title: Validation of a method for noninvasive prenatal testing for fetal aneuploidies risk and considerations for its introduction in the Public Health System.
abstract: OBJECTIVE
  The aim of this study was to validate noninvasive prenatal testing (NIPT) for fetal aneuploidies by whole-genome massively parallel sequencing (MPS).
  
  
  METHODS
  MPS was performed on cell-free DNA (cfDNA) isolated from maternal plasma in two groups: a first set of 186 euploid samples and a second set of 195 samples enriched of aneuploid cases (n = 69); digital PCR for fetal fraction (FF) assessment was performed on 178/381 samples. Cases with <10 × 106reads (n = 54) were excluded for downstream data analysis. Follow-up data (invasive testing results or neonatal information) were available for all samples. Performances in terms of specificity/sensitivity and Z-score distributions were evaluated.
  
  
  RESULTS
  All positive samples for trisomy 21 (T21) (n = 43), trisomy 18 (T18) (n = 6) and trisomy 13 (T13) (n = 7) were correctly identified (sensitivity: 99.9%); 5 false positive results were reported: 3 for T21 (specificity = 98.9%) and 2 for T13 (specificity = 99.4%). Besides FF, total cfDNA concentration seems another important parameter for MPS, since it influences the number of reads.
  
  
  CONCLUSIONS
  The overall test accuracy allowed us introducing NIPT for T21, T18 and T13 as a clinical service for pregnant women after 10 + 4 weeks of gestation. Sex chromosome aneuploidy assessment needs further validation due to the limited number of aneuploid cases in this study.
authors.affiliation: a Diagnostic Genetics Unit, Careggi University Hospital , Firenze , Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Gerundino
authors.affiliation: a Diagnostic Genetics Unit, Careggi University Hospital , Firenze , Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Giachini
authors.affiliation: a Diagnostic Genetics Unit, Careggi University Hospital , Firenze , Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Contini
authors.affiliation: a Diagnostic Genetics Unit, Careggi University Hospital , Firenze , Italy.
authors.forename: Matteo
authors.initials: M
authors.lastname: Benelli
authors.affiliation: a Diagnostic Genetics Unit, Careggi University Hospital , Firenze , Italy.
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Marseglia
authors.affiliation: a Diagnostic Genetics Unit, Careggi University Hospital , Firenze , Italy.
authors.forename: Costanza
authors.initials: C
authors.lastname: Giuliani
authors.affiliation: a Diagnostic Genetics Unit, Careggi University Hospital , Firenze , Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Marin
authors.affiliation: a Diagnostic Genetics Unit, Careggi University Hospital , Firenze , Italy.
authors.forename: Genni
authors.initials: G
authors.lastname: Nannetti
authors.affiliation: a Diagnostic Genetics Unit, Careggi University Hospital , Firenze , Italy.
authors.forename: Ermanna
authors.initials: E
authors.lastname: Lisi
authors.affiliation: a Diagnostic Genetics Unit, Careggi University Hospital , Firenze , Italy.
authors.forename: Fiammetta
authors.initials: F
authors.lastname: Sbernini
authors.affiliation: b Prenatal Diagnosis Unit, Azienda Sanitaria Firenze , Firenze , Italy.
authors.forename: Enrico
authors.initials: E
authors.lastname: Periti
authors.affiliation: b Prenatal Diagnosis Unit, Azienda Sanitaria Firenze , Firenze , Italy.
authors.forename: Adalgisa
authors.initials: A
authors.lastname: Cordisco
authors.affiliation: c Department of Obstetrics and Gynecology , Reproductive Medicine and Prenatal Care Unit, Misericordia Hospital , Grosseto , Italy.
authors.forename: Enrico
authors.initials: E
authors.lastname: Colosi
authors.affiliation: c Department of Obstetrics and Gynecology , Reproductive Medicine and Prenatal Care Unit, Misericordia Hospital , Grosseto , Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: D'ambrosio
authors.affiliation: d Division of Molecular and Reproduction Diagnostics , Misericordia Hospital , Grosseto , Italy.
authors.forename: Marta
authors.initials: M
authors.lastname: Mazzi
authors.affiliation: d Division of Molecular and Reproduction Diagnostics , Misericordia Hospital , Grosseto , Italy.
authors.forename: Maya
authors.initials: M
authors.lastname: Rossi
authors.affiliation: a Diagnostic Genetics Unit, Careggi University Hospital , Firenze , Italy.
authors.forename: Lucia
authors.initials: L
authors.lastname: Staderini
authors.affiliation: a Diagnostic Genetics Unit, Careggi University Hospital , Firenze , Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Minuti
authors.affiliation: e Medical Genetics Unit, Azienda Sanitaria Firenze , Firenze , Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Pelo
authors.affiliation: f Department of Molecular Medicine and Medical Biotechnology University "Federico II" , Napoli , Italy.
authors.forename: Rita
authors.initials: R
authors.lastname: Cicatiello
authors.affiliation: g Department of Gynecology and Obstetrics , University "Federico II" , Napoli , Italy.
authors.forename: Giuseppe Maria
authors.initials: GM
authors.lastname: Maruotti
authors.affiliation: g Department of Gynecology and Obstetrics , University "Federico II" , Napoli , Italy.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Sglavo
authors.affiliation: f Department of Molecular Medicine and Medical Biotechnology University "Federico II" , Napoli , Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Conti
authors.affiliation: a Diagnostic Genetics Unit, Careggi University Hospital , Firenze , Italy.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Frusconi
authors.affiliation: a Diagnostic Genetics Unit, Careggi University Hospital , Firenze , Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Pescucci
authors.affiliation: a Diagnostic Genetics Unit, Careggi University Hospital , Firenze , Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Torricelli
mesh_terms: D000782:Aneuploidy; D002474:Cell-Free System; D015331:Cohort Studies; D004247:DNA / Q000097:blood*; D004314:Down Syndrome / Q000097:blood*; D005260:Female; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D011247:Pregnancy; D011248:Pregnancy Complications / Q000097:blood; D011296:Prenatal Diagnosis / Q000379:methods*; D011634:Public Health; D012680:Sensitivity and Specificity; D018709:Statistics, Nonparametric
chemical_list: D004247:DNA
keywords: Circulating cell-free fetal DNA; fetal trisomy; first-trimester screening; massively parallel sequencing; maternal plasma