Cruciate thinning of the zona pellucida for more successful enhancement of blastocyst hatching in the mouse

Implantation rates remain low following human in-vitro fertilization (IVF). Suboptimal culture conditions may limit the ability of embryos to hatch as blastocysts, and artificial opening of the zona pellucida has been proposed as a means to promote subsequent hatching (assisted hatching). Such techniques must have minimal adverse effects on the embryos, while maximizing the potential for an embryo to hatch fully as a blastocyst. In a mouse model, we compared embryonic development after zona drilling, and cruciate thinning of the zona (CTOZ) intended to simulate the natural thinning of the zona pellucida. Using acidic Tyrode's solution both zona drilling and cruciate-thinning were performed on day 3 morulae. On day 4 the rates of complete hatching of blastocysts were 0/165, 24/172 and 72/175 in control, zona drilled and thinned groups respectively (P < 0.0001). On day 5 the rates of complete hatching in the same groups were 20/165, 54/172 and 120/175 respectively (P < 0.00001) and by day 6, 66/165, 74/172 and 130/175 respectively (P < 0.00001). The rate of arrest at the morula stage was 24/172 versus 8/175 in the zona drilled and thinned groups respectively (P < 0.005), whilst the rate of arrest at the blastocyst stage was 21/172 versus 14/175 respectively (NS). Hence cruciate thinning of the zona appears less detrimental at the morula stage than zona drilling, but eventual rates of arrest at the blastocyst stage were comparable. Both techniques significantly increased the rate of hatching, but zona drilling did not guarantee complete hatching. Less acid Tyrode's medium was needed to perform zona thinning, and acid was unable to enter the perivitelline space while the integrity of the zona pellucida was retained; consequently the viability of blastomeres was not compromised by contact with acid Tyrode's and the intact zona pellucida following cruciate thinning retained all of its protective functions. Therefore, it seems that cruciate thinning of the zona acts optimally to promote both early and complete hatching in the mouse. © 1992 Oxford University Press.