Detection and characterization of circulating cell-free fetal DNA (cffDNA) from maternal circulation requires an extremely sensitive and precise method due to very low cffDNA concentration. In our study, droplet digital PCR (ddPCR) was implemented for fetal RHD genotyping from maternal plasma to compare this new quantification alternative with real-time PCR (qPCR) as a golden standard for quantitative analysis of cffDNA. In the first stage of study, a DNA quantification standard was used. Clinical samples, including 10 non-pregnant and 35 pregnant women, were analyzed as a next step. Both methods’ performance parameters—standard curve linearity, detection limit and measurement precision—were evaluated. ddPCR in comparison with qPCR has demonstrated sufficient sensitivity for analysing of cffDNA and determination of fetal RhD status from maternal circulation, results of both methods strongly correlated. Despite the more demanding workflow, ddPCR was found to be slightly more precise technology, as evaluated using quantitative standard. Regarding the clinical samples, the precision of both methods equalized with decreasing concentrations of tested DNA samples. In case of cffDNA with very low concentrations, variance parameters of both techniques were comparable. Detected levels of fetal cfDNA in maternal plasma were slightly higher than expected and correlated significantly with gestational age as measured by both methods (ddPCR r = 0.459; qPCR r = 0.438).

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We report a rare case regarding an oral mass (epignathus) with intracranial extension originally noted antenatally at 16 weeks´ gestation. Early second trimester diagnosis of small potentially serious lesion is difficult and needs expert 2D and perhaps 3D sonographic re-evaluation over time. Postmortem MRI and pathologic examination of the fetus confirmed an oral teratoma with bilateral ventricular dilatation, corpus callosum agenesis, and a neuroepithelial intracranial cyst. The relevant literature regarding this anomaly is reviewed.

These findings suggest the possible participation of miRNAs in Down syndrome impaired placentation and connected pregnancy pathologies. © 2016 John Wiley & Sons, Ltd.

Background and objectives The aim of the study was to optimize routine non‐invasive prenatal detection of fetal RHD gene from plasma of RhD‐negative pregnant women (the median of gestational age was 25 weeks, range 10–38) to detect RhD materno‐fetal incompatibility and to avoid the redundant immunoprophylaxis. Materials and methods Initially only one exon of RHD gene (exon 10) was investigated in 281 plasma samples (144 verified after delivery), in the second phase three RHD exons (5, 7, 10) were analyzed in 246 samples of plasma and maternal genomic DNA (204 verified) by real‐time PCR method. Detection of Y‐chromosomal sequence DYS‐14 and five X‐chromosomal insertion/deletion polymorphisms was used to confirm the fetal cfDNA detectability in plasma. Specific polymorphisms in RHD gene were detected by sequence‐specific primer PCR in nine samples. Results When only the RHD exon 10 was tested, 2·8% of verified samples were false positive and 3·5% false negative. With three RHD exons (5, 7, 10) and maternal genomic DNA testing, only one case was false negative (0·5%). Nine samples were inconclusive due to RHD‐positive results in maternal genomic DNA. These samples were analyzed for specific mutations in RHD gene. Combination of both methods for fetal cfDNA verification succeeded in 75% of tested group. Conclusion Implementation of analysis of three RHD exons and maternal genomic DNA to routine practice lowers dramatically the ratio of false positive and negative results. This method enables more accurate determination of fetal RHD status with the reduction of unnecessary medical care and RhD immunoprophylaxis.