AUTHOR=van ‘t Hof Liseanne J. , Kapsenberg Johanna M. , Drabbels Jos J.M. , van der Meeren Lotte E. , Roelen Dave L. , Eikmans Michael , van der Hoorn Marie-Louise P. 

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

JOURNAL=Frontiers in Immunology

VOLUME=Volume 16 - 2025

YEAR=2025

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2025.1575086

DOI=10.3389/fimmu.2025.1575086

ISSN=1664-3224

ABSTRACT=IntroductionMaternal-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).MethodCervical 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.ResultsPurity and yield of HLA-G+β-hCG+CK-7+ cells after TRIC failed to match pre-isolation HLA-G+ cell counts, despite protocol optimization. FACS revealed a fetal HLA genotype. In contrast, only the maternal HLA genotype was detected in TRIC-isolated cells.ConclusionEVT 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.