Timing and duration of alpha-minimum essential medium exposure during preimplantation development determine long-term metabolic phenotypes in mice.

The environment experienced during preimplantation development can influence long-term health. Continuous culture of mouse preimplantation embryos in α-minimum essential medium (αMEM) for 48 h induces the established "MEM mouse" phenotype, but the critical exposure window and required duration remain unclear. Here, 2-cell embryos were cultured for 48 h in mCZB alone (mCZB), in αMEM for the first 24 h followed by mCZB (M/C), in mCZB for the first 24 h followed by αMEM (C/M), or in αMEM for the full 48 h (MEM). Blastocyst development, lineage allocation, fetal and placental growth, postnatal growth, survival under high-fat diet (HFD), and glucose tolerance were evaluated. Continuous 48 h exposure reduced blastocyst formation, and late or sustained αMEM exposure reduced Cdx2-positive trophectoderm cell number. After embryo transfer, in vitro culture groups showed increased placental and birth weights compared with natural mating controls, although possible litter/recipient effects should be considered. Under HFD challenge, MEM offspring developed overweight-associated impaired glucose tolerance, M/C offspring showed severe HFD intolerance with body-weight loss and reduced survival, and C/M offspring showed a tendency toward impaired glucose handling without marked overweight. Our findings suggest that the timing and duration of αMEM exposure during preimplantation development influence long-term metabolic phenotypes. Sustained 48 h exposure was associated with the overweight-related phenotype, whereas temporally restricted exposures were associated with different postnatal responses.