Morphological and molecular markers are correlated with maturation-competence of human oocytes

Abstract

Does the position of the germinal vesicle (GV) in human oocytes correlate with molecular and morphological parameters as well as with maturation-competence? The position of GV in human oocytes correlates with density of microtubule (MT) filaments, concentration of Fyn, nucleolus localization and the ability of the oocytes to complete maturation following GV breakdown (GVBD). Our knowledge is confined to oocytes of young mice where maturation-competence is correlated with a central GV and regulated by MTs and the presence of a chromatin ring. Fyn kinase is localized at the spindle and cortex of mouse oocytes and plays a role in both maturation and MT stabilization. Spatial localization of the GV and nucleolus (central or peripheral), the presence of a chromatin ring, the localization of Fyn, MT density and oocyte maturation were assessed in 153 human oocytes, 335 oocytes from young mice (2-month-old) and 146 oocytes from old mice (12-month-old). GV human oocytes were donated by consenting female patients (n = 57), 21-45-year-old undergoing IVF/ICSI. As a control, GV mouse oocytes were collected from female mice after injection of pregnant mares' serum gonadotrophin. Human and mouse GV oocytes allocated for immunocytochemistry were fixed on day of retrieval, stained with specific antibodies and imaged using a confocal laser-scanning microscope. Human and mouse oocytes allocated for maturation were incubated for 48 and 24 h, respectively. GVBD and extrusion of the first polar body (PBI) were assessed using differential interference contrast optics. GV location was peripheral and independent of age in 69.9% of the human oocytes, but GV location did vary with age in mice oocytes; it was central in 89.9% of the oocytes retrieved from young-mice and peripheral in 52.1% of the oocytes retrieved from old mice (P < 0.05). A central GV, whether in human or mouse oocytes, was highly correlated with a central nucleolus, absence of Fyn at the GV and a dense MT network (P < 0.05), whereas a peripheral GV correlated with peripheral nucleolus, presence of Fyn at the GV and a flimsy MT network. After 48 h in culture, no degeneration was observed in human central-GV oocytes, however, 12/95 (12.6%) of the peripheral-GV oocytes degenerated (P < 0.05). No correlation was observed between GV position and presence of a chromatin ring. The percentage of human oocytes that extruded the PBI after completing GVBD was significantly higher (73.7%) in central than in peripheral-GV oocytes (45.8%; P < 0.05). In mice oocytes, central location of the GV correlated with maturation competence in young (P < 0.05) but not old mice. The fact that the human GV oocytes used in this study were exposed to gonadotrophic stimulation but failed to mature in vivo might be a sign of their low quality and this should be considered when drawing conclusions from the data. Furthermore, our observation that only peripheral-GV human oocytes were degraded may indicate that they are of a lower quality than central-GV human oocytes. We suggest that the central location of GV within the oocytes, which is associated with an absence of Fyn at the GV and the presence of thick filamentous MTs in the ooplasm, may serve as a predictor of successful maturation and provide new insights for the use of IVM.