Several recent studies reported the sensitivity, specificity, and PPV of NIPT for T21, T18, and T13 screening. However, to date, no large-scale clinical studies have been conducted to assess the efficiency of NIPT for detecting SCA. To the best of our knowledge, this is the largest study (45,773 cases) to evaluate NIPT for SCA. According to our data, the overall PPV of SCA detected by NIPT was 40.56%, within the range of PPV for SCA screening between 30 and 60% [12, 13, 15, 18, 19]. When categorized by individual SCAs, the PPVs were 12.5% for 45,X, 51.72% for 47,XXX, 66.67% for 47,XXY, and 83.33% for 47,XYY, which were similar to those reported by other studies [1, 13, 20]. However, in our study, we noticed that different pregnancy characteristics showed different PPVs; for example, the PPV of SCA in AMA pregnancy cases (50.79%) was higher than that in all cases (40.56%). Moreover, the overall termination rate was 50% for SCA (when mosaic cases were included) and 41.38% (when mosaic cases were not included); the termination rates for foetal SCA were 85.71% for 45,X, 20% for 47,XXX, 73.08% for 47,XXY, and 10% for 47,XYY [13].
Our study showed that NIPT performed better in predicting sex chromosome trisomies than monosomy X. This may be due to the following. [1] There are 1098 genes on the X chromosome and 78 genes on the Y chromosome, and 58 genes are homologous genes on both sex chromosomes. The majority of the genes (29 genes) are at the ends of the sex chromosomes. [2] The low guanosine-cytosine content of the X chromosome leads to highly variable amplification of the X chromosome. [3] The nonrandom inactivation of the X chromosome in placental tissue might be the reason for the low PPV of Turner syndrome, with the paternal X chromosome tending to be inactivated in XX female trophoblasts [1, 13]. In addition, it was reported that there is an age-related X chromosome loss in normal female white blood cells, which may influence the effectiveness of predicting foetal 45,X [13], although this was not observed in our study.
The PPV of SCA is lower than that of other common chromosome aneuploidies. The reason is that sex chromosome abnormalities are less prevalent [15]. Wang et al. reported that 8.6% of positive results for SCA were due to maternal mosaicism [21], which was later confirmed by other studies [1, 13, 22]. With the combination of NIPT and maternal peripheral blood karyotype analysis, we found that approximately 12.5% of the discordant NIPT SCA results were due to maternal mosaicism in our study. Previous studies have demonstrated that the identification of maternal karyotypes tends to decrease the rate of false-positive SCA and can offer an explanation for the false-positive results for SCA [13, 14, 23]. With regard to the discordance between NIPT and invasive prenatal testing, another reason is confined placental mosaicism (CPM), which occurs in approximately 1–2% of all pregnancies [12]. The origin of most cell-free foetal DNA (cffDNA) in the maternal plasma is mostly the apoptosis of placental cells from the cytotrophoblast [24]. The mosaicism degree reduces the effective cffDNA concentration in maternal plasma, thus affecting the performance of NIPT in detecting foetal aneuploidies. Grati et al. determined the potential contribution of CPM to the NIPT false‐positive rate, demonstrating that chromosomes 13 and X were more likely to be associated with CPM than chromosomes 18 and 21 [25]. In current study, postnatal placental analysis was preformed in one case with CPM. This case illustrates that extensive cytogenetic analysis can be required to identify CPM, and that patients should be counseled regarding the possibility of discordant NIPT results. However, obtaining the placenta for clinical cases remains challenge for clinical laboratories preforming analysis of discordant NIPT results. Our results verified the value of determining the maternal karyotype and examination of placental tissue in increasing the accuracy of reporting NIPT results for chromosomes X and Y; however, further studies are needed to provide more clinical data in support of this premise.
Despite the prenatal diagnosis of foetal SCA, some pregnant women were willing to continue their pregnancies, and there were differences in the rates of pregnancy termination between different types of SCAs. Pregnant women whose foetuses were 45,X or 47,XXY were more eager to terminate their pregnancies than those whose foetuses were 47,XXX or 47,XYY, which was consistent with other studies [13, 26]. Gruchy et al. [27] reported that with regard to Turner syndrome, the rate of pregnancy termination was closely related to the 45,X karyotype, mosaic karyotype, and structural abnormalities of the X chromosome. In our study, pregnant women whose foetuses were 45,X with mosaicism of 23.08% had a stronger tendency to continue their pregnancies, while those with mosaicism of 28.38%, 48.53%, 59.38%, 76.67%, and 80.49% were more inclined to terminate their pregnancies. Current studies report that almost all pregnant women carrying foetuses with 45,X decide to terminate their pregnancy [28]. To some extent, the parental decisions for pregnancy termination may have been related to the types of SCA, level of prenatal genetic counselling, history of infertility, parental and social acceptance, economic condition, and related factors.
