Improving vitrification efficiency of human in vitro matured oocytes by the addition of LEA proteins

Vitrification cryopreservation of oocytes is an enabling technology for assisted reproductive technology. However, many factors in the vitrification process, such as the toxicity of cryoprotectant, osmotic stress, ice crystal formation during rewarming, will cause fatal damage to oocytes, thus affecting the embryo developmental potential and subsequent clinical outcomes. Therefore, oocyte vitrification still faces significant challenges. Recent studies have shown that LEA protein has the potential to improve oocyte cryopreservation, while the molecular mechanism by which it exerts protective effects is still unclear. Therefore, we have systematically investigated the effects of LEA proteins on the vitrification cryopreservation and their molecular mechanisms. Results revealed that the proteins improved the developmental potential of human oocytes following cryopreservation, mostly by downregulating FOS genes and reducing oxidative stress. Moreover, the synergistic cryoprotection of LEA proteins by inhibiting the formation of ice crystals was given full play. This study provides a new strategy for high-quality cryopreservation of human oocytes. The SMART (Switching Mechanism at 5' End of RNA Template) technique was used to build the library. Using the total RNA of a single cell as a template, the first-strand cDNA was synthesized and amplified to obtain a sample sufficient for sequence analysis. According to the experimental requirements, a control group (0M), 100 µg/mL AfrLEA2 group, 100 µg/mL AfrLEA3m group and fresh group were set up with 3 replicates in each group, and a total of 12 human oocytes were collected from the corresponding condition groups. After washing each oocyte twice in PBS, a volume of less than 5 µl of cells and fluid was aspirated and quickly transferred under a microscope to a clean 200 µl PCR (RNase-free) tube containing lysate, and each sample was placed on dry ice and frozen immediately after the addition of lysate for the next sample. The PCR tubes were grouped and numbered with a marker, and the centrifuge tubes were tightly wrapped with Parafilm sealing film to keep the samples in a frozen state. This step was performed on dry ice as much as possible.