Abstract

Vitrification is a method for long-term biological sample cryopreservation that transforms cells into a glass-like state by cooling without causing intra- and extra-cellular ice formation, which is a major driver of cell cryoinjury. Compared to slow freezing, another conventional cryopreservation method, vitrification is simple, cost- effective and does not require a complex programmable freezer. Vitrification has been increasingly used to cryopreserve gametes and embryos for fertility preservation in assisted reproductive technology (ART). Moreover, vitrification of individual follicles followed by in vitro maturation (IVM) has emerged as a new fertility preservation method, particularly for childhood cancer patients who have no mature oocytes available for harvesting and for patients who cannot undergo ovarian tissue transplantation after cryopreservation because of the risk of reintroducing malignant cells. However, vitrification of individual follicles has been challenging because intact follicles have a more complex structure and larger size than individual oocytes or early embryos. Traditional oocyte/embryo vitrification methods are not optimized for individual follicles, and have been shown to compromise the qualities of follicles or oocytes, partly by damaging the gap junction between follicular cells or the transzonal projections (TZP) between the oocyte and cumulus cells. In our previous studies, we developed a closed vitrification method for cryopreserving ovarian tissues that was modified for individual follicles. Furthermore, using an alginate hydrogel encapsulated in vitro follicle growth (eIVFG) system, we have recently demonstrated that compared to freshly-harvested follicles, vitrified follicles have normal follicle and oocyte reproductive outcomes as well as comparable expression levels of several genes that are essential for gonadotropin-dependent folliculogenesis and oogenesis. However, it is unknown whether vitrification preserves the molecular signatures of folliculogenesis at the whole transcriptomic level, which is the primary research focus in this study.