Chromosomal analysis of meiotic stages of human oocytes matured in vitro: benefits of protease treatment before fixation.
OBJECTIVE
To modify Tarkowski's air-dry technique for mouse oocytes to develop a rapid, consistent procedure for human oocytes that enables accurate scoring of meiotic stage.


METHODS
Meiotically immature human oocytes, obtained after oophorectomy, were cultured for various periods and then subjected to Tarkowski's air-dry procedure (n = 104) or to our modified procedure (n = 175) that used a brief exposure to protease (20 to 40 seconds) before fixation.


METHODS
Air-dried oocytes were assessed for readability and for whether they contained overspread or overlapping chromosomes. In addition, discrete meiotic stages in human oocytes were identified.


RESULTS
Our protease procedure significantly increased readability of air-dried oocytes (96% versus 79% readable for protease versus Tarkowski, respectively; P less than 0.001) by significantly reducing the number of preparations with either overscattered (0.7% versus 3.4% for protease versus Tarkowski, respectively, P less than 0.05) or overlapping (1.3% versus 18% for protease versus Tarkowski, respectively, P less than 0.001) chromosomes.


CONCLUSIONS
Protease exposure of oocytes, combined with a modification of Tarkowski's procedure, resulted in high quality air-dries of human oocytes. This rapid and reliable procedure should have clinical application in in vitro fertilization programs for meiotic assessment of oocytes failing to fertilize.
Racowsky
C
C
Department of Obstetrics and Gynecology, University of Arizona, Tucson 85724.
Kaufman
M L
ML
Dermer
R A
RA
Homa
S T
ST
Gunnala
S
S
D000705:Anaphase; D002478:Cells, Cultured; D016922:Cellular Senescence; D002843:Chromatin / Q000648:ultrastructure; D002874:Chromosome Mapping; D010450:Endopeptidases / Q000494:pharmacology*; D005404:Fixatives; D006801:Humans; D008540:Meiosis; D008677:Metaphase; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*
D002843:Chromatin; D005404:Fixatives; D010450:Endopeptidases