Increased METTL3-mediated m(6)A 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 N(6)-methyladenosine (m(6)A) 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 m(6)A levels and major m(6)A methyltransferases/demethylases mRNA levels in mid-secretory endometrium from normal and RIF women were examined by colorimetric m(6)A quantification strategy and quantitative real-time PCR, respectively. The effects of METTL3-mediated m(6)A 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 m(6)A 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 m(6)A 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 m(6)A modification represents an adverse impact on embryo implantation by inhibiting HOXA10 expression, contributing to the pathogenesis of RIF.