Assessment of a new in vitro maturation system for mouse and human cumulus-enclosed oocytes: three-dimensional prematuration culture in the presence of a phosphodiesterase 3-inhibitor.

BACKGROUND: Controlling nuclear maturation during oocyte culture might improve nuclear-cytoplasmic maturation synchrony. In the present study, the quality of mouse and human cumulus-enclosed oocytes (CEOs) was examined after a two-step culture consisting of a three-dimensional prematuration culture (3D-PMC), followed by in vitro maturation (IVM).

METHODS: Mouse and human CEOs were embedded in an extracellular matrix (collagen-gel Type I). The gels containing the CEOs were cultured in medium with a phosphodiesterase 3-inhibitor (PDE3-I; cilostamide 1 microM) for 24 h. Afterwards, CEOs were removed from the gel and washed away from inhibitor then underwent IVM. The optimal concentration of collagen (diluted 1:2 versus not-diluted) was first determined in the mouse model. Cytoplasmic maturation after IVM of human and mouse oocytes was assessed in relation to fertilization and embryonic developmental capacity.

RESULTS: The diluted form of collagen was better for supporting the structure of the expanding CEOs and meiotic competence of the oocytes. Electron microscopy in combination with Lucifer Yellow dye coupling assay revealed that oocyte-cumulus cell connections could be preserved during 3D-PMC. Percentages of mouse 2-cell embryos after IVF were higher in the 3D-PMC group compared with in vitro controls and 2D-PMC oocytes, but lower compared with in vivo controls. In the human model, percentages of polar body-extruded oocytes were significantly higher in the 3D-PMC group compared with conventionally matured oocytes. The 3D-PMC also had a beneficial effect on embryonic development on Day 3 post-ICSI.

CONCLUSIONS: Applying a 3D-PMC in the presence of a PDE3-I preserves oocyte-cumulus cell connections and influences oocyte developmental capacity.