Round spermatid injection (ROSI) is an in vitro fertilisation method used to generate offspring from infertile male patients lacking mature spermatozoa. Despite the healthy development of these embryos, ROSI results in lower birth rates than conventional intracytoplasmic sperm injection (ICSI), which uses fully matured spermatozoa. Previous studies have alluded to epigenetic differences and associated altered chromatin structures being responsible for the observed reduction in birth rates. In this study, Masatoshi Ooga and colleagues profile fertilised mouse ROSI and ICSI embryos using ATAC- and RNA-sequencing to characterise epigenetic and chromatin discrepancies. They identify a repressive methylation mark, tri-methylation of histone H3 at lysine 27 (H3K27me3), found at a higher level only in ROSI embryos, which decreases chromatin accessibility and thus impairs gene expression. It is known that major epigenetic remodelling occurs during spermatozoa maturation (spermiogenesis), and the authors show there that this includes loss of H3K27me3. Therefore, paternal H3K27me3 marks from round spermatids, which have not undergone spermiogenesis, are inherited into ROSI embryos. Consequently, gene transcription profiles of ROSI embryos differ from those in ICSI embryos during zygotic genome activation. Thus, this paper highlights the importance of epigenetic regulation in fertilisation and embryogenesis, and provides a potential explanation for the reduced success rate of ROSI embryo development.