PREGNANCY ACHIEVED WITH PRONUCLEAR-STAGE EMBRYOS THAT WERE CRYOPRESERVED AND THAWED TWICE - A CASE-REPORT

Purpose: Our purpose was to determine if pronuclear-stage embryos (2PN ) could be thawed, then frozen again with subsequent survival and clea vage after thawing. Methods: A simplified cryopreservation protocol wa s used in which a slow cooling program is started at the seeding tempe rature of -6 degrees C in an alcohol-bath controlled-rate freezer 1,2- Propanediol (1.5 M) was added to embryos before cooling. A fast thawin g technique at room temperature was used. The cryoprotectant was remov ed in one step using a 1 M sucrose solution. Results: Three months aft er refreezing, the three 2PN embryos were thawed and all three cleaved after 24 hr in culture. Following embryo transfer a pregnancy was ach ieved and a healthy full-term baby girl was born. Conclusions: This is the third case reported of successful pregnancies after transfer of h uman embryos that were frozen twice before transfer and the first case where the second freeze occurred at the pronuclear stage. This is als o the first successful refreezing of human embryos using a simplified freezing and thawing technique with one-step addition and removal of c ryoprotectant.