Ultrastructure of Human Preovulatory Oocyte in Assisted Reproduction Protocols


Stefania A. Nottola *,

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In the field of assisted reproduction technology oocyte cryopreservation protocols have not been fully optimized yet and overall clinical success remains relatively suboptimal. Further experimental (morphological biochemical biomolecular) and clinical studies seem thus necessary in order to better comprehend the effects on human oocytes of all factors associated with freezing and ultimately tailor the best protocol for human oocyte cryopreservation. Our aims were to evaluate the ultrastructure of human preovulatory oocytes frozen-thawed (F/T) using different protocols of cryopreservation and to compare the data obtained with the morphology usually shown by fresh (control) oocytes. In detail our study investigated the ultrastructural features of oocytes subjected to various procedures of slow cooling and vitrification. Particular emphasis has been given on how the vitrification process impacts the ultrastructural morphology of the mature oocyte. In fact vitrification currently offers interesting perspectives in the field of oocyte cryopreservation being judged more tolerable than slow cooling by the oocyte. Despite this very little is known about the fine morphology of vitrified-warmed oocytes and the available studies are almost totally concerning non-human species. F/T oocytes were vitrified using two alternative devices (cryoloop and cryoleaf). By light microscopy both fresh and F/T oocytes were generally rounded 90-100 microns in diameter provided with an ooplasm showing a uniform texture and surrounded by a continuous zona pellucida. Sparse microvacuolization was only occasionally detected in both fresh and F/T oocytes at the same extent. Transmission electron microscopy (TEM) evaluation confirmed the very sporadic incidence of ooplasmic vacuolization in fresh and F/T oocytes. By TEM oocyte organelles mostly consisted of mitochondria-smooth endoplasmic reticulum (M-SER) aggregates varying in shape size and location. In particular in more than a half of the F/T oocytes observed M-SER aggregates appeared slender in shape and smaller in size than those observed in fresh oocytes; in spite of this a proper mitochondrial fine structure was generally maintained in all these samples. In the remaining F/T oocytes and in fresh oocytes as well both small and large aggregates were found randomly distributed in the ooplasm. Finally numerous F/T oocytes displayed a non-homogeneous distribution of microvilli and a reduced complement of cortical granules (CGs) – the latter particularly evident in those oocytes vitrified using the cryoloop device. In conclusion a) the virtual absence of ooplasmic microvacuolization seems the most relevant marker of quality in vitrified-warmed oocytes irrespective of the device utilized; b) CG pattern appears better preserved in cryoleaf-supported oocytes; c) the finding of underdeveloped M-SER aggregates and altered microvilli emphasize the need of further ultrastructural studies on human vitrified oocytes. Funds were provided by Italian Ministry of Education University and Research (university grants) and by the Italian National Health Institute.