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A modified cryoloop vitrification protocol in the cryopreservation of mature mouse oocytes
Zengyan Wang1, Zhengyi Sun, Ying Chen
1Department of Gynecology and Obstetrics, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing, China.
Abstract:
In this study, we examined a modified cryoloop vitrification protocol in the cryopreservation of mature mouse oocytes. The mature mouse oocytes were first vitrified and then warmed up in a modified cryoloop vitrification medium [15% ethylene glycol (EG) + 15% dimethyl sulphoxide (ME2SO) + 5.8 mg/ml Ficoll 400 (F) + 0.58 mol/l sucrose (S)]. These oocytes were later studied along with fresh oocytes, which served as the control group.Based on the post-warm-up incubation time, the oocytes in the study group were divided into three subgroups: 0 h, 1 h and 2 h. We then examined the configurations of spindles and chromosomes, the fragmentation of DNA, and the oocyte's ability to be fertilized and developed into blastocysts. By evaluating the vitrified oocytes' morphology, we confirmed that 601 out of 612 (98.2%) oocytes survived this protocol. The percentage of oocytes with normal spindle and chromosome configurations in the study groups 0 h, 1 h and 2 h were all quite similar to each other and not statistically different from that of the control group. Similar results were also observed in the percentage of oocytes containing fragmented DNA. The fertilization rate and blastocyst formation rate of the thawed oocytes were not statistically different from that of the control group either. However, if not handled properly (too much remnant medium on oocytes in the process of freezing or too long a time of oocytes in the vitrification medium before freezing), the cryopreserved oocytes could show dramatic difference from the control group in terms of the morphologically survival rate, the configuration of the spindles and chromosomes, and the DNA fragmentation. In conclusion, when followed correctly, this modified cryoloop vitrification protocol had little effect on the survival rate and development potential of mature mouse oocytes.
Insights
This study shows a modified cryoloop vitrification protocol effectively cryopreserves mature mouse oocytes. Proper handling ensures high survival and developmental potential, comparable to fresh oocytes.
Area of Science:
- Reproductive Biology
- Cryobiology
- Developmental Biology
Background:
- Cryopreservation of oocytes is crucial for assisted reproductive technologies.
- Vitrification protocols aim to minimize ice crystal formation during cryopreservation.
- Optimizing vitrification media and techniques is essential for maintaining oocyte viability.
Purpose of the Study:
- To evaluate a modified cryoloop vitrification protocol for mature mouse oocytes.
- To assess the impact of this protocol on oocyte survival, spindle-chromosome configuration, DNA integrity, fertilization, and blastocyst development.
- To identify critical handling steps that may affect cryopreservation outcomes.
Main Methods:
- Mature mouse oocytes were vitrified using a modified cryoloop protocol with a specific vitrification medium (15% EG + 15% ME2SO + 5.8 mg/ml Ficoll 400 + 0.58 mol/l sucrose).
- Vitrified and warmed oocytes were analyzed at 0, 1, and 2 hours post-warming.
- Evaluations included spindle-chromosome configuration, DNA fragmentation, fertilization rates, and blastocyst formation rates, with fresh oocytes serving as controls.
Main Results:
- The modified cryoloop vitrification protocol resulted in a high survival rate (98.2%) of mature mouse oocytes.
- No significant differences were observed in spindle-chromosome configuration, DNA fragmentation, fertilization rates, or blastocyst formation rates between vitrified-warmed oocytes and the control group when the protocol was followed correctly.
- Improper handling, such as excess remnant medium or prolonged exposure to the vitrification solution, led to significantly poorer outcomes.
Conclusions:
- The modified cryoloop vitrification protocol is effective for cryopreserving mature mouse oocytes with minimal impact on their viability and developmental potential.
- Adherence to precise handling procedures is critical for successful oocyte cryopreservation using this method.
- This protocol offers a reliable option for oocyte cryopreservation when executed correctly.
