Genetic diagnosis using polymerase chain reaction and fluorescent in-situ hybridization analysis of biopsied cells from both the cleavage and blastocyst stages of individual cultured human preimplantation embryos
| Autor: | M. Wall, A.L. Muggleton-Harris, S.J. Pickering, A.M. Glazier |
|---|---|
| Rok vydání: | 1995 |
| Předmět: |
Genetic Markers
Male Blastomeres X Chromosome Biology Preimplantation genetic diagnosis Y chromosome Polymerase Chain Reaction law.invention law Culture Techniques Prenatal Diagnosis Y Chromosome medicine Humans Blastocyst In Situ Hybridization Fluorescence Polymerase chain reaction Repetitive Sequences Nucleic Acid Retrospective Studies Chromosome Aberrations Hybridization probe Rehabilitation Genetic Diseases Inborn Obstetrics and Gynecology Embryo culture Embryo Blastomere Molecular biology Globins medicine.anatomical_structure Oligodeoxyribonucleotides Reproductive Medicine embryonic structures Female DNA Probes |
| Zdroj: | Human Reproduction. 10:183-192 |
| ISSN: | 1460-2350 0268-1161 |
| DOI: | 10.1093/humrep/10.1.183 |
| Popis: | Cultured human preimplantation embryos have been used to develop methods which allow preimplantation genetic diagnosis (PGD) analyses by polymerase chain reaction (PCR) and fluorescent in-situ hybridization (FISH) on biopsied blastomeres and trophectoderm cells from the same embryo. An experimental design is described and experiments undertaken, which demonstrate the feasibility of extending biopsy and PGD procedures currently in use. We have shown that dual-stage biopsies are possible, and that the PCR and FISH analyses of the biopsied cell samples are effective. One to two blastomeres were biopsied from an 8- to 10-cell embryo and processed for the simultaneous PCR amplification of a beta-globin and a cytosine adenine (CA) repeat sequence, or a Y chromosome sequence. FISH procedures were also used to detect the presence of Y chromosome markers. The biopsied cleavage-stage embryo can be cultured to the blastocyst stage, where the serial biopsy of three to five mural trophectoderm cells provides two further cell samples. These can be used to repeat and/or undertake additional PGD analyses. The biopsied blastocyst is either used to confirm earlier diagnoses, or placed in culture for a further 4-24 h. Maintenance of a blastocoele cavity, hatching and formation of an outgrowth demonstrates continuing viability following the dual-stage biopsy procedures. The PCR DNA amplification procedures are effective at the cellular level for both biopsied blastomeres and mural trophectoderm cells. The FISH techniques have shown a definitive Y signal in 50% (one out of two) and 100% (two out of two) of the biopsied blastomeres and 72% (two out of three, four out of five and 7 out of 10) for the trophectoderm cell nuclei. Preliminary experiments have demonstrated that the FISH preparations can be re-amplified to improve the signal, and dual fluorescent procedures using the X and Y probes are effective. A retrospective PCR analysis has also been undertaken on preparations of biopsied cells which were previously used for PGD analysis by FISH. |
| Databáze: | OpenAIRE |
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