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The present pilot study investigates whether an abnormal miRNA profile in NIPT plasma samples can explain the finding of a low cell-free DNA (cfDNA) fetal fraction (cfDNAff) in euploid fetuses and non-obese women. Twelve women who underwent neoBona® NIPT with a normal fetal karyotype were studied. Six with a cfDNAff &lt, 4% were matched with a control group with normal levels of cfDNAff &gt, 4%. Samples were processed using the nanostring nCounter® platform with a panel of 800 miRNAs. Four of the maternal miRNAs, miR-579, miR-612, miR-3144 and miR-6721, had a significant abnormal expression in patients. A data filtering analysis showed that miR-579, miR-612, miR-3144 and miR-6721 targeted 169, 1, 48 and 136 placenta-specific genes, respectively. miR-579, miR-3144 and miR-6721 shared placenta-specific targeted genes involved in trophoblast invasion and migration pathways (IGF2R, PTCD2, SATB2, PLAC8). Moreover, the miRNA target genes encoded proteins localized in the placenta and involved in the pathogenesis of pre-eclampsia, including chorion-specific transcription factor GCMa, PRG2, Lin-28 Homolog B and IGFBP1. In conclusion, aberrant maternal miRNA expression in circulating plasma could be a source of dysregulating trophoblast invasion and migration and could represent a novel cause of a low cfDNAff in the sera of pregnant women at the time of NIPT analysis.

Panorama Plus (Natera), a single-nucleotide polymorphism- (SNP-) based approach that relies on the identification of maternal and fetal allele distributions, allows the detection of common aneuploidies and also incorporates a panel of 5 microdeletions including Di George syndrome. We report here the first case of Di George syndrome detected by NIPT in Italy; blood was drawn at 12 weeks’ gestation. The patient had an amniocentesis to confirm the diagnosis by MLPA (multiplex ligation-dependent probe amplification) and an ultrasound aimed to detect the features associated with the syndrome. A right aortic arch and suspect of thymus atrophy were detected, but not other severe malformations typical of the disease. The patient terminated the pregnancy at 17 weeks. NIPT allowed an early screening of Di George syndrome. As the patient was at low risk, it is likely that an ultrasound would have missed the condition.

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Objective: Our objective was to retrospectively evaluate whether the levels of cell-free DNA (cfDNA) fetal fraction differed in the first trimester of pregnancies between controls and those who subsequently developed early- or late-onset fetal growth restriction (FGR). Methods: This was a case–control study conducted between May 2015 and May 2018 in 231 low-risk women who had received first trimester screening for major fetal aneuploidies (Panorama, Natera, San Carlos, CA, USA). Early- and late-onset FGR developed in 5 and 16 women, respectively, according to Delphi criteria. Multiples of median (MoM) were used to evaluate the differences in cfDNA fetal fraction between cases and controls. cfDNA fetal fraction was adjusted for gestational age (from 10 + 0 to 13 + 6 gestational weeks) and maternal weight (43–96 kg). Results: The median cfDNA fetal fractions for controls and early- and late-onset FGR were 1.00 (interquartile range [IQR] 0.89–1.12), 0.69 (IQR 0.44–0.84) and 0.93 (IQR 0.83–1.03) MoM, respectively. Statistically lower cfDNA fetal fraction MoM values were observed only in patients with early-onset FGR (Kruskal–Wallis test with Dunn post hoc test). In a 1:35 ratio (one case of early-onset FGR: 35 controls), the mean observed rank of 2.00 ± 2.23 in the cases was significantly lower than the expected 18.97 ± 10.17 (p < 0.001). Conclusions: Low-risk pregnancies that developed early-onset FGR had lower cfDNA fetal fractions than did the matched controls. This result is consistent with the placental dysfunction typical of early-onset FGR. For possible clinical use, the cfDNA fetal fraction would yield a better predictive value if adjusted for maternal weight, since maternal weight affects both cfDNA fetal fraction and the occurrence of FGR.

Objective: To verify the hypothesis that aberrant placental mRNA genes related to cardiogenesis can be detected in maternal plasma at the second trimester of pregnancy. Methods: NanoString technology was used to identify aberrant genes, comparing 39 women carrying a fetus with a congenital heart defect (CHD) to 31 controls at 19–24 weeks of gestation. The genes with differential expression were subsequently tested using real time polymerase chain reaction. Linear discriminant analysis (LDA) was used to combine all the mRNA species with discriminant ability for CHD. A multivariable receiver operating characteristic (ROC) curve having the estimated discriminant score as an explanatory variable was generated. Results: Six genes with differential expression, namely FALZ, PAPP-A, PRKACB, SAV1, STK4 and TNXB2, were found. The ROC curve yielded a detection rate of 66.7% at a false positive rate of 10%. A higher discriminant score (>75th centile) was reached for 14 CHD cases (82.4%) and only 1 control (5.8%). Two cases (11.8%) of heart rhythm disorders also yielded a discriminant score value >75th centile. Conclusion: These data represent a step forward in the screening of CHDs. Additional studies are needed to detect more mRNAs with discriminant ability and to move the first trimester screening.

Objective The aim of this study was to present a new method for fetal Kell genotyping by means of the allelic discrimination of K1 and K2 in real-time polymerase chain reaction (PCR). Methods Real-time quantitative polymerase chain reaction incorporating an allele-specific primer was developed for detecting the K allele of KEL. Results By means of this method, the K1/K2 genotype was able to be determined in all blood samples analyzed. Results using cell-free fetal DNA (cffDNA) from two Kell-negative pregnant women confirmed the Kell-positive genotype of fetuses. The real-time PCR analysis also allowed the determination of the fetal fraction using the quantification of Kell-positive DNA. Conclusion An efficient and reliable strategy for Kell genotyping is herein presented. The method was optimized on cffDNA to create a non-invasive prenatal test which could be routinely used for the prevention of hemolytic disease of the fetus and the newborn (HDFN).