Modified Cryo-Loop Vitrification Yields Superior Post-Thaw Survival and Development of Rhesus Monkey Blastocysts

Abstract

Objective: Evaluation of the loop vitrification procedure for preservation of blastocysts by comparing results using 2 different cryoprotectant mixtures to earlier results with controlled rate cooling. Vitrification using a monofilament loop has a high cooling rate enabling concentrated cryoprotectents to achieve a glass-like state without ice crystals.Design: A prospective study in a primate model at a regional primate research center.Methods: Rhesus monkey blastocysts were produced by ICSI of mature oocytes retrieved from cycling females exposed to follicular stimulation with recombinant human hormones. Healthy blastocysts of comparable development and quality were randomly divided between vitrification procedures. For procedure A, the final freezing solution before plunging into liquid nitrogen (LN) was 20% DMSO and 20% with 0.65 M sucrose and 10 mg/ml Ficoll (Lane et al, F&S, 1999); for procedure B, the final solution was 25% glycerol and 25% ethylene glycol (Donnay et al, Anim Repro Sci, 1998). The slow freeze procedure (Menezo et al, F&S, 1992) had a final solution of 9% glycerol and 0.2 M sucrose in vials which were cooled at −0.3° C/min to −30° C after seeding at −8° C. After > 48 hrs in LN, all thawing was done in concentrated sucrose media with successive dilutions before co-culture on BRL cells.Results: Of 12 blastocysts vitrified via Procedure A, 4 (33%) survived the thaw process with minimal blastomere lysis and one of these hatched but did not attach. Vitrification with Procedure B of 13 blastocysts resulted in 11 embryos surviving the thaw (85%; p<0.05), with 10 hatching. Further culture of 6 observed attaching and growing out. Our prior results with the slow freezing procedure involved 22 blastocysts of which 8 (36%) survived the thaw and hatched.Summary: A modified cryoloop vitrification procedure with a final cryoprotectant concentration of 25% glycerol and 25% ethylene glycol has shown promising results for cryopreservation of rhesus monkey blastocysts. This simple, rapid, and inexpensive procedure is now undergoing embryo transfer trials to demonstrate in vivo developmental potential.Supported by NIH RR12804, A142709, and Ares Advanced Technology, Inc. Objective: Evaluation of the loop vitrification procedure for preservation of blastocysts by comparing results using 2 different cryoprotectant mixtures to earlier results with controlled rate cooling. Vitrification using a monofilament loop has a high cooling rate enabling concentrated cryoprotectents to achieve a glass-like state without ice crystals. Design: A prospective study in a primate model at a regional primate research center. Methods: Rhesus monkey blastocysts were produced by ICSI of mature oocytes retrieved from cycling females exposed to follicular stimulation with recombinant human hormones. Healthy blastocysts of comparable development and quality were randomly divided between vitrification procedures. For procedure A, the final freezing solution before plunging into liquid nitrogen (LN) was 20% DMSO and 20% with 0.65 M sucrose and 10 mg/ml Ficoll (Lane et al, F&S, 1999); for procedure B, the final solution was 25% glycerol and 25% ethylene glycol (Donnay et al, Anim Repro Sci, 1998). The slow freeze procedure (Menezo et al, F&S, 1992) had a final solution of 9% glycerol and 0.2 M sucrose in vials which were cooled at −0.3° C/min to −30° C after seeding at −8° C. After > 48 hrs in LN, all thawing was done in concentrated sucrose media with successive dilutions before co-culture on BRL cells. Results: Of 12 blastocysts vitrified via Procedure A, 4 (33%) survived the thaw process with minimal blastomere lysis and one of these hatched but did not attach. Vitrification with Procedure B of 13 blastocysts resulted in 11 embryos surviving the thaw (85%; p<0.05), with 10 hatching. Further culture of 6 observed attaching and growing out. Our prior results with the slow freezing procedure involved 22 blastocysts of which 8 (36%) survived the thaw and hatched. Summary: A modified cryoloop vitrification procedure with a final cryoprotectant concentration of 25% glycerol and 25% ethylene glycol has shown promising results for cryopreservation of rhesus monkey blastocysts. This simple, rapid, and inexpensive procedure is now undergoing embryo transfer trials to demonstrate in vivo developmental potential. Supported by NIH RR12804, A142709, and Ares Advanced Technology, Inc.