Genome-wide DNA Methylation Study In ART-conceived Mice And Their Following Descendants

Part I Behavioral, morphological and genome-wide DNA methylation evaluation in IVF-conceived mouse and their cross-bred descendantsObjective:By establishing the IVF-conceived mouse model, we want to gain a prospective understanding of the marriage safety between ART-born children in human population, reveal the possible effect of IVF on the epigenetic inheritance, and privide theoretical foundation for empirical study in human population.Materials and methods:1. Mice model derived from in vitro fertilizationand embryo transfer (IVF-ET) were built and crossed. Naturally conceived (NC) mice were used as the control group. Effects of IVF on the spatial learning and memory capability at Fl generation were examined by Morris water maze at 6 weeks old. After Morris water maze were finished, Fl male mice were bred to F1 female mice to get the Fl generation;2. Effects of IVF on the spatial learning and memory capability at F2 generation were detected as did in F1 generation. After Morris water maze test, organs, including brain, heart, liver, lung, stomach, intestine, kidney, spleen, testis and ovary were excised, weighted and stained with hematoxilyn and eosin (H&E). The effects of IVF on development were detected by checking the specific gravity of the organs from male and in the female mice at 7 week;3. Genome-wide DNA methylation status were investigated in central nervous system (CNS) at F1,2 generation and naturally conceived mice by using the MM8 CpG promoter microarray;4. Eight concomitant hypermethylated promoters in F1 and F2 generation (Cryga, Fgfl, NosS, Mb, Myog, Notch3, Th, Vavl) and four unique hypermethylated promoters (Col9a2, Fgf6, Lck, SlcSa1) only in F2 generation, which associated with organ development function were further validated by bisulfite genomic sequencing;5. Expression of bisulfite sequencing confirmed genes(Fgfl, Nos3, Notch3, Th, Vavl in Fl, F2 generation and Col9a2, Fgf6, Slc5al in F2 generation) were analyzed by quantitative real-time RT-PCR in order to evaluate the relationship between promoter methylation and gene expression.Result(s):1. IVF-born mice model were successfully established;2. No differences in learning and memory ability including incubation period and the swimming distances were detected by the water maze test between IVF-born mice (F1), their cross-bred F2 generation and naturally conceived littermates;3. No statistically significant reduction or increase in the weight of total body. Obvious phenotypic abnormalities and defects, such as small eye, brachyury, short ear extra toes from first week to 6-7week old were not fount in F1 and F2 generation, but significant decrease in gravity of spleen in Fl generation mice was observed when compared with naturally conceived littermates;4.225 CpG islands and 191 promoters were hypermethylated at Fl generation. In contrast, only 22 CpG islands and 28 promoters showed a trend towards hypomethylation.196 CpG islands and 213 promoters were hypermethylated and 69 CpG islands,56 promoters were hypomethylated in F2 generation. A comparison of F1 to F2 epigenome identified 113 concomitant hypermethylated promoters and 143 hypermethylated CpG islands;5. Fgfl, Nos3, Notch3, Col9a2, Fgf6 and Slc5al were found with a gain in DNA methylation as validated by bisulfite genomic sequencing in F1 and F2 generation, which confirmed the results obtained by MeDIP-CHIP. No significant differences in Cryga, Mb, Myog in F1, F2 generation and Lck in F2 generation were found, which suggest they were false positive loci; 6. Fgfl, Nos3, Notch3, Col9a2, Fgf6 and Slc5al displayed lower levels of expression, which further validated their methylation status, but expression of Th and Vavl were not disturbed by their hypermethylated promoters, indicating that some genes were not susceptible to perturbation by promoter methylation.Conclusion(s):1. Learning and memory ability in IVF-conceived mice and their cross-bred F2 generation were not affected;2. Even the growth and development of Fl, F2 generation were not disturbed, IVF can slightly modify the epigenome at the central nerve system in F1 generation;3. IVF-induced DNA methylation aberration can be transmitted from Fl to F2 generation, and de novo aberrant DNA methylation occurred in F2 generation;4. Our findings suggest that there are some epigenetic risks in the marital events between ART-born children in human population and the exact mechanism need further investigation. Part II The inheritance of IVF-induced aberrant DNA methylation in IVF