The Genomics Research Of Offspring Conceived Through Assisted Reproductive Technology

IntroductionSince the application of in-vitro fertilization(IVF)nearly 30 years ago,over three million babies have been conceived through assisted reproductive technology(ART) worldwide,and increasing worry is focused on the health problem of ART-conceived offspring now.Amplified risks associated with ART has been reported,including spontaneous abortion,low or very low birth weight,small for gestational age,major malformations,cerebral palsy and so on.Recently,imprinting disorders including Beckwith-Wiedemann syndrome and Angelman's syndrome attract most attention with respect to the safety of ART. Findings of different case series and case-control studies have consistently suggested an increased risk of imprinting disorders in pregnancies after ART.Differential expression of the paternal and maternal 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 methylation reprogramming of imprinted genes.Therefore,ART is likely to induce inappropriate methylation of imprinted genes and disrupts imprinting.The relationship between ART procedures and expression of imprinted genes in offspring still remains unclear.Experimental evidences in animals and embryonic stem cells indicate that manipulation and in vitro embryo culture may lead to disrupted genomic 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 stably maintained during the ART procedure.To clarify the genomic status in offspring conceived through ART,karyotyping and detection of Y-chromosome microdeletion were performed to evaluate the risks of mutation after ART procedure firstly.Then a general mRNA expression survey of whole genome and imprinted genes was carried out using Affymetrix Oligo microarray to estimate the risks of mRNA expression alteration after ART.The imprinted genes differentially expressed between the two groups were selected as candidate genes for following detections.Finally the monoallelic expression of the candidate genes was detected and further the relationship between DNA methylation and imprinting instability was investigated.Partâ… Clinical mutation detection in male offspring conceived through assisted reproductive technologyObjective:To investigate the risks of gene mutation after ART for couples with comparable genetic backgrounds.Methods:Ninety-seven male children whose fathers had normal spermatogenesis were recruited,including 19 babies conceived through IVF,18 babies conceived through intracytoplasmic sperm injection(ICSI),and 60 naturally conceived babies, as well as the babies' fathers.Peripheral and umbilical cord blood samples were collected.Karyotype and the Y-chromosome microdeletion of 13 Y-specific markers covering four azoospermia factor(AZF)subregions were detected.Results:All children had a normal 46,XY karyotype,but de novo Y-chromosome microdeletions were identified in 1(5.3%)of 19 IVF offspring and in 3(16.7%)of 18 ICSI offspring.The incidence of de novo Y-chromosome microdeletion in male children conceived through ICSI or IVF was statistically significantly higher than that in those conceived naturally(10.8%vs.0).In four babies with microdeletion,one was complicated with hypospadias.Conclusion:Our results,for the first time,indicate that risks of gene mutation may increase in the ART offspring,even though their fathers have normal spermatogenesis and genetic background.Hence,intense attention should be placed on genetic safety in the ART children,and the benefits and risks of ART should be balanced gingerly.Partâ…¡Expression profile of global genome and imprinted genes in offspring conceived through assisted reproductive technologyObjective:To investigate the expression profile of whole genome and imprinted genes in ART-conceived offspring and pick the key genes differentially expressed for further study.Methods:Gene expression profiling was completed using Affymetrix U133 Plus 2.0 oligonucleotide microarrays in 8 babies conceived through ART and 8 controls.The genes differentially expressed were identified using significance analysis of microarray(SAM)and further analysed by hierarchical clustering,GO(gene ontology)analysis and Pathway analysis.Gene expression data were validated by quantitative real-time reverse-transcription polymerase chain reaction(RT-PCR)in 40 ART babies and 40 controls.Results:Comparison of the gene profiles between ART babies and controls identified 159 differentially expressed genes.Hierarchical clustering showed that the gene profiles of ART babies and naturally conceived babies were distinct.GO analysis revealed that some genes involved in growth and development were down regulated in ART offspring.Pathway analysis highlighted several pathways including the toll like receptor signal pathway.To validate the gene expression data,eight nonimprinted genes and nine most discriminatory imprinted genes were investigated by real-time RT-PCR,which found that six nonimprinted genes and three imprinted genes were differentially expressed.Conclusion:This study provided the general expression profile of whole genome and imprinted genes in ART-conceived offspring.These data provide a useful basis for understanding the molecular events leading to the increased risks in ART-conceived offspring and could be considered as potential targets for further study.Partâ…¢Imprinting status of PEG10,L3MBTL and PHLDA2 in offspring conceived through assisted reproductive technologyObjective:To elucidate the allele specific expression of PEG10,L3MBTL and PHLDA2 in ART-conceived offspring.Methods:Sixty ART-conceived babies and 20 naturally conceived babies were involved.The allele-specific expression was detected using direct sequencing after PCR and RT-PCR.Results:Both PEG10 and L3MBTL did not reveal biallelic expression in naturally conceived offspring blood,but in ART-conceived offspring,loss of imprinting(LOI) of PEG10 was observed with the frequency of 31.8%and LOI of L3MBTL was observed with the frequency of 5.6%.PHLDA2 was biallelic expressed in ART-conceived and naturally conceived offspring.Conclusion:The imprinting of PEG10 and L3MBTL was not disrupted in most of the ART-conceived offspring,but some cases displayed loss of imprinting,indicating the instability of imprinting in umbilical cord blood of ART-conceived offspring.Partâ…£DNA methylation status of PEG10 and L3MBTL in ART-conceived offspringObjective:To investigate the DNA methylation status of CpG islands of PEG10 and L3MBTL in ART-conceived offspring,and elucidate the relationship between abnormal methylation of CpG and imprinting defects.Methods:Four babies conceived through ART and 2 babies conceived naturally were involved.Sodium bisulfite sequencing was applied to detect the methylation levels at CpG islands of PEG10 and L3MBTL.Results:All CpGs of PEG10 were almost completely unmethylated in umbilical cord blood in both ART-conceived offspring and naturally conceived offspring.In the naturally conceived offspring,all the CpGs of L3MBTL were moderately methylated. The methylation levels of some CpGs in the ART-conceived offspring were lower than naturally conceived.In one ICSI-conceived offspring,all CpGs were completely unmethylated and monoallelic expression was disrupted.Conclusion:Monoallelic expression of L3MBTL was associated with differential methylation of CpGs,indicating a regulatory role for DNA methylation at these loci.