Cervical trophoblasts for non-invasive single-cell genotyping and prenatal diagnosis

Abstract Objective We aimed at developing a method to recover trophoblastic cellsfrom the cervix through a completely non-invasive approach and obtaining a genetic proof of their fetal nature implying that they can be used for non-invasive prenatal diagnosis (NIPD). Methods We studied obstetrical samples from 21 pregnant women between 8 and 12 weeks of gestation scheduled for chorionic villus samplingor undergoing elective termination of pregnancy. A cytobrush was used to extract cells from the external parts of the cervix and transferred to 10 ml of preservative solution. Cells were layered on filters with 8 microns pores using the ISET system (Isolation by SizE of Tumor/ Trophoblastic cells) and stained. Putative fetal cells were collected by single cell laser-assisted microdissectionand identified as fetal or maternal cells by Short Tandem Repeat genotyping. NIPD was blindly performed on 6 mothers at risk of having a fetus with Cystic Fibrosis or Spinal Muscular Atrophy. Results Trophoblastic cellswere recovered from all tested cervical samples with a frequency of 2–12 trophoblastsper 2 ml. NIPD was blindly obtained and verified in 6 mothers at risk of having a fetus with Cystic Fibrosis or Spinal Muscular Atrophy. Discussion Although larger confirmation studies are required, this is the first report providing a solid proof of principle that trophoblastscan be consistently and safely recovered from cervical samples. Since they are a source of pure fetal DNA, i.e. fetal DNA not mixed with maternal DNA, they constitute an ideal target to develop NIPD of recessive diseases, which is a technical challenge for methods based on cell free DNA.