Single‐Embryo RNA Sequencing Reveals Transcriptomic Alterations in Blastocysts Derived From IVM or Vitrified Oocytes

In vitro production (IVP) of bovine embryos often results in lower developmental competence compared to in vivo-derived embryos. This disparity is further exacerbated by oocyte cryopreservation (vitrification). This study aimed to investigate the molecular basis of reduced embryo quality by profiling the transcriptomic impact of in vitro maturation (IVM) and vitrification on bovine blastocysts. Day 7 blastocysts from three groups were analyzed: (1) in vivo: control embryos produced entirely in vivo, (2) IVF: embryos from fresh oocytes subjected to IVM and in vitro fertilization, and (3) VIVF: embryos from vitrified-thawed oocytes subjected to IVM and fertilization. Whole-blastocyst transcriptome profiling was performed using single-embryo RNA sequencing (SUPeR-seq; n = 3 blastocysts per group) and analyzed without cellular dissociation. Developmental competence was significantly reduced in the VIVF group (~ 11. 8% blastocyst rate) compared to the IVF group (~ 41. 5%) and in vivo controls. Transcriptome analysis revealed 362 differentially expressed mRNAs in VIVF embryos and 78 in IVF embryos compared to in vivo controls. Genes such as ATP1A1, GOT1L1, and QSOX1 were significantly altered, influencing pathways related to oxidative phosphorylation, lysosome activity, and chromatin organization. In addition, 694 DE-lncRNAs in the VIVF group and 187 in the IVF group were identified. Exonic circRNAs (608) were detected with differential expression and enrichment in signaling and epigenetic pathways, including Hedgehog signaling and stem cell pluripotency regulation. Integrated network analysis revealed coordinated dysregulation across mRNA, lncRNA, and circRNA layers, suggesting disruption of multi-layered regulatory circuits. Overall, this study presents a detailed transcriptomic landscape of bovine blastocysts affected by IVM and vitrification.