Comparison of effects of zona drilling by non-contact infrared laser or acid Tyrode's on the development of human biopsied embryos as revealed by blastomere viability, cytoskeletal analysis and molecular cytogenetics

Abstract

Use of a non-contact infrared laser (IRL) or acid Tyrode's for zona drilling before embryo biopsy was compared by assessing blastomere viability using various fluorescent markers or culture of the single biopsied blastomere, and, by cytoskeletal and molecular cytogenetic analysis of the biopsied embryos following culture to the blastocyst stage. There was no significant difference in the proportion of biopsied embryos that showed no damage in both the biopsied blastomere and in the remaining embryo (acid Tyrode's: 75% versus IRL: 68%), or in the proportion of single biopsied blastomeres that divided in culture (P > 0.05). However, single biopsied blastomeres from laser drilled embryos showed a greater tendency to form miniblastocysts. The proportion of laser or acid Tyrode's biopsied embryos that reached the blastocyst stage by day 6 was similar, although evident earlier (day 5) in the laser biopsied embryos. Spindle abnormalities at the blastocyst stage included tripolar and tetrapolar spindles, but their incidence was not significantly different from controls. In addition, no significant difference was observed in the incidence of chromosomal abnormalities and mosaicism between the two groups. It is concluded that using an IRL at a safe working distance does not cause adverse immediate or longer term effects on the development of human biopsied embryos, although damage can occur if drilling within this distance is unavoidable. Acid Tyrode's drilling can also cause damage, and tended to retard blastocyst development.

Are zona pellucida laser drilling and polar body biopsy safe for in vitro matured oocytes?

“…Two small studies (25 and 15 oocytes) of human oocytes [41] and one study of mouse oocytes (n=154) [32] have been published and suggest that laser ZP drilling is safe. Blastomere viability has been studied [33] and confirmed that the non-contact laser procedure had no impact, using two fluorescent markers of cell viability: CFSE, which binds covalently in the cytoplasm of viable cells, and propidium iodide, which is excluded from viable cells but can enter cells with damaged plasma membrane and bind to double-stranded DNA. Here, as described by Montag et al [32] who studied the impact of first polar body biopsy at the zygote stage, we used 4 criteria of oocyte quality after treatment.…”

“…It was first studied after mouse blastomere biopsy [31] and polar body biopsy [32], and secondly after human blastomere biopsy [33]. The use of non-contact infrared laser for zona drilling before embryo biopsy seems to have no impact on preimplantation development to the blastocyst stage, or on pregnancy rate.…”

Preimplantation genetic diagnosis and the biopsy technique: Important considerations

“…Independently from the procedure chosen, a biopsy involves two steps: 1) zona pellucida (ZP) drilling, which can be performed mechanically, chemically or by laser energy [18][19][20], and 2) cell/s removal.…”

Nuclear organisation in totipotent human nuclei and its relationship to chromosomal abnormality

“…Human embryos: preparation for cytogenetic analysis Preimplantation embryos were generated by IVF using standard protocols and embryo biopsy was carried out as previously described, removing one or two cells per embryo that were then processed for FISH analysis (Chatzimeletiou et al, 2005a). Following selection of embryos that appeared chromosomally normal on the basis of the diagnosis from the biopsied cells, embryo transfer proceeded.…”

“…Embryo nuclei were fixed to poly-L-lysine-coated glass slides through exposure to 0.01 M HCl and 0.1% Tween 20 as described previously (Harper et al, 1994;Chatzimeletiou et al, 2005a). Regular replenishing of this solution until the point that the zona pellucida (if present) and cytoplasm were dissolved preceded air-drying onto the poly-L-lysine slide then PBS washing and dehydration in an ethanol series.…”