Non-invasive cell-free DNA-based approach for the diagnosis of clinical miscarriage: A retrospective study.

OBJECTIVE: To evaluate cell-free DNA (cfDNA) testing as a non-invasive approach to detecting aneuploidies in clinical miscarriages. DESIGN: A retrospective cohort study of women with pregnancy loss. SETTING: Hospitals and genetic analysis laboratories. POPULATION OR SAMPLE: Pregnancy losses in the period 2021-2022. METHODS: Results derived from non-invasive cfDNA testing (Veriseq NIPT Solution V2) of maternal blood and invasive analysis of products of conception (POC) (Ion ReproSeq) compared in 120 women who suffered a miscarriage. MAIN OUTCOME MEASURES: Concordance rate results, cfDNA testing performance, non-informative rate (NIR) and fetal fraction (FF). RESULTS: We found no significant differences in the NIR between invasive (iPOC) and non-invasive (niPOC) analysis of POC (10.0% [12/120] versus 16.7% [20/120]). Of 120 samples, 90 provided an informative result in iPOC and niPOC groups (75%). cfDNA analysis correctly identified 74/87 (85.1%) samples (excluding triploidies). Sensitivity and specificity were 79.4% and 100%, respectively; all discordant cases were female. A binomial logistic model suggested fetal sex as the only variable influencing the concordance rate (P = 0.035). A Y-chromosome-based FF estimate allowed the optimal reclassification of cfDNA of non-informative male fetuses and a more accurate evaluation of cfDNA testing performance. The difference between the two FF estimates (native algorithm and Y-chromosome-based) suggests that female non-concordant cases may represent non-informative cases. CONCLUSIONS: Cell-free DNA-based testing provides a non-invasive approach to determining the genetic cause of clinical miscarriage.

Cell-free fetal DNA testing performance and fetal fraction estimation are not affected in ART-conceived pregnancies.

STUDY QUESTION: Does ART-based conception influence fetal fraction (FF) estimation and cell-free fetal DNA (cffDNA) testing performance? SUMMARY ANSWER: Mode of conception (ART versus natural) does not impact FF estimation or cffDNA test informativity rates. WHAT IS KNOWN ALREADY: Pregnancies achieved via ART are increasing, and cffDNA testing is displacing traditional prenatal screening methods due to its high sensitivity and specificity and noninvasive nature. However, conflicting data exist on cffDNA testing performance and FF in ART pregnancies compared with natural pregnancies. STUDY DESIGN, SIZE, DURATION: We performed a case-control study that included 21 558 consecutive pregnancies (spontaneous, n = 15 707; ART, n = 5851). ART-conceived pregnancies were stratified into two groups according to oocyte origin. Samples were collected from April 2015 to September 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study included women from different centers worldwide. Blood samples were drawn from the 10th week of gestation onward. Massive parallel whole-genome sequencing was used to analyze cffDNA content in blood plasma. Two different types of technologies (single-end and paired-end) were applied because of analysis technology changes made by the sequencing provider over time. FF was determined using different methods depending on the type of technology used. Cases with an FF <2% or with failure in any quality control metrics were classified as noninformative. An analysis of covariance model was selected to identify which qualitative (sequencing methodology, mode of conception, type (i.e. multiplicity) of gestation and age (women >35 or <35 years old)) and quantitative (gestational age, BMI) variables were predictors of FF value. Multinomial logistic regression was used to evaluate whether the mode of conception impacted cffDNA testing performance. MAIN RESULTS AND THE ROLE OF CHANCE: A univariate t-test demonstrated no significant differences in FF values between ART (median FF = 9.2%) and spontaneous pregnancies (median FF = 9.2%). Also, a multivariate analysis showed that the mode of conception, did not strongly impact the percentage of FF. ART-treated women showed a lower incidence of high-risk cffDNA results compared to women who conceived naturally, specifically for trisomy (T)21 (0.7% versus 1.3%, P = 0.001) and T18 (0.1% versus 0.3%, P = 0.001). A multivariate model stratified by type of aneuploidy suggested that these differences were conditioned by oocyte origin, especially for the T21 risk classification (P < 0.0001). False-positive rates (FPRs) were significantly higher in the ART population, mainly for T13 (P = 0.001) and sexual chromosome aneuploidies (SCAs; P < 0.001). A multivariate model suggested that the differences observed in SCAs were caused by sequencing modality rather than by mode of conception. Likewise, ART-treated women who used their own oocytes had a higher probability of a false positive for T13 (P = 0.004). LIMITATIONS, REASONS FOR CAUTION: Our study lacks follow-up data for low- and high-risk cases of both ART-conceived and naturally conceived pregnancies. Therefore, the results comparing FPR in both populations should be interpreted carefully. Also, collecting information about different ART modalities and regarding preimplantation genetic testing for aneuploidy treatments would help draw definite explanations for the trends observed in this study. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study that demonstrates, with a large sample size, that FF is not influenced by mode of conception, demystifying the notion that patients undergoing ART have a higher probability of noninformative cffDNA testing results. Multivariate models stratified by oocyte origin and type of aneuploidy demonstrated that ART-conceived pregnancies do not have a higher probability of classification as a high-risk pregnancy in prenatal testing. This information is especially valuable to clinicians and genetic counselors when informing patients about the risks and limitations of cffDNA testing in ART pregnancies. STUDY FUNDING/COMPETING INTEREST(S): This study was financially supported by Igenomix Lab S.L.U. All authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Inherited unbalanced reciprocal translocation with 3q duplication and 5p deletion in a foetus revealed by cell-free foetal DNA (cffDNA) testing: a case report.

