Non-invasive Isolation Of Endocervical Trophoblast Cells To Recover Fetal Genetic Information For Prenatal Testing

BackgroundThe traditional invasive methods used to obtain fetal DNA for prenatal diagnosis carry certain risks of infection or abortion.Therefore,the collection of trophoblast cells from the cervix has been proposed as a noninvasive alternative for prenatal testing.This method is possible because cervical trophoblast cells contain complete fetal genome information,and this method can be performed regardless of maternal age,gestational weeks,weight;hence,the method has attracted research attention in recent years.Studies have used trophoblast cells isolated from transcervical cell(TCC)samples to detect common fetal chromosome aneuploidy and monogenic diseases such as spinal muscular atrophy and cystic fibrosis by sequencing analysis.However,there are excessive maternal cells in TCC samples.The effective isolation of trophoblast cells from TCC samples is important for downstream detection and analysis,but fast,effective,and reproducible methods are lacking.In recent years,technology for noninvasive prenatal detection of monogenic diseases has developed rapidly,but research on the detection of monogenic genetic diseases from trophoblast cells isolated from TCC samples is still in the exploratory stage,and there are still many difficulties with this method.ObjectiveThe main objectives of this study are as follows:(1)establish a method for rapid,effective,and reproducible enrichment of trophoblast cells from TCC samples in order to provide sufficient cells for downstream detection and analysis;(2)investigate the fetal origin of trophoblast cells,which will lay an important foundation for downstream detection and analysis;and(3)explore the feasibility of noninvasive prenatal testing of single gene genetic diseases based on trophoblast cells.Methods(1)The surface markers of female genital tract cells were identified by consulting existing literature,and their expression levels in the cervical epithelial cell line,JEG3 cell line,and TCC samples were identified by flow cytometry so as to screen the specific markers of maternal cells.(2)The RNA of the cervical epithelial cell line and the JEG3 cell line were sequenced and analyzed by RNA-seq technology.Based on the results of transcriptome sequencing,potential markers of cervical epithelial cells were preliminarily screened according to the principles of cell membrane expression,relatively high expression abundance,and commercial antibodies,and then specific markers were screened by flow cytometry.(3)Based on the screened maternal cell specific markers and the extravillous trophoblast(EVT)specific marker HLA-G,a new strategy combining negative and positive selection(CNPS)was designed to isolate cervical trophoblast cells.The enrichment efficiency of CNPS was preliminarily identified by JEG3 cell line+TCT incorporation samples.(4)TCC samples were collected from three women,all of whom had a normal singleton pregnancy with a male fetus.The candidate EVT cells enriched by CNPS were sorted by flow cytometry,and then all the sorted single cells were subjected to whole genome amplification(WGA).The WGA products were sequenced and analyzed with low coverage,and the male fetal cells were identified according to the sequencing results.(5)Low coverage sequencing of EVT cells was performed to confirm their identity as high-quality EVT cells,and then the high-quality EVT cells and cord blood gDNA were subjected to whole genome sequencing(WGS)to evaluate their coverage rate,single nucleotide variant(SNV)and insertion and deletion(Indel)variation at the whole genome level,and to explore the feasibility of noninvasive prenatal testing for single gene genetic diseases.Results(1)The results of flow cytometry showed that the expression of leukocyte surface common antigen CD45 on the surface of the cervical epithelial cell line and the JEG3 cell line was only 0.01%and 0.0%,respectively,whereas it was 13.1%in TCC samples.Therefore,CD45 was included as a marker for leukocyte negative screening in TCC samples.(2)Based on the results of transcriptomics,7556 genes were differentially expressed between cervical epithelial cell lines and JEG3 cell lines,among which 3757 genes were significantly upregulated and 3799 genes were significantly downregulated.According to the principle of cell membrane expression,relatively high expression abundance,and commercial antibodies,and the functions of differentially expressed genes,seven potential cervical epithelial cell markers were preliminarily screened:CD44,CD227,CD82,CD9,CD49c,CD58,and HLA-A.According to flow cytometry,the expression levels of CD44 and CD227 in cervical epithelial cell lines and TCC samples increased significantly,but there was either no expression or very low expression in the JEG3 cell lines,which met the requirements of negative selection of maternal cervical epithelial cells.(3)The proportion of JEG3 cells+TCT mixed with 0.1%cells can be increased to 8.2%by using CNPS,which is significantly higher than the enrichment efficiency of magnetic beads coupled with HLA-G antibody positive selection alone(0.3%).(4)As previously mentioned,in this study,three TCC samples were collected from women with singleton pregnancies with male fetuses.The WGA products of the GM12878 female cell line and GM50166 male cell line were used for low coverage sequencing,and the threshold of RsrPKMy was set according to the low coverage sequencing analysis results to distinguish gender differences.When RsrPKMy value>10 and coverage>30%,the candidate EVT cells are of male fetal origin,and the coincidence rate of male fetal identification is 100%.(5)The male fetal derived cells with low coverage sequencing analysis unique map ratio>30%and coverage>30%were defined as high-quality cells.Seven,seven,and six cells with high data quality were selected from the three samples for subsequent WGS.The WGA-WGS coverage of EVT cells was found to be as high as 94.15%.The distribution of reads on each chromosome of single cells in three samples showed a similar distribution trend compared with the corresponding cord blood gDNA WGS sequencing results.The isolated EVT cells have detection ability for SNVs and Indels at the whole genome level that is similar to those of cord blood gDNA.The results showed that EVT cells isolated from TCC samples recovered fetal genome as much as possible.ConclusionsThis study preliminarily established a CNPS enrichment method,which can provide sufficient EVT cells for downstream detection.The coincidence rate of identifying male fetal cells based on the threshold of RsrPKMy can reach 100%.Through deep depth whole genome sequencing of the WGA amplification products of isolated high-quality EVT cells,the fetal genomic profiling is relatively complete and has a high coverage.The isolated EVT cells have detection ability for SNVs and InDels at the whole genome level that is similar to those of cord blood gDNA.The preliminary results show that EVT cells isolated from TCC samples have potential clinical value for noninvasive prenatal testing of monogenic diseases.