Strengths and limitations
The major strength of this study was the consecutive observational
approach and prospective collection of blood samples. Previously,
several studies have reported the disturbance of NIPT by residual cfDNA
of twin demise [11, 21, 22]. However, very few studies demonstrate
the longitudinal effect of residual cfDNA after twin demise. We
successfully established the consecutive profiles of residual cfDNA
changes after twin demise by densely testing blood samples until birth,
which provides the robust evidence to elucidate an important question:
how long the placenta of a deceased co-twin can release cfDNA into the
maternal circulation? Another strength of this study was a combination
of three different methods of calculating fetal fractions. This helps
compare the total fetal fraction measured by FF-QuantSC, the fetal
fraction of the demised co-twin measured by relative coverage of the
trisomy chromosome, and in two cases (patient 3 and 4) the fetal
fraction of the survived euploid fetuses measured by Y-chromosome.
The limitations of this study included a small sample size and the
restriction of selecting DCDA twins with a trisomy co-twin to simulate a
natural twin demise. Due to the small sample size, statistical analysis
could not be conducted. For the ease of analyzing the individual fetal
fraction of different co-twins, we only used dizygotic twins in our
study. It is not clear if the same observations can be replicated in
monozygotic twins, especially monochorionic twins who have a
significantly higher risk of fetal demise [23, 24]. Moreover, the
results obtained using the reduced trisomy co-twin might not fully
represent natural twin demise, which can be caused by many other reasons
with or without chromosomal abnormalities [7, 25]. Besides, in our
study, women opted for reduction at 14 to 17 gestational weeks, thus
missing the opportunities to observe residual cfDNA at early pregnancy
when most vanishing twins occur. Lastly, because of the difficulties
during clinical practice, the time points of sample collection in each
woman were not identical. In one case (patient 2), the blood sample
before the reduction was missing.