Survival and post-warming in vitro competence of human oocytes after high-security closed system vitrification

Abstract
Introduction: Oocyte cryopreservation has become common practice in many infertility clinics. Most centers use open devices for vitrification and obtain survival rates over 90% and fertilization and pregnancy rates comparable to those obtained in fresh cycles. A drawback of using open systems is the risk of cross-contamination during liquid nitrogen storage. In order to prevent contamination, the use of high-security closed straws (CBS-VIT HS) is an option. The aim of this study was to optimize the procedure for human oocyte cryopreservation in a high-security closed system in terms of survival, fertilization and embryo development.
Material and Methods: The study was approved by the Local and the Federal Ethical Committee. Embryos were created for research after informed consent by the gamete donors. Two vitrification methods and two warming methods, based on the procedures by Irvine Scientific®, were compared in two experiments on human sibling oocytes using CBS-VIT HS closed straws. In the first vitrification method (V1), oocytes were placed immediately in equilibration solution (ES) for 10 minutes. In the second protocol (V2) one droplet of Hepes-buffered Human Tubal Fluid was merged with two ES droplets before moving the oocytes to a fresh ES droplet for 10 minutes. The major difference between the two warming methods was the first step. In the first warming method (W1) oocytes were placed in a 25 µl droplet of preheated (37°C) thawing solution (TS) that was put at room temperature for 1 minute once the oocytes were inside the droplet. In the second warming method (W2) oocytes were placed in a 150 µl TS droplet for 1 minute at 37°C.
In the first experiment, the V1 was combined with the W1 or W2. The second experiment combined the V1 or V2 with W2. The endpoints of both experiments were oocyte survival, fertilization and embryo quality on day 3 and day 6. Statistical analysis was performed by Wilcoxon Signed-Rank test.
Results: In the first experiment with V1, 92 sibling oocytes from 16 donors were used. The rates of survival (65.9% vs 85.4%), fertilization (65.5% vs 85.4%), good-quality day 3 embryos (46.7% vs 77.3%) and good-quality blastocysts (33.3% vs 69.2%) were all significantly higher (P <0.05) after using W2.
For 11 of these 16 donors, the results of 26 vitrified (V1)/warmed (W2) oocytes were compared with their 63 fresh sibling oocytes. Fertilization rate and proportion of good-quality day 3 and day 6 embryos was 76.0% vs. 77.8%, 71.4% vs. 76.2% and 77.8% vs. 64.0% for vitrified and fresh oocytes respectively. No significant differences (P > 0.05) were observed between oocytes warmed with W2 and their fresh sibling counterparts.
In the second experiment with W2, 41 sibling oocytes from 7 donors were used. The rates of survival, fertilization and good-quality day 3 and day 6 embryos were 90.0% vs. 85.7%, 77.8% vs. 72.2%, 76.9% vs. 84.6% and 77.8% vs. 100% for V1 and V2 respectively. No significant differences (P > 0.05) were observed between the two vitrification methods.
Conclusion: The results show that 90% survival rate can be achieved with high-security closed vitrification when the oocytes are immediately warmed in a large droplet at 37°C for 1 minute. When compared to fresh sibling oocytes, these vitrified/warmed oocytes showed the same fertilization rate and in-vitro development. Gradual exposure to cryoprotectants did not provide an added value to the survival rate or in-vitro developmental competence, as compared to direct exposure to cryoprotectants