BACKGROUND: Preimplantation genetic diagnosis (PGD) usually involves blastomere biopsy 3 days post-insemination (p.i.), followed by genetic analysis and transfer of unaffected embryos later on day 3 or 4. We evaluate a strategy involving embryo biopsy on day 3 p.i., genetic analysis on day 4 and, following culture in blastocyst sequential media, transfer of unaffected embryos on day 5 p.i. METHODS: PGD cycles were initiated in 15 couples at risk of transmitting beta-thalassaemia major. Oocyte retrieval and ICSI were performed according to standard protocols. Embryo culture used blastocyst sequential media. Embryos were biopsied on day 3 p.i. using acid Tyrode's for zona drilling, and the single blastomeres were genotyped by a protocol involving nested polymerase chain reaction and denaturing gradient gel electrophoresis analysis. RESULTS: Forty of 109 (37%) embryos biopsied on day 3 p.i. developed to blastocysts by day 5 p.i., with at least one blastocyst available for transfer in 12 cycles (80%). Genotype analysis characterized 51/109 (47%) embryos unaffected for beta-thalassaemia major, of which 28 were blastocysts. Transfer of 37 day 5 p.i. embryos (blastocysts and non blastocysts) initiated eight clinical pregnancies. Implantation rate per embryo transferred was 12/37 (32%). CONCLUSIONS: Embryo biopsy on day 3, followed by delayed transfer until day 5 p.i. offers a novel and effective strategy to overcome the time limit encountered when performing PGD, without compromising embryo implantation.
BACKGROUND: Preimplantation genetic diagnosis (PGD) usually involves blastomere biopsy 3 days post-insemination (p.i.), followed by genetic analysis and transfer of unaffected embryos later on day 3 or 4. We evaluate a strategy involving embryo biopsy on day 3 p.i., genetic analysis on day 4 and, following culture in blastocyst sequential media, transfer of unaffected embryos on day 5 p.i. METHODS: PGD cycles were initiated in 15 couples at risk of transmitting beta-thalassaemia major. Oocyte retrieval and ICSI were performed according to standard protocols. Embryo culture used blastocyst sequential media. Embryos were biopsied on day 3 p.i. using acid Tyrode's for zona drilling, and the single blastomeres were genotyped by a protocol involving nested polymerase chain reaction and denaturing gradient gel electrophoresis analysis. RESULTS: Forty of 109 (37%) embryos biopsied on day 3 p.i. developed to blastocysts by day 5 p.i., with at least one blastocyst available for transfer in 12 cycles (80%). Genotype analysis characterized 51/109 (47%) embryos unaffected for beta-thalassaemia major, of which 28 were blastocysts. Transfer of 37 day 5 p.i. embryos (blastocysts and non blastocysts) initiated eight clinical pregnancies. Implantation rate per embryo transferred was 12/37 (32%). CONCLUSIONS: Embryo biopsy on day 3, followed by delayed transfer until day 5 p.i. offers a novel and effective strategy to overcome the time limit encountered when performing PGD, without compromising embryo implantation.