Human embryo cryopreservation does not affect DNA methylation epigenetic reprogramming.

Introduction: It has been shown that procedures associated with assisted reproductive
technologies may interfere with epigenetic reprogramming of oocytes
and embryos.
After fertilisation the mammalian embryo undergoes a gradual genomewide
DNA demethylation, which results in relatively low DNA methylation
levels at the morula stage. Subsequently, DNA methylation is re-established
from the blastocyst stage onward. This de novo DNA methylation is set up by
de novo DNA methyltransferases, DNMT3a and DNMT3b.
We investigated if the process of embryo cryopreservation on day 3 (using
a slow-freezing DMSO protocol) affects the expression levels of DNMT3a and
DNMT3b or global DNA methylation levels.
Material and Methods: Spare human preimplantation embryos from infertility
treatments at our centre were donated for research after informed consent
of the patients and with the approval of the Local and the Federal Ethical Committee
for scientific research on human embryos in vitro. The embryos had
been cryopreserved on day 3 and were thawed using a slow-thawing DMSO
protocol (n = 34). At day 6, this group was classified into a subgroup of good
quality embryos (n = 15) and a subgroup of delayed and arrested embryos
(n = 19). The control group consisted of 'fresh' embryos (n = 10) that were
of good quality, but were affected after preimplantation genetic diagnosis, or
embryos of suboptimal quality that did not fulfil the criteria for transfer or
cryopreservation. All embryos were cultured in vitro for 6 days in sequential
medium.
We compared the global DNA methylation level as well as the expression
levels of de novo DNA methyltransferases DNMT3a and 3b in cryopreserved
and fresh embryos at day 6 of preimplantation development. The analysis was
done by immunofluorescence with antibodies against 5'methyl-cytosine (Eurogentec)
in single staining experiments or against the DNMT3a and DNMT3b
protein (Santa Cruz) in double staining experiments. Embryos of similar stages
were stained with normal serum of the same host species as an internal negative
control for the primary antibody. Fluorescence was visualized by confocal laser
scanning and further analysis was done with Volocity imaging software. The
fluorescence intensity (FI) per embryo was measured in all embryonic cells and
expressed as the sum of all intensities over the embryonic size. Finally the mean
FI for the global DNA methylation or DNMT3a and DNMT3b levels was calculated
per embryo (sub)group. Data of the fresh embryo group or the subgroup of
delayed and arrested cryopreserved embryos were relatively quantified against
the mean FI of the good quality cryopreserved embryo subgroup (mean FI = 1).
Results: No difference was observed between the mean FI of DNA methylation
levels (1.2 ± 0.1) in fresh embryos compared to levels in good quality cryopreserved
embryos. Similar DNMT3a (1 ± 0.3) and DNMT3b (0.8 ± 0.2) levels
were found in the fresh embryo group versus the good quality cryopreserved
embryos.
The mean FI of DNA methylation (1.1 ± 0.4) levels in delayed and arrested
cryopreserved embryos was similar to the levels of good quality cryopreserved
embryos as were the mean FI of DNMT3a (1 ± 0.3) and DNMT3b (0.8 ± 0.3).
Conclusions: These results show that at day 6, the de novo DNA methylation
levels and enzyme expression levels are reinstated in a timely manner in
human blastocysts that developed from embryos cryopreserved on day 3 using
a slow-freezing DMSO protocol. The cryopreservation process does not seem
to interfere with DNA methylation epigenetic reprogramming.

Publication year:2011

Keywords:DNA methylation