The Whole Genome Methylation Pattern Of CpG Island And Promoter For Human Gamete And Preimplantation Embryos Or Morulas From In Vitro Maturation Oocytes

Primordial germ cells to produce mature gametes, fertilized zygote genome restart is the two phase of the most important human and mammalian life cycle.In former studies, quantitative analysis were performed on whole genome methylation sites in human and mammalian gametes and early embryos,which confirmed that the producing of human and mammals mature gametes, fertilized zygotes and DNA methylation during the preimplantation embryonic development are of dynamic change. The dynamic change pattern of methylation provides an important theoretical basis for us to understanding the gene expression and regulation of human early embryo of and the inhibiting effects of transposons. Although RRBS method can accurately quantified 5-mC and 5-hmC respectively, but the coverage is low (CpG island 10% of the methylation sites).Qiao-Jie and some researchers used the method of whole genome bisulphite sequencing (WGBS) to detect the methylation sites changes of cell mass in the blastcyst with relatively large number of cell (ICM) and after planting embryonic genome-wide. But they did not elucidated genome-wide methylation sites changes of gamete and embryo of early blastocyst. MeDIP-Chip was performed in this study, which can cover almost all promoter and CpG island, to analysis the dynamic change of whole genome CpG island and promoter region methylation in various developmental stages of human sperm, oocytes and preimplantation embryos. The work helps understanding the CpG island and promoter methylation pattern of dynamic change and adjustment mechanism of methylation dynamic change and it’s function during early embryonic development.In the period of Metaphase (prophase I,MI), oocyte begins to demethylation, after birthlate thick line of secondary oocyte re-establish methylation patterns to a during development of the primordial follicles transform to antral follicles, and completed before fertilization. The immature oocytes were collected from antral follicles phase, after 24-48h in vitro matured, be fertilized when reach maturity. But the studies have shown that it is not enough for oocyte to complete reconstruction of methylation before it matured, which will bring about the emergence of various abnormal in the latter part of the embryonic development. The immature oocyte which from in vitro fertilization (IVF) patients’ small follicle, can get mature oocytes after IVM, when it get fertilization we can successfully obtain clinical pregnancies after embryo transplantation. However, in each study the ratio of the pregnancy rate are very different. And even some studies suggested that the embryos obtained from this part of immature oocytes are of poor quality and low development potential, the pregnancy rate post transplantation is very low. That is why some researchers doubted its utilization value. From this, the utilization value of immature oocytes, which obtained form ovulation induction patients, is controversial. This study will do mached analysis to IVM morula and in vivo maturation morula in CpG islands and promoter of whole genome, which is a critical period of zygotic genome starts activation. It provides an evaluation of the possible mechanisms of low development potential of IVM embryos and their safety from the perspective of epigenetics..Part 1:the Map of Human Gametes and Preimplantation Embryos Whole-genome CpG Island and Promoter methylation[Objective]Previous researches have confirmed that the DNA methylation of producing of human and mammals mature gametes, fertilized zygotes and the preimplantation embryos are dynamic change. But the studies covered only 10% methylation sites of the CpG island. In this study, I will use the method of MeDIP-Chip, which can cover almost all genome promoters and CpG islands (most methylation events occur region),to analysis the dynamic change of whole genome CpG island and promoter region methylation in human sperm,oocytes and various developmental stages of preimplantation embryonic. We also want to understanding the CpG island and promoter methylation pattern of dynamic change and adjustment mechanism of methylation dynamic change and it’s function during early embryonic development.[Methods]Sperm, oocytes, cleavage stage embryos used in this study were from donation of volunteers and assisted reproductive patients in the Third Affiliated Hospital of Guangzhou Medical University, from July 2010 to July 2013. And Hospital Ethics Committee approved the collection.GroupingThey were divided into 8 groupsbased on developmental stages of specimens.① Sperm group,30 specimens;② MⅡ oocytes were 25; ③2PN period zygote were 25; ④The number of 4-cell stage embryos were 5;⑤The number of 8-cell stage embryos were 4;⑥ Morula embryos were 6; ⑦5 ICM (Inner cell mass) of blastosyst;⑧ 5 TE (Trophoblastic cells) of blastocyst. DNA extractionWe will extract sperm DNA by the method of difference lysis and combine Genomic DNA urification Kit (Promega);②-⑥groups:Removed of the zona pellucida and adhesive granulosa cell of Oocytes and early embryo with the method of Tyrode’s solution, then lyse cell and extracted DNA after moved it into PCR tube, ⑦ and ⑧roups:To separate ICM and TE using attenuated PAP pipe, then moved into the PCR tube cracking cells to collected DNA. (3) (MeDIP-Chip)Using ultrasonic broke the DNA of each group into random size fragments about 200-1000 bp. Afterimmunoprecipitated, purified by rat 5-methyl monoclonal antibody, use the WGA Kit that labeled by Cy3 and Cy5 to Whole Genome Amplification. Then hybridized to a microarray with Methylation 3x720k promoter and CpG islands which containing the NimbleGen human DNA. And scanning byGenePix 4000B microarray scanner.