Polscope analysis of meiotic spindle changes in living metaphase II human oocytes during the freezing and thawing procedures.
BACKGROUND
The clinical efficacy of the current methods used for cryopreservation of metaphase II human oocytes is low. Meiotic spindle disorders are thought to be largely responsible for this situation.


METHODS
Supernumerary fresh metaphase II human oocytes were cryopreserved in 1,2-propanediol with 0.1 M sucrose using a slow freezing/rapid thawing programme. Meiotic spindles were analysed in these living metaphase II oocytes at sequential steps of the freezing and thawing procedures with the use of a computer-assisted polarization microscopy system (Polscope).


RESULTS
The meiotic spindle was detected in all 56 oocytes (from 16 patients) before freezing and remained visible in all these oocytes throughout the preparation for freezing up to the time that they were loaded into cryopreservation straws. Immediately after thawing, the spindle was visible in 35.7% of oocytes, but it disappeared in all of the thawed oocytes during the subsequent washing steps. However, the spindle reappeared in all surviving thawed oocytes after washing (57.4%), by 3 h of incubation at 37 degrees C in culture medium.


CONCLUSIONS
The current techniques of oocyte freezing and thawing inevitably cause meiotic spindle destruction. All spindles observed in thawed oocytes result from post-thaw reconstruction.
Rienzi
L
L
Centre for Reproductive Medicine, European Hospital, Via Portuense 700, 00149 Rome, Italy. rienzi.laura@libero.it
Martinez
F
F
Ubaldi
F
F
Minasi
M G
MG
Iacobelli
M
M
Tesarik
J
J
Greco
E
E
D002470:Cell Survival; D002478:Cells, Cultured; D015925:Cryopreservation; D003470:Culture Media; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D008677:Metaphase; D008859:Microscopy, Polarization; D009865:Oocytes / Q000502:physiology / Q000648:ultrastructure*; D008941:Spindle Apparatus / Q000648:ultrastructure*; D013696:Temperature; D007507:Therapeutic Irrigation; D013997:Time Factors
D003470:Culture Media