E H Kontogianni1, D K Griffin, A H Handyside. 1. Human Embryology Laboratory, Institute of Obstetrics and Gynaecology, Royal Postgraduate Medical School, London, England.
Abstract
INTRODUCTION: 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. MATERIALS AND 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.
INTRODUCTION: 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. MATERIALS AND 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.
Authors: D K Griffin; A H Handyside; J C Harper; L J Wilton; G Atkinson; I Soussis; D Wells; E Kontogianni; J Tarin; S Geber Journal: J Assist Reprod Genet Date: 1994-03 Impact factor: 3.412
Authors: I Soussis; J C Harper; E Kontogianni; T Paraschos; D Packham; A H Handyside; R M Winston Journal: J Assist Reprod Genet Date: 1996-03 Impact factor: 3.412