Altered cytoplasmic maturation in rescued in vitro matured oocytes

Abstract

Do culture conditions affect cytoplasmic maturation in denuded immature non-GV oocytes? The maturation rate of denuded non-GV oocytes is not affected by culture media, but in vitro maturation seems to alter the mitochondrial membrane potential, endoplasmic reticulum (ER) and actin cytoskeleton compared with in vivo maturation. In vitro maturation of denuded immature non-GV oocytes benefits cycles with poor in vivo MII oocyte collection, but maturation levels of non-GV oocytes are only scored by polar body extrusion. Since oocyte maturation involves nuclear as well as cytoplasmic maturation for full meiotic competence, further knowledge is needed about cytoplasmic maturation in in vitro culture. This basic research study was carried out between January 2017 and September 2018. A total of 339 denuded immature non-GV oocytes were cultured in SAGE 1-Step (177) or G-2 PLUS (162) for 6–8 h after retrieval, and 72 in vivo matured MII oocytes were used as controls. Cultured immature non-GV oocytes were scored for polar body extrusion and analysed for mitochondrial membrane potential (ΔΨm), ER clusters, cortical granules number and distribution, spindle morphology and actin cytoskeleton organization. The obtained parameter values were compared to in vivo matured MII oocyte parameter values. The maturation rates of oocytes cultured in G-2 PLUS and SAGE 1-Step were similar (65% vs 64.2%; P = 0.91). The differences observed in cortical granule density were not statistically significant. Also spindle morphometric parameters were mostly similar between in vitro and in vivo matured MII oocytes. However, the number of ER clusters, the ΔΨm and the cortical actin thickness showed significant differences between in vivo MII oocytes and denuded immature non-GV oocytes cultured in vitro until meiosis completion. Frozen-thawed oocytes together with fresh oocytes were used as controls. Due to technical limitations (fixation method and fluorochrome overlap), only one or two parameters could be studied per oocyte. Thus, a global view of the maturation status for each individual oocyte could not be obtained. Characterization of in vitro matured oocytes at the cellular level will help us to understand the differences observed in the clinical outcomes reported with rescue IVM compared to in vivo MII oocytes and to improve the culture methods applied. This work was supported by intramural funding of Clinica Eugin and by the Torres Quevedo Program to A.F.-V. from the Spanish Ministry of Economy and Competitiveness. No competing interests are declared.