BACKGROUND: Since 2011, screening maternal blood for cell-free foetal DNA (cffDNA) fragments has offered a robust clinical tool to classify pregnancy as low or high-risk for Down, Edwards, and Patau syndromes. With recent advances in molecular biology and improvements in data analysis algorithms, the screening's scope of analysis continues to expand. Indeed, screening now encompassess additional conditions, including aneuploidies for sex chromosomes, microdeletions and microduplications, rare autosomal trisomies, and, more recently, segmental deletions and duplications called copy number variations (CNVs). Yet, the ability to detect CNVs creates a new challenge for cffDNA analysis in couples in which one member carries a structural rearrangement such as a translocation or inversion. CASE PRESENTATION: We report a segmental duplication of the long arm of chromosome 3 and a segmental deletion of the short arm of chromosome 5 detected by cffDNA analysis in a 25-year-old pregnant woman. The blood sample was sequenced on a NextSeq 550 (Illumina) using the VeriSeq NIPT Solution v1 assay. G-band karyotyping in amniotic fluid only detected an abnormality in chromosome 5. Next-generation sequencing in amniocytes confirmed both abnormalities and identified breakpoints in 3q26.32q29 and 5p13.3p15. The foetus died at 21 weeks of gestation due to multiple abnormalities, and later G-band karyotyping in the parents revealed that the father was a carrier of a balanced reciprocal translocation [46,XY,t(3;5)(q26.2;p13)]. Maternal karyotype appeared normal. CONCLUSION: This case provides evidence that extended cffDNA can detect, in addition to aneuploidies for whole chromosomes, large segmental aneuploidies. In some cases, this may indicate the presence of chromosomal rearrangements in a parent. Such abnormalities are outside the scope of standard cffDNA analysis targeting chromosomes 13, 18, 21, X, and Y, potentially leading to undiagnosed congenital conditions.

Chromosome Y as a marker for sex discrepancies in patients with organ transplants: a case report.

BACKGROUND: Organ transplantations cause discrepancy in results from cell-free DNA (cfDNA) testing, but scientific literature is scarce. CASE: A 33-year old gravida underwent cfDNA testing, which showed high levels of Y chromosome (ChrY) in the maternal bloodstream. The ChrY pattern was comparable to an adult male reference. As a result, cfDNA testing was only informative for autosomes. Routine 20-week ultrasound scan showed no structural alterations and the presence of female external genitalia. Post-clinical research revealed that the patient received a bone marrow transplant from a male donor several years before. Fluorescence in situ hybridization showed that 100% of nuclei analysed from the patient's lymphocytes presented a ChrY. CONCLUSION: This case demonstrates ChrY can be used as a marker to avoid sex discrepancies in certain patients with organ transplants.

Should vanishing twin pregnancies be systematically excluded from cell-free fetal DNA testing?

OBJECTIVE: To demonstrate the feasibility of cell-free DNA (cfDNA) testing in vanishing twin (VT) pregnancies in routine clinical practice. METHODS: Our study included 24 874 singleton and 206 VT consecutive pregnancies. Cell-free DNA was analyzed by massively parallel sequencing. Both aneuploidy analysis (chromosomes 13,18, 21, X, and Y) and fetal fraction estimation were performed according to an Illumina algorithm. Contaminant DNA contribution from the demised co-twin was studied in detail. RESULTS: VT pregnancies exhibited a higher prevalence of screen-positive cases (5.8% vs 2.5%), sex discrepancies (10.2% vs 0.05%), and false positive rates (FPR) (2.6% vs 0.3%) than singleton pregnancies. However, their incidence was significantly lower in tests performed after the 14th week (screen-positive cases: 3.1%; sex discrepancies: 7.8%; and FPR: 0.8%). Among the 12 cases in which cfDNA was performed at two time points, fading of contaminating cfDNA was observed in four cases with a sex discrepancy and in one false positive for trisomy 18, resulting in a final correct result. CONCLUSIONS: Our data suggest VT pregnancies could be included in cfDNA testing as long as it is applied after the 14th week of pregnancy. However, future studies to validate our findings are needed before including VT cases in routine clinical practice. Once established, unnecessary invasive procedures could be avoided, mitigating negative emotional impact on future mothers.