| Objective:To investigate the effects of embryo vitrification to the methylation status of H19/Igf2 differentially methylated domain (DMD) and the expression of relative genes in the mouse fetuses and placentas.Methods:There were three groups:Group A, Group B, and Group C. The D14 fetuses and placentas from copulation of normal male and female mice were taken as the Group A (the first control group); those from superovulation, in-vitrio fertilization, in-vitro culture and embryo transfer were taken as the Group B (the second control group); those from superovulation, in-vitrio fertilization, in-vitro culture, embryo vitrification and embryo transfer were taken as the Group C (the study group). Extract genomic DNA and total RNA from the fetuses and placentas, the methylation of H19/Igf2 DMD were analyzed by cloning and sequencing after DNA bifulfite treatment; the expression quantity of H19 and Igf2 were analyzed by Real Time RT-PCR.Results:There are 51 fetuses and placentas from 5 pregnant ICR females in the Group A.10 pseudopregnant ICR females underwent successful embryo transfer, from which we obtained 53 fetuses and placentas from the the Group B and 50 fetuses and placentas from the Group C. Hypermethylated clones from fetuses in the Group A, the Group B and the Group C were of 51.4%±1.13% 37.79%±0.76% and 20.16%±0.97%, respectively. Compared with the Group A, loss of methylation was found in the H19/Igf2 DMD of the fetuses from the Group B and the Group C (P<0.05), but more severe in the Group C (P＜0.05). Hypermethylated clones from placentas in the Group A, the Group B and the Group C were 53.24%±1.11%,32.49%±1.24% and 36.87%±0.97%, respectively. Compared with the Group A, loss of methylation was found in the Group B and the Group C (P<0.05), the latter two have no significant difference (P>0.05). The H19ΔΔCt in fetuses of the above three groups were 0± 0.41,-3.53±0.43 and -1.77±0.32, respectively; the relative expression quantities were 1.04±0.16,8.01±0.15 and 2.2±0.12, respectively. Compared with the Group A, the H19 expression was significantly increased in the Group B and the Group C; however, the quantity in the Group C was less than that detected in the Group B (P<0.05). The H19ΔΔCt in placentas of the above three groups were 0±0.37,2.44±0.56 and 3.79±0.81, respectively; the relative expression quantities were 1.13±0.11,0.23±0.13 and 0.08±0.17, respectively. Compared with the Group A, the H19 expression was significantly decreased in the Group B and the Group C (P<0.05), more severe in the Group C (P＜0.05). The Igf2ΔΔCt in fetuses of the above three groups were 0±0.29,2.01±0.43 and 1.21±0.24, respectively; the relative expression quantities were 1.07±0.11,0.30±0.03 and 0.56±0.08, respectively. Compared with the Group A, the Igf2 expression was significantly decreased in the Group B and the Group C (P＜0.05), however the Igf2 relative expression quantity in the Group C was more than that in the Group B (P<0.05). The Igf2ΔΔCt in placentas of the above three groups were 0±0.37,-1.48±0.23 and -1.69±0.45, respectively; the relative expression quantities were 1.02±0.18,2.92±0.03 and 3.14±0.04,respectively. Compared with the Group A, the Igf2 expression was significantly increased in the Group B and the Group C (P<0.05), the differences between the latter two groups were not significant (P>0.05).Conclusion For D14 fetuses, embryo vitrivication and warming aggravated the loss of methylation in H19/Igf2 DMD caused by procedures such as Ovulation induction, In-Vitro-Manipulation and so on, however compensated the expression disorders of H19/Igf2; for placentas, it didn't affect the methylation of H19/Igf2 DMD and Igf2 expression quantity significantly, however aggravated the H19 expression decrease by the procedures in the Group B.
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