In clinical practice, AMA pregnant women are willing to accept NIPT as a noninvasive and accurate screening method. In this study, the proportion of AMA pregnant women was the second highest in the NIPT screening population, reaching 36.97%. Among them, NIPT detected 108 cases of SCA; 63 cases underwent amniocentesis, and 32 (50.79%) cases were confirmed to be true-positive. Of all the confirmed SCA cases, 55.17% (32/58) were AMA pregnant women. The study showed that NIPT could be used as a useful screening test for SCA in AMA pregnant women, which was similar to the results reported by Zheng et al. [29]. The differences in the frequency of SCA were statistically significant among the age groups, and the frequency was significantly higher in the > 39 years age group (P < 0.05). And the frequency of SCA in the AMA group was significantly higher than in the non-AMA group. We may conclude that AMA may be a risk factor for SCA. Therefore, genetic counselling, combined with serological screening tests and B-ultrasound detection of abnormalities, should be fully carried out for AMA pregnant women. The frequencies of 47,XXX and 47,XXY were significantly correlated with maternal age, whereas the frequencies of 45,X and 47,XYY did not show significant correlations. Although there was no statistical significance regarding the frequency of 47,XYY and maternal age, the frequency of 47,XYY decreased with maternal age in the advanced aged group. To determine whether there is a correlation between maternal age and the frequency of a foetus with 47,XYY, further studies with larger sample sizes are warranted [2]. The risk of 47,XXX increased with AMA, which was consistent with the findings by Zhu et al. [2]. Previous studies showed that for 45,X syndrome, the maternal age coefficient is negative, implying a decreasing frequency among older mothers [2]. However, this was not consistent with our results. Understanding the frequency of SCA has proven valuable in counselling couples who seek advice about the risk of foetal sex chromosome abnormalities with AMA and are considering the option of prenatal diagnosis and termination of pregnancy.
This study has a few limitations. First, the sensitivity, specificity, and negative predictive value were not calculated. New-borns with SCA usually appear phenotypically normal. Therefore, it was difficult to confirm the results of SCA without karyotype analysis during the neonatal period. Second, the number of SCA cases in our study was not large enough to discuss its frequency across different age groups. Therefore, more pregnancies must be evaluated to further understand the association between maternal age and foetal SCA.
Recent studies have demonstrated that early interventions such as postnatal hormone therapy, physical therapy, and occupational therapy could have positive effects on the behavioural phenotype or neurodevelopmental outcomes if applied earlier to SCA patients [9, 30]. Prenatal screening and diagnosis of SCA can provide the opportunity for early intervention, comprehensive postnatal management, and improve the quality of life of the affected child [10]. Sex chromosome abnormalities are more common than major trisomies at birth, and neonates are often phenotypically normal [31]. Conventional prenatal screening cannot be used to directly identify sex chromosome abnormalities that can only be identified using postnatal karyotyping, which in turn may delay the treatment of SCA patients. NIPT allows prenatal screening of SCA. The application of NIPT can allow pregnant women to have an alternate option to invasive prenatal testing for the identification of foetal sex chromosome abnormalities. However, there are still some issues that require further consideration. Due to the existing false-positive rate of NIPT screening for SCA, the number of unnecessary invasive prenatal diagnoses may increase, especially for 45,X [13]. Nevertheless, the benefit of detection for foetal SCA outweighs the risk related to invasive procedures. Some pregnant women may decide to terminate their pregnancies if chromosomal abnormalities are incidentally discovered by SCA screening, which involves ethical issues regarding the mild phenotype of the SCA and the potential increase in the rate of sex selection [13].
With the development of sequencing technology, recent methods have higher accuracy than earlier methods which used to rely on chromosome dosage changes only. Huang et al. [32] presented a novel silicon-based nanostructured microfluidic platform (Cell Reveal™) for capturing circulating fetal nucleated red blood cells (fnRBC) and extravillous cytotrophoblasts (EVT) for cell-based noninvasive prenatal diagnosis (cbNIPD). This method used a microfluidic device coated with specific antibodies to capture the related antigen covered cells. Through the method, the foetal cells could be isolated from the maternal circulation and foetal DNA could be used efficiently to detect copy number abnormalities [32]. In addition, another method, named cell-based noninvasive prenatal testing, could be used to detect the subchromosomal abnormalities (≥ 1 Mb) in foetal cells by using low-coverage shotgun next-generation sequencing [33], which was an ideal way to balance the depth and accuracy of sequencing. Moreover, whole-genome NGS methylomic analysis was also a method to provide a version of the placental methylome from the maternal plasma that could be effective for chromosomal abnormalities testing [34]. All together, NIPT’s research on chromosomal abnormalities has been expanding and deepening, not only staying on the chromosome dosage changes.