124 A THREE-DIMENSIONAL IN VITRO IMPLANTATION MODEL WITH NONHUMAN PRIMATE EMBRYOS AND EXTRACELLULAR MATRIX UNDER VARIOUS CULTURE CONDITIONS

Abstract

The need for blastocyst culture and post-implantation embryo research has emerged in the past few years. Our objective is to evaluate a novel in vitro model to study implantation and placenta formation in vitro with rhesus macaque embryos under various culture conditions. A novel nonhuman primate in vitro 3-D system can provide cues for implantation and interaction with the extracellular environment not available in 2-D planar models. Optimization of such a model can be tested with diverse culture environments. We developed and evaluated an in vitro 3-D implantation model utilizing IVF-derived, blastocyst-stage rhesus macaque embryos embedded in 3-D Matrigel droplets cultured with different feeder cells and media. Signs of implantation including enlargement of the embryo mass, invasion and proliferation of trophectoderm cell layers, cystic formation, and cellular outgrowths derived from the embryo were initiated within the first week post-embedding. Trophoblast structures with protrusion and branches growing from the surface of embryo implants were observed. Immunohistochemical staining for chorionic gonadotropin (CG) combined with immunoassays for CG and progesterone indicated differentiation of trophoblastic cell lineages. In addition, we found morphological factors, such as proliferation of embryonic and extraembryonic structures, as well as initiation of protrusions interacting with the extracellular matrix, to predict successful establishment of prolonged embryo development. We further evaluated effects of different types of feeder cells and media combinations, and found that a combination of BRL, Ishikawa cells, and human uterine fibroblasts, provided an optimized culture microenvironment to promote peri-implantation embryo development and hormone secretion including CG and progesterone. In conclusion, we have established a 3-D in vitro system modeling implantation initiation, and demonstrating the capability of the embryo to interact with the extracellular matrix. Further studies will facilitate the methodology of peri-implantation blastocyst culture and accelerate our understanding of nonhuman primate embryo development, with potential for insights into early pregnancy loss and related pathologies. The present study and future directions may be extended to provide retrospective views on blastocyst selection for embryo transfer in assisted reproductive technology.

This study was funded by NIH grants RR000167, RR21876, and HD053926.