Methylation Status Of Two Imprinting Control Regions At Human Chromosome 11p15.5 Of The Offspring Conceived Through Assisted Reproductive Technology

IntroductionAssisted reproductive technology (ART) have been widely used over the past three decades to help infertile couples conceive. Over 3,500,000 babies have been conceived through ART woridwide, the number is growing. Recent studies on the ARTconceived population have raised concern about the possible risks of these techniques, in particular with regard to increased incidence of growth and developmental disorders. Amplified risks associated with ART has been reported, including spontaneous abortion, low or very low birthweight, small for gestational age, major malformations, cerebral palsy and so on. Study on the safety of ART has becoming to a hot-spot to genetist and researchers working on reproduction.Recently, imprinting disorders including Beekwith-Wiedemann Syndrome and Angelman Syndrome attract most attention with respect to the safety of ART. Although current evidence does not allow definite conclusions to be drawn,findings of different case series and case-control studies have consistently suggested an inereased risk of imprinting disorders in pregnaneies after ART.Differential expression of the Paternal and matmal alleles of imprinted genes requires regulated DNA methylation.ART procedures, including hormone-induced superovulation, in vitro fertilization and embryo culture, manipulate gametes and embryos during gametogenesis, fertilization and pre-implantation stages, which is crucial for methytation reprogramming of imprinted genes.Therefore, ART is likely to induce in appropriate methylation of imprinted genes and disrupts imprinting.On the other hand, the couples suffering infertility might spread the disabled fertility problem to their offspring by ART treatment.The relationship between ART procedures and expression of imprinted genes in offspring still remains unelear.Experimental evidences in animals and embryonic stem cells indicate that manipulation and invitro embryo culture may lead to disrupted genomie imprinting.However, several studies in human found no detectable alteration of imprinted gene expression, indicating that the imprints, which have been set during gametogenesis, are stabely maintained during the ART procedure.Till now,we can see most of studies on ART offspring are focusing on clinical case control cohort report and imprinting disorders, but few studies on imprinted genes of phenotypic normal ART offspring have been done, so more molecular biology studies are suggested to confirm the safety of ART.Human chromosome band 11p15.5 includes a cluster of genes that are imprinted.Dysregulation of this gene cluster is associated with the overgrowth and tumor predisposition syndrome, BWS, isolated hemihyperplasia, SRS, and transient neonatal diabetes mellitus.Here we investigate the expression levels of IGF2 and H19, which are very important in the control of fetal and postnatal growth,and test imprinting and methylation status of two important imprinting control regions at chromosome 11p15.5, which regulate the two main domains of 11p15.5, to evaluate the safety of ART.Part1 Expression levels of IGF2 and H19 of the offspring conceived throught assisted reproductive technologyObjective:To explore the expression levels of IGF2 and H19 gene of ART offspring and natural conceived offspring, evaluate the growth and developmental condition and safety of ART offspring.Methods:A total of 58 newborns conceived by ART and 32 newborns conceived naturally from 2008 to 2009 at the Nanfang Hospital, Guangzhou, China, were chosen for our studies. Cord-blood samples (10-15 mL)were collected in EDTA tubes from the umbilical cords of the infants. Maternal weight, height, age, past and present medical history, babies’gender, birth weight (BW), record of birth (including mode and indication for delivery) and gestational age were recorded. Testing their expression levels of imprinted genes IGF2 and H19 by real-time PCR. One-way ANOVA and SPSS 16.0 were used to do satistical work.Results:1.A major difference between the two sample sets concerns the frequency of twin pregnancies. Due to multiple embryo transfer following ART twin pregnancies are very frequent in ART group, while only one twin pregnancy was among the samples obtained from the spontaneously conceived group (p<0.05). Accordingly, children born after ART had a slightly lower gestational age and were lighter and smaller at birth compared to the spontaneously conceived children (p<0.05). In addition, maternal age was higher in the ART groups (p<0.05)2. There existed significant difference between ART group and naturally conceived group with the expression levels of IGF2 (p<0.05);the difference of the expression levels of H19 was also significantly (p<0.05).which might be both due to twin pregnancy since there were no significant difference between naturally conceived singletons and ART singletons with the expression levels of IGF2 and H19 gene (p>0.05)3. There exsited no significant difference between IVF group and ICSI group with the expression levels of IGF2,so did fresh cycle group and frozen/thawn group (p>0.05).But as to H19 gene, the former was significant (p<0.05),the latter was not (p>0.05)4. The correlation between birth weight and expression levels of H19 gene was significantly (p<0.05), so was gestational age and expression levels of IGF2 gene (p<0.05).The correlation of expression levels between the two imprinted genes was significantly too (p<0.05)Conclusion:High twin pregnancy rate of ART is likely to result in preterm birth and low birth weight, which might has some connection with changing of expression levels of imprinted genes as well, although birth defects haven’t been found in this study, we still need further study to focus on the safety of ART in the long run.Part2 Imprinting and methylation status of two imprinting control regions at chromosome 11p15.5 in offspring conceived through assisted technologyObjective:To explore the imprinting and methylation status of two important control regions at human chromosome 11p15.5, evaluate the probability of abnormal imprinting and methylation happening to ART offspring.Methods:A total of 61 newborns conceived by ART and 30 newborns conceived naturally from 2008 to 2009 at the Nanfang Hospital, Guangzhou, China, were chosen for our studies. Cord-blood samples (10-15 mL)were collected in EDTA tubes or heparin sodium tubes from the umbilical cords of the infants. Maternal weight, height, age, past and present medical history, babies’gender, birth weight (BW), record of birth (including mode and indication for delivery) and gestational age were recorded.Methylation specific PCR(MSP) was used to detect methylated and unmethylated alleles. Combined bisulphite restriction analysis(COBRA) and bisulphite sequencing(BSP) were chosen for detecting imprinting and methylation status of the two imprinting control regions.Results:1 Abnormal imprinting and hypo-methylation at H19 ICR were found to happen to three dizygotic twins conceived after intracytoplasmic sperm injection (ICSI).Near upon 66%(14/22) of cases with low birth-weight were found to have a single C/T polymorphism(SNP) (H19 8271, GenBank accession no. AF125183) at H19 ICR, calculated with nearly 25%(13/43) of ART twins compared with 10%(3/30) of naturally conceived singletons and 0%(0/18) of ART singletons.There existed significant difference between cases with low birthweight and cases with normal birthweight (p<0.05)2.COBRA did found no cases with apparent abnormal imprinting of KCNQ1 ICR while BSP found 6 cases with low birthweight(including 2 naturally conceived singletons) and 1 ICSI singleton with normal birthweight in hyper-methylation status of KCNQ1 ICR.3.All of the cases showed normal imprinting at IGF2 DMR which detected by MSP and COBRA.Conclusion:The abnormal imprinting/methylation probably results from an imprint erasure defect in the paternal germ line and not the in vitro fertilization/ICSI procedure. Imprinting/methylation test on paternal sperm is suggested to confirm our imagination. Additional larger study is considered to determine whether the C/T SNP detected in our study is associated with growth regulation.Part3 Karotype of offspring conceived by assisted reproductive technologyObjective:To investigate the karyotype of ART offsping, evaluate genetic risk of this group.Methords:48 ART offspring including 30 conceived through IVF,18 conceived through ICSI were recruited as study group,20 naturally conceived newborns were collected as compared group.Karyotype was detected in a standard way.Results:All cases had normal karyotype,including 35 male cases with 46,XY and 33 female cases with 46,XX.Conclusion:We consider that the children conceived by ART with no birth defects have few chances to carry abnormal karyotype, a larger number of case study is suggested to support our imagination.