How a mammalian embryo determines and arrives at its attachment site has been studied for decades but our understanding of this process is far from complete. Using confocal imaging and image analysis, we evaluate embryo location along the longitudinal oviductal-cervical axis of murine uteri. Our analysis reveals three distinct pre-implantation phases: a) Embryo entry; b) Unidirectional movement of embryo clusters; and c) Bidirectional scattering and spacing of embryos. We show that unidirectional clustered movement is facilitated by a mechanical stimulus of the embryo and is regulated by adrenergic uterine smooth muscle contractions. Embryo scattering, on the other hand, depends on embryo-uterine communication reliant on the LPAR3 signaling pathway and is independent of adrenergic muscle contractions. Finally, we demonstrate that uterine implantation sites in mice are neither random nor predetermined but are guided by the number of embryos entering the uterine lumen. These studies have implications for understanding how embryo-uterine communication is key to determining an optimal implantation site necessary for the success of a pregnancy.

How a mammalian embryo determines and arrives at its site of attachment is a mystery that has puzzled researchers for decades. Additionally, in multiparous species, embryos face a unique challenge of achieving adequate spacing to avoid competition for maternal resources. Using our enhanced confocal imaging and 3D image reconstruction technology, we evaluate murine embryo location in the uterus along the longitudinal oviductal-cervical axis. Our analysis reveals three distinct pre-implantation stages: a) Embryo entry; b) Unidirectional movement of embryo clusters; and c) Bidirectional scattering and spacing of embryos. We show that unidirectional movement of embryo clusters is facilitated by a mechanical stimulus of the embryo as a physical object and is regulated by adrenergic uterine smooth muscle contractions. Embryo scattering, on the other hand, relies on embryo-uterine communication reliant on the LPAR3 signaling pathway and is independent of adrenergic muscle contractions. We propose that the presence of embryo clusters in the uterine horn provides an opportunity for the uterus to sense and count the embryos, followed by scattering and spacing these embryos along the given length of the horn. Thus, uterine implantation sites in mice are neither random nor predetermined but are guided by the number of embryos entering the uterine lumen. These studies have implications for understanding how embryo-uterine communication is key to determining an optimal implantation site, which is necessary for the success of a pregnancy. Significance StatementIn mammals that carry multiple offspring in one gestation, embryos seemingly acquire even embryo spacing. Such even distribution would imply a guided interaction between the mother and the fetus very early on in pregnancy to allow favorable pregnancy outcomes. Thus, it is essential to understand quantitatively if and when such a uniform distribution of embryos is established. Further, uncovering the physical and biological mechanisms that allow for such equal distribution of embryos, will improve our understanding of early pregnancy events and provide for novel targets for improving pregnancy success in case of infertility and artificial reproductive technologies as well as to develop non-hormonal therapies for contraception.