Background While mammalian embryos can adapt to their environments, their sensitivity overshadows their adaptability in suboptimal in vitro conditions. Therefore, the environment in which the gametes are fertilized or to which the embryo is exposed can greatly affect the quality of the embryo and consequently its implantation potential. Objectives Since providing an optimal culture condition needs a deep understanding of the environmental effects, and regarding the fact that normal morphology fails to be a reliable indicator of natural embryo development, the current study aimed at comparing in vivo - and in vitro -derived blastocysts at the molecular level. Materials and Methods In vivo and in vitro mouse blastocysts were obtained by flushing the uterine horns and in vitro fertilization/culture, respectively. Normal blastocysts of both groups were evaluated in terms of hatching rate and expression of three lineage-differentiation-, apoptosis-, and implantation-related genes. Results The hatching rate was lower in In vitro fertilization (IVF)-produced blastocysts in comparison with that of the in vivo counterparts. More importantly, the study results indicated significant changes in the expression levels of eight out of ten selected genes, especially Mmp-9 (about -10.7-fold). The expression of Mmp-9 in trophoblast cells is required for successful implantation and trophoblast invasion. Conclusions The current study, in addition to confirming that the altered gene expression pattern of in vitro -produced embryos resulted in normal morphology, provided a possible reason for lower implantation rate of in vitro -produced blastocysts regarding the Mmp-9 expression.

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