Interpretations
At the first two to three weeks after fetal reduction, there were discordant observations of fetal fraction changes, as two cases showed a bland increment of cfDNA release of demised co-twin while two other cases showed a slight decline. After that, all cases demonstrated consistent patterns of fast increase of a fetal fraction of demised co-twin, and reached the top level at 7-9 weeks after reduction and the cfDNA disappeared at 12-16 weeks after a demise or even longer. This indicates that the fetal demise induced increased release of residual cfDNA into maternal circulation for a substantial period. A recent study by Bevilacqua et al. may observe the a similar phenomenon [26], in which the fetal aneuploidy risk of NIPT after the selective reduction of a trisomy co-twin also displayed the ‘rise-and-drop’ changes in two cases.
Recently, Boyd described the histopathological changes of the placenta in intrauterine demise [27]. After fetal demise, maternal perfusion and fetal blood flow pressure disappear, which leads to the compromise of villous capillary integrity and endothelial karyorrhexis, eventually resulting in fibrosis avascular villi and solid fabrin deposition of intervillous space. Meanwhile, villous edema and intravillous hemorrhage accompanied by maternal inflammation could also occur to increase organ autolysis [27]. It was believed that intravascular karyorrhexis and multifocal villous obliteration might occur soon after fetal demise, whereas extensive obliteration of stem villi and have the level of villous fibrosis may take 14 days or more [27, 28]. Thus, we speculated that the initial discordant change patterns of residual cfDNA released from demised co-twin reflects the complicity of placental autolysis immediately after the cease of fetal blood flow and maternal perfusion. In contrast, the afterward rapid increase of residual cfDNA release was the result of extensive villous fibrosis and stem villi obliteration occurred at least 14 days after fetal demise. Also, it appeared that the fetal fraction of the surviving euploid fetus increased a lot faster when the release of residual cfDNA from the demised fetus declined at 7-9 weeks after reduction. This may reflect that the normal development and cfDNA emitting of the surviving fetus is influenced by the demised fetus.
Gestational age at fetal demise might have important roles in determining how long the residual cfDNA of the demised fetus can last. Gromminger et al. reported a vanishing twin that cfDNA of the demised co-twin only contributed to a small proportion of total fetal fraction, presumably because of early pregnancy demise [22]. However, another case reported by Niles et al. demonstrated different findings, in which a co-twin demised at 6+1 weeks showed persistent false positive NIPT results for at least 15 weeks after fetal demise [29]. Our study added no evidence since the lack of samples with fetal demise at early pregnancy. Hence, further studies are still needed to explain the effect of gestational age on residual cfDNA clearance.
The fetal demise of a co-twin has been known as an important factor causing false positive NIPT results [30, 31]. It has been estimated that a substantial proportion of false positive NIPT results ranging from 15% to 33% might be caused by the fetal demise of unaware twin pregnancies [32-34]. A key concern for clinical practice of providing NIPT service is how long the residual cfDNA from the demise co-twin remains in maternal circulation, and whether NIPT should be provided if the fetal demise of a co-twin is identified. Two previous studies have provided preliminary evidence. Curnow et al. demonstrated in five vanishing twins that the fetal cfDNA from vanished twins was detectable at eight weeks after demise [21]. However, their study was based on retrospective NIPT data of samples known with a fetus deceased at seven to eight weeks, and thus provided no follow-up of the rest of pregnancy. Bevilacqua et al. also used fetal reduction of a trisomic co-twin as a model for natural demise, and reported the prospective cfDNA testing results in seven cases [26]. However, due to minimal sampling frequency, although they found transiently detectable aneuploidy signals at three to eight weeks following reduction, they could not conclude the predictable patterns of aneuploidy risk and fetal fraction. In contrast, our study demonstrated that after fetal reduction, there was a consistent pattern of the change of fetal trisomy T-score and fetal fraction.
Both the fetal fraction of demised fetus and the trisomy T-score increased to a peak level at seven to nine weeks after reduction, possibly because of the increased rate of placental tissue autolysis after fetus deceased. Although continuous decline after eight weeks post-reduction, the detectable fetal fraction of demised fetus and trisomy T-score still lasted from 12 to 16 weeks after reduction, and even up to 20 weeks post-reduction. This finding is consistent with a sporadic case showing NIPT false positive results of fetal sex and aneuploidy at 15 weeks after a demised co-twin [29]. However, this phenomenon has not been thoroughly proven. Based on our results, residual cfDNA of fetal demise remained in maternal circulation had a long-lasting effect on NIPT by inducing false positive results for a long time of pregnancy. Therefore, the current data does not support providing NIPT to pregnancies given the identification of fetal demise.