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FISH for Pre-implantation Genetic Diagnosis

  • Paul N. Scriven
  • Caroline Mackie OgilvieEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 659)

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 individual spermatocytes (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 sporadic chromosome aneuploidy (also known as PGS or PGD-AS) in order to try and improve the efficiency of assisted reproduction; however, due to the unacceptably low predictive accuracy of this test using FISH, it is not recommended for routine clinical use.

This chapter describes the selection of suitable probes for single-cell FISH, assessment of the analytical performance of the test, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to PGD in a clinical setting.

Key words

Fluorescence in situ hybridization Pre-implantation genetic diagnosis PGD Sex determination Translocations Chromosome aneuploidy 

References

  1. 1.
    Hsu, L. Y., Benn, P. A., Tannenbaum, H. L., Perlis, T. E., Carlson, A. D. (1987) Chromosomal polymorphisms of 1, 9, 16, and Y in 4 major ethnic groups: a large prenatal study. Am. J. Med. Genet. 26, 95–101.PubMedCrossRefGoogle Scholar
  2. 2.
    Shim, S. H., Pan, A., Huang, X. L., Tonk, V. S., Varma, S. K., Milunsky, J. M., Wyandt, H. E. (2003) FISH variants with D15Z1. J. Assoc. Genet. Technol. 29, 146–151.PubMedGoogle Scholar
  3. 3.
    Knight, S. J. and Flint, J. (2000) Perfect endings: a review of subtelomeric probes and their use in clinical diagnosis. J. Med. Genet. 37, 401–409.PubMedCrossRefGoogle Scholar
  4. 4.
    Thornhill, A. R., deDie-Smulders, C. E., Geraedts, J. P., Harper, J. C., Harton, G. L., Lavery, S.A., Moutou, C., Robinson, M.D., Schmutzler, A.G., Scriven, P.N., Sermon, K.D., Wilton, L.; ESHRE PGD Consortium (PGS) (2005) ESHRE PGD Consortium ‘Best practice guidelines for clinical preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS)’. Hum. Reprod. 20, 35–48.PubMedCrossRefGoogle Scholar
  5. 5.
    Delhanty, J. D., Harper, J. C., Ao, A., Handyside, A. H., Winston, R. M. (1997) Multicolour FISH detects frequent chromosomal mosaicism and chaotic division in normal preimplantation embryos from fertile patients. Hum. Genet. 99, 755–760.PubMedCrossRefGoogle Scholar
  6. 6.
    Bahce, M., Cohen, J., Munné, S. (1999) Preimplantation genetic diagnosis of aneuploidy: were we looking at the wrong chromosomes? J. Assist. Reprod. Genet. 16, 176–181.PubMedCrossRefGoogle Scholar
  7. 7.
    Trussler, J. L., Pickering, S. J., Ogilvie, C. M. (2004) Investigation of chromosomal imbalance in human embryos using comparative genomic hybridization. Reprod. Biomed. Online 8, 701–711.PubMedCrossRefGoogle Scholar
  8. 8.
    Scriven, P. N., Handyside, A. H., and Ogilvie, C. M. (1998) Chromosome translocations: segregation modes and strategies for preimplantation genetic diagnosis. Prenat. Diagn. 18, 1437–1449.PubMedCrossRefGoogle Scholar
  9. 9.
    Estop, A. M., Van Kirk, V., and Cieply, K. (2005) Segregation analysis of four translocations, t(2;18), t(3;15), t(5;7), and t(10;12), by sperm chromosome studies and a review of the literature. Cytogenet. Cell Genet. 70, 80–87.CrossRefGoogle Scholar
  10. 10.
    Mackie Ogilvie, C., and Scriven, P. N. (2002) Meiotic outcomes in reciprocal translocation carriers ascertained in 3-day human embryos. Eur. J. Hum. Genet. 10, 801–806.PubMedCrossRefGoogle Scholar
  11. 11.
    Pellestor, F., Andréo, B., Arnal, F., Humeau, C., and Demaille, J. (2003) Maternal aging and chromosomal abnormalities: new data drawn from in vitro unfertilized human oocytes. Hum Genet. 112, 195–203.PubMedGoogle Scholar
  12. 12.
    Shi, Q., and Martin, R. H. (2000) Aneuploidy in human sperm: a review of the frequency and distribution of aneuploidy, effects of donor age and lifestyle factors. Cytogenet. Cell Genet. 90, 219–226.PubMedCrossRefGoogle Scholar
  13. 13.
    Tarkowski, A. K. (1966) An air drying method for chromosome preparations from mouse eggs. Cytogenetics 5, 394–400.CrossRefGoogle Scholar
  14. 14.
    Munné, S., Lee, A., Rosenwaks, Z., Grifo, J., and Cohen, J. (1993) Diagnosis of major chromosome aneuploidies in human preimplantation embryos. Hum. Reprod. 8, 2185–2192.PubMedGoogle Scholar
  15. 15.
    Harper, J. C., Coonen, E., Ramaekers, F. C. S., Delhanty, J. D. A., Handyside, A. H., Winston, R. M. L., and Hopman, A. H. N. (1994) Identification of the sex of human preimplantation embryos in two hours using an improved spreading method and fluorescent in situ hybridisation using directly labelled probes. Hum. Reprod. 9, 721–724.PubMedGoogle Scholar
  16. 16.
    Dozortsev D. I., and McGinnis K. T. (2001) An improved fixation technique for fluorescence in situ hybridization for preimplantation genetic diagnosis. Fertil. Steril. 76, 186–188.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Cytogenetics DepartmentGuy’s HospitalLondonUK
  2. 2.Guy’s and St Thomas’ Centre for Preimplantation Genetic DiagnosisGuy’s HospitalLondonUK

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