A diagnostic feasibility study on screening for trisomy 21 and XY chromosomes via cervical smear testing in early pregnancy

Abstract Objective To evaluate the technical feasibility of isolating fetal trophoblasts and detecting fetal chromosomal anomalies (trisomy 21 and XY chromosomes) using cervical smear samples obtained during routine prenatal care, and to explore the potential of this approach as a preliminary step towards a cost-effective alternative to current non-invasive prenatal testing methods. Study design Prospective cohort study with fluorescence in situ hybridization (FISH) analysis of cervical smear samples for chromosomal screening. Place and duration Department of Obstetrics and Gynecology, Caspian International Hospital, Azerbaijan, conducted in 2024. Methods Fifty pregnant women between 5 and 15 gestational weeks underwent cervical smear collection via cytobrush during routine prenatal visits. Samples were processed for the isolation of extravillous trophoblasts (EVTs). Successful isolation of EVTs was confirmed by morphological assessment and the presence of HLA-G markers. Samples were then analyzed using FISH methodology with probes specific for chromosomes 21, X, and Y. Ultrasound confirmation of fetal sex and nuchal translucency measurements was performed for initial correlation (Interim Correlation Standard). Automated scanning systems and manual verification were employed for accurate chromosomal analysis. Results Extravillous trophoblasts were successfully isolated in all 50 samples (100% success rate (95% CI: 92.9%-100%)). XY chromosomes were detected in 17 cases (34%), with ultrasound confirming male sex in 6 of 7 eligible cases (85.7% concordance). XX chromosomes were identified in 33 cases (66%), with ultrasound confirming female sex in 12 of 14 cases (85.7% concordance). No trisomy 21 cases were detected in this cohort, precluding the calculation of sensitivity for Trisomy 21 detection. One case presented with nuchal translucency ≥ 3 mm and was referred for amniocentesis, with subsequent normal karyotype confirmed by amniocentesis. FISH analysis demonstrated 100% specificity for chromosomal detection with no false-positive results for the sex chromosomes when correlated with the interim ultrasound standard. Conclusion Cervical smear-based FISH analysis represents a promising non-invasive, cost-effective approach for early fetal chromosomal screening that leverages existing clinical infrastructure. While demonstrating excellent specificity for sex chromosome determination, and technical feasibility of EVT isolation, the absence of Trisomy 21 cases in this pilot cohort limits the assessment of sensitivity for aneuploidy detection. A key limitation of this feasibility study is the use of ultrasound as an interim standard for sex determination; we lacked definitive postnatal follow-up data. Definitive validation will require a subsequent publication with full newborn follow-up data. Larger multicenter studies with known aneuploidy cases are essential to validate sensitivity for trisomy 21 detection and establish clinical implementation protocols. This methodology offers potential advantages in resource-limited settings and could complement current prenatal screening strategies as a preliminary screening tool.