Materials and methods

This systematic review was conducted following the PRISMA guidelines.
Search strategy
PubMed, Web of Science, and Scopus were the databases searched. The keywords used were “cell-free fetal DNA”, “non-invasive prenatal testing”, “aneuploidy”, and “trisomy”. An advanced search was conducted, combining: keywords, synonyms, MeSH equivalents, Boolean Operators (“AND”, “OR”), and type of study (only primary research articles were accepted). The full search strategy for each of the databases is available in Appendix S1 . The only filter applied was the publication date (2017 or later, after the last best review). The final search was conducted on 18/09/2023.
Study selection and eligibility criteria
Firstly, duplicate articles were eliminated from the bulk retrieval generated by the search. Next, articles were screened by title and abstract. The remaining articles were fully reviewed and assessed according to the eligibility criteria.
The eligibility criteria were as follows. Only primary research articles were included. Studies including pregnant women with a singleton pregnancy, who underwent a NIPT by cffDNA were considered eligible. Furthermore, the cffDNA results must have been compared to confirmatory prenatal or postnatal tests. The abnormalities included were the most common autosomal trisomies, as described above, and SCAs.
Studies including diseases other than those previously stated were excluded. Multiple pregnancies (including vanishing twins) were also excluded, as they are commonly associated with inconclusive cffDNA results.
Data extraction
Data was collected manually from the eligible studies, following the screening process.
The methodological characteristics of the studies were extracted: primary author, publication date, type of study, country, duration of study, number of participants, participants characteristics (maternal and gestational ages), cffDNA sequencing technology used and diagnostic confirmation technique. Table S1 summarizes these characteristics.
The statistical results comparing cffDNA to the confirmed (gold-standard) diagnoses of each study were collected for each aneuploidy: T21 – T18 – T13 – SCAs (45,X0 – 47,XXY – 47,XXX – 47,XYY). The positive predictive value (PPV) is presented as the key outcome variable in this systematic review as it was the common metric across the 14 studies, presented in Table 1 (for autosomal trisomies) and Table 2 (for SCAs). The prevalence of a positive NIPT test outcome was also collected for each of the studies (see Table 3 for autosomal trisomies and Table 4 for SCAs). The rate of inconclusive results was also collected.
When the overall PPV of several aneuploidies was given, the individual PPV for each aneuploidy was calculated when the data was available. As for the high-risk NIPT result prevalence, it was either directly provided or calculated as a percentage by dividing the number of high-risk (positive) cffDNA test results by the total number of samples tested.
Quality assessment and risk of bias
The study selection, quality assessment and data extraction were carried out following the PRISMA guidelines. The Critical Appraisal Skills Programme (CASP) checklists were used to assess the risk of bias.