium is one of the most dynamic tissues in human body. Under the action of steroids during sexual cycle, endometrium undergoes cyclic developmental changes and highly ordered differentiation, leading to be receptive to blastocyst implantation ∼6 days after ovulation and remains receptive for 4 days (cycle days [20][21][22][23][24] [4,5]. Endometrial receptivity deficiency may be the critical factor for women with recurrent implantation failure (RIF) who have high-quality embryos but undergo repeated implantation failure following in vitro fertilization-embryo transplantation (IVF-ET) treatment [6]. Several different signaling pathways and their associated genes have been demonstrated to be involved in the adjustment of endometrial receptivity [5,7], in which homeo box A10 (HOXA10) has emerged as an important and well-characterized biomarker. The expression of HOXA10 is dynamic through the menstrual cycle and significantly increased at the time of implantation with increased progesterone levels [8,9]. The gene HOXA10 has a highly conserved homeodomain that specifically recognizes the TTAT sequence in the promoter of downstream target genes, leading to expression changes of target genes, including β3-integrin (ITGB3) and empty spiracles homeobox 2 (EMX2) [10][11][12]. Studies have shown that ITGB3 expression is directly up-regulated by HOXA10 [10], whereas EMX2 expression is inhibited by HOXA10 [12]. ITGB3 is a transmembrane glycoprotein that presents on the surface of cells, which participates in cell adhesion and cell-surface-mediated signaling during embryo implantation [13][14][15]. EMX2 is a crucial transcription factor necessary for reproductive tract differentiation and development, but changes in endometrial EMX2 expression levels usually lead to abnormalities of the endometrium [16,17]. Accumulating studies indicate that decreased HOXA10 expression contributes to the failure of embryo implantation [18][19][20][21]. However, the expression of HOXA10 and its underlying mechanisms for epi-transcriptomic regulation of HOXA10 in RIF remain to be characterized. N 6 -methyladenosine (m 6 A) is the most abundant internal modification in messenger RNAs (mRNAs). The m 6 A modification is catalyzed by "writers" methyltransferases, including methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), RNA binding motif protein 15 (RBM15) and Wilms tumor 1-associated protein (WTAP). Meanwhile, m 6 A modification can be removed by "erasers" demethylase, such as fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5). In addition, m 6 A "readers" are responsible for recognition of the m 6 A modification [22]. Members of every classes of m 6 A regulators cooperatively participate in the regulation of mRNA stability and translation, affect gene expression output and thus play an important role in physiological and pathological conditions [22][23][24][25][26]. In the m 6 A methyltransferase complex, METTL3 functions as the the catalytic core while METTL14 serves as the RNA-binding platform [27]. METTL3 is a transferase that methylates mRNA, identifies methylated mRNA, and regulates mRNA translation. Recently, accumulating studies have identified multiple roles and molecular mechanisms associated with METTL3 in various biological processes [28][29][30]. However, whether METTL3mediated m 6 A modification is involved in the regulation of endometrial receptivity and how this relates to RIF remains unclear.
Herein, we found that both the levels of global mRNA m 6

Patient samples and ethical approval
The patients enrolled in this study were recruited from in vitro fertilization unit of Reproductive Medicine Center of the Affiliated Changzhou Maternity and Child Health Care Hospital of Nanjing Medical University. All the endometrial samples were collected with written informed consent of the patients, and approval from the Scientific Research Ethics Committee was obtained for this study (2020103). The mid-secretory phase endometrial tissues were collected by endometrial biopsy from normal women and women with RIF according to the criteria described previously [19]. The normal control group was composed of women who were infertile due to male factors and proved to be fertile after the IVF-ET treatment. RIF was defined as the absence of implantation following two fresh or frozen embryo replacement cycles, during which at least four embryos with good quality were transferred to uterus. Women with endometriosis, adenomyosis, hydrosalpinx, uterine malformation, endometrial polyps or autoimmune disease were not included. The details of these patients are summarized in Table 1.

Cell culture
The human endometrial adenocarcinoma cell line Ishikawa was purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and maintained in MEM medium (Thermo Fisher Scientific, Waltham, MA, USA). The human placental choriocarcinoma cell line BeWo was purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in Ham's F-12K (Kaighn's) medium (Thermo Fisher Scientific). These mediums contain 10% fetal bovine serum (FBS; Thermo Fisher Scientific), 100 U/mL penicillin, and 100 mg/mL streptomycin (HyClone, South Logan, UT, USA). Cells were incubated at 37°C with 5% CO 2 . Actinomycin D (S8946; Selleck Chemicals) was added for the indicated times at a final concentration of 10 μg/mL for the transcription inhibition assay.