(4) Statistical data of chip methylationPeak Score:-log10 (P-value) of peak on the probe can reflect the the degree of methylation.Peakvalue:log2 (IP/Input)-ratio of the median that measured by probe, n is the peak valuewhen Peak Score≥2.[results](1) The purity of DNA extraction and the DNA fragments that enrichment in immunoprecipitation are consistent with experiments.(2) Unique dynamic changes pattern of human gametes and pre-implantant embryonic genome CpG island methylation.①The degree of sperm whole-genome CpG island methylation was the highest (sperm,n=15604), after fertilization, the original nucleus gradually DE methylation (2PN, n=3744), the degree of whole-genome CpG islands methylation of 4cell reached the lowest (4cell, n=2826), after 8cell the number of CpG island methylation begin to gradually rebuilding (8cell,n=3073). In the stage of morula, CpG island methylation levels increased further (morula,n=5374),until ICM CpG island methylation levels which in the stage of blastocyst close to oocytes level (IVM,n=5706), but TE CpG island methylation level is between the oocyte (oocyte,n=6062)and sperm (TE,n=8376).②Dynamic changes region of preimplantation embryonic whole-genome CpG island methylation was mainly between hypermethylation (Peak Mvalue≥0.7) and hypomethylated (Peak Mvalue<0.4).③There are three dramatic period in the change of Preimplantation embryonic whole Genome CpG island methylation degree. The trend of this change in the first period is sperm, the transition form after-fertilization oocyte to 2PN mainly is the reduction of methylation degree (rapid demethylation), in the second stage morula transform to blastocyst, methylation rapid rebuild, at the third phase the degree of methylation reconstruction process of TE which is produced after the process of blastocyst formation that cells transform to ICM and TE differentiate is greater than ICM.④The dynamic change condition of CpG island methylation in Intragenic, intergenic and Promotor regions:the dynamic change of whole-genome CpG island methylation is mainly caused by CpG island methylation which belong to the promoter region dynamically change.73.7% methylation signal of sperm is from promoter region,oocytes is 60.8%, zygotes of 2PN stage is 57.9%,4 cell period embryos is 52.2%,8cell period embryos is 50.3%, morula stage embryos is 68.8%, blastocyst ICM is 66.6%, trophoblastic cells is 66.8%. The change of CpG island methylation in intergenic and Intragenic region is not so obvious as in Promotorregion.(3) Dynamic changes human preimplantation embryonic whole-genome promoter methylation level①The methylation degree of sperm’s whole-genome is the highest in promoter region (sperm,n=10634), after fertilization, the original nucleus gradually de-methylation(2PN,n=5951),in the period of 4cell (4cell,n=6451),on the contrary it slightly elevated but still lower than the level of the oocyte genomic methylation levels in promoter region (oocyte,n=6678),after that it gradually began to demethylation until the morula stage to a minimum (morula,n=5374),after forming a blastocyst and differentiated to ICM it rising slightly (ICM,n=5723),the methylation degree reconstructed by differentiated TE cell is the highest (TE,n=7323) and between oocytes and sperm.②Dynamic changes of preimplantation embryonic whole-genome promoter methylation was main caused by the change of the region of hypermethylation (Peak Mvalue≥0.7) promoter and hypomethylated promoter (Peak Mvalue<0.4)③Human preimplantation embryonic genome-wide promoter methylation dynamic change is mainly caused by the dynamic change of HCP regional level LCP regional methylation.The proportion of genome-wide promoter HCP that be detected in 4cell period is lowest(40.2%),then it gradually increase, HCP proportion of ICM (58.1%) and TE (59.5%) of blastocyst stage that be detected is between oocytes (75.2%)and sperm cells(53%).The LCP proportion of promoter be detected is on the contrary with it, LCP proportion in 4cell period is the highest (38.8%). ICP distribution of Genome promoter be detected is not so obvious.Conclusion(1) The unique dynamic changes pattern of preimplantation embryonic whole-genome CpG island methylation:after fertilization, the original nucleus gradually demethylation, methylation degree of whole-genome CpG island reached the lowest in 4cell period, then gradually re-establish methylation until ICM of blastocyst stage close to oocytes level,and TE is between oocytes and sperm.(2) There are three dramatic change stages of the methylation changes of preimplantation embryonic CpG island, in different development period. Main stage of CpG island methylation erasure is in 4cell period, but Whole Genome is in 2 cell period.(3) Dynamic changes of whole-genome CpG island methylation is mainly caused by the dynamic changes of methylation of CpG islands thatin Promotor region.