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Updated: Aug 19, 2025
Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay
Published on: January 8, 2012
Isolation of single circulating trophoblasts from maternal circulation for noninvasive fetal copy number variant
Anna Doffini1, Claudio Forcato1, Chiara Mangano1
1A. Menarini Biomarkers Singapore Pte Ltd, R&D department, Singapore, Singapore.
Objective:
To develop a multi-step workflow for the isolation of circulating extravillous trophoblasts (cEVTs) by describing the key steps enabling a semi-automated process, including a proprietary algorithm for fetal cell origin genetic confirmation and copy number variant (CNV) detection.
Methods:
Determination of the limit of detection (LoD) for submicroscopic CNV was performed by serial experiments with genomic DNA and single cells from Coriell cell line biobank with known imbalances of different sizes. A pregnancy population of 372 women was prospectively enrolled and blindly analyzed to evaluate the current workflow.
Results:
An LoD of 800 Kb was demonstrated with Coriell cell lines. This level of resolution was confirmed in the clinical cohort with the identification of a pathogenic CNV of 800 Kb, also detected by chromosomal microarray. The mean number of recovered cEVTs was 3.5 cells per sample with a significant reverse linear trend between gestational age and cEVT recovery rate and number of recovered cEVTs. In twin pregnanices, evaluation of zygosity, fetal sex and copy number profiling was performed in each individual cell.
Conclusion:
Our semi-automated methodology for the isolation and single-cell analysis of cEVTS supports the feasibility of a cell-based noninvasive prenatal test for fetal genomic profiling.
