Results
Search results
A total of 1209 articles were retrieved from the initial database
searches, and 512 duplicates were eliminated. Following the screening of
titles/abstracts, 624 publications were excluded and 61 articles were
retrieved for full review and eligibility assessment. Finally, 14
articles were considered eligible for inclusion (see the PRISMA
flowchart; Figure S1).
Included study characteristics
Table S1 summarizes the methodological characteristics of the
included studies. Of the 14 studies included in this review, 13 were
retrospective and 1 was prospective. Of the 13 retrospective studies, 12
consisted of observational or retrospective cohort studies, and 1 was a
nested case-control study The cohort size of the studies ranged from 200
to 93 048. The maternal age was unknown in 2 studies and the
interquartile range was the only information given in one study. Ten
studies used massive parallel sequencing (MPS) technology as the DNA
sequencing method. All the positive NIPT screening cases were supported
by prenatal or postnatal karyotyping, provided that the participants
consented to the procedure, and they were not lost to follow-up. In 4
studies, both autosomal trisomies and SCAs were studied, whereas only
autosomal trisomies were studied in 4 articles, and only SCAs in 6
articles. All in all, 8 studies for autosomal trisomies and 10 for SCAs
were gathered.
Methodological quality of
included studies
The CASP tool was used to assess the risk of bias. A summary of the
results is presented in Table S2 and described in Appendix
S2 .
Analysis of Autosomal
Trisomies: 21, 18, and 13
Out of the 14 included studies, 8 studies evaluated the autosomal
trisomies 21, 18 and 13. The results are summarised in Table 1 .
The cohort sizes range between 200 and 40311 participants (Table
S1 ). Of the 8 studies, the highest prevalence of a positive cffDNA test
result found for T21 was of 5.5% (Table 3).
In general the studies concurred in that T21 was found to have the
highest PPVs as compared to T18 and T13 (Table 1). Three studies showed
a 100% PPV for T21. Another 2 studies reported PPVs between 90-99%
whilst PPVs of 80% to 90% were seen in 2 studies.
The lowest relative overall PPVs were reported for T13. A PPV of 0%,
followed by 14.3% were the lowest value reported across the 8 studies.
Results were quite heterogenous, with PPVs as high as 100%, and a
number of results between 20-90% also
reported17,19,22. One study reported no cases of
trisomy 13, and consequently did not present its PPV.
For T18, the results were intermediate between those of T21 and T13. Lee
DE et al. (2019) and Serapinas D et al. (2020) reported
PPVs of 100%. Alyafee Y et al. (2021) found an 80% PPV. The
remaining 4 studies had results below 60%: 59%, 58.7%, 48.2% and
0%.
Among the 8 studies that reported on the autosomal trisomies, 1 study
omitted the individual PPVs and only included the overall PPV of the 3
trisomies, with a result of 64.7%16. However, a PPV
of 100% was reported when the cases with multiple clinical indications,
rather than just one, and including abnormal ultrasound findings for
NIPT, were exclusively accounted for.
Analysis of Sex Chromosome
Aneuploidies
Out of the 14 included studies, 10 studies involved analysis of SCAs.
The results are summarised in Table 2 .
The number of participants per study ranged from 862 to 93048
(Table S1 ). The prevalence of a high-risk NIPT is presented
individually for each SCA in 6 studies, while only the overall PPV for
all SCAs is available for 4 studies (Table 4). Of all these studies, the
highest prevalence of a positive cffDNA test is 0.6%.
In comparison to the other SCAs, the lowest PPVs were associated with
Turner syndrome (all PPVs were <30%). The PPVs for
Klinefelter syndrome ranged from 25% - 77.8%. In the case of triple X
syndrome, 4 studies reported PPVs from 29.7% - 51.7%, whilst two
studies reported a PPV of 100%. Finally, studies which screened for
Jacob’s syndrome demonstrated heterogenous results. Two studies returned
PPVs of 100%, with others in the range of 60% - 83.3%. One study
returned a low PPV of 27.3%. It is pertinent to note that of the two
studies reporting a PPV of 100%, the latter study only identified one
case that bore the 47,XYY SCA.
