GM-CSF perturbs cell identity in mouse pre-implantation embryos

Abstract

Growth factors became attractive candidates for medium supplementation to further improve the quality of embryo culture and to mimic in vivo nutrition. Granulocyte macrophage colony-stimulating factor (GM-CSF) is a cytokine influencing the maternal-fetal interface and supporting placental development in mouse and human. It is expressed in epithelial cells of the endometrium under the regulation of estrogens. The factor is already in clinical use and a large clinical trial showed that, if supplemented to an embryo culture medium, it leads to increased survival of embryos, especially in women with previous miscarriages. Animal and cell culture studies on isolated trophectoderm cells support an effect mainly on cellular expansion. Aim of this study was to investigate, if the supplementation of GM-CSF either in a human ART medium or in a mouse optimized medium, leads to a change in cell number and cell lineages in the early pre-implantation mouse embryo. Our data shows that mouse GM-CSF increased total cell numbers with increasing concentrations. This increase of cell number has not been found in embryos cultured in ART media with or without human GM-CSF (hGM-CSF) or in a mouse medium supplemented with different concentrations of hGM-CSF. The changes were caused by a marked difference in TE and primitive endoderm cell numbers but not due to a change in epiblast cell numbers. Additionally, results show an ectopic expression of NANOG among trophectoderm cells in both, human ART media (with and without GM-CSF) and at increasing concentrations in the mouse and the human GM-CSF supplemented media. In conclusion, we could show that GM-CSF has an effect on cell identity in mice, which might probably also occur in the human. Therefore, we would like to rare awareness that the use of supplements without proper research could bare risks for the embryo itself and probably also in the post-implantation phase.

Fig 1. Effect of mGM-CSF: Comparison of absolute cell numbers.

Fig 2. Representative picture of a blastocyst (E4.5) with ectopic expression of NANOG.

Fig 3. Percentage of occurrence of blastocysts cultured in medium containing mGM-CSF exhibiting a double staining of the cell lineage markers CDX2 and NANOG in trophectoderm cells.

Fig 4. Percentage of occurrence of blastocysts cultured in ART media and medium containing hGM-CSF exhibiting a double staining of the cell lineage markers CDX2 and NANOG in trophectoderm cells.