From the results described above, we found that NIPT could be applied on the VTS pregnancies. Despite the low PPV and relative high testing failure rate, which may be due to low number samples in our cohort, the best strength of our research is that we found that sex chromosomes of the demised fetal seriously interfere with the accuracy of NIPT results even the time interval of fetal demise and NIPT sampling is longer than 10 weeks, but this phenomenon could disappear when the time interval is longer than 15 weeks.
The main cause of VTS is believed to be chromosomal aberrations of the demised fetus. The placenta of the aborted fetus with abnormal chromosome is not completely absorbed, so its DNA may affect the NIPT, causing no call, false-positive results or discordant phenotypic sex [12].
Some past studies declared that VTS should be excluded from cffDNA testing. However, existing studies recommend a more differentiated approach for the VT population as long as pregnancy women and laboratory are appropriately informed, they suggest that NIPT can successfully detect common autosomal aneuploidies in pregnancies affected by a VT but with a higher rate, including our data here[13–16].
Some researchers found that VTS was related to a false-positive NIPT result [17, 18]. One study revealed that VT could cause 13% false-positive NIPT cases (7/54) [19]. Some other researches even found that co-twin demise, however, results in as much as 42.1% of confirmed NIPT results[20].
Our present study fully demonstrated the applications of NIPT in VTS pregnancies significantly improve the false-positive rate (5/5,100%) than that of routine NIPT clinical samples [21, 22]. The five positive NIPT results are all about sex chromosomes. According to our local database [23], when the chrY < 0.007%, the fetus is recognized as female, however, when the chrYz > 5 (chrYz ≤ 5 represents female) simultaneously, the analysis system may indicate Sex Chromosome Abnormality (SCA), a re-sampling is needed. When the result of the re-sampling was still SCA, the invasive prenatal diagnosis is recommended. For cases 1, 4, 5, 6 and 3 (three initial samplings), the chrY < 0.007% but the chrYz > 5, thus the analysis system could not explicitly estimate chromosome X or Y aneuploidies. When the sex of the two twins is inconsistent, the deceased fetus will affect the determination of the sex of the surviving fetus by NIPT. Researchers declared that the majority of the cases of discordant suspected sex results on US and NIPT results were due to VT[24]. It was generally reported that when the demised fetus is male and the ongoing fetus is female, it is easy to misjudge the viable fetus as male [25, 26].
In here, the viable fetuses are all girls, so we suspected that the demised fetuses are all male fetuses, continuous release of male fetal DNA caused the discrepancy of chrY < 0.007% but the chrYz > 5.
In order to reduce the risk of false-positive, sex discrepancy and test failure, the timing of sampling for these VT pregnancies needed to be noticed. Lots of literatures declared that the demised fetal cffDNA is tentatively assumed to be detectable up to 15 weeks after fetal demise[13, 18, 27] Yang Zou et al in 2021 found that re-sampling after 15 weeks of gestation could reduce the most risks of negative effects of VT[28]. From our case 3, we could learn that as time extended, the effect of demised fetal could disappear. When the time interval of fetal vanish and re-sampling is longer than 15 weeks of gestation age, we could get accurate NIPT results. Therefore, VT patients should be counseled about the risks of early testing. Further studies are needed to determine the optimal timing of NIPT in VT pregnancies in consideration of individual differences.
In our series, the overall fetal fraction (FF) is similar to that of other’s study. The numerical value is between those of the singleton pregnancies and twin pregnancies with no VT [29]. When the FF is lower than 3.5%, a no-call result occurred. Possibly extending the time of re-sampling of NIPT or an ultrasound is recommended. Although NIPT on pregnant women with VTS may have, its negative exclusion ability is strong, which can reduce unnecessary invasive prenatal diagnosis. The effect of VTS on NIPT results can be reduced by extending the interval of re-sampling.
Even though, we have some new findings here. Nevertheless, there are several limitations in the current study. Firstly, there are no positive results about trisomies 21, 18 and 13, which are the main target chromosomes. Secondly, the number of positive cases is small, which may due to the insufficient overall sample size. And also, these cases are all false-positives, which signify laboratory quality control needs to be strict. Thirdly, only one sample successfully displayed normal result after re-testing or re-sampling, the time interval of VT and re-sampling is unconvincing.