descendantsObjective:To determine the mode of epigenetic inheritance in IVF-induced aberrant DNA methylation and find the way they got transmitted to their following descendants, which could pave a road for investigating the precipitating factor of epigenetic disorders in human.Materials and methods:1. Female and male IVF-born F1 generation mice were mated to wild-type male and female mice to get the female-and male-line-derived F2 generation;2. Three concomitantly hypermethylated promoters loci (Fgfl, Nos3, Notch3) that were previously found in IVF-born mice and their F2 generation and three loci (Col9a2, Fgf6, Slc5a1) that found hypermethylated only in F2 generation were examined in female-line and male-line derived F2 generation;3. Expressions of these genes(Fgfl, Nos3, Notch3, Col9a2, Fgf6 and Slc5al) in male-and female-line-derived F2 generation were analyzed by quantitative real-time RT-PCR.Results:1. In female-line derived F2 generation, Fgfl, Notch3 and three de novo hypermethylated promoters indicated normal methylation status in central nerve system, but Nos3 was still hypermethylated and displayed disturbed levels of mRNA;2. In male-line-derived F2 generation, Fgfl, Nos3, Notch3 and three de novo hypermethylated promoters (Col9a2, Fgf6, Slc5a1) have come to normal methylation status, and all of the six loci did no show significant difference in the methylation values and their expression were not disturbed.Conclusions:1. Hypermethylated have been repaired by some pathways in male-line-derived F2 generation at the following phases of development in order to assure the integrity of the epigenome and IVF-induced aberrant DNA methylation might not be transmitted through male-line gametes;2. Epigenetic disturbances induced by IVF, such as aberrant methylation, can not be completely eliminated in the maternal germ line in mice, which were responsible for the epigenetic inheritance to their following generation. The risk of animals with maternally transmitted methylation aberrations depends on the efficiency of maternal epigenome repair following after fertilization. Partâ…¢Nos3 methylationo in female-line F3 generation and evaluation of DNA methylation status at differentially methylated regions (DMRs) in IVF-conceived newborn twinsObjective:To study the aberrant methylated Nos3 in female-line F3 generation. The effects of in-vitro fertilization (IVF) on the stability of DNA methylation at differentially methylated regions (DMRs) in IVF-conceived twins were investigated.Materials and methods:1. Nos3, which was found hypermethylated in IVF-born mice and can be transmitted through female gamete, was further investigated in female-line F3 generation; Its expressions was detected by real-time RT-PCR;2.59 pairs of twins were recruited, including 29 pairs conceived through IVF and 30 pairs naturally conceived twins. Umbilical cord blood samples were collection after cesarean section. DNA was extracted from umbilical cord blood. Two maternally methylated regions (KvDMR1 and PEG1) and one paternally methylated region (H19/IGF2 DMR) were analyzed using bisulfite-based technologies.Results:1. The methylation tendency of Nos3 has come to normal pattern at F3 generation. No differences were detected in the expressions of Nos3 in F3 generation;2. The median methylation percentages of IVF-conceived at PEG1 were 5 comparable to naturally conceived controls, and no significant differences were found (P=0.103) There was a trend toward hypomethylation in 3 children from 3 separate pairs of twins out of 29 (5.08%) pairs of IVF children who had methylation levels slightly lower than 21%, but 1 child out of 30 pairs (1.67%) of control children also displayed hypomethylation (Fisher's exact test,_P=0.611). One IVF child had an H19/IGF2 DMR methylation level of 68%. However, one naturally conceived case with a methylation level slightly higher than 70%was also found. Statistical analysis did not reveal significant differences in the methylation percentages of the H19/IGF2 DMR (P=0.103).Conclusion:1. Aberrant hypermethylated Nos3, which was found in IVF-born Fl generation and their female line F2 generation, has been corrected at F3 generation;2. Our results suggest no significant increase in imprint variability at these DMRs, but KvDMRl showed slightly more variable levels of methylation in IVF cases than in spontaneous cases, and the greater variance in the IVF kids has a biologically meaningful consequence and is still sources of concern for future investigation. Large samples of study are needed to systematically assess the potential epigenetic risk in IVF-conceived twins.