Cryoloop vitrification yields superior survival of Rhesus monkey blastocysts

BACKGROUND: Vitrification using the cryoloop procedure was evaluated for pr eservation of non-human primate blastocysts by comparing survival results f rom two different cryoprotectant mixtures with prior results from controlle d rate cooling. METHODS: Rhesus monkey blastocysts were produced by intracy toplasmic sperm injection of mature oocytes from cycling females stimulated with recombinant human hormones. Morphologically well-formed blastocysts w ere divided between Procedure A (2.8 mol/l dimethylsulphoxide and 3.6 mol/l ethylene glycol with 0.65 mol/l sucrose and 25 mu mol/l Ficoll in TALP-HEP ES with 20% fetal bovine serum (TH20)) and Procedure B (3.4 mol/l glycerol and 4.5 mol/l ethylene glycol in TH20). After >48 h in liquid nitrogen, the removal of cryoprotectants was accomplished in the presence of a 3-step se ries of decreasing sucrose concentrations in TH20. Surviving embryos were c o-cultured on buffalo rat liver cells. RESULTS: Of 16 blastocysts vitrified via Procedure A, 38% survived with minimal lysis and only one hatched in c ulture; in contrast, of 33 blastocysts vitrified by Procedure B, 85% surviv ed and 71% hatched. Of 22 blastocysts cryopreserved by conventional slow co oling, 36% survived and 6% hatched. Transfer into three recipients, each wi th two embryos vitrified with Procedure B, resulted in a successful twin-te rm pregnancy. CONCLUSION: Modified cryoloop vitrification with a final solu tion of 3.4 mol/l glycerol and 4.5 mol/l ethylene glycol is a promising pro cedure for preserving Rhesus monkey blastocysts that is simple, rapid, and inexpensive.