Lei Cai,1,* Mingwei Lv,2,3,* Jianbo Wei,1 Chang Liu,4 Yuehan Li,1 Zhiqi Liao,1 Tianhui Li,5 Hanwang Zhang,1 Ling Xi,2,3 Cong Sui1 

1Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, People’s Republic of China; 2Department of Gynecological Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China; 3National Clinical Research Center for Obstetrics and Gynecology, Cancer Biology Research Center, Key Laboratory of the Ministry of Education, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China; 4Reproductive Medicine Center, Department of Obstetrics and Gynecology, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medicine School, Nanjing, 210000, People’s Republic of China; 5State Key Laboratory of Digestive Disease, Li Ka Shing Institute of Health Sciences, Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong

*These authors contributed equally to this work

Correspondence: Cong Sui, Email csui0904@163.com

Background: Recurrent implantation failure (RIF) presents a crucial obstacle to in vitro fertilization success. Previous research has shown that small extracellular vesicles (EVs) from endometrial RIF patients hinder embryo development, yet the underlying mechanism and potential solutions remain largely unexplored. In this study, we aimed to investigate the effectiveness of miR-218-5p as a molecular factor in RIF-EVs. Our findings revealed that miR-218-5p disrupted mouse embryo development, and this effect could be reversed by engineered extracellular vesicles (E-EVs) containing anti-miR-218-5p.
Methods: The percentage of blastocyst development and hatching rates, embryo morphology, and the total cell number were measured. RNA-sequencing was used to analyze transcriptional changes in embryos post miR-218-5p agomir treatment. The abnormal segregation genes of trophectoderm (TE) and inner cell mass (ICM) were visualized via qRT-PCR and immunofluorescence staining. The E-EVs were using the EVs derived from Human Umbilical Cord Mesenchymal Stem Cells (HUMSCs). Characteristics of the EVs were measured using Western blotting, nanoparticle tracking analysis, and transmission electron microscopy. EVs internalization was visualized using BODIPY TR ceramide staining.
Results: Mouse embryos were arrested at the morula stage and demonstrated reduced blastocyst and hatching rates following miR-218-5p agomir treatment (P < 0.001). Essential transcription factors for TE and ICM, such as Cdx2, Yap1, Sox2, Nanog, Tead4, were reduced at the mRNA level in the miR-218-5p treated morula. This was accompanied by decreased Cdx2 protein levels at the 8– 16-cell stage (P < 0.001) and disruption of co-localization of Yap1 and Cdx2. The blastocyte rate was increased by anti-miR-218-5p-encapsulated E-EVs compared with miR-218-5p group (P < 0.001).
Conclusion: This study offers valuable insights into the potential role of miR-218-5p in RIF and presents. The utilization of engineered vesicles containing anti-miR-218-5p may present a promising avenue for patients facing challenges with RIF.
Plain Language Summary: The phenomenon of recurrent implantation failure (RIF) is a common occurrence in patients undergoing the assisted reproductive technology (ART) process. It intensifies the strain on patients, but as of now, there is no universally accepted definition, cause, or therapeutic strategy. RIF can result from various factors, and recently, the impact of small vesicles released from cells called extracellular vesicles (EVs) has garnered increasing attention in the disease process of RIF. However, there is still a lack of mechanistic evidence and therapeutic recommendations. In this study, we discovered that miR-218-5p is one of the functional molecules inside the RIF-EVs, and it disrupts the development of the embryo into a blastocyst. Furthermore, we found a deeper mechanism related to the distribution of the formation of the trophectoderm (the embryo’s outer cell). This provides a plausible explanation for one form of embryonic disorder associated with RIF. Most significantly, our work introduces a novel therapeutic approach, employing engineered EVs encapsulating anti-miR-218-5p, which effectively mitigates the deleterious effects of miR-218-5p. This research provides a novel and non-invasive insight into potential therapeutic strategies for RIF.

Keywords: recurrent implantation failure, extracellular vesicles, microRNA, pre-implantation embryo, engineered extracellular vesicles