Quantitative RT-PCR reveals tuberous sclerosis gene, TSC2, mRNA degradation following cryopreservation in the human preimplantation embryo

Abstract

The use of cryopreserved human embryos in gene expression studies provides an additional source to the scarce embryos available for research. To validate their use we have implemented a quantitative RT-PCR to characterize the levels of the tuberous sclerosis, TSC2 gene in fresh and frozen-thawed human embryos. Frozen embryos were thawed using two different clinical protocols. In fresh embryos 9.95 fg of TSC2 cDNA was present in the unfertilized oocyte, which was comparable to the level on day 2 of preimplantation development. On day 3 there was a significant drop (P < 0.001) to 6.8 fg, followed by an increase in cDNA levels to 10.8 fg (P < 0.01) on day 6 at the expanded blastocyst stage. Day 2 frozen embryos possessed 50% less (P < 0.001) TSC2 mRNA in comparison to the fresh embryos using thawing protocol one (from frozen to 37°C and 25% less TSC2 mRNA (P < 0.01) with thawing protocol 2 (from frozen to room temperature). After culturing day 2 frozen embryos for an additional day they showed mRNA levels comparable with fresh day 3 embryos. There was no significant difference in the levels of TSC2 mRNA between fresh and frozen day 3 human embryos with either thawing protocol. This study demonstrates that cryopreservation does affect the normal pattern of gene expression during human preimplantation development, and that intact frozen-thawed embryos are not equivalent to their non-frozen counterparts. Furthermore human embryos frozen on day 2 appear to be more susceptible to temperature change than embryos frozen on day 3.