Among the 10 studies that reported on SCAs, 3 studies reported only the
overall PPV, without specifying the PPV of each individual SCA. They all
presented PPVs < 50%.
Analysis of Discordant (false
positives; FP and false negative; FN) results
The incidence of FP and FN cases were not uniformly reported across the
14 studies.
Although the number of FP cases was documented in 13 of the studies, the
cause of the result was only confirmed in 4 studies, while the others
offered a generic (non-specific) explanation. Ma L et al. (2018)
proved that a case wrongly screened as XYY was later confirmed as a
microdeletion. Placental mosaicism was a cause for one T18 FP case,
whilst one T13 FP case was confirmed postnatally by placental
investigation and karyotyping of cord blood, which explains the 0% PPVs
found in this study for T18 and T13. Furthermore, 2 maternal mosaicism
cases were confirmed by maternal karyotyping in a 2021 study. Two 47,XXX
FP cases were identified, secondary to undiagnosed maternal 47,XXX SCA,
as confirmed by maternal blood karyotype. The final study presented PPVs
of 100% across all the identified aneuploidies, and therefore no FP
were detected.
In general, FN were poorly reported, or not reported at all in most of
the studies. A small number of studies proffered some basic explanation
(without differentiating between FP and FN), whilst others specifically
described the FN outcomes of their studies. Our analysis shows that no
FN were identified in 5 studies. However, placental mosaicism was
confirmed as a source of FN in a single study, in which 3 cases of T18
were identified by CVS with quantitative fluorescent polymerase chain
reaction.
Inconclusive results
Reporting of the percentage of no-call (or inconclusive) results was
absent in 5 studies. Nevertheless, 10 of the 14 studies stated that the
main cause for no-call results was an insufficient cffDNA fraction
(defined as a fetal fraction of < 4% in 9 studies). In one
study, it was defined as < 2.8%, and the median fetal
fraction in the no-call group was significantly lower (3.1%) than in
the group with a conclusive result (9.1%).
In addition to an insufficient cffDNA fraction, other reasons cited for
the no-call results were: unusually high total free DNA, failure to pass
the quality control measures and sequencing run failure.
In most cases, the sample was successfully rerun, giving a valid result
and a much lower final no-call rate compared to that of the first sample
run. In one study, 94% of the no-call results were successfully rerun
with the same sample, whereas 3 cases required a third rerun to obtain
results. Similarly, there were originally 509 samples with a no-call
result in a 2020 study, which was reduced to 46 samples after retesting.
As for the 2021 study by Luo Y et al ., 38 participants had a
definitive no-call, as opposed to 468 participants after the first
sample run.
Discussion:
Main findings
The results have demonstrated that NIPT with cffDNA is reliable in the
screening of autosomal trisomies. Overall, T21 had the most promising
results, whereas T13 had the least promising. NIPT is also useful for
SCAs screening, although it has yielded poorer results overall than
those for the autosomal trisomies. Further work is required to improve
PPV of SCAs, especially for 45,X0. All study authors agreed that
confirmation by invasive procedures are necessary in all cases of
high-risk cffDNA results.
Interpretation of the results
Aneuploidies
Amongst the autosomal trisomies, T21 had the highest and T13 had the
lowest PPVs. Amongst the SCAs, the lowest PPVs were found for 45,X0. For
each aneuploidy, the results oscillated greatly between the studies.
It is important to highlight that the patients with high-risk NIPT
results who declined further invasive testing could have significantly
impacted the resulting PPV, either in-creasing it if they were true
positives or decreasing it if they were false positives.
Discordant results
Most of the studies only listed some general causes of false results.
Mosaicism can be of maternal or fetal origin, given that as maternal age
increases, a natural loss of the X chromosome results in a maternal
cell-free DNA with less X fragments, and that for SCAs, many cases can
have multiple cell lines. Another vastly mentioned cause of discordant
results is the presence of a vanishing twin, which justifies classifying
this factor as an exclusion criteria in our study.
