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Key Clinical Message One of the confounders in noninvasive prenatal testing (NIPT) is the vanishing twin phenomenon. Prolonged contribution to the maternal Cell‐free DNA (cfDNA) pool by cytotrophoblasts representing a demised, aneuploid cotwin may lead to a false‐positive outcome for a normal, viable twin. We show that a vanishing trisomy‐14 twin contributes to cfDNA for more than 2 weeks after demise.

The practice of prenatal screening is undergoing important changes as a result of the introduction of genomic testing technologies at different stages of the screening trajectory. It is expected that eventually it will become possible to routinely obtain a comprehensive 'genome scan' of all fetuses. Although this will still take several years, there are clear continuities between present developments and this future scenario. As this review shows, behind the still limited scope of screening for common aneuploidies, a rapid widening of the range of conditions tested for is already taking shape at the invasive testing stage. But the continuities are not just technical; they are also ethical. If screening for Down's syndrome is a matter of providing autonomous reproductive choice, then why would providing the choice to have a full fetal genome scan be something entirely different? There is a clear need for a sustainable normative framework that will have to answer three challenges: the indeterminateness of the autonomy paradigm, the need to acknowledge the future child as an interested stakeholder, and the prospect of broad-scope genomic prenatal screening with a double purpose: autonomy and prevention.

Noninvasive prenatal testing (NIPT) validation studies show high sensitivity and specificity for detection of trisomies 13, 18, and 21. False negative cases have rarely been reported. We describe a false negative case of trisomy 13 and another of trisomy 18 in which NIPT was commercially marketed directly to the clinician. Both cases came to our attention because a fetal anatomy scan at 20 weeks of gestation revealed multiple anomalies. Karyotyping of cultured amniocytes showed nonmosaic trisomies 13 and 18, respectively. Cytogenetic investigation of cytotrophoblast cells from multiple placental biopsies showed a low proportion of nontrisomic cells in each case, but this was considered too small for explaining the false negative NIPT result. The discordant results also could not be explained by early gestational age, elevated maternal weight, a vanishing twin, or suboptimal storage or transport of samples. The root cause of the discrepancies could, therefore, not be identified. The couples involved experienced difficulties in accepting the unexpected and late-adverse outcome of their pregnancy. We recommend that all parties involved in caring for couples who choose NIPT should collaborate to clarify false negative results in order to unravel possible biological causes and to improve the process of patient care from initial counseling to communication of the result.

The existence of cell free fetal DNA, derived from apoptotic syncytiotrophoblasts, in the maternal circulation during pregnancy has opened new possibilities of non-invasive prenatal diagnosis, such as fetal RhD genotyping, prenatal diagnosis of hemoglobinopathies and fetal sexing in X-linked disorders like hemophilia. These diagnostic applications are however hampered by the lack of a generic control marker for circulating fetal DNA, which is especially cumbersome early in pregnancy. Fetal DNA concentration is extremely low and can be less then 5 genome equivalents (geq)/mL in the first trimester. It gradually increases during pregnancy, but greatly varies between women, from 15 to 150 geq/ml at 30th week of pregnancy. A negative test result can therefore be due to lack of fetal DNA in the PCR tube or to absence of the mutation/polymorphism in the fetus. Fetal DNA control assays based on Y-chromosome sequences can be used in only 50% of the pregnancies. We have previously developed a set of RQ-PCRs based on del-ins polymorphisms as fetal identifier, which has been introduced in routine diagnostics. However, in up to 5–10% of cases no suitable paternal marker can be identified and also the workload is considerable. Recently, it has been demonstrated that the promoter of RASSF1A gene is hyper-methylated in the placenta and hypo-methylated in maternal blood cells (Chiu et al., Am J Pathol.2007;170:941). This methylation pattern allows the use of methylation-sensitive restriction enzyme digestion with BSTU1, for detecting the placental, hence fetal-derived hyper-methylated RASSF1A sequences in maternal plasma. We wanted to implement this potential fetal DNA identifier, RASSF1A, in our diagnostic tests. Initial results however showed weak positive results in virtually all male plasmas. This could be due to incomplete digestion of hypo-methylated leukocyte derived DNA or to the presence in plasma of hypermethylated DNA from other sources. To increase the efficiency of digestion we performed a double digestion by adding another enzyme Hha1, which adds two extra restriction sites within the PCR target DNA sequence. Pilot experiments on DNA derived from male plasma showed decreased amplification signals, suggesting that the previously observed weak signals were derived from incompletely digested hypo-methylated DNA. We selected enzymes working in the same buffer. The optimal reaction time and temperature were established (1 hour at 37 °C with 10U of each enzyme). Applying this protocol, no amplification or only spurious amplification with high Ct values (mean Ct value of the positive wells: 43.3) were observed with DNA isolated from 2 ml of plasma obtained from non-pregnant women or male (n=20). In contrast, all samples obtained from pregnant women (n= 20) showed positive amplification signals in all wells tested (n=87, 3 or 5 replicates), with clearly lower Ct values (mean 41.04 ± 1.54) The beta-actin data showed in all samples complete digestion. In conclusion: Our results indicate that a real time PCR assay on RASSF1a sequence can be used as universal fetal identifier for non-invasive fetal genotyping assays, if it is tested after double digestion with BSTU1 and Hha1. It is to be expected that the availability of this control assay will lead to wider implementation of prenatal genotyping assays based on cell-free fetal DNA.