Endocervical trophoblast for interrogating the fetal genome and assessing pregnancy health at five weeks

European Journal of Medical Genetics 62 (2019) 103690

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Endocervical trophoblast for interrogating the fetal genome and assessing pregnancy health at five weeks

T

Leena Kadama, Chandni Jaina, Hamid Reza Kohan-Ghadrb, Stephen A. Krawetza,c, Sascha Drewlob, D. Randall Armanta,d,∗ a

Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI, United States Department of Obstetrics, Gynecology and Reproductive Biology, College of Human Medicine, Michigan State University, Grand Rapids, MI, USA c Centre for Molecular Medicine and Genetics, Wayne State University, Detroit, MI, United States d Department of Anatomy and Cell Biology, Wayne State University, Detroit, MI, United States b

A B S T R A C T

Prenatal testing for fetal genetic traits and risk of obstetrical complications is essential for maternal-fetal healthcare. The migration of extravillous trophoblast (EVT) cells from the placenta into the reproductive tract and accumulation in the cervix offers an exciting avenue for prenatal testing and monitoring placental function. These cells are obtained with a cervical cytobrush, a routine relatively safe clinical procedure during pregnancy, according to published studies and our own observations. Trophoblast retrieval and isolation from the cervix (TRIC) obtains hundreds of fetal cells with > 90% purity as early as five weeks of gestation. TRIC can provide DNA for fetal genotyping by targeted next-generation sequencing with single-nucleotide resolution. Previously, we found that known protein biomarkers are dysregulated in EVT cells obtained by TRIC in the first trimester from women who miscarry or later develop intrauterine growth restriction or preeclampsia. We have now optimized methods to stabilize RNA during TRIC for subsequent isolation and analysis of trophoblast gene expression. Here, we report transcriptomics analysis demonstrating that the expression profile of TRIC-isolated trophoblast cells was distinct from that of maternal cervical cells and included genes associated with the EVT phenotype and invasion. Because EVT cells are responsible for remodeling the maternal arteries and their failure is associated with pregnancy disorders, their molecular profiles could reflect maternal risk, as well as mechanisms underlying these disorders. The use of TRIC to analyze EVT genomes, transcriptomes and proteomes during ongoing pregnancies could provide new tools for anticipating and managing both fetal genetic and maternal obstetric disorders.

1. Introduction Safe access to fetal tissue in ongoing pregnancies is the ultimate goal of noninvasive perinatal testing as it could provide valuable information about both the fetal and pregnancy health status. Analysis of cell free DNA has significantly furthered the field of noninvasive prenatal genetic testing but suffers from limitations of a low fetal DNA fraction, requiring extensive bioinformatic analysis to interpret. Analysis of placental cells collected at the onset of pregnancy has the potential to both, provide access to the genetic status of the developing fetus and alert clinicians to women who will eventually develop placental insufficiencies. The emergence of this information could enable the development of new approaches for targeted management of at-risk pregnancies. 1.1. Cervical trophoblast cells to assess fetal and placental status

towards decidua and myometrium, anchoring the placenta, (ii) remove the smooth muscle cells of the uterine spiral arteries, ensuring adequate perfusion of maternal blood into the placenta to support the growing fetus, and (iii) remodel and invade the uterine glandular epithelium, securing histotrophic nutrition for the developing embryo before the blood supply is established (Weiss et al., 2016a; Moser et al., 2010, 2015). Indeed, dysregulation of EVT invasion has been associated with miscarriage or early pregnancy loss (EPL) and the placental disorders preeclampsia (PE), preterm birth (PTB) and intrauterine growth restriction (IUGR) (Ball et al., 2006; Khong et al., 1986, 1987; Reister et al., 2006; Brosens et al., 1972; DiFederico et al., 1999). The pathophysiology of these disorders is an active area of research, but is challenged due to the inaccessibility to the placenta during pregnancy. Research has been limited to animal models, cell lines or explanted placental tissue procured post-delivery. Access to EVT cells that migrate from the placenta into the maternal reproductive tract and accumulate in the cervix offers an exciting avenue to probe ongoing pregnancies.

