FISH for Pre-implantation Genetic Diagnosis

Abstract

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a cohort generated by assisted reproduction technology (ART). This selection may be required because of familial monogenic disease (e.g. cystic fibrosis), or because one partner carries a chromosome rearrangement (e.g. a two-way reciprocal translocation). PGD is available for couples who have had previous affected children, and/or in the case of chromosome rearrangements, recurrent miscarriages, or infertility. Oocytes aspirated following ovarian stimulation are fertilized by in vitro immersion in semen (IVF) or by intracytoplasmic injection of an individual spermatozoon (ICSI). Pre-implantation cleavage-stage embryos are biopsied, usually by the removal of a single cell on day 3 post-fertilization, and the biopsied cell is tested to establish the genetic status of the embryo. Fluorescence in situ hybridization (FISH) on the fixed nuclei of biopsied cells with target-specific DNA probes is the technique of choice to detect chromosome imbalance associated with chromosome rearrangements, and to select female embryos in families with X-linked disease for which there is no mutation-specific test. FISH has also been used to screen embryos for spontaneous chromosome aneuploidy (also known as PGS or PGD-AS) in order to try and improve the efficiency of assisted reproduction; however, the predictive value of this test using the spreading and FISH technique described here is likely to be unacceptably low in most people's hands and it is not recommended for routine clinical use. We describe the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to PGD in a clinical setting.

Benefits and drawbacks of preimplantation genetic diagnosis (PGD) for reciprocal translocations: lessons from a prospective cohort study

“…The median number of eggs collected was 13 (range 4-39) and the median number of embryos biopsied was 6 (range [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. In our study the number of oocytes was not an important predictor for a live-birth pregnancy (OR 1.0, 95% CI 0.92-1.10, P ¼ 0.960).…”

“…19,20 In brief, a standard long stimulation protocol for controlled ovarian stimulation was followed by IVF, or ICSI when the patient had suboptimal semen characteristics, then biopsy of one or two cells from cleavage-stage embryos 3 days after normal (2PN) fertilisation for testing, followed by embryo transfer on day 4 (included in the subgroup analysis) and day 5 (the most recent cycles and not included in the subgroup analysis). A maximum of three embryos were transferred.…”

High-throughput Physical Mapping of Chromosomes using Automated <em>in situ</em> Hybridization

“…The details about isolating mitotic chromosomes from mosquitoes are given elsewhere 15 . Although regular squash preparations are sufficient for many purposes including FISH 16 and immunostaining 17 , the high-pressure method not only lowers potential variance from one slide to the next, but also increases overall chromosome quality, leading to higher detail when mapping chromosomes. This procedure can also be used for preparing polytene chromosome membrane slides for laser-capture microdissection.…”

Shallow whole genome sequencing is well suited for the detection of chromosomal aberrations in human blastocysts

“…In the past, fluorescent in situ hybridization has been widely used to screen for chromosomal imbalances in the embryos (3). With fluorescent in situ hybridization, the chromosomes involved in the translocation can be evaluated for unbalanced rearrangements at the single cellular level.…”

“…With fluorescent in situ hybridization, the chromosomes involved in the translocation can be evaluated for unbalanced rearrangements at the single cellular level. However, for every single case, an individual laboratory genetic work-up is needed to optimize the protocol before starting the PGD procedure (3,4).…”