Fetal ploidy status in pregnancy loss evaluated by cell-free fetal DNA in maternal blood versus direct sequencing of the pregnancy tissue

Abstract Study question Is cffDNA in maternal blood applicable for fetal ploidy evaluation in early pregnancy loss, and what are the strengths and limitations of the method. Summary answer With a sensitivity of ∼85% for aneuploidies and ∼93% specificity, cffDNA-based testing is a robust method for evaluation of fetal ploidy status in PL. What is known already Pregnancy loss (PL) is an under investigated condition without precise prognostic models or evident treatments. From previous studies using karyotype and chromosomal microarray analysis it is described that approximately half of PLs are caused by fetal aneuploidy, but international guidelines still refrain from recommending routine genetic evaluation of PL. Traditional fetal cytogenetics are expensive and dependent on collection of pregnancy tissue containing fetal or chorionic tissue to avoid inconclusive results and maternal contamination. Consequently, chromosomal investigation of the lost fetus is lacking in most literature about PL and couples are currently not getting an explanation of their loss. Study design, size, duration As a part of the prospective Copenhagen Pregnancy Loss (COPL) cohort, 1000 consecutive women with PL (spontaneous miscarriage, missed miscarriage or anembryonic sac) before GA 22 weeks were recruited between Nov 12, 2020, and May 1, 2022. Results from the first 333 women were used to validate cffDNA-based testing compared with direct sequencing of the collected pregnancy tissue. Additional 667 women were included to evaluate cffDNA performance and result distribution in 1000 PLs Participants/materials, setting, methods Blood for cffDNA-based testing were drawn in STRECK © tubes before treatment was initiated or within 24 h after complete passage of pregnancy tissue. Pregnancy tissue was collected from the vacuum system if surgical treated or by home collection if medically treated and classified as fetal, chorionic villi or unknown tissue and sequenced at University of Copenhagen. Genome wide cffDNA-based testing and fetal fraction of DNA by SeqFF was performed at Hvidovre Hospitals NIPT Center. Main results and the role of chance Among invited candidates, the participation rate was 73%. Mean maternal age was 33.9 years (SD 5.2). Gestational age at inclusion ranged from 35 days to 149 days measured from last menstrual period with a mean of 70.5 days (SD 16.5), or 10 weeks+1 day. Mean maternal BMI was 24.6 kg/m² (SD 4.6) and 233 (23%) conceived after fertility treatment. In 19 (6%) of the initial 333 women, collection of the pregnancy tissue failed for practical or psychological reasons. DNA was isolated from fetal tissue or chorionic villi in 228 (73%) cases, or from unknown tissue in 86 (27%) cases and thereby at a high risk of being maternal contaminated. The sensitivity of cffDNA-based testing compared with the pregnancy tissue sequencing result was 85% (95% CI 79–90), specificity of 93% (88–96), accuracy of 89% (85–92), and Cohen’s coefficient 0.78 showing substantial agreement.112 (11%) of 1000 total cffDNA analyses were inconclusive due to low fetal fraction (SeqFF<0.015 or < 0.025) or low sequencing quality. In instances of a conclusive result, 446 (50%) were euploid, 405 (46%) were aneuploid, and 37 (4%) contained multiple aneuploidies. The most common abnormal karyotypes identified were trisomy 16 (n = 91), monosomy X (n = 86), and trisomy 22 (n = 41). Limitations, reasons for caution The used platform for cffDNA-based testing does not report fetal polyploidy, uniparental disomy, and copy number variants found in approximately 10% of early PLs. Moreover, blood for cffDNA-based testing must be drawn with the pregnancy tissue in situ or within 24 hours limiting the window for testing. Wider implications of the findings Considering the difficulties of pregnancy tissue collection, it is relevant to introduce a pregnancy tissue-independent alternative. The validity of cffDNA-based testing for fetal ploidy status found in this study shows the potential of the method to improve clinical management and research in the field of PL. Trial registration number H-18024745