Quantification of m 6 A RNA methylation
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), followed by the purification of polyadenylated mRNA using Dynabeads mRNA Purification Kit (Thermo Fisher Scientific) according to manufacturer's protocol. An m 6 A RNA Methylation Assay Kit (Abcam, Cambridge, MA, USA) was used to evaluate the m 6 A content of total RNA according to the manufacturer's instructions, as previously reported [31]. Equal amounts of total RNA (200 ng) were bound to strip wells using a RNA high binding solution. The m 6 A was captured and detected using the specific capture antibody and detection antibody. Then, the detected m 6 A signal was enhanced using enhancer solution, and quantified colorimetrically after adding color developing solutions by reading the absorbance at a wavelength of 450 nm in a microplate spectrophotometer.

Dot blotting assay
A dot blotting assay was performed essentially as previously reported [32]. Total RNA or poly (A) + mRNA was isolated as described above. Equal amounts of total poly (A) + mRNA samples (2 μg) were denatured at 65°C for 5 min. Then the samples were loaded onto nylon membranes (GE Healthcare, USA) with ice-cold 20× saline sodium citrate solution (Sigma Aldrich) in a dot blot apparatus (Bio-Rad, USA). The membranes were then UV-crosslinked for 5 min, blocked with 5% non-fat milk for 1 hour, incubated with an m 6 A antibody (1:400; ab151230, Abcam) overnight at 4 °C and horseradish peroxidase-conjugated anti-rabbit IgG for 1 hour at room temperature, and finally detected with a 3,3'-diaminobenzidine peroxidase substrate kit. At the same time, the same poly (A) + mRNA (2 μg) samples were spotted onto membranes, UV-crosslinked twice, stained with 0.02% methylene blue in 0.3 M sodium acetate for 2 hours, and washed with ribonuclease-free water for 5 hours, followed by the scanning to indicate the total content of input RNA.

Quantitative real-time PCR (qRT-PCR)
Total RNA was lysed using TRIzol reagent and used for the synthesis of cDNA with a One-Step RT-PCR Kit (Thermo Fisher Scientific). Reactions of qRT-PCR were performed using the ABI Vii7 system (Applied Biosystems, USA). GAPDH was used as a housekeeping gene. Relative gene expression was calculated by the 2 -△△CT cycle threshold method [33]. The primers used for qRT-PCR analysis are listed in Table 2.

In vitro embryo implantation assay
We used multicellular spheroids of human placental choriocarcinoma BeWo cells co-cultured with a confluent monolayer of endometrial adenocarcinoma Ishikawa cells as an in vitro model of embryo attachment [19]. First, a single-cell suspension of BeWo cells was placed in a 35 mm 2 culture dish pre-coated with an anti-adhesive polymer poly-2-hydroxyethyl methacrylate (Sigma Aldrich

Statistical analysis
Data are presented as mean ± SD of at least three independent experiments. Statistical analyses were performed using GraphPad Prism 9 software (La Jolla, CA, USA). Differences between group means were evaluated with the Student's t-test or one-way analysis of variance. P < 0.05 shows a statistical significance.

Upregulation of m 6 A modification and METTL3 in the endometrial tissues of women with RIF
To explore the potential role of m 6 A modification in RIF, we first examined global m 6 A levels in total RNA from mid-secretory phase endometrial tissues of normal and RIF women by colorimetric m 6 A quantification strategy. We found that endometrial m 6 A levels were significantly increased in the RIF patients than in the controls (Fig. 1a). This increase was further confirmed by dot blotting assay (Fig. 1b). Then, we detected the mRNA levels of major m 6 A methyltransferases (METTL3, METTL14, RBM15, WTAP and VIRMA) and demethylases (FTO and ALKBH5) in the normal and RIF endometrial tissues. The m 6 A methyltransferases (METTL3, METTL14, and RBM15) and demethylase FTO were significantly increased in the RIF endometrial tissues compared with  normal controls, while the m 6 A methyltransferase WTAP was significantly decreased in the RIF endometrial tissues (Fig. 1c). However, there were no significant differences in the mRNA levels of VIRMA or ALKBH5 between normal and RIF patients. Considering the increased m 6 A modification in RIF endometrium and the catalytic abilities of these m 6 A regulators, we selected METTL3 as the candidate molecule for further studies of aberrant m 6 A modification in RIF. Protein levels of METTL3 in the RIF endometrial tissues were significantly increased in comparison to normal controls (Fig. 1d), similar to the result obtained from qRT-PCR.

