Intracellular ice formation (IIF) is the primary cause of cell death during cryopreservation. IIF prevention strategies typically optimize the cooling and warming rates using cryoprotectants. The introduction of trehalose into cells has been shown to significantly enhance cryosurvival rates
however, how intracellular trehalose influences the dynamics of ice formation remains unclear. To address this knowledge gap, this study used an innovative electroporation-based method to efficiently deliver trehalose directly into oocytes and systematically explored its intracellular effect on ice formation in oocytes. Although the trehalose solution exhibited a strong ice-regulating capacity, its efficacy in controlling the intracellular ice dynamics was significantly limited in the extracellular environment. However, intracellular delivery of trehalose significantly inhibited intracellular ice nucleation and growth. Specifically, oocytes treated with 0.15-0.3 M intracellular trehalose exhibited a delayed initial IIF temperature (-55.9 °C) and reduced cumulative probability of cells with IIF (0.52) during cooling to -120 °C. After thawing, the proportion of oocytes with no significant volume change increased to 33.3%, and the extent of cellular damage was significantly alleviated. Collectively, our findings provide theoretical support for the application of intracellular trehalose for cryopreservation and highlight its efficiency in enhancing oocyte cryopreservation, thereby extending its practical applications to reproductive medicine, biobanking, and other fields relying on high-quality cryopreservation.