Introduction: MicroRNAs (miRNAs) are important regulators of many biological functions, including embryo implantation and development. Recently, it has been reported that miRNAs in biofluids are predictive for physiological and pathological processes. Objectives: In this study, we aim to investigate whether the miRNAs secreted by human embryos in culture medium can be used as embryonic biomarkers. Methods: The culture media were prospectively collected from embryos of patients at reproductive medicine center with informed consent. A high-throughput miRNA sequencing method was applied to detect the miRNA profiles in the human embryo culture media. After bioinformatics analysis and screening of differentially expressed miRNAs, quantitative real-time polymerase chain reaction (qRT-PCR) assay was subsequently performed to further confirm the sequencing results with mixed samples. Furthermore, we performed droplet digital PCR (ddPCR) to verify the target miRNAs at single sample level. Receiver operating characteristic (ROC) analyses were performed for differentially expressed miRNAs. Results: Compared with embryos with failed pregnancy, the embryos with successful pregnancy secreted different miRNA profiles into the culture media, which were predicted to be involved in multiple biological processes. Validated by droplet digital polymerase chain reaction (ddPCR), the expression of hsa-miR26b-5p and hsa-miR-21-5p in the culture media of cleavage embryos with successful pregnancy was significantly lower than that of embryos with failed pregnancy. Moreover, the Receiver Operating Characteristic (ROC) curve analysis indicated that hsa-miR-26b-5p and hsa-miR-21-5p could serve as potential biomarkers for reproductive outcomes. Conclusion: Together, our findings highlight the important predictive potential of miRNAs secreted by human embryos in culture media, which is meaningful for non-invasive embryo selection in assisted reproductive technology. (C) 2021 The Authors. Published by Elsevier B.V. on behalf of Cairo University.