Successful development in vitro of hamster 8-cell embryos to ‘zona-escaped’ and attached blastocysts: assessment of quality and trophoblast outgrowth

Abstract

The peri-implantation development involves zona escape (hatching) of blastocysts and their attachment and proliferation. These events are difficult to studyin vivo, so in this study hamster 8-cell embryos were cultured through the hatched and attached blastocyst stages using different formulations of hamster embryo culture medium (HECM)-2. Supplementation of succinate, amino acids, vitamins (inositol, pantothenate, choline chloride) and bovine serum albumin (BSA) to HECM-2 supported 100% development of ‘zona-escaped’ blastocysts. In this medium (designated as hatching, i.e. HECM-2h) all blastocysts invariably deflated and escaped from focally lysed zonae, which underwent complete dissolution. In their presence, pre-morula stage embryos also escaped from zonae. Omission of BSA from HECM-2h failed to support zona escape whereas that of vitamins reduced zona escape (34.0% 7.0). Blastocysts with the potential to undergo zona escape in HECM-2h were of high quality as they had a higher mean cell number (MCN) than the MCN of those developing in BSA-free HECM-2h (35.2 1.6 v. 24.3 1.1). Cell allocation (i.e. trophectoderm to inner cell mass ratio) in blastocysts remained unaltered in both media (2.6 0.2 v. 2.7 0.2). Supplementation of 10% bovine fetal serum (BFS) to HECM-2h was detrimental to the development of blastocysts (22.3% 7.4) and none of them underwent zona escape. Interestingly, BFS was required either as a supplement to the medium or as a coating on dishes for azonal blastocysts to attach (≥70%) and exhibit trophoblast (TB) outgrowth (30.3 × 10³ 2.9 × 10³ µm² at 48 h). These results show that HECM-2h supports maximal development of zona-escaped blastocysts with the potential to attach and exhibit TB outgrowth, and there is a developmental stage-specific requirement for serum during peri-attachment in hamster development.