Non-invasive prenatal testing (NIPT) by random massively parallel sequencing of maternal plasma DNA for multiple pregnancies is a promising new option for prenatal care since conventional non-invasive screening for fetal trisomies 21, 18 and 13 has limitations and invasive diagnostic methods bear a higher risk for procedure related fetal losses in the case of multiple gestations compared to singletons. In this study, in a retrospective blinded analysis of stored twin samples, all 16 samples have been determined correctly, with four trisomy 21 positive and 12 trisomy negative samples. In the prospective part of the study, 40 blood samples from women with multiple pregnancies have been analyzed (two triplets and 38 twins), with two correctly identified trisomy 21 cases, confirmed by karyotyping. The remaining 38 samples, including the two triplet pregnancies, had trisomy negative results. However, NIPT is also prone to quality issues in case of multiple gestations: the minimum total amount of cell-free fetal DNA must be higher to reach a comparable sensitivity and vanishing twins may cause results that do not represent the genetics of the living sibling, as described in two case reports.

Late-breaking abstract presentation during the plenary session at the ISPD conference, Tuesday, 14 July 2015.Funding sources: This work was funded by Life Codexx AG. Conflicts of interest: WH is an employee and shareholder of LifeCodexx AG. SG, US and JB are employees of LifeCodexx AG. CvK is a member of the Supervisory Board of LifeCodexx AG and shareholder of LifeCodexx AG. The other authors declare no conflict of interest.Non-invasive prenatal testing (NIPT) for common fetal trisomies has been radically changing prenatal diagnosis.1 Several hundreds of thousands of tests are being performed worldwide every year. Until today the fetal fraction in cell-free maternal plasma DNA is the most critical determinant for a successful NIPT analysis which is affected by certain parameters such as maternal weight 2 or the presence of an aneuploidy. 3 No other pregnancy-related factors, crucial for a reliable NIPT analysis, have been identified yet. We provide the first report describing an effect of drugs on circulating plasma nucleic acids and subsequent NIPT results. Low molecular weight heparin (LMWH) is widely used as anticoagulants in placenta-mediated pregnancy complication. 4 We show that in pregnant women on LMWH medication the cell-free plasma DNA contains a higher proportion of small DNA fragments featuring an unusually high guanosin-cytosin (GC) content than in unaffected women. This apparently biases NIPT results so that they cannot be interpreted correctly and even may provide false results. For such cases our report provides guidance that blood sampling for NIPT should ideally occur right before the next application of LMWH when the LMWH level is lowest in blood. Within three months of the year 2014, we performed a total of 1614 routine NIPT analyses (Method in the Supplementary Appendix). In 12 samples (0.74%), results could not be interpreted correctly due to an elevated GC content of higher than 44.0% in comparison to the average GC content of 42.0% estimated for the majority of analyzed plasma samples (data not shown). In correlation with the elevated GC content the z-score of chromosome 18 increased whereas the z-scores for chromosome 13 and chromosome 21 decreased. Thus, not assessing the GC content of a sample as a quality criterion of NIPT leads to falsepositive results for trisomy 18 or false negative results for trisomy 13 or trisomy 21. Repeat analysis on the routine backup blood samples (taken at the same time of the first blood sample) failed for the same reason. The clinical records of the patients with failed analyzed samples revealed that nine of these twelve women were on LMWH prophylaxis. The indications for the LMWH medication were thrombosis prophylaxis, protein C deficiency, risk of pulmonary embolism and repeated miscarriages. For five of these women (Table 1 and Table S1 in the Supplementary Appendix), an additional blood sampling was carried out right before the next pending LMWH injection, when the plasma level from the previous injection was lowest. Then, routine NIPT of these pre-LM...

Our newly established non-invasive prenatal test allows detection of fetal trisomies 13, 18, and 21 with high accuracy in a population in Germany and Switzerland.

Our results confirm previous reports that massively parallel sequencing of cell-free fetal DNA allows the reliably noninvasive detection of trisomy 21 from maternal blood with the potential to enhance test selectivity and specificity by bioinformatic means. According to our preliminary results, targeted sequencing might be an alternative strategy to detect chromosomal aneuploidies besides trisomy 21.

The authors wish to make the following corrections to this paper [1]: On page 683 at the end of Section 3.2. lines 13–14, the word “no” is missing. The correct sentence should be: “There has been no evidence of false-negative NIPT results so far in the pregnancies included in this study.”[...]