In cattle, approximately 40% of all pregnancies are lost early during the first stages of pregnancies. The high rate of gestational loss is usually due to inefficient communication between the conceptus and mother. Thus, reducing embryonic losses through better understanding of embryonic signals can contribute significantly to the in vitro embryo production system by increasing efficiency and reducing costs to producers in a wide range of production systems not only in Brazil, but throughout the world. In this away, to overall aim is to establish a 3D culture system to test in vitro conceptus-maternal interactions during early stages of pregnancy in cattle. Additionally, to establish and validate a micro-fluidics system to test maternal stressors in vitro. Briefly, the endometrium will be dissected from the uterine horn ipsilateral to the corpus luteum. Dissected tissue will be incubated in digest solution and the resulting cell pellet, containing epithelial and stromal cells, will re-suspended in culture media. The heterogeneous cell population will be seeded at 1×105 cells/mL into 75 cm2 culture flasks. In parallel, cumulus-oocyte complexes (COCs) containing compact cumulus cell layers and homogeneous cytoplasm will be selected and matured in vitro in groups of 15-20 COCs. After 22-24h of maturation the COCs will be fertilized with the thawed semen from the same bull and batch and prepared according to the Percoll gradient technique. The IVF blastocysts will cultured with endometrial cells in a conventional 2D monolayer culture versus a 3D cell culture system. Additionally, the cells and blastocysts will be also culture in a micro-fluidic device system to test the effect of maternal stressors on conceptus-maternal interactions in vitro. All the cultures will be incubated at 38°C in a humidified incubator with 5% CO2 in air. In this study, we will analyze the influence of culture system in the gene expression of endometrial cells and in blastocysts cultured in a conventional 2D monolayer culture versus a 3D on impact of cell function by genome-wide expression profiling using RNA-sequencing. Also, will be analyzed the ability of this Micro-fluidic Device System to support early embryo development as well as effects of exposure to conceptus-derived proteins will be assessed.
News published in Agência FAPESP Newsletter about the scholarship: