Assessing the chromosome copy number in metaphase II oocytes by sequential fluorescence in situ hybridization

Aneuploidy in oocytes is the main cause of failed embryo implantation and of miscarriage. At present, only limited data on the prevalence of aneuploidy in freshly collected human oocytes are available and all studies have been performed with conventional methods for karyotyping. In this feasibility study, multiple-hybridization fluorescence in situ hybridization (FISH) was evaluated as an alternative method to determine the number of chromosomes in oocytes.

Fifty-two spare oocytes were collected from 23 patients treated with gonadotropins for intrauterine insemination or intracytoplasmic sperm injection. A conventional dual color FISH approach using mixtures of chromosome-specific standard alpha-satellite probes was applied consecutively to the chromosomes of the same metaphase II oocyte. Mixtures of three to six probes were designed in order to allow chromosome identification based on signal color and centromeric index.

One hybridization cycle was possible in 52 uninseminated metaphase II oocytes, two hybridizations in 43 oocytes (82.7%), three hybridizations in 30 oocytes (57.6%), four hybridizations in 27 oocytes (51.9%), and five hybridizations in 15 oocytes (28.8%). Altogether, 591 chromosomes could be marked (47.4% of the entire chromosome complement, 11.4 chromosomes per oocyte). The most important single factor contributing to technical failure was loss of the oocyte from the slide.

This feasibility study demonstrates that multiple-hybridization FISH can be used for the assessment of a larger proportion of the chromosome complement in oocyte as compared to previous studies based on FISH.