CHROMATIN QUALITY AS A CRUCIAL FACTOR FOR THE SUCCESS OF FLUORESCENT IN SITU HYBRIDIZATION ANALYSES OF UNFERTILIZED OOCYTES, POLAR BODIES AND ARRESTED ZYGOTES
Zhivkova RS1,*, Delimitreva SM1, Toncheva DI2, Vatev IT1
*Corresponding Author: Ralitsa S. Zhivkova, 1, St. Georgi Sofiiski Str., Laboratory of IVF and Preimplantation Embryology, Department of Biology, Medical Faculty, Medical University of Sofia, 1431, Sofia, Bulgaria; Tel.: +3592-91-72-678; Fax: +3592-91-72-660; E-mail: zhivkova@medfac.acad.bg
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Abstract

Material that is supernumerary or unsuitable for in vitro fertilization (IVF) procedures is used for basic and for IVF-related research. Despite the disadvantages of such cells, they have contributed much to our understanding of the mechanisms and prevalence of different abnormalities.

Fifty-four human unfertilized oocytes, 34 arrested bipronuclear zygotes and 15 polar bodies were fixed for analysis on the third day after in vitro insemination and were subjected to fluorescent in situ hybridization (FISH) with probes for chromosomes 18, 21, X and Y (centromere for 18, X, Y and locus-specific for 21). The aim of the study was the comparison of FISH efficiency in differently condensed chromatin.

The success of FISH analysis was over 60% of analyzed cells and it was dependent on the chromatin changes (condensation and/or fragmentation) during the culture period before cell fixation. Chromatin ageing was the crucial factor for the reduced success of FISH in both oocyte chromosomes (60.0%) and pronuclei (61.76%). The chromatin of second polar bodies (PBII), and premature chromosome condensation (PCC) of the sperm chromatin in oocytes was more suitable for FISH analysis (FISH success 75.0% in PBII and 64.29% in PCC)] with both centromere and locus-specific probes.

These results revealed the significance of early signs of in vitro cell ageing for the success of FISH analysis and for the interpretation of results in case of analysis of unfertilized human ova, polar bodies and arrested zygotes.

Keywords: Chromatin ageing, Fluorescent in situ hybridization (FISH), Oocyte, Polar body, Zygote




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