Noninvasive Prenatal Test By Sequencing Of Maternal Plasma Cell-free DN A In First Trimester For Advanced Maternal Age Pregnancies

ObjectiveThis study is to evaluate the performance of noninvasive prenatal t esting (NIPT) by massively parallel sequencing (MPS) of maternal plasma cell-free fetal DNA (cffDNA) for trisomies21,18,13and sex chromoso mal aneuploidies among advanced maternal age group in the first trimest er and validate its clinical efficacy and practical feasibility.MethodsA prospective, blinded study was performed between May2012and Augu st2013in Peking Union Medical College Hospital. Eligibility criteria included advanced maternal age women, singleton pregnancies,8to13we eks of gestation. We collected peripheral blood samples into Cell-free D NA BCTTM tubes (Streck, Omaha, NE) and only a single blood draw was perm itted. Detailed clinical information for each patient and sample was re corded on the study database. Fetal aneuploidies associated with chromo some21,.18,13, X and Y were detected by12-plex the flux Hisq2000pla tform of their maternal DNA plasma samples. Chorionic villus sampling (CVS) is the first recommendation for pregnancies with advanced materna1age consented to undergo an invasive prenatal diagnosis. Karyotyping was offered to all women to confirm both NIPT positive and negative res ults. NIPT and invasive prenatal diagnosis was conducted double blindly on each sample free of charge, at Berry Genomics, Beijing and the Peki ng Union medical college hospital Prenatal Diagnosis Center, respective ly. The birth outcome of each pregnancy refusing invasive prenatal diagn osis was followed up by clinical record or telephone. Measurement of fe tal DNA fraction in male pregnancies was based on a standard formula wh ich defines the%of Y chromosome reads in each male plasma sample rela tive to the%of Y chromosome reads in the maternal plasma of euploid f emale pregnancies. For aneuploid samples, fetal DNA fraction was calcul ated by the difference of genome percentage of the abnormal chromosome between the test sample and the reference samples.Results1. A total of two-hundred and fifteen blood samples were prospective ly collected and its plasma samples were sequenced, among which five cases of (2.3%) have no followed-up data; thirty-four cases refused to undergo confirmatory invasive prenatal diagnosis by either CVS o r amniocentesis due to the small but significant risk of miscarriage and/or infection (15.8%); one case’s blood sample taken for NIPT di d not meet QC standard due to haemolysis (0.5%). Thus, we conducted analysis on180samples.2. The average maternal age was37.33±2.19years (between35and45years). The blood samples were collected between8and13weeks of gestation (average9.67±1.214weeks of gestation).3. seven abnormal chromosome karyotypes were diagnosed. These seven samples included two cases of T21, one case of T18, one case of T13, one case of trisomy11, one case of47, XXY, one case of mosaicism (45,X[16]/47,XXX[14]); the positive rate was3.9%. A11cases of T21, T18and T13were correctly identified by sequencing. The NIPT resul ts of three fetuses who had abnormal karyotypes of T11,47, XXY and45,X[16]/47,XXX[14] were normal.4. NIPT using maternal plasma detected five cases of fetal chromosom al aneuploidy. These five samples included two cases of T21, one cas e of T18, one case of T13and one case of45, X. The positive rate wa s2.8%(5/180). All the T21, T18and T13samples identified by NIPT matched those diagnosed by Karyotyping. The only discordant result w as where karyotyping diagnosis was normal but the sequencing was45, X. Therefore, sequencing yielded one false positive45, X sample.5. For chromosomes21,18and13, NIPT achieved a detection sensitiv ity, specificity, positive prediction value (PPV) and negative predi ction value (NPV) of100%; no false negative or positive cases. For sex chromosome aneuploidy, there was one false positive (0.6%) and t wo false negative (1.1%) samples.6. The overall false positive rate (FPR) was0.6%(one45, X). Other c hromosomal diseases are so complex that noninvasive test fails detec ting.7. Fetal fractions of two cases (27.13%,20.61%) were too high. Thus, in secondary analyses, we examined the relationship between fetal D NA fraction and gestational age, using the NIPT data from101of180maternal plasma samples associated with a male pregnancy. The media n fraction of fetal cell free DNA in maternal plasma of the remained cases is8.58%. We found no significant association between fetal f raction and gestational age from8weeks through to13weeks. Plots of fetal DNA fraction versus maternal weight for all101samples sho wed a general trend towards an inverse relationship with an standard coefficient value of-0.438(p<0.001).ConclusionThe FPR of NIPT by MPS of maternal plasma cffDNA to screen of T21, T18and T13between eight and thirteen weeks’gestation is very low. An d the detection sensitivity, specificity, PPV and NPV of NIPT detecting T21, T18and T13is100%. It initially proves clinical efficacy and pr actical feasibility of NIPT to test T21, T18and T13as a screening pro tocol in the first trimester for women with advanced maternal age.