(4) The unique dynamic change pattern of whole-genome promoter methylation of human preimplantation embryonic:after fertilization, the original nucleus gradually demethylation, in the period of 4cell,on the contrary it slightly elevated but still lower than the level of the oocyte,after that it gradually began to demethylation until the morula stage to a minimum, after forming a blastocyst and differentiated to ICM it rising slightly, the methylation degree reconstructed by differentiated TE cell is the highest and between oocytes and sperm. Genome-wide promoter methylation dynamic change is mainly caused by the dynamic change of HCP regional level LCP regional methylation.Part2 Methylation Patterns Compare between IVM and IVOMorula CpG Island and Promoter Region[Objective]Immature oocytes that obtained from in vitro fertilization (IVF) patients’small follicles can get matured after in vitro culture in 24-48h. When matured oocytes are fertilized, we can obtain clinical pregnancy by embryo transplantation.However, the ratio of pregnancy rate in all studies is falls far short. And even some studies suggest that the embryos obtained form this part of immature oocytes are poor quality, and with low development potential, after transplantation the pregnancy rate is very low. That is why some researchers questioned its utilization value. From this, the utilization value of immature oocytes, which obtained form ovulation induction patients, is controversial. Furthermore the immature oocytes removed during antral follicles phase, after 24-48h cultured in vitro,it can be fertilized when reach maturity. But the studies have shown that it is not enough for oocyte to complete reconstruction of methylated before it mature, which will bring about the emergence of various abnormalin the latter mbryonic development. This study will do mached analysis to IVM morula and in vivo maturation morula of whole genome CpG islands and promoter which in the period of cell fusion which is a critical period of zygotic genome starts activation.From the perspective of epigenetics to evaluate the possible mechanisms of low development potential of IVM embryos and the safety of it.[Methods](1) Experimental subjectsExperimental group:from 2012 January to 2013 November, patients with ICSI and short-term fertilization in patients with stage MⅠ oocyte 168, part of IVM zygotes for morula culture, to extraction of morula DNA for methylation chip experiment; part of IVM zygotes for blastocyst culture, to observation the developmental potential of IVM embyos.Control group:select 100 MⅡ oocytes from ICSI patients that Male semen is less weak sperm and no woman’s factors from 2012 to 2013.MeDIP-Chip control group is from the morula of group 6 in the first part of the experiment.(2)Embryo culture:Placed MⅠ oocytes into three gas incubator that low oxygen partial pressure.Mature Oocyte were ICSI fertilized, and culture morula and blastocyst. The control group’embyros were cultured for whole embryo blastocysts culture to MⅡ oocytes after fertilized.(3) Removal of the zona pellucida and adhesive granulosa cell of morula that from IVM and IVO’s with the method of Tyrode’s solution, then lyse cell and extract DNA after moved it into PCR tube, for MeDIP-Chip experiment, with the method that the same to first part experiment.(4) Statistical data of chip methylationPeak Score:-log10 (P-value) of peak on the probe can reflect the the degree of methylation.PeakMvalue:log2 (IP/Input)-ratio of the median that measured by probe, n is the peak valuewhen Peak Score≥2.[Results]1.There are 168 IVM oocytes, and the rate of vitro maturation is 83.3%,there was no statistical difference (P>0.05) between the two groups about 2PN rate, cleavage rate and Morula formation rate. But the high quality blastocyst formation rate of IVM group (11.4%) is significantly lower than the rate of IVO group (46.7%) (P<0.01).2. TO CpG island1) To CpG island the proportion of hypermethylation of IVM morula is higher than than IVO morula,but hypomethylation region was lower than the control group.2) The number of signals from the IVM morula methylation(n=3281)is more than the number from IVO morula (n=611).3) In the regions of Intragenic, intergenic and Promotor the PeakMvalue average value of IVM morula is higher than IVO morula.3.To Promotor aspect1) To Promotor the same to the CpG island region, hypermethylation proportion of IVM morula is higher than IVO morula, however, the region of hypomethylation is lower than control group.2) The number of signals from the IVM morulamethylation (n=3447)is more than the number from IVO morula (n=2744).[Conclusions]1. There was no statistical difference between the two groups about 2PN rate, cleavage rate and Morula formation rate. But the high quality blastocyst formation rate of IVM group is significantly lower than the rate of IVO group.2.There is a large difference on the aspects of whole genome CpG island and methylation of promoter region between morulas that from vitro mature oocytes and vivo mature oocytes. CpG islands hypermethylation level of IVM morula is higher than the level of the IVO morula. And promoter region hypermethylation level of IVM morula is higher than the level of the IVO morula.This difference may be the main reason that result in poor developmental potential and low pregnancy rate. So this part of the oocytes should be cautious in clinical use.