A number of studies confirmed the origin of the discordant results:
microdeletion, undiagnosed maternal SCA and mosaicism. These are all
well-known causes and are in accordance with a prior systematic review,
which found mosaicism to be amongst the main reasons of discordant
results.
Although the causes of FP were not verified in most studies, as they
would require further testing, such as placental investigation, the
number of FP cases were detailed, as this value is needed to calculate
the PPV.
With regards to FN, they were poorly reported, or not reported at all.
No FNs were identified in 5 studies. However, the underestimation of FNs
is very likely given that confirming FNs would require a karyotype of
the fetus/newborn, following a negative cffDNA test. A phenotypical
postnatal follow-up would not be sufficient to discard an aneuploidy,
especially for SCAs as they are phenotypically diverse and can be
undetectable at birth. In fact, historically, 75-90% of patients with
SCAs remain undiagnosed in their lifetime.
Inconclusive results
The main cause for no-call results was an insufficient cffDNA fraction.
The cut-off value was different in one study due to the different DNA
sequencing technique used (Single nucleotide polymorphisms technology)
(see Table S1), which is claimed to provide accurate results with fetal
fractions ≥2.8% (as opposed to ≥4% by MPS) and yet the median fraction
was of 3.1% in the no-call group .
The no-call results were missing in 5 studies, possibly because an
inconclusive result could have been immediately discarded, or
successfully automatically rerun. In one study, all the fetal fractions
were ≥4% (4-31%), which may explain the absence of no-call results.
Consequently, the fetal fraction significantly impacts cffDNA testing.
While in most cases the sample was successfully rerun, a second blood
draw was sometimes necessary, contrary to the recommendation of the
American College for Medical Genetics and Genomics32.
Interpretation of the results in the context of other evidence
Over the years, although various systematic reviews assessing the
usefulness of cffDNA in prenatal screening have been published, not many
focused on the value of cffDNA testing for autosomal trisomies and for
SCAs, and on their comparison. In concordance with our results, a 2017
systematic review and bivariate meta-analysis concluded that NIPT by
cffDNA is an accurate tool in the screening of autosomal trisomies and
45,X0, and that FN and inconclusive results were poorly reported. A 2017
meta-analysis on cffDNA demonstrated that cfDNA screening in singleton
pregnancies could detect >99% of fetuses with T21, 98% of
those with T18 and 99% of those with T13; however, the number of
reported SCA cases was too low to be accurately. Also consistent with
our findings, a recent systematic review found that NIPT is a reliable
screening test for SCAs.
Strengths and limitations
This systematic review was conducted according to the PRISMA 2020
guidelines. A thorough review of the available articles related to the
topic was performed on 3 databases.
Compared with a 2017 systematic review and meta-analysis, in which prior
studies were included, other factors related to cffDNA were investigated
and a meta-analysis of the results was conducted. However, the PPV, a
relevant indicator of accuracy, was not reported. Furthermore, of all
the SCAs, only 45,X0 was investigated. The reason for the fewer studies
in our systematic review is due to the focus on aneuploidies, excluding
all the other uses of cffDNA.
This review has some limitations. Firstly, PPV was the only metric used
and therefore presents its own biases. Secondly, more larger scale
studies are needed. Thirdly, given that pregnancy outcome was not an
objective of this review, this aspect was not considered. In addition,
the differences in the DNA sequencing techniques used in each study were
not considered. Finally, there were publications that were not
retrieved, despite our best attempts to contact the authors.
Implications for clinical
practice
The findings of this review cautiously support the use of cffDNA testing
as a screening test for T21, T18, T13, 45,X0, 47,XXY, 47,XXX, and
47,XYY. However, despite the high PPV, we do not recommend its use as a
diagnostic test, and should be followed by an invasive confirmatory
diagnostic technique.