The extravillous trophoblast (EVT) lineage of placental cells plays a crucial role in establishing and anchoring the placenta in the uterine cavity. The EVT cells (i) invade through the uterine interstitium

∗

Corresponding author. C.S. Mott Center for Human Growth & Development, Wayne State University, 275 East Hancock Street, Detroit, MI, 48201-1405, USA. E-mail address: [email protected] (D.R. Armant).

https://doi.org/10.1016/j.ejmg.2019.103690 Received 2 March 2019; Received in revised form 1 June 2019; Accepted 5 June 2019 Available online 18 June 2019 1769-7212/ © 2019 Elsevier Masson SAS. All rights reserved.

European Journal of Medical Genetics 62 (2019) 103690

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characteristics of trophoblast cells residing in cervix. A gap existed in understanding how trophoblast cells are deposited in the cervix, far from their primary location in the placenta, which raised further questions about their similarity to the EVT in the growing placenta and their reliability in reflecting placental health status in ongoing pregnancies. Recently, Moser et al. provided evidence that during trophoblast invasion of the decidua basalis EVT cells penetrate the uterine glands at the lateral margins of the placenta and are carried by secretions into the uterine cavity where they eventually accumulate in the cervix (Moser et al., 2018). It was suggested that the trophoblast cells residing in the endocervical canal are endoglandular in origin. However, further studies are required to confirm these observations. The cells obtained by TRIC have been characterized as EVT-like based on expression of EVT markers, including HLA-G (Imudia et al., 2009, 2010; Bulmer et al., 2003). Bulmer et al. stained and confirmed expression of both villous and EVT markers in cells obtained by transcervical lavage, raising the possibility that more than one trophoblast subtype resides in the cervix (Bulmer et al., 1995). Cells isolated by TRIC express the cytotrophoblast markers human chorionic gonadotropin beta (β-hCG) and cytokeratin 7 (KRT7), as well as the EVT proteins integrin α1 (ITGA1), platelet endothelial cell adhesion molecule 1 (PECAM1) matrix metalloproteinase 9 (MMP9) and cadherin 5 (CDH5) that are associated with cell invasion (Table 1) (Bolnick et al., 2014a). They do not express markers associated with syncytiotrophoblast located in the chorionic villi, including placenta specific beta-glycoprotein 1 (PSG1), cadherin 1 (CDH1) and integrin α6 (ITGA6) (Bolnick et al., 2014a). It should be noted that the TRIC protocol is based on enrichment with anti-HLA-G antibody, which selects for cells of the EVT lineage, an advantage for development of downstream testing to assess placental pathologies. Further research is needed to fully ascertain the diversity of trophoblast sub-types available from cervical specimens. It is significant that the cells obtained by TRIC are similar to EVT cells thought to be defective in their known function during placentation, leading to perinatal diseases, including EPL, PE, PTB and IUGR. Pilot studies have been conducted to explore the utility of the EVT cell population obtained by TRIC to detect placental pathologies by investigating their expression of proteins associated with perinatal disease (Table 2). Fritz et al. examined a set of proteins associated with uteroplacental insufficiency in trophoblast cells obtained by TRIC between 5 and 10 weeks of gestational age (GA) and found significant differences in six of the seven proteins between control term pregnancies matched for GA at the time of cervical sampling and those ending in an EPL, suggesting that the molecular profiles of the EVT cells correlates with pregnancy health status (Fritz et al., 2015b). Furthermore, a survey of the same proteins in EVT cells obtained by TRIC at the beginning of gestation from 41 pregnancies demonstrated similar discrimination between term pregnancies without complications and those that developed IUGR or PE at a much later GA (Bolnick et al., 2016). In these two studies, the expression galectin 14 (LGALS14), placental growth factor (PGS) and pregnancy associated plasma protein-A (PAPPA), placental proteins known to be downregulated in IUGR and PE, were significantly reduced in the cervical EVT cells isolated from pregnancies that later developed insufficiencies. Expression of known placental biomarkers of PE/IUGR, FMS-like tyrosine kinase (Flt), endoglin (ENG) and alpha fetoprotein (AFP) were elevated significantly in cervical EVT cells from women who later developed IUGR/PE as compared to the normal controls. These studies provide direct evidence that EVT lineage cells obtained by TRIC are indeed altered at the molecular level early in pregnancy, as suspected for EVT cells residing in the placenta in pregnancies developing uteroplacental insufficiency. Therefore, the broader molecular profiles of cells isolated by TRIC could potentially be useful for investigating perinatal disease and for developing new clinical approaches to assess risk at the outset of pregnancy. A detailed understanding of the relationship between EVT cells in the

