Prospective Safety Evaluation Of Assisted Reproductive Technology And Study Of DNA Methylation Alterations In The Cerebrum From The Offspring Of The Mice Conceived From IVF-ET

Part I Prospective safety evaluation of assisted reproductive technologyObjective:To compare the pregnancy outcome between assisted reproductive techniques (ART) (in vitro fertilization and embryo transfer, IVF-ET and intracytoplasmic sperm injection, ICSI) conceived and natural conceived women, in order to evaluate the safety of ART.Material and Methods:The ART group included women who become pregnancy after IVF or ICSI and fresh embryo transfer in Women’s hospital, School of Medicine, Zhejiang University during the period from 2009 to 2013.The control group included the naturally conceived women who were diagnosed pregnant in the same hospital by urine hCG test in the corresponding period and wanted to keep pregnancy.We designed a prospective cohort study to compare the information of ingravidation, pregnancy and neonate between ART conceived and natural conceived women.Methods of the survey:Telephone, QQ, WeChat, E-mail, Outpatient consultation, medical records, etc.The follow-up time and frequency:First investigation:ART Group:from positive blood hCG test within 14 days after the embryo implantation; Control Group:from the first visit of positive urine pregnant test.Telephone conversations to find out whether the patients had any early abortion, ectopic pregnancy or something else in 12 gestational weeks. Make sure if the patients had induced labor or late abortion in 28 gestational weeks. Make a telephone calls per twice-weekly over 36 gestational weeks.Terminations of the survey:Abortion, ectopic pregnancy, hydatidiform mole, stillbirth, premature delivery or live birth was diagnosed.Results:The abortion rates of IVF and ICSI subgroup, ART group and control group were 19.64%,20.33%,19.82% and 13.19% respectively. The incidence of pregnancy loss in ART group was significantly higher than that of control group.The incidences of early pregnancy loss in IVF subgroup, ICSI subgroup, ART group and control group were 17.71%,17.87%,17.75% and 10.36% respectively. The incidence of early pregnancy loss in ART group was significantly higher than that of control group.We also found that the incidences of late pregnancy loss in IVF subgroup, ICSI subgroup, ART group and control group was 2.34%,2.99%,2.51% and 3.14% respectively. The incidence of late pregnancy loss in IVF group was significantly lower than that of control group.The results showed that the incidences of ectopic pregnancy in IVF subgroup, ICSI subgroup, ART group and control group were 6.10%,4.11%,5.59% and 1.09% respectively. The incidence of late pregnancy loss in IVF group was significantly lower than that of control group.The incidences of preterm labor and preterm labor of singletons in ART group were significantly higher than that of control group. The incidence of twin pregnancy was also significantly higher in ART group than that of control group. But the incidence of preterm labor of multi-pregnancy was comparable between the two groups.The rates of preterm labor in IVF subgroup, ICSI subgroup, ART group and control group were 20.25%,17.79%,19.61% and 5.09% respectively. The incidence of preterm labor of singletons was higher in IVF subgroup than in ICSI subgroup without statistic significance. The increase in both subgroups had statistic significance.The incidence of preterm labor of multi-pregnancy was lower in IVF subgroup than in ICSI subgroup without statistic significance and there was no significant difference between the two subgroups and the control group.The total incidence of caesarean section (c-section) and the incidence of c-section for singletons were significantly higher in ART group than those of control group.The incidences of lower birth weight in ART group, IVF subgroup and ICSI subgroup were significantly higher than that of control group. The incidence of lower birth weight in IVF subgroup was higher than ICSI subgroup without statistic significance. The incidences of lower birth weight of both term and preterm singletons in ART group were lower than those of control group without statistic significance.Conclusions:The incidences of pregnancy loss, ectopic pregnancy, preterm labor, preterm labor of singletons, multiple pregnancy, c-section and lower birth weight in ART group were all higher than those of control group.Part Ⅱ Study of DNA methylation alterations in the cerebrum from the offspring of the mice conceived from IVF-ETObjective:This study was designed to investigate the DNA methylation status of nonimprinting genes in cerebrum of mice born through in vitro fertilization (IVF) and its second generation, the continuity of altered epigenetic modification from weaning period to adult period of them was also studied.Material and Methods:The females C57BL/6J were super-ovulated then matured metaphase Ⅱ oocytes were collected from the oviducts of super-ovulated females. After IVF, embryos were transferred into the uterine horns of pseudo-pregnant female mice to obtain the IVF mice. Then the IVF mice were crossed with IVF mice or naturally conceived (NC) mice to obtain F2 generation, male derived F2 generation, female derived F2 generation. Nonimprinting genes Col18a1, Cdc51, Zfp191 and Zfp639 in IVF mice cerebrum, which were hypermethylated in our previous methylation arrays, were selected, their DNA methylation and mRNA expression levels of IVF mice and all kind of F2 generations(at 3 weeks and 10 weeks age) were analyzed by pyrosequencing and real-time PCR respectively.Results:In cerebrum of the four nonimprinting genes we selected, in IVF mice both at 3 weeks and 10 weeks age, we found that DNA methylation showed a significant rising trend, and gene expression of Coll8al, Cdc51 and Zfp191 showed down-regulated trend when compared with NC mice. In all kind of F2 generations both at 3 weeks and 10 weeks age, DNA methylation of all the nonimprinting genes showed a significant rising trend, gene expression of Col18a1, Cdc51 and Zfp191 showed down-regulated trend when compared with NC mice. Only for Zfp639, gene expression at 3 weeks age of IVF mice, male derived F2 mice, and female derived F2 mice was down-regulated when compared with NC mice, gene expression of F2 generation at 3 weeks age and all generations at 10 weeks age was up-regulated when compared with NC mice.Conclusions:Our study clarified that IVF would change the methylation pattern of mouse cerebrum, and the alteration can be inherited by the following descendents. DNA methylation aberration at weaning IVF mice was maintained in adult IVF mice, what is more, the analog alterations was found in its all kind of F2 generations.