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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INTRODUCTION

Genetic investigations of human oocytes are an important source of knowledge about chromosomal aberrations of the embryo, which is the major factor determining the success of assisted reproductive techniques. Female meiosis is a major cause for aneuploidy via two mechanisms: non division of bivalents during the first division and predivision of chromatids during the second division [1]. These meiotic errors can be revealed by genetic analysis of first and second polar bodies (PBI, PBII), respectively.

Even in the absence of a male infertility factor, some human oocytes do not undergo fusion with sperm cells and remain unfertilized. Anomalies of human fertilization, such as premature chromosome condensation (PCC) and polyspermy, are attributed to asynchrony between meiotic chromosomal and cytoplasmic maturity of the oocyte. The PCC of the sperm chromatin is due to preserved activity of maturation promoting factor in the ooplasm [2,3]. This status is irreversible and no pronuclei can be formed. Ooplasm immaturity is related to unreliable blockage of polyspermy because of ineffective cortical reaction during fertilization [4].

Anomalies in pronuclear formation are associated with poor prognosis for the success in in vitro fertilization and embryo transfer (IVF-ET) practice. An abnormal number of pronuclei indicates abnormal ploid status of the embryo and transfer of mono-pronuclear or three-pronuclear zygotes is avoided [5,6]. Transfer of bi-pronuclear (2PN) zygotes on the third day after insemination is controversial because typically developing embryos at this time are at the 4-cell stage. The delayed 2PN stage can be caused, in some cases, by fluctuations of normal fertilization but most frequently is a sign of fertilization failure [7,8].

Preimplantation genetic analyses of oocytes, polar bodies (PBs) and zygotes were recently performed in order to reveal their chromosomal status and development potential. Despite the numerous studies, the significance of eventual correlation between chromosomal status of such cells and their restricted developmental potential is a question that still remains open [9-14]. For PBs and blastomeres, genetic analyses can also be used for preimplantation screening in assisted reproductive programs in order to prevent the transfer of chromosomally abnormal embryos.

The selection of an appropriate method for the preimplantation genetic analysis of single cells depends on the purpose of investigation and the characteristics of the chromatin. Classical karyotyping of the oocyte metaphase chromosomal set has long been used [15-17]. However, it is technically difficult, time-consuming and dependent on good chromosome spreading. The high degree of chromosome condensation in the oocyte and PBI is a known phenomenon and the chromosome identification using banding technique is unreliable [18]. Classical karyotyping of zygotes and blastomeres is possible only after cytostatic pretreatment [9,19,20], which makes the method even more time-consuming and unsuitable for diagnosis before embryo transfer. Interphase chromatin of PBII, pronuclei and blastomeres and metaphase chromosomes of oocytes and PBI can be analyzed by molecular cytogenetic methods [21-25].

Fluorescent in situ hybridization (FISH) is currently accepted as an appropriate technique for preimplantation genetic analyses [26-28]. It is fast, uncomplicated and suitable for assay of metaphase and interphase chromatin. Fluorescent in situ hybridization is presumed to overcome the difficulty of chromosome spreading and, for that reason, to be appropriate for unfertilized oocytes and polar bodies. For the interphase chromatin, FISH allows analysis without cytostatic pretreatment. Up to 12 chromosomes have been detected in two or three rounds of hybridization on the same cell using sets of directly labeled fluorescent probes [12,29,30]. In clinical applications, biopsy and fixation of polar bodies or blastomeres is performed immediately after oocyte retrieval, fertilization or embryo formation. In these cases, FISH is the most appropriate and efficient technique because of the uncomplicated and time-saving procedure that uses commercially available directly labeled probes for all human chromosomes.

Material that is supernumerary or unsuitable for IVF procedures is useful in basic and IVF-related research. Despite the disadvantages of these cells, the results have contributed much to our understanding of the mechanisms and prevalence of different abnormalities. Human oocytes and preimplantation embryos with poor quality or excessive number are used for analysis. The changes associated with in vitro cell ageing started at this time and their first manifestation is chromatin condensation and/or fragmentation. Little is known about the potential impact of these processes on performance of FISH [31,32]. The present study aimed to compare the efficiency and success of FISH in human unfertilized oocytes, arrested zygotes and polar bodies which differ in chromatin condensation and quality.




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