Application of Trophoblast in Noninvasive Prenatal Testing

The application of trophoblast cells in maternal peripheral blood or cervical exfoliated cells for prenatal diagnosis began in the 1980s and 1990s...


Trophoblast Cell Collection Method
Collecting Trophoblast Cells from Maternal Peripheral Blood: As early as 1893, Schmorl [4] discovered trophoblast cells in the lungs of corpse pregnant women with eclampsia. According to the literature, all three types of trophoblast cells can be found in the peripheral blood of the mother, mainly the trophoblast cells outside the villi5. In 1997, loke, YW et al. [1] reported that the trophoblast cells existed in the maternal blood circulation by the extracellular trophoblast cell-specific antibody HLA-G and FISH detection. The mechanism is speculated as follows; Extravillous trophoblast cells have powerful invasive migration functions. At 8 and 12 weeks of gestation, it can invade the spiral arteries in the endometrium and myometrium, initiate the vascular remodeling and lead to changes in the shape and function of spiral arteries, such as vasodilation, reduction of vascular muscular layer, reduction of resistance, etc [5][6][7]. These changes increase blood flow from the uterus to the placenta by 40 times to ensure adequate blood perfusion of the placenta [8]. In conclusion, extravillous trophoblasts can invade the spiral artery and appear in the maternal peripheral blood.
In addition, some scholars also found these two types of cells in maternal peripheral blood by using cytotrophoblast specific antigen H315 and syncytiotrophoblast specific antigen ft1-41. 13.
Mouawia et al. [9] reported that more than half of trophoblast cells in maternal peripheral blood were larger than 15 microns in diameter. Currently, the most ideal specific antibodies for placental trophoblast cells are hla-g, beta-hcg and CK [10][11][12].
It has been reported that trophoblast cells can be collected from maternal peripheral blood at 5 ~ 12 weeks of gestation and 20 weeks at last [9,13], which is consistent with the time of trophoblast invasion and extension into the spiral artery during the establishment of the maternal-fetal cycle. Depending on the different screening and isolation methods, the number of trophoblast cells captured is different. There are 1-6 trophoblast cells per ml of peripheral blood sample [14,15]. The proportion of nucleated cells in maternal peripheral blood is 1/106 ~ 107 [16].

Collection of Trophoblast from Maternal Cervical
Exfoliated Cell: In 1971, Shettle [17] first reported the presence of trophoblast cells in cervical mucus of pregnant women, and successfully identified the sex of 10 fetuses by using Y chromosome fluorescence staining. The mechanism which trophoblasts reach the uterine cavity and cervical canal is unclear. It is speculated from the occurrence of villi that the time to collect the trophoblast cells through the cervix is from the invasive growth of villi to the end of uterine cavity disappearance and closure of the internal cervical os [18].
The methods of obtaining cervical exfoliated cells in early pregnancy include: Mucus suction, cell brush or cotton swab, cervix lavage ,intrauterine lavage, etc. Different methods have different success rates [11,[19][20][21][22][23][24][25][26][27] (Table 1). When sampling with cotton swab, It is easy for cells to be involved in cotton fiber and not easy to fall off from cotton swab , which leads to the disadvantage of sampling. At present, this method is basically not adopted. The positive rate and the number of trophoblast cells collected by lavage and suction are slightly higher [12], but there is a risk of uterine infection caused by stimulation of blastocyst, which may cause miscarriage or poor pregnancy. Retrieval of trophoblast cells from the endometrial canal using a cytobrush can be considered minimally invasive. The office procedure is essentially a Pap smear, and can be performed successfully between 5 and 20 weeks GA [12]. Pap smears are recommended during pregnancy, and several studies that surveyed approximately 1900 pregnant women found the cytobrush procedure to be safe and associated with no serious adverse outcomes [26,28]. Although cell brush sampling is the safest method, it is most susceptible to the influence of maternal cells and residual sperm and its genetic material, which leads to a wide range of success rate of target cells. The earliest time of collecting trophoblast cells through the cervix by different methods was 5 ~ 7 weeks of pregnancy, and the latest time was 13 ~ 20 weeks of pregnancy [12,18,29]. It is reported that the number of trophoblast cells collected from the cervix at 9 weeks of gestation decreased, which is statistically significant compared with that collected at 6, 7 and 8 weeks of gestation [30].
In addition, the number of trophoblast cells collected through the cervix was not affected by maternal age or obesity [12]. The time of collecting trophoblastic cells from the cervix was basically the same as that from maternal peripheral blood. In normal pregnant cervical exfoliated cells, the proportion of trophoblast cells collected by cell brush is about 1:200011, which is far more than the number of trophoblast cells collected from peripheral blood [31].
To sum up, on the one hand, trophoblast cells are derivatives of mitosis of zygote, which can accurately reflect fetal genetic characteristics to some extent, on the other hand, the existence of placental trophoblast cells in maternal peripheral blood and cervical exfoliated cells has been confirmed by many scholars, and the cell size and morphological characteristics are obvious. It is easy to identify and isolate trophoblast cells with ideal specific antibodies, and the integrity of DNA of these cells is verified by terminal transferase labeling, which contains all the genetic material of the fetus and is not affected by multiple pregnancy [32]. Therefore, it is feasible to collect placental trophoblast cells from maternal peripheral blood or cervical mucus for noninvasive prenatal test.

Separation of Trophoblast Cells:
The Jing et al's research [34], the success rate of trophoblast cells in differential adherent culture was 95%, and the gender of 90% of the fetuses was successfully identified. The culture success rate of the cells in the direct culture control group was 96%, but only 38% of fetuses' gender was correctly identified.