1.2. Cervical trophoblast for studying fetal genome EVT cells primarily localize to the placenta and the maternal uterine wall at the site of implantation. However, evidence has been presented that a small number of these EVT cells are displaced and can be found in the reproductive tract (Shettles, 1971; Imudia et al., 2009; Moser et al., 2018). Historically, several attempts have been made to collect these cells and then utilize them for studying the fetal genome. The premise was that the trophoblast cells originate from the embryo and hence reflect the fetal genome. A variety of methods including but not limited to cervical swabs, cervical mucus aspiration, uterine lavage and cervical canal lavage were used to retrieve fetal cells from the maternal reproductive tract and study fetal genetics (Chaouat et al., 1994; Adinolfi et al., 1995; Bussani et al., 2007; Massari et al., 1996; Bulmer et al., 2003). Shettles used quinacrine dye to identify Y bodies in cervical swabs, whereas Griffith-Jones et al. used PCR to identify Y-chromosome derived sequences from cervical samples (Shettles, 1971; Griffith-Jones et al., 1992). The strategy was extended to include detection of female fetuses with the introduction of PCR based sequencing for short tandem repeat (STR) sequences in the genome (Adinolfi et al., 1995; Massari et al., 1996; Kingdom et al., 1995; Pfeifer et al., 2016). But a major obstacle that impeded progress was the need for a method to consistently isolate the trophoblast cells from the vast maternal cell population present in cervical or uterine specimens. Current modalities available for fetal genetic assessment include the invasive amniocentesis and chorionic villus sampling (CVS), as well as noninvasive analysis of cell-free fetal DNA from maternal blood, which are available beginning late in the first trimester (10–15 weeks of gestation). While the invasive methods allow comprehensive genetic analysis, they post a significant risk to the pregnancy. Only the noninvasive approach has the advantage of reduced risk to the pregnancy, but it is limited in scope to detecting aneuploidies and large deletions. Further, these approaches specifically detect genetic abnormalities in the fetus, with no means of assessing the health of the pregnancy. The isolation of EVT cells from cervical specimens can be reproducibly achieved by trophoblast retrieval and isolation from the cervix (TRIC), which uses immunomagnetic nanoparticles to select for cells that express the EVT protein human leukocyte antigen G (HLA-G) (Imudia et al., 2009; Bolnick et al., 2014a). TRIC provides > 200 EVT cells with > 90% purity and offers an approach to access cells of fetal origin for prenatal screening as early as 5 weeks of gestation (Bolnick et al., 2014a; Imudia et al., 2010; Fritz et al., 2015a; Jain et al., 2016). We further reported that the EVT cells retrieved by TRIC can in fact be used to genotype the fetus at single nucleotide resolution (Jain et al., 2016). Using the targeted next generation sequencing platform, Forenseq™ (Illumina), 59 small tandem repeats (STRs) and 94 single nucleotide variants (SNVs) were sequenced using DNA from cells isolated by TRIC (fetal DNA) and compared to DNA from the corresponding placentas and mothers. The fetal DNA was identical to the placental sequences and differed from the maternal sequences, but shared at least one allele with maternal DNA at every locus, validating the expected parent-offspring relationship. The average fetal DNA fraction of 92.2% was significantly greater than that achieve with cell free DNA technology (Jain et al., 2016). Thus, TRIC reproducibly and noninvasively isolates high-purity, fetal trophoblast cells from cervical specimens as early as 5 weeks of gestation. This advance paves the way for development of both targeted and whole genome-based prenatal genetic screening platforms for early and reliable detection of fetal genetic abnormalities. 1.3. Cervical trophoblast for interrogating placental health The analysis of trophoblast cells for fetal genotyping and prenatal screening is being extensively studied. Their utility in evaluating and monitoring pregnancy health has been relatively unexplored largely due to a lack of knowledge about the origin and biological 2

