Objective: To investigate whether endometrial extracellular vesicles (EVs) from patients with recurrent implantation failure (RIF) attenuate the growth and invasion of embryos. Design: In vitro experimental study. Setting: University-affiliated hospital. Patient(s): Ten RIF patients and seven fertile women. Interventions(s): Endometrial cells isolated from endometrial tissues obtained from patients with RIF and fertile women were cultured and modulated in vitro via hormones. Conditioned medium was collected for EV isolation. Main Outcome Measure(s): EVs secreted by endometrial cells of patients with RIF (RIF-EVs) or fertile women (FER-EVs) were characterized with the use of Western blotting, nanoparticle tracking analysis, and transmission electron microscopy. EVs from the two groups were co-cultured with 2-cell murine embryos. Fluorescence-labeled EVs were used to visualize internalization by embryos. Following co-culture, blastocyst and hatching rates were calculated. Blastocysts were stained with diamidino-2-phenylindole to count the total cell number, and the hatched embryos were used to test invasion capacity. Result(s): RIF-EVs and FER-EVs are bilayered vesicles similar to 100 nm in size and enriched with TSG101, Alix, and CD9. EVs were internalized within 12 hours. The blastocyst rates in the RIF-EV groups were significantly decreased compared with the FER-EV groups at 5, 10, and 20 mu g/mL. The hatching rates and total cell numbers of blastocysts also were decreased significantly in the RIF-EV groups compared with the FER-EV groups at 10 and 20 mu/mL. Moreover, the invasion capacity of hatched embryos decreased significantly in the RIF-EV group. Conclusion(s): Endometrial EVs from patients with RIF attenuate the development and invasion of embryos. ((C) 2020 by American Society for Reproductive Medicine.)