Archival fixed and analysed preimplantation human embryonic cells: a DNA resource for retrospective PCR analysis at the cellular level.
Biopsies of human embryonic cell preparations previously analysed by cytogenetic and/or fluorescent in-situ hybridization (FISH) chromosome probes provide a unique reference DNA resource for the archival preimplantation genetic diagnosis (PGD) of the transferred embryo. DNA polymerase chain reaction (PCR) may be utilized on these fixed cell preparations to verify equivocal FISH/PGD results. Retrospective PCR screens of the genotype of biopsied embryonic cell(s) may be of benefit in the case of a suspected genetic mutation. Currently, carrier detection or linkage analysis is often not possible because of early death of the fetus, or of patients with a lethal disease. Alternatively, fixed/stained 'failed fertilized' oocytes provide a resource to extend genetic analysis to infertile patients. A successful research is described which minimizes loss of individual analysed fixed/stained oocytes, metaphase chromosomes, and embryonic cell samples. Initial DNA amplification takes place in situ using a modified PCR protocol. Comparative cellular studies using primer sets previously used for PGD analyses show that 65% of the preparations amplified unequivocally using the modified protocol and primers for a CA repeat motif gene sequence, in comparison with 81% using the original PCR protocol. With further refinement and optimization, the methods outlined have the potential to retrospectively screen archival fixed chromosomes, gametes, and embryonic cells for clinical application, and enable the further study of the fixed human preimplantation embryo at the morphological, cell and molecular level.
Smith
T A
TA
Division of Obstetrics and Gynecology, UMDS, St Thomas's Hospital, London, UK.
Wall
M B
MB
Muggleton-Harris
A L
AL
D001706:Biopsy; D001755:Blastocyst / Q000737:chemistry*; D002970:Cleavage Stage, Ovum / Q000737:chemistry*; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification*; D004624:Embryo Transfer; D005260:Female; D030342:Genetic Diseases, Inborn / Q000175:diagnosis / Q000235:genetics; D006801:Humans; D008297:Male; D009865:Oocytes / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D011296:Prenatal Diagnosis / Q000379:methods; D012189:Retrospective Studies
D004247:DNA