Ultra-rapid freezing of mouse oocytes lowers the cell number in the inner cell mass of 5 day old in-vitro cultured blastocysts.

Abstract

We demonstrated previously that ultra-rapid freezing of mouse oocytes with 3.5 M dimethylsulphoxide (DMSO) decreased cell numbers in day 5 in-vitro cultured blastocysts. In the present study we counted cell numbers of trophectoderm (TE) and inner cell mass (ICM) separately following differential labelling of TE with propidium iodide (red) and ICM with bisbenzimide (blue). Blastocysts were from four groups of oocytes: (i) cumulus-enclosed; (ii) hyaluronidase-treated cumulus-free; (iii) cumulus-free and exposed to 3.5 M DMSO; and (iv) cumulus-free and ultrarapidly frozen with 3.5 M DMSO. Mean (+/-SD) blastocyst cell numbers were 54.7 +/- 22.0, 51.1 +/- 17.3, 52.3 +/- 13.1 and 40.4 +/- 18.4, respectively. Mean TE cell numbers were 31.7 +/- 18.2, 28.9 +/- 13.3, 31.2 +/- 13.3 and 26.2 +/- 16.5 while mean ICM cell numbers were 23.0 +/- 9.4, 22.2 +/- 9.4, 21.1 +/- 7.3 and 14.2 +/- 7.3, respectively. Blastocyst and ICM cell numbers were significantly lower in the group derived from ultra-rapidly frozen oocytes compared with all other groups. Significantly more blastocysts had < or = 32 cells and in blastocysts with > 64 cells a lower mean percentage of ICM was found. Ultra-rapid freezing of mouse oocytes with 3.5 M DMSO can thus lead to day 5 in-vitro cultured blastocysts with significantly decreased ICM cell numbers. The residual ICM cell number in affected blastocysts may not reach a critical mass sufficient for successful postimplantation development.