Despite significant advances that have been made in human in vitro fertilization (IVF), a pregnancy can only be established in 35 % of IVF-cycles. To date, selection of human embryos for transfer is based on morphological embryo scoring. Prolonged culture (to day 5 (blastocyst) instead of day 3) can increase implantation rates after transfer, but may cause epigenetic changes in embryos or imprinting disorders in babies born after IVF. Recently, our group has demonstrated that extracellular vesicles (EVs) and their cargo are important regulators of embryo development and can be isolated in a non-invasive way from media in which embryos have been cultured (spent media). First, we hypothesized that we can identify miRNAs derived from EVs secreted by single embryos in day 3 and day 5 spent media, which have the potential to serve as biomarker for successful pregnancy in humans. Second, we hypothesized that epigenetic changes are more obvious in human embryos cultured for 5 days than for 3 days, by identifying differential expression of DNA methyltransferases in embryonic EVs. To this end, we will retrospectively compare the contents (miRNAs and DNA methyltransferases)of EVs isolated from day 3 and day 5 spent media from embryos that did or did not initiate a pregnancy. As such, we may be able to identify biomarkers for implanting embryos already after 3 days of culture. Moreover, we may confirm that more methylation changes are occurring during prolonged culture of human embryos.