I Pfeifer1, A Benachi2, A Saker1, J P Bonnefont3, H Mouawia1, L Broncy1, R Frydman4, M L Brival5, B Lacour6, R Dachez7, P Paterlini-Bréchot8. 1. INSERM Unit 1151 - Team 13, Paris Descartes University, Paris, France. 2. Department of Gyncology, Antoine Béclère Hospital, Clamart, France. 3. Laboratory of Medical Genetics, Necker-Enfants Malades Hospital, Paris, France. 4. Department of Reproduction, Foch Hospital, Suresnes, France. 5. Department of Maternity, Les Lilas, France. 6. Laboratoiry of Biochemistry A, Necker-Enfants Malades Hospital, Paris, France. 7. Alfred Fournier Institute, Paris, France. 8. INSERM Unit 1151 - Team 13, Paris Descartes University, Paris, France; Laboratoiry of Biochemistry A, Necker-Enfants Malades Hospital, Paris, France. Electronic address: patriziapaterlini@gmail.com.
Abstract
OBJECTIVE: We aimed at developing a method to recover trophoblastic cells from 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 sampling or 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 microdissection and 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 cells were recovered from all tested cervical samples with a frequency of 2-12 trophoblasts per 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 trophoblasts can 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.
OBJECTIVE: We aimed at developing a method to recover trophoblastic cells from 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 sampling or 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 microdissection and 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 cells were recovered from all tested cervical samples with a frequency of 2-12 trophoblasts per 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 trophoblasts can 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.
Authors: Chandni V Jain; Leena Kadam; Marie van Dijk; Hamid-Reza Kohan-Ghadr; Brian A Kilburn; Craig Hartman; Vicki Mazzorana; Allerdien Visser; Michael Hertz; Alan D Bolnick; Rani Fritz; D Randall Armant; Sascha Drewlo Journal: Sci Transl Med Date: 2016-11-02 Impact factor: 19.319