METTL3 overexpression impairs embryo attachment in vitro
To further investigate the effects of METTL3-mediated m 6 A modification on embryo attachment, we established METTL3 overexpression cell models in Ishikawa cells by lentivirus. The efficiency of overexpressing METTL3 at the mRNA and protein levels were verified by qRT-PCR (Fig. 2a) and Western blotting (Fig. 2b), respectively. We found that METTL3 overexpression significantly enhanced total m 6 A levels in Ishikawa cells, as indicated in the colorimetric m 6 A quantification assay (Fig. 2c) and dot blotting assay (Fig. 2d). In an vitro model of a confluent monolayer of Ishikawa cells co-cultured with BeWo spheroids, METTL3 overexpression significantly decreased the ration of BeWo spheroids attachment (Fig. 2e). As HOXA10 is a well-characterized marker of endometrial receptivity and a critical upstream regulator of ITGB3 and EMX2, we then evaluated the expressions of HOXA10 and its downstream targets in METTL3-overexpressing Ishikawa cells. Notably, significant decreases in both HOXA10 mRNA (Fig. 2a) and protein (Fig. 2b) levels were observed in METTL3-overexpressing Ishikawa cells. Moreover, the METTL3-overexpressing Ishikawa cells exhibited a lower expression of ITGB3 and a higher expression of EMX2 compared with the control cells ( Fig. 2a and b). Collectively, METTL3

METTL3 epigenetically decreases HOXA10 expression
We next investigated the mechanism by which METTL3 participates in the regulation of HOXA10 and its downstream targets expressions, contributing to the impairment of embryo implantation. We first assessed the mRNA levels of HOXA10 in endometrial tissues from RIF and normal subjects. The mRNA levels of HOAX10 were significantly decreased in the endometrial tissues from RIF patients (Fig. 3a). Then, we analyzed transcriptome m 6 A mapping data by an online m 6 A modification site predictor (http:// www. cuilab. cn/ sramp/) and found that at least 11 m 6 A residues were located across the HOXA10 sequence. Consistently, METTL3 overexpression significantly enhanced m 6 A methylation of HOXA10 mRNA in Ishikawa cells (Fig. 3b). When we generated a mutated METTL3 (D395A and W398A) construct with disordered enzymatic activity, as described previously [35], we found that the mutated METTL3 (D395A and W398A) failed to elevate the m 6 A methylation of HOXA10 mRNA in Ishikawa cells (Fig. 3b). The levels of HOXA10 mRNA and protein were both decreased by wild-type METTL3, but not mutated METTL3 (D395A and W398A) in Ishikawa cells (Fig. 3c and d). Furthermore, the decay rate of HOXA10 mRNA was accelerated rapidly by wild-type METTL3, but not mutated METTL3 (D395A and W398A), in the transcription inhibition assay (Fig. 3e). These results demonstrated that METTL3 epigenetically decreases HOXA10 expression.

HOXA10 overexpression rescues METTL3-impaired embryo attachment in vitro
To determine whether decreased HOXA10 expression was responsible for the reduced ration of BeWo spheroids attachment upon METTL3 overexpression in Ishikawa cells, we attempted to rescue this phenotype by overexpressing HOXA10. The efficiency of METTL3 and HOXA10 overexpression were verified by qRT-PCR (Fig. 4a) and Western blotting (Fig. 4b). As above, METTL3 overexpression induced corresponding changes in the expressions of HOXA10, ITGB3 and EMX2, whereas HOXA10 overexpression dramatically enhanced the expression of ITGB3 and decreased the expression of EMX2 despite in the METTL3-overexpressing Ishikawa cells ( Fig. 4a and b). We further found that overexpression of METTL3 dramatically increased total m 6 A levels with or without HOXA10 overexpression in Ishikawa cells ( Fig. 4c and d). In the vitro model of a confluent monolayer of Ishikawa cells co-cultured with BeWo spheroids, HOXA10 overexpression significantly reversed the METTL3-decreased ration of BeWo spheroid attachment (Fig. 4e). Collectively, these results suggested that METTL3 impaired embryo attachment in vitro in a HOXA10-dependent manner.

