Mechanistic foundations of the metaphase II spindle of human oocytes matured in vivo and in vitro.

Abstract

Are morphometric and morphological parameters of the metaphase II (MII) spindle of human oocytes matured in vivo or in vitro predictive of chromosome alignment on the metaphase plate? Morphometric spindle parameters were very comparable between oocytes matured in vivo and in vitro and were unable to predict chromosome alignment, while a flattened shape of both poles was positively associated with chromosome displacement from the metaphase plate. The relationship between MII spindle morphometry and chromosome alignment has only been sporadically investigated in human oocytes. The possible implications of spindle pole morphology are totally unrecognized. Morphometric and morphological analysis of the MII spindle of donated supernumerary human oocytes (N = 93) aimed at investigating possible associations between novel microtubule parameters and chromosome arrangement. MII oocytes from three sources were analysed: (i) stimulated cycles matured in vivo (ivo-MII), (ii) leftover cumulus-free germinal vesicle oocytes from stimulated cycles matured in vitro (lgv-MII) and (iii) immature cumulus-cell oocyte complexes (COCs) recovered from in vitro maturation (IVM) cycles and matured in vitro (ivm-MII). Oocytes were fixed and stained for tubulin, chromatin and actin. Optical sections were collected at 0.3 μm intervals by high-performance confocal microscopy and three-dimensionally reconstructed for assignment of specific spindle and chromosomal properties. Spindle pole morphology was classified as either focused or flattened depending on whether microtubule ends were more or less convergent, respectively. Optical density measurements were generated to estimate microtubule abundance in chromosome to pole domains proximal and distal to the oolemma. In ivo-MII oocytes, the sizes (mean ± SD) of major and minor axes were 11.8 ± 2.6 and 8.9 ± 1.7 μm, respectively, while maximum projection was 88.8 ± 29.5 μm(2). Very comparable values of these parameters were found in lgv-MII and ivm-MII oocytes. Double-focused spindles were rarely found (3.1%), unlike those with a double-flattened conformation (47.7%). Spindles with both focused and flattened poles amounted to almost half of the sample set (49.2%), but in this subgroup it was very infrequent (4.6%) to observe the flattened pole oriented towards the oolemma. Overall, differences in the relative proportions of pole morphology categories in ivo-MII, lgv-MII and ivm-MII oocytes were not statistically significant. For both the distal and proximal spindle hemidomains, optical intensity profiles were also comparable between ivo-MII, lgv-MII and ivm-MII oocytes. None of the morphometric parameters (major and minor axes, their ratio, maximum projection, distances of the metaphase plate from the poles) was associated with chromosome alignment on the metaphase plate or arrangement inside and outside the spindle. Importantly, a double-flattened outline of pole morphology was positively associated with the displacement of one or more chromosomes from the metaphase plate. Moreover, when a flattened pole was oriented towards the oolemma, a higher rate of chromosome displacement was observed. The findings of the study will require confirmation by further in-depth analysis and extension of the database, especially regarding the relationship between microtubule abundance and chromosome arrangement. Furthermore, considering the high number of comparisons, the observed statistical differences will require future 'ad hoc' analysis. Collectively, this work provides a robust database for future research on the human oocyte cytoskeleton, and contributes to a better definition of oocyte quality in assisted reproduction technology. Also, these data support the notion that IVM does not affect spindle morphometry and morphology. Part of this work was supported by a grant awarded by the Italian Ministry of Labour, Health and Social Policies. The authors have no conflicts of interest to declare. Not applicable.