Microfluidic Chip Technology: Microfluidic chip technology
integrates basic operations such as sample preparation, reaction, separation and detection into a micron scale chip. One example is Abnova's CytoQuest™ CR microfluidic system. Microfluidic chip micron-sized channel is suitable for single cell introduction, manipulation, reaction, separation and detection, and is more suitable for samples of peripheral blood to separate and capture trophoblast cells.

Identification of Trophoblast Cells
Fetal origin identification of isolated cells is an essential step for prenatal diagnosis with trophoblast cells, and it is also the key to achieve accurate prenatal diagnosis. The isolated cells were  [1,18]; NDOG1 recognizes syncytiotrophoblast cells [30]; FT141.1 recognizes all trophoblast cells [22]. β-hCG is a specific protein secreted by trophoblast cells, which has been used to identify the purity of isolated cells [35]. Anti-CK-7 antibodies are low in specificity, and the false negative result caused by the specific antibody can be counteracted by the combined staining of anti-ck-7 antibody and high specific antibody [11]. Insufficient antibody specificity or sensitivity of antibodies and antigen destruction caused by pretreatment of specimens will reduce the efficiency of cell identification by immunohistochemistry. It is necessary to explore combinations of antibodies with strong specificity and high sensitivity, or to combine with molecular biological analysis to offset the lack of methodology. be performed by using the fluorescence peak ratio of the given STR site [30]. The use of QF-PCR for prenatal diagnosis eliminates the need to culture and expand fetal cells and allows rapid diagnosis of some selected chromosomal anomalies (13,18,21, and X and Y). Compared with standard karyotyping, FISH and QF-PCR can be exquisitely sensitive for the analysis of TCC and IUL samples.

Application of Trophoblast Cells in Prenatal Diagnosis Early Application of Trophoblast Cells for Noninvasive Prenatal Testing
In the early stage, due to the lack of rare cell separation RhD negative blood group after RhD gene amplification, and the prediction accuracy of fetal Rh blood group was less than 50%.

Progress in NIPT of Trophoblast Cells with the Development of Isolation and Capture of Rare Cells
The development of isolation and capture technology of rare cells and molecular biology technology has provided new ideas and new models for the application of trophoblast cells in NIPT.
Brechot [32] used laser capture microdissection technology to separate trophoblast cells from peripheral blood of pregnant mother (4-12 weeks gestation). After genome-wide amplification, STR classification was used to identify whether the cells were fetal origin. NIPT was carried out in 63 pregnant women with progressive spinal muscular atrophy (SMA, 31 cases) or cystic fibrosis (CF, 32 cases). 14 suffering fetuses were detected, and confirmed by chorionic biopsy results. The sensitivity of diagnosis was 95% (confidence interval 76.8-100%) and the specificity was 95% (confidence interval 92.7-100%).
Breman A.M [32] used microfluidic technology to sort CK+ and CD45-trophoblast cells in maternal peripheral blood and captured 0.36 trophoblast cells per milliliter of peripheral blood on average.
After whole-genome amplification, the fetal origin was determined by STR classification. After whole-genome amplification of isolated trophoblast cells, chromosome aneuploidy (13/18/21/X/Y) and 2.7Mb microdeletions were detected by arrayCGH and Next Generation Sequencing for the first time.
Jain, C. V 10 collected cervical mucus from 20 pregnant women (5-20 weeks gestation) that before induced abortion, sorted trophoblast cells by MACS technique, and developed a "nuclear separation method" to remove most of the extracellular maternal DNA fragments. The Next Generation Sequencing was conducted (detection sites include 94 SNPs and 59 STRs distributed on all chromosomes), and Next Generation Sequencing results of aborted fetal cells were used as gold standard controls to calculate the proportion of maternal contamination. The results showed that the purity of fetal DNA was 92.2% ± 6.5% in the early pregnancy (the earliest is 5 weeks gestation), and the correct rate of fetal genotype was 100%. However, for the current NIPT based on fetal free nucleic acid, the proportion of fetal DNA in maternal peripheral blood free DNA is only 4% to 10% at the 10th week of pregnancy. Their results suggested that cervical trophoblast cells can provide an earlier and more accurate prenatal genetic screening method.
Sifakis S et al. [39] used cell brush to obtain cervical mucus from 11-13 weeks pregnant women，and accurately diagnosed 5 fetuses with Down's syndrome through FISH analysis. The largest number of triploid cells in a single sample was up to 27. Biron-shantal et al. [40] strengthened the understanding of cervical trophoblast cells in early pregnancy. By comparing the fish hybridization results of cervical specimens and placental tissues in early pregnancy, it was found that in normal pregnancy, there were polyploid cells caused by intranuclear replication of trophoblast cells, but most of them were tetraploid, which would not lead to false-positive triploid.
Pfeifer et al. [26] successfully screened 3 cystic fibrosis and 3 spinal muscular atrophy fetuses from 21 cervical specimens of 8-12 gestational weeks pregnant women by single cell gene expression profiling. In addition, the researchers also used the number of cervical trophoblast cells or the difference in proteins expressed by the cells to predict adverse pregnancy outcomes [11].

Conclusion
Compared with NIPT which is based on fetal free nucleic acid, specific antibodies and being easy to identify and separate. Moreover, the number of trophoblast cells obtained by cervical collection is more than that obtained by peripheral blood collection, and it is easy to obtain enough fetal genetic material. With the continuous development of molecular biology, placental trophoblast cells will cover more studies on birth defects in NIPT in the future.