Three Years Experience WithRoutine Vitrification Of HumanPronuclear Stage Oocytes



Introduction: Cryopreservation of human oocytes and embryos are mandatory tools leading increased cumulative outcome of artifical reproduction techniques while decreasing costs. Vitrification is a cryopreservation technique that leads a glasslike solidification, and rapid freeze of cells and/or tissues. Nowadays it is claimed to be the future of cryopreservation of human embryos due to improved survival rates and clinical outcomes. This study is conducted at a university clinic to asses the safety and efficiency of vitrification of human zygotes as a routine management.
Materials and Methods: This study consisted of 106 cycles of cryopreserved embryo transfer (cryoET) and vitrification of 2PN stage zygotes of 82 patients who underwent IVF/ICSI at department of gynaecology and obstetrics, University of Luebeck/Germany between March 2004 and July 2007. Depending on the case additional PN zygotes were prepared for vitrification. The PN zygotes (all zygotes of the same patient together for time saving) were incubated in Equilibration solution (ES) comprising 7.5% Ethylene Glycol (EG) and 7.5% Dimethy Sulfoxide (DMSO) in Ham´s F-10 medium supplemented with 20% serum for 8-10 min (according to the time needed for reexpansion of the zygote) at room temperature. After an initial shrinkage and recovery, they were then aspirated and placed into the Vitrification Solution (VS) (15% EG + 15% DMSO+0.5M Sucrose) in Ham´s F-10 medium supplemented with 20% serum for a period not more than 60sec at room temperature. After having observed that Cytoplasmic shrinkage has been taken place, zygotes were aspirated and placed on the tip of the Cryotop. No more than two zygotes were placed on each Cryotop. Cooling of the zygotes was done by direct contact with fresh clean liquid nitrogen (LN2). The Cryotops were capped under the LN2 to seal and protect the vitrified material before cryostorage for at least two months. Warming of zygotes was performed by placing the Cryotop in Thawing Solution (TS) (1M Sucrose) for a period not more than 60sec. at a temperature of 37 C and then in to Dilution Solution (DS) (0.5M Sucrose) for three min. followed by another DS of 0.25M Sucrose for additional 3 min. at room temperature. The rewarmed zygotes were washed 8-10 times in culture medium before incubation or culture Sage medium under oil for 24 hours prior to embryo transfer. The survival was assessed morphologically at the day of embryo transfer.
Results: The mean age of the patients was 32.18 (standard deviation+4.47) and the cause of infertility was either male factor infertility or in combination with tubal infertility who were not complicated with hydrosalpinx. A total of 849 PN stage zygotes were vitrified between March 2004 and July 2007. During this era 103 cycles of cryopreserved embryo transfer were completed. Totally 339 PN zygotes were warmed and warming procedure resulted in a 91.15% survival rate (309 PN zygotes). A three times higher pregnancy rate (36.8%) was obtained when compared to the results of slow rate freezing method (10.2%) which was previously used for a long time in the same centre. In conclusion vitrification of Abstract of the 8th Royan International Twin Congress, Tehran, Iran, 5-7 September 2007 24 Yakhteh Medical Journal, Vol 9, Sup 1, Summer 2007 human zygotes at PN stage seems to be a successful and reliable method with favourable outcomes which can be recommended as a routine technique of cryopreserved of human embryos.
Conclusion: Routine vitrification of human zygotes is an efficient and reliable method of cryopreservation of zygotes and seems to improve clinical outcomes of artificial reproductive techniques.