Non-invasive Prenatal Aneuploidy Testing:the Specific Impact Of Chromosome Aneuploidies

The research is divided into the following two parts:First,The specific impact of sex chromosome aneuploidies following noninvasive prenatal testing;Second,false positive non-invasive prenatal testing results due to vanishing twins.1.The specific impact of sex chromosome aneuploidies following noninvasive prenatal testing.Objective To evaluate the positive predictive value of sex chromosome aneuploidy in non-invasive prenatal testing(NIPT)and to analyse the possible causes leading to false positive results.Methods From November 2016 to March 2018,the gene testing center of Qingdao Women's and Children's Hospital carried out NIPT for 23,984 singleton pregnant women.The cases with high risk of sex chromosome aneuploidy were provided with genetic counseling and informed consent,and amniocentesis prenatal diagnosis or newborn chromosome karyotype examination were advised.Peripheral blood chromosome karyotyping of pregnant women and placental fluorescence in situ hybridization(FISH)were carried out for the false positive cases.Furthermore,cases without aneuploid abnormalities of sex chromosome are counted into the false positive groups.A 1:1matched studies were performed in the paired cases with the same detection batch,the same gestational week,BMI±5 and NIPT low risk.Copy number variation sequencing(CNV-seq)were carried out in both false positive and paired groups through maternal white blood cell.Paired chi-square test(McNemer c2 test)and linear regression were used to analyze the difference between CNV number and fragment sizes,and the relationship between CNV and Z score of X chromosome in NIPT in both groups.Results1.The positive predictive value of NIPT for sex chromosome aneuploid is 31.7%(39/123),and for ChrX(-),ChrX(+),ChrX(+)Y,ChrY(+),ChrX(-)Y are 17.5%,60.0%,56.5%,100%,0%,respectively.2.123 pregnant women in total had underwent invasive prenatal diagnosis orchromosome karyotyping for their newborns,as consequnces,84 were proven as NIPT false positive for sex chromosome aneuploidy.Among these 84 cases,8 pregnant women(8/84)were found with sex chromosome aneuploidy abnormalities,and 1 case with X chromosome structural abnormality.3.In the false positive cases with normal peripheral blood karyotype,21 has accept placenta FISH examination after delivery,and 1 case showed localized chimerism of placental chromosome aneuploidy(1/21).4.Among 76 pregnant women with no aneuploid abnormality of sex chromosome in the false positive group,11 were found to harbor CNVs of X chromosome,with fragments ranging from 0.1 to 11 Mb,4 of which harbor a CNV larger than 1 Mb.In matched group,8 cases were found to harbor CNVs on X chromosome,all of these CNVs were less than1 Mb.By paired chi-square analysis,there was no significant difference in X chromosome copy number variation between the two groups.Linear regression analysis of cases with CNV greater than 1Mb showed that there was a positive significant correlation between the Z score of X chromosome and CNV size(R2=0.99,P < 0.05).Conclusion1.The positive predictive value of NIPT for sex chromosome aneuploidy abnormalities is31.7%.Therefore,invasive diagnosis is essential for pregnant women whose NIPT indicates aneuploidies.2.Maternal sex chromosome aneuploidy abnormalities,maternal sex chromosome copy number variation and confined placental mosaicism are important factors leading to sex chromosome false positive results in NIPT,accounting for about 9.5%(8/84),4.76%(1/21)and 5.26%(4/76),respectively.3.Sex chromosome copy number variation of pregnant women is one of the causes of NIPT false positive,which is related to CNV size.4.For pregnant women whose NIPT sex chromosome aneuploidy abnormality,clinicians should suggest to perform chromosome karyotype analysis and gene chip/CNV-seq detection at the same time,so as to exclude the possibility of pathogenicity of sex chromosome copy number variation in fetus.2.False positive non-invasive prenatal testing results due to vanishing twins.Objective To explore the mechanism and duration of false positive results of non-invasive prenatal testing(NIPT)caused by vanishing twins.Methods To detect the variation of cell-free fetal DNA fraction before and after the fetal death and explore its influence on the results of NIPT at different gestationalweeks.Prenatal dignosis was also carried out on amniotic fluid sample derived from the survivor twin.After birth,the two placentas and papyraceous fetus were obtained to ascertain the definitive genetic diagnosis and pathologic changes through fluorescence in situ hybridzation,fluorescence quantitative PCR and histopathological examination.Eight cases of vanishing twins leading to discordant NIPT results were reviewed for determining the duration of this influence.Results The vanishing twin has led to immediate flooding of cfDNA into the maternal plasma due to necrotic cytotrophoblasts,which in turn caused increased release of fetal DNA in a short time.However,this did not change the NIPT result for a period of time.The tissue and chorionic villi of perished fetus presented extensive degenerative necrosis.Conclusion The false positive NIPT result caused by vanishing twins may be attributed to continuous release of DNA fragments into the maternal plasma by fetuses.The influence of the vanished twins,which may lead to discordant NIPT results,can last for at least 7~8 weeks but no more than 12~14 weeks during the first and second trimester.