Advanced maternal age (AMA) is known to be related to the decrease in the quality and quantity of oocytes. Oocyte vitrification is now considered an established assisted reproductive technology for fertility preservation. However, it remains unclear whether the oocytes in older women are more sensitive to various insults during vitrification. Thus, we evaluated whether AMA affects cellular and molecular features and developmental outcomes of oocytes after vitrification in mice. The oocytes were grouped as young fresh (YF), young vitrified/warmed (YV), aged fresh (AF), and aged vitrified/warmed (AV). The survival rate of AV oocytes was significantly lower than that of YV oocytes. The rates of fertilization, cleavage, and blastocyst formation of AV oocytes were significantly lower than those of other groups. AV oocytes were represented as aberrations in mitochondria distribution, microvacuole size, and autophagosome formation, leading to delayed embryo development in mice. This delay was associated with a reduced number of total cells and trophectoderm in the blastocyst developed from AV oocytes. Collectively, AMA exaggerates the vulnerability of oocytes to cryo-damage that occurs during vitrification in mice, suggesting that the current vitrification protocols optimized for oocytes from young females should be modified for oocytes from aged women.

The open carrier system (OC) is used for vitrification due to its high efficiency in preserving female fertility, but concerns remain that it bears possible risks of cross-contamination. Closed carrier systems (CC) could be an alternative to the OC to increase safety. However, the viability and developmental competence of vitrified/warmed (VW) oocytes using the CC were significantly lower than with OC. We aimed to improve the efficiency of the CC. Metaphase II oocytes were collected from mice after superovulation and subjected to in vitro fertilization after vitrification/warming. Increasing the cooling/warming rate and exposure time to cryoprotectants as key parameters for the CC effectively improved the survival rate and developmental competence of VW oocytes. When all the conditions that improved the outcomes were applied to the conventional CC, hereafter named the modified vitrification/warming procedure using CC (mVW-CC), the viability and developmental competence of VW oocytes were significantly improved as compared to those of VW oocytes in the CC. Furthermore, mVW-CC increased the spindle normality of VW oocytes, as well as the cell number of blastocysts developed from VW oocytes. Collectively, our mVW-CC optimized for mouse oocytes can be utilized for humans without concerns regarding possible cross-contamination during vitrification in the future.