P-248 The meiotic spindle is preserved during freezing but post-thawing rehydration causes its destabilization in human eggs
Authors: Holubcova Z , Kyjovská D , Otevřel P , Kloudová S 
Published: June 2023

Abstract

Study question
Is the human egg spindle integrity impaired by vitrification and thawing procedures?

Summary answer
Cryoprotective-caused dehydration stabilizes the MII spindle, but the re-entry of water during thawing induces depolymerization of microtubules, thus promoting instability of the division apparatus.

What is known already
Freezing sperm and preimplantation embryos have become routine IVF procedures with excellent clinical results, whereas cryopreservation of oocytes remains problematic. The major factor underlying the human oocytés notorious propensity to cryoinjury is the temperature sensitivity of the meiotic spindle, the fidelity of which is critical for faithful post-fertilization development. Most studies focus on evaluating vitrified-thawed oocytes' post-fertilization outcome, but behavior of the meiotic spindle during the freezing process has received only a little attention.

Study design, size, duration
The experimental study involved a total of 165 human oocytes donated for research. The presence and morphology of the meiotic spindle were examined by polarized light microscopy (PLM) and fluorescent confocal imaging to map out the MII spindle dynamics during the vitrification-thawing procedure.

Participants/materials, setting, methods
A total of 114 women gave informed consent for their surplus immature oocytes to be used in this project. Oocytes that completed maturation in vitro were subjected to spindle imaging (Oosight) and vitrified using a Kitazato/Cryotop open system. The PLM-positive/negative oocytes were fixed at 6 steps of the vitrification-thawing protocol and 2 hours after thawing (15 oocytes in each subgroup). Microtubules and DNA were fluorescently labeled to inspect chromosome-spindle configuration.

Main results and the role of chance
Time-course experiments showed that PLM-detectable bipolar spindle remained intact in all but one oocyte (44/45) fixed during pre-vitrification equilibration. Notably, the PLM signal became more prominent after adding a cryoprotectant that displaced intracellular water. In contrast, the MII spindle signal progressively disappeared during thawing. Specific tubulin labeling revealed that the microtubule mass was still present in most (41/45) oocytes from the vitrified-thawed sample group. However, the division apparatus tended to lose its bipolarity and disintegrate (8/45) during the washing steps. This trend was accented in the group of oocytes that lacked PLM-detectable spindles before freezing. Here, only a minority of cells (11/45) undergoing rehydration displayed characteristic chromosome-spindle configuration. The difference in eggś capability to ensure spindle bipolarity was apparent even 2 hours after thawing when 13/15 of initially PLM-positive but only 7/15 PLM-negative oocytes exhibited normal-shaped spindles. Based on these data, we hypothesize that the transient disappearance of the PLM spindle signal during warming may be attributed to the restoration of microtubule dynamics and spindle destabilization. The speed and efficiency of spindle reconstruction seem to depend on the overall egǵs fitness.

Limitations, reasons for caution
This research study involved a limited number of hormonally-primed oocytes that extruded a polar body in vitro shortly after retrieval. The obtained imaging data represent snapshots of the process. A technically challenging live imaging study is needed to complement the description of the oocyte spindle dynamics during post-vitrification recovery.

Wider implications of the findings
Our results demonstrate that meiotic spindles in frozen-thawed eggs are not products of de novo spindle assembly but are built on preexisting microtubule mass. Suboptimal handling and reducing the interval between thawing and ICSI may enhance unavoidable spindle instability and thus compromise the developmental potential of vitrified eggs.

Trial registration number
not-applicable