Comparative Characteristics of Morphological Feature and Localization of Meiotic Spindle in Human Oocytes before and after Cryopreservation

Using only oocyte morphological parameters is not enough to predict the quality and implantation potential of the future embryo. As well known, the presence of the first polar body is not always a criterion for oocyte maturity. Evidence of oocyte metaphase II stage is the presence of second meiotic division spindle which can be visualized by a polarizing microscope. However, there is no classification of morphological characteristics and localization of meiotic division spindle in the scientific data. This classification would allow predicting oocyte cryoresistance and quality of embryos obtained from them. The disturbances of the meiotic division spindle of metaphase II stage can lead to abnormal segregation of chromosomes after fertilization of cryopreserved oocytes and as a result to aneuploidy of embryos. The aim of this work is to determine the influence of cryopreservation by vitrification method on the morphology and localization of second meiotic division spindle of human oocytes. We have studied and developed a classification of the oocyte's meiotic spindle apparatus morphological forms and its location relative to the first polar body to achieve this goal. An experimental study was performed on 84 donor oocytes of women who took part in infertility treatment programs by ART. The structure and localization of the meiotic spindle in native and cryopreserved human oocytes was studied using the Oosight polarization system (Hamilton Thorne, USA). The meiotic spindle structure was evaluated and classified. The spindle's grade characteristics are as follows: A - compact rhomboid spindle with defined edges, B - modified spindle form with blurred edges, C - weak visualization, D - spindle on the 1stpolar body border and cytoplasm (telophase I), E - spindle not visualized, G – increased in a size. The location was graded as the follows: “a” - 0°-20° due to the 1stpolar body, “b”- 21°-45°,“c”- 46° - 90°,“d”- 91° - 180°. The criteria for the morphological features and localization of the meiotic division spindle of human oocytes was first ascertained. After cryopreservation, the survival rate of oocytes was 95.2%. Evaluation of meiotic division spindle morphology enabled us to establish that 45.2% of freshly isolated oocytes had a compact spindle, but after cryopreservation the amount of such oocytes decreased to 28.6%. The decrease in oocyte quantity in the spindle of category A was due to changes in the spindle structure. After cryopreservation, appeared oocytes of category E (with the absence of meiotic spindle) up to 7.1%. This can indicate damage of meiotic spindle. The amount of oocytes with category D spindle (at the telophase stage) which also appeared was 9.5%. This shows premature activation of oocyte after freeze-thawing. After cryopreservation localization changes were found spindle cells in 19 (22.6%). There was a significant decrease in the number of oocytes with a spindle in category a - from 40,5% to 21,4%. It has been established that cryopreservation factors can lead to morphological characteristic changes of second meiotic division spindle and its destruction. It was shown that cryopreservation factors can induce premature oocyte activation which can lead to adverse consequences for the future embryo quality.