Identifying the sex of human preimplantation embryos in X-linked disease: amplification efficiency of a Y-specific alphoid repeat from single blastomeres with two lysis protocols.
BACKGROUND
Preimplantation diagnosis involves detecting genetic defects in one or two blastomeres biopsied from cleavage stage embryos following a vitro fertilization (IVF). For X-linked recessive disease, identification of the sex of embryos allows transfer of only unaffected females. To examine how critical the preparation of the single blastomere is for amplification of a Y chromosome specific repeat sequence using the polymerase chain reaction (PCR), the incidence of amplification failure has been examined following two lysis protocols.


METHODS
Amplification of a Y alphoid repeat sequence from single blastomeres disaggregated from cleavage stage embryos was examined after either (1) lysis in distilled water and freeze-thawing twice or (2) a two-step lysis protocol involving an initial treatment in potassium hydroxide and dithiothreitol. Some of the embryos had been previously sexed by cleavage-stage biopsy and fluorescent in situ hybridization with X- and Y-specific probes.


RESULTS
Amplification failure occurred in 6 of 50 (12%) and 4 of 60 (7%) single blastomeres from male embryos following lysis in distilled water or using the two-step protocol, respectively. Conversely, amplification from contaminating DNA occurred in 5 of 63 (8%) single blastomeres from female embryos and 6 of 94 (6%) of control medium-blanks.


CONCLUSIONS
The incidence of amplification failure was improved but not eliminated using the two-step lysis protocol. At least two cells, therefore, would be necessary for accurate identification of males by amplification of Y-specific repeat sequences alone. Nevertheless, this protocol for preparing cleavage-stage blastomeres is likely to give more consistent amplification of any unique or repeat sequences.
Kontogianni
E H
EH
Human Embryology Laboratory, Institute of Obstetrics and Gynaecology, Royal Postgraduate Medical School, London, England.
Griffin
D K
DK
Handyside
A H
AH
D001757:Blastomeres / Q000737:chemistry* / Q000187:drug effects; D002458:Cell Fractionation / Q000379:methods*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D004229:Dithiothreitol / Q000494:pharmacology; D005069:Evaluation Studies as Topic; D005260:Female; D005307:Fertilization in Vitro; D005615:Freezing; D005808:Genes, Recessive; D005819:Genetic Markers; D006801:Humans; D006878:Hydroxides / Q000494:pharmacology; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D008832:Microchemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D017680:Potassium Compounds / Q000494:pharmacology; D011296:Prenatal Diagnosis / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid; D012732:Sex Determination Analysis / Q000379:methods*; D012743:Sex Preselection / Q000379:methods*; D014867:Water / Q000494:pharmacology; D014960:X Chromosome / Q000235:genetics; D014998:Y Chromosome / Q000235:genetics*
D005819:Genetic Markers; D006878:Hydroxides; D017680:Potassium Compounds; D014867:Water; D004247:DNA; D004229:Dithiothreitol; C029943:potassium hydroxide