Korean J Fertil Steril.  2000 Mar;27(1):47-58.

Developmental competence and Effects of Coculture after Crypreservation of Blastomere-Biopsied Mouse Embryos as a Preclinical Model for Preimplantation Genetic Diagnosis

• Kim JG
• Lee JY
• Kim SH
• Jee BC
• Suh CS
• Choi YM
• Moon SY

Abstract

OBJECTIVE
The effects of cryopreservation with or without coculture on the in vitro development of blastomere-biopsied 8-cell mouse embryos were investigated. This experimental study was originally designed for the setup of a preclinical mouse model for the preimplantation genetic diagnosis (PGD) in human.
METHODS
Eight-cell embryos were obtained after in vitro fertilization (IVF) from F1 hybrid mice (C57BLfemale symbol/CBAmale symbol). Using micromanipulation, one to four blastomeres were aspirated through a hole made in the zona pellucida by zona drilling (ZD) with acid Tyrode's solution (ATS). A slow-freezing and rapid-thawing protocol with 1.5M dimethyl sulfoxide (DMSO) and 0.1M sucrose as cryoprotectant was used for the cryopreservation of blastomere- biopsied 8-cell mouse embryos. After thawing, embryos were cultured for 110 hours in Ham's F-10 supplemented with 0.4% bovine serum albumin (BSA). In the coculture group, embryos were cultured for 110 hours on the monolayer of Vero cells in the same medium. The blastocyst formation was recorded, and the embryos developed beyond blastocyst stage were stained with 10% Giemsa to count the total number of nuclei in each embryo.
RESULTS
The survival rate of embryos after cryopreservation was significantly lower in the blastomere-biopsied (7/8, 6/8, 5/8, and 4/8 embryos) groups than in the non-biopsied, zona intact (ZI) group. Without the coculture, the blastocyst formation rate of embryos after cryopreservation was not significantly different among ZI, the zona drilling only (ZD), and the balstomere-biopsied groups, but it was significantly lower than in the non-cryopreserved control group. The mean number of cells in embryos beyond blastocyst stage was significantly higher in the control group (50.2+/-14.0,) than in 6/8(26.5+/-6.2), 5/8(25.0+/-5.5), and 4/8(17.8+/-7.8) groups. With the coculture using Vero cells, the blastocyst formation rate of embryos after cryopreservation was significantly lower in 5/8 and 4/8 groups, compared with the control, 7/8, and 6/8 groups. The mean number of cells in embryos beyond blastocyst stage was also significantly lower in 4/8 group (25.9+/-10.2), compared with the control (50.2+/-14.0), 7/8 (56.0+/-22.2), and 6/8 (55.3 +/-25.5) groups.
CONCLUSION
After cryopreservation, blastomere-biopsied mouse embryos have a significantly impaired developmental competence in vitro, but this detrimental effect might be prevented by the coculture with Vero cells in 8-cell mouse embryos biopsied one or two blastomeres. Biopsy of mouse embryos after ZD with ATS is a safe and highly efficient preclinical model for PGD of human embryos.

MeSH Terms

Animals
Biopsy
Blastocyst
Blastomeres
Coculture Techniques*
Cryopreservation
Dimethyl Sulfoxide
Embryonic Structures*
Fertilization in Vitro
Herpes Zoster
Humans
Mental Competency*
Mice*
Micromanipulation
Preimplantation Diagnosis*
Prostaglandins D
Serum Albumin, Bovine
Sucrose
Survival Rate
Vero Cells
Zona Pellucida
Dimethyl Sulfoxide
Prostaglandins D
Serum Albumin, Bovine
Sucrose