Prolonged persistence of fetal cfDNA after in utero fetal demise: Cases involving a demised anomalous twin during pregnancy

Objective

To determine the impact of a vanished twin in non-invasive prenatal testing (NIPT) during pregnancies with a demised anomalous twin.

Methods

Serial blood samples were obtained from four cases of twin pregnancies with in utero demise of the anomalous (trisomy 21 (T21) or mosaic T21) twin. Genotyping, targeted sequencing, and whole genome sequencing was used to determine twin-specific fetal fractions and fragment sizes, zygosity, as well as the T21 z-score in both an invasive and non-invasive approach (FetalQuant).

Results

After fetal demise, cfDNA from the demised anomalous twin was persistently detected, lasting more than four months in the longest series. The demised twin’s fetal fraction remained > 4% at least two months afterwards, with minimal size profile change and a z-score all > 8. The presence of a twin with T21 can be predicted by pairing the z-score of each sample with each twin’s fetal fraction and calculating its D score, which is its difference to the theoretical linear relationship between z-score and T21 fetal fraction. The non-invasive determination of this D score and zygosity can be used to predict whether or not a positive z-score is due to a vanished twin by also comparing with the known number of fetuses in utero.

Conclusion

These case series are the first to demonstrate prolonged persistence of cfDNA from a demised twin. This study confirms the vanishing twin phenomenon as a source of false positives in NIPT. However, we also propose a non-invasive method to predict the presence of a vanished twin to ameliorate this false positive source.