Cervical EVT isolation for non-invasive fetal HLA typing in early pregnancy is limited by purity and maternal cell contamination; a methodological comparison

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Introduction: Maternal-fetal HLA compatibility influences pregnancy outcome, including preeclampsia risk. Cervical extravillous trophoblasts (EVT) in early pregnancy provide a non-invasive source for fetal genome acquisition, potentially enabling fetal HLA typing for obstetric risk assessment. This study aimed to achieve fetal HLA typing through EVT isolation using HLA-G-coupled nanoparticle immunomagnetic separation (TRIC) and fluorescence-activated cell sorting (FACS). Method: Cervical samples from 32 pregnant women were collected by cytobrush. Saliva and umbilical cord blood (n=13) served as maternal and fetal HLA genotype controls, respectively. Cervical samples from non-pregnant women, primary cultured EVT, and cryo-sectioned term placentas served as controls for cell phenotype, protein expression, and effect of fixation. FACS and TRIC were applied to isolate EVT from maternal cells, followed by RSSO-PCR for HLA typing. EVT presence pre- and post-isolation was determined through HLA-G, β-hCG, and Cytokeratin-7 (CK-7) expression. TRIC was optimized by improving antibody-binding-efficiency, and comparing three (nano)beads types and two magnets. Results: cell counts, despite protocol optimization. FACS revealed a fetal HLA genotype. In contrast, only the maternal HLA genotype was detected in TRIC-isolated cells. Conclusion: EVT counts and maternal cell contamination limit reliable fetal HLA typing from cervical samples. Refining non-invasive EVT isolation techniques may enable fetal HLA typing to be included in risk assessment of pregnancy complications.