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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.

The goal of this study was to gain a better understanding of the impact of vanishing twins (VTs) on the incidence of discrepant cfDNA testing results. To do so, the researchers analyzed a series of cases of patients with VT to determine the extent to which this compromised cfDNA testing results.This was an observational, retrospective study that took place between April 2015 and October 2018 at the Prenatal Department of Igenomix Spain Lab. The study included 25,080 consecutive pregnancies that underwent cfDNA screening for fetal aneuploidy for chromosomes 13, 18, 21, and sex chromosomes. Singleton pregnancies or VT were classified based on clinical information provided by the referring provider. False-positive cfDNA screening was defined as a screen-positive case by cfDNA with a normal karyotype obtained after an amniocentesis, chorionic villus sampling, or at birth. Both aneuploidy and fetal fraction were determined according to an Illumina algorithm.In this cohort, cfDNA screening was performed at somewhat later gestational ages in VT pregnancies than singleton pregnancies (VT: 14.9 ± 3.2 weeks vs singleton: 13.4 ± 2.7 weeks; P < 0.0001). Maternal age was also significantly higher in the VT group compared with the singleton group (VT: 37.1 ± 3.9 years vs singleton: 36.2 ± 4.5 years; P = 0.002). In terms of absolute values, 97.4% of cases in the VT population had concordant results for aneuploidy, compared with 99.7% singleton pregnancies ( P = 0.1). In all, 94.2% (n = 194) of VT pregnancies had a screen negative cfDNA testing result compared with 97.5% (n = 24,240) of the singleton population ( P = 0.003). There was a significantly increased prevalence of sex discrepancies observed in the VT group compared with the singleton group (VT: 10.2% [n = 21] vs singleton: 0.05% [n = 12]; P < 0.0001). A significantly increased number of false-positive results was also observed in the VT group compared with the singleton group (VT: 2.6% [n = 5] vs singleton: 0.3% [n = 80]; P < 0.0001). Last, among the 12 cases where cfDNA was performed at 2 different time points, fading of contaminating cfDNA was observed in 4 cases with a sex discrepancy and in 1 false-positive for trisomy 18, which resulted in a final correct result.Limitations of this study include incomplete follow-up for singleton pregnancies. In addition, more VT cases are needed to determine whether a delay in testing could be helpful in the reduction of the false-positive rate, eventual false-negative results cannot be excluded, and the researchers did not specify how many weeks should pass between a first screen-positive cfDNA testing result and a repeat test.The data from this study suggest that VT pregnancies could consider cfDNA testing if it is applied after the 14th week of pregnancy and with appropriate genetic counseling. Future studies are needed before including VT cases in routine clinical practice. Once this is put in place, unnecessary procedures could be avoided, which could decrease the negative impact on pregnancy.

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.

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 &lt;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 &gt;35 or &lt;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 &lt; 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 &lt; 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.

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.

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BackgroundSince 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 presentationWe 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. 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.ConclusionThis 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.