Human Reproduction, Vol 12, 2011-2018, Copyright © 1997 by Oxford University Press
E Martini, SP Flaherty, NJ Swann, D Payne and CD Matthews
Chromosomal aberrations are the major cause of pre- and post- implantation
embryo wastage and some studies suggest that half of all human conception
have a chromosomal abnormality. Analysis of gametes provides information on
the origin of these chromosomal aberrations. The purpose of this study was
to develop a reliable multi-probe fluorescence in-situ hybridization (FISH)
procedure that would enable us to investigate aneuploidy in unfertilized
oocytes subjected to intracytoplasmic sperm injection (ICSI). Oocytes were
spread with HCl and Tween 20 solution, and then two rounds of triple-probe
FISH were performed on each oocyte using directly-labelled centromeric
probes: chromosomes 1, 7, 15 (overnight hybridization); chromosomes 1, X, Y
(2 h hybridization). After the first round, the slides were counterstained
and evaluated, and the positions of FISH signals were recorded. For the
second round, the counterstain was removed and the second probe cocktail
was applied. The chromosome 1 probe was an internal control for the two
hybridization procedures, while the Y chromosome probe was used to detect
sperm DNA. To evaluate the method, a total of 79 oocytes from 27 patients
were studied. Of these, 67 (84.8%) were successfully spread and 97% of
these oocytes exhibited discernible FISH signals. Upon lysis, oocytes
exhibited one or more DNA fragments (mean 1.9, range 1-3). Of the 65
analysable oocytes, 17 (26.2%) displayed a normal haploid chromosome
constitution with paired spots for the two chromatids. A further 23 oocytes
(35.4%) showed an ambiguous chromosome complement due to an abnormal number
of DNA fragments which may have resulted from loss of DNA during spreading
or to an abnormal oocyte, while 25 oocytes (38.4%) displayed aneuploidy for
one or more of the chromosomes studied. In conclusion, this new approach is
a quick and efficient method with which numerical chromosomal abnormalities
in human oocytes can be studied; interpretation of the patterns of DNA
fragments and FISH signals requires further clarification.
ARTICLES
Analysis of unfertilized oocytes subjected to intracytoplasmic sperm injection using two rounds of fluorescence in-situ hybridization and probes to five chromosomes
Department of Molecular Cell Biology and Genetics, University of Maastricht, The Netherlands.
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