Confirmation of diagnosis in preimplantation genetic diagnosis (PGD) through blastocyst culture: preliminary experience

Abstract

Three cases of preimplantation genetic diagnosis (PGD) (two for sexing and one for aneuploidy screening) are presented. Embryo biopsy was performed at day 3 and diagnosis was established with fluorescent in situ hybridization (FISH). Embryos not used for replacement were cultured in sequential media for blastocyst development. Blastocyst rate was 39.3 per cent. Confirmations of diagnosis were established with FISH in blastocysts and arrested embryos. Mosaicism was observed in 7/8 blastocysts (mean number of cells analysed: 55.5) and 5/8 arrested embryos. The percentage of abnormal cells was 17.1 per cent for blastocysts and 54 per cent for arrested embryos. Polypoid cells were observed in 4/8 blastocysts. Confirmation of diagnosis at the blastocyst stage is a useful tool in PGD.

Discordance among blastomeres renders preimplantation genetic diagnosis for aneuploidy ineffective

“…Previous reports have implicated mosaicism as a source of discordance among blastormeres and have suggested that mosaicism is the major drawback in the efficiency and reliability of preimplantation genetic diagnosis [17,18]. Chromosomally abnormal cells can coexist with normal cells and mosaic embryos can develop to morphologically [27] and chromosomally [20] normal blastocysts in vitro.…”

“…A major pitfall of PGD for aneuploidy screening with a single blastomere biopsy is the existence of mosaicism [17][18][19][20][21]. Chromosomally normal and abnormal blastomeres can coexist within the same early-cleavage-stage embryo [19].…”

“…Chromosomally normal and abnormal blastomeres can coexist within the same early-cleavage-stage embryo [19]. Mosaicism is common in human embryos generated in vitro and can lead to erroneous results [17,18]. A recent report described the rate of mosiacism among day 3 embryos to be 50% [21].…”

Use of cross-species in-situ hybridization (ZOO-FISH) to assess chromosome abnormalities in day-6 in-vivo- or in-vitro-produced sheep embryos

“…As the blastomeres divide and the embryos develop to the blastocyst stage, diploidYpolyploid mosaicism appears more frequently (Bielanska et al 2002). DiploidY tetraploid mosaicism was noted in 3Y26% of early morula stage human IVP embryos while the figure reached 23Y74% in blastocysts (Veiga et al 1999, Ruangvutilert et al 2000. Our observation on sheep embryos is comparable to those on human embryos (Veiga et al 1999, Ruangvutilert et al 2000.…”

Gamete and Embryo Manipulation

“…As such, the biopsy of 6 to 10 cells from the outer layer of the trophectoderm is less likely to have a detrimental effect on the blastocyst's mass of the developing fetus which originates from the inner cell mass (ICM). 282,283,291 The technique of zona slitting (similar to partial zona dissection) can be used to open the zona pellucida with a microneedle. 282 The blastocyst zona can also be opened using a laser.…”