Discussion
With the application of high-throughput sequencing for m 6 A mapping in RNA, the understanding of its internal regulatory mechanism is being revealed. At present m 6 A has been recognized as the most prevalent internal modification in mRNAs. The m 6 A modification in mRNAs could influence mRNA stability and splicing, translation efficiency, nuclear output, and selective polyadenylation. The m 6 A modification is maintained by three different groups of RNA binding proteins, including m 6 A writers, erasers and readers. Dynamic and reversible nature of m 6 A modification makes it play a key role in cellular communications [22][23][24][25][26][27][28][29][30][31][32]. In the current study, we found that the levels of m 6 A-modified RNAs and the critical methyltransferase METTL3 were significantly upregulated in the endometrial tissues of RIF. METTL3 overexpression inhibited the endometrial receptivity biomarker HOXA10 expression and impaired the embryo attachment in vitro. These results suggested that METTL3-mediated m 6 A modification is an important determinant of embryo implantation, and that increased METTL3 expression might contribute to the pathogenesis of RIF. Deregulation of m 6 A modification has been recently implicated in endometrial diseases [36][37][38]. Jiang et al. [36] analyzed the expressions of 20 m 6 A regulators in 34 normal, 127 eutopic, and 46 ectopic samples of endometrium tissue from different menstrual cycle phases which were merged from public microarray datasets of endometriosis, and found that most m 6 A methylation regulators in endometriosis were abnormal in the eutopic vs. normal endometrium, including decreased METTL3/METTL14/ RBM15/FTO and increased ALKBH5. Moreover, METTL3 expression in endometriosis was reduced in the ectopic vs. eutopic endometrium while FTO expression was elevated. Functional, co-expression, correlation analyses of proliferative phase endometrial tissues from adenomyosis vs. controls found that decreased METTL3 expression in adenomyosis led to declining total m 6 A levels and the downstream increased insulin-like growth factor-1 (IGF1) and D-Dopachrome Tautomerase (DDT); and it revealed that IGF1 and DDT might correlate with epithelial cell proliferation and migration, both of which are involved in the pathogenesis of adenomyosis [38]. In addition, ∼70% of endometrial tumors exhibited reductions in m 6 A methylation that are due to either METTL14 mutation (R298P) or decreased METTL3 expression. Reductions in m 6 A methylation decreased the negative AKT regulator PHLPP2 expression and increased the positive AKT regulator mTORC2 expression, which contributed to increased proliferation and tumorigenicity of endometrial cancer cells through the activation of AKT pathway [37]. In the present study, increased METTL3 expression and m 6 A levels were found in the endometrial tissues from RIF. Overexpression of METTL3 increased m 6 A methylation, and impaired embryo attachment in vitro by inhibiting the endometrial receptivity biomarker HOXA10 expression. These studies suggest that m 6 A methylation is involved in the pathogenesis of endometrial diseases, including endometriosis, adenomyosis, endometrial cancers and RIF, all of which shares some characteristics with each other. However, different status and pattern of m 6 A methylation and its regulators may be found in the endometrial tissues from different endometrial diseases, different menstrual cycle phases, and even from different sites in the same patient.
Embryo implantation is a subtle and complicated process that requires accurate communication between high-quality embryos and receptive endometrium under the action of maternal hormones and their downstream molecules [1][2][3]. During ∼6 days after ovulation, ovarian estrogen and progestin cooperatively induces the morphological and physiological changes of epithelial cells in the endometrium and secretion of various cytokines. These transformations cause the uterus to be receptive to blastocyst implantation [4,5]. The transcription factor homologue HOXA10 has emerged as an important and well-characterized biomarker of endometrial receptivity. The expression of HOXA10 in the uterus depends on the stage of menstrual cycle, which is significantly increased in the mid-secretory phase, corresponding to the implantation time and the increase of progesterone level [8,9]. Both estrogen and progestin independently and synergically elevated the expression of HOXA10 in endometrium [8]. In turn, HOXA10 regulates endometrial acceptance and decidualization activation or compression by downstream markers specific to the window of implantation [10][11][12][18][19][20][21]. Abnormal expression of HOXA10 and its downstream target genes leading to decreased endometrial receptivity are closely related to female infertility in the patients with gynecological diseases, such as endometriosis [11,20,21], adenomyosis [39,40], and hydrosalpinx [41]. The importance of maternal HOXA10 expression in embryo implantation has been demonstrated by a targeted disruption of the Hoxa10 gene in mice. Female mice with deletion of Hoxa10 gene were infertile due to endometrial receptivity defects [42]. Small ubiquitin like-modifier 1 (SUMO1) inhibited HOXA10 protein stability and transcriptional activity via sumoylation at the evolutionarily conserved lysine 164 residue in the endometrium of women with RIF, which impairs endometrial receptivity and embryo implantation [19]. In our study, we found that wildtype METTL3, but not mutated METTL3 (D395A and W398A), decreased the expression of HOXA10 due to increases in the m 6 A methylation of HOXA10 mRNA in Ishikawa cells. Enforced expression of HOXA10 in Ishikawa cells effectively rescued the impairment of METTL3 on the embryo attachment in vitro. Jiang et al. [19] found that increased SUMO1-modified HOXA10 expression without changes of HOXA10 expression was detected in the mid-secretory endometrium of women with RIF; however, increased m 6 A content in total RNA with decreased HOXA10 expression was found in our study. The difference may be due to individual difference and limitation of sample size.

Conclusions
In conclusion, increased m 6 A content in total RNA with high METTL3 expression was found in the mid-secretory endometrium of women with RIF compared with that of the control fertile women. METTL3 catalyzed the m 6 A methylation of HOXA10 mRNA and repressed the expression of HOXA10 leading to the impairment on embryo attachment in vitro. However, global RNA m 6 A methylation in the endometrium from women with RIF during the window of implantation is not restricted to METTL3/HOXA10, and further studies are required to investigate the function of m 6 A methylation in endometrial receptivity and embryo implantation.