The Research Of Trinucleotide Repeat Dynamic Mutations In The Offspring Coneeived Through Assisted Reproductive Technology

BackgroundSince the birth of Louise Brown,the world's first in vitro fertilization(IVF) baby 30 years ago,an estimated 3.5 million children have been born worldwide after treatment with assisted reproductive technology(ART).The combined estimates from the countries reporting to United States and European ART registries alone indicate that 500,000 treatment cycles were undertaken in 2004,resulting in approximately 105,000 deliveries.As ART is increasingly used to overcome infertility,the health of the children conceived by ART has always been concerned.Two meta-analyses were published in 2005 both addressed the association between ART and birth defects.Some hold a viewpoint implied that infants conceived with use of ICSI or IVF have twice as high a risk of a major birth defect as naturally conceived infants.There have been findings suggesting that some birth defects occur more often among infants conceived with ART.Although the mechanism is not clear,couples considering ART should be informed of all potential risks and benefits. Since the stages of gametogenesis and preimplantation embryo development are the most critical periods which epigenetic reprogramming occurs,the procedures of ovarian stimulation,assisted fertilization and other in-vitro manipulation in ART might cause the disruption of DNA or chromatin modifications.Publications from Europe, the United States,and Australia have shown increased incidences of imprinting disorders,such as Beckwith Wiedemann syndrome(BWS),Angelman syndrome and retinoblastoma.More than 90%of children with BWS that were born after ART had imprinting defects,compared with 40-50%of children with BWS and conceived naturally.Moreover,ART-derived offspring with an increased the risk of karyotypic abnormalities has been confirmed by several research groups.Later,higher incidence of de novo Y-chromosome microdeletion in male children conceived through IVF or ICSI compared with those conceived naturally has been also reported.However,the accumulating evidences of association of ART with de novo genetic aberrations are controversy.Caperton and his colleagues used a transgenic mouse model to examine the effects of ART on the frequency and spectrum of point mutations in mid-gestation mouse fetuses,and they did not find significant difference occurred in the frequency of de novo point mutations in transferred DNA between the ART and naturally-derived offspring.In 2009,a research performed by Riccaboni's team showed the mutation frequency of cystic fibrosis transmembrane conductance regulator gene(CFTR) in ART-offspring is similar to that in the general population.However,de novo mutations at any one site typically occur at a frequency of only 1 in 10,000-1,000,000 genomes. It is impractical to use data from common structural genes to evaluate the influences on gene mutation in ART-derived offspring.Within the closing decade of the twentieth,14 neurological disorders were shown to result from the expansion of trinucleotide repeats in the coding regions or regulatory regions of the genes.This type of DNA alteration is called dynamic mutation,which demonstrates as unstable trinucleotide repeat expansion or contraction in those special genes.Compared with other gene loci,the frequency of dynamic mutation is much higher.Copy number alteration of trinucleotide repeat within a certain range in those genes will not result in any phenotypie change.The frequencies of dynamic mutation should be a relatively sensitive index for analyzing DNA instability.Therefore,in view of the safety of ART offspring,for further investigation the effects of ART on genetic mutation,the frequency of dynamic mutation in the babies conceived by ART were detected and analyzed with those conceived naturally as control.ObjectiveTo investigate the possible changes of frequency of trinucleotide repeat dynamic mutation in the offspring conceived through ART in comparison with matched controls from spontaneous pregnancies.Materials and Methods7 major dynamic mutation genes are chose as target genes,ATN1,AR,ATXN1, ATXN3,Huntington,DMPK,FMR-1.The target genes can cause the disorders are as follows:dentatoubral and pallidoluysian atrophy(DRPLA),spinal bulbar muscular atrophy(SBMA),spinocerebellar ataxia typeⅠ(SCA1),machado-joseph disease (MJD),huntington's disease(HD),myotonic dystrophy(DM) and fragile X syndrome (FraX).One hundred and twenty-one families were recruited,including 45 families with their babies conceived through IVF,16 families with babies conceived through ICSI, and 60 naturally conceived babies,as well as the babies' parents.Genomic DNA sequences in trinucleotide repeat genes were amplified by polymerase chain reaction with Ta Ka Ra LA Taq DNA polymerase and GC buffer.And after agarose gel and non-denaturing polyacrylamide gel electrophoresis combined with DNA sequencing, trinucleotide repeat dynamic mutations of gene ATN1,AR,ATXN1,ATXN3,Huntington, DMPK,FMR-1 were detected in those families.ResultsThe intergenerational dynamic mutation was detected in 17(37.78%) of 45 IVF families,2(12.50%) in 16 ICSI families and total 19(31.15%) of 61 ART families. Compared with that(23.33%,14 in 60) in the families with naturally conceived babies, there were no significant differences(P＞0.05).Besides,median of mutated trinucleotide repeat copy number in the total 7 dynamic mutation genes was 3.00 (1.00～8.00) in ART group,which is no statistically different from that in the control group.Conclusion(1) ART,including IVF and ICSI,has little significant influence on the mutation frequency of trinucleotide repeat dynamic mutation genes.(2) All subjects,including all parents and children,copy number of their trinucleotide repeat are within the normal range.Compared with naturally conceived babies,we didn't find increased copy number of trinucleotide repeat in ART-conceived babies in our researches.(3) Summarily,ART has little significant influence on dynamic mutation or stability of DNA sequence.With respect to maintenance of genetic integrity among intergeneration,ART appears to be safe.