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Table 1 Comparison of trophoblast subtype-specific protein expression in cervical EVT cells isolated by TRIC with villous trophoblast and EVT cells residing in the placenta. Cytokeratin 7 (KRT7) and chorionic gonadotropin beta subunit (β-hCG) are expressed in all trophoblast lineage subtypes. From published data (Bolnick et al., 2014a).

2. Materials and methods

Table 2 Relative expression of known disease marker proteins in EVT cells isolated by TRIC. Cervical specimens were obtained from women who later had a normal term pregnancy (controls) or developed perinatal disease leading to an early pregnancy loss (EPL), preeclampsia or intrauterine growth restriction (IUGR). Arrows indicate the direction of change from controls in pregnancies with the indicated disorders. From published data (Fritz et al., 2015b; Bolnick et al., 2016).

2.1. Cervical specimen collection Pregnant patients (n = 48) were recruited in the GA range of 5–20 weeks with written informed consent as approved by the Wayne State University institutional review board. Cervical specimens were obtained with a cytobrush and immediately fixed in ThinPrep (Imudia et al., 2009), preserving proteins and nucleic acids, including RNA. Prior to cervical EVT isolation, specimens were cross-linked with 2% paraformaldehyde in PBS for 10 min before washing the cells with PBS three times by centrifugation (400×g) and resuspension. The EVT cells were isolated by immunomagnetic separation of HLA-G-positive cells according to the published TRIC protocol (Bolnick et al., 2014b). Both the enriched EVT cells and EVT-depleted maternal cells were collected. 2.2. RNA extraction RNA from the formaldehyde-treated cells was de-crosslinked using a Qiagen FFPE RNA kit with incubations at 56 °C for 15 min and 80 °C for 15 min, according to the manufacturer's instructions. The RNA was treated with DNase for 15 min prior to RNA extraction. Isolated RNA was quantified, and its purity assessed using an Agilent 2100 Electrophoresis Microfluidics Analyzer with the RNA Pico Kit (Agilent Technologies).

placenta and cervix would further establish their utility for investigating the pathophysiology of placental insufficiency and for development of new disease biomarkers. In the present study, RNA was analyzed in EVT cells obtained by TRIC using quantitative reverse transcription and polymerase chain reaction (RT-qPCR) and next-generation sequencing of RNA (RNA-seq) to determine if RNA of the cervical EVT cells was distinct from that of the maternal cells and characteristic of an EVT phenotype.

2.3. Library preparation After pregnancy outcomes were determined from patient records, a cohort of pregnancies (n = 9) with uncomplicated term deliveries (≥37 weeks) was identified and selected for library preparation. Libraries 3

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Table 3 Transcripts assayed by qPCR. Gene

Forward Primer

Reverse Primer

HLA-G KRT7 CDH5 MMP9 ITGA6 CDH1 CD83 CSF1 DDX39A RBM15 TLR1 AMBP FGF23 KCNQ1 NUNDT9 TRPC7

GTG TGG TAC TTT GTC TTG AGG A GGT CAG CTT GAG GCA CTG AAG AAC TGG CCC TTG TGA C CGT CGA AAT GGG CGT CT TTG GAC TCA GGG AAA GCT ATT G CTG AGG ATG GTG TAA GCG ATG TCC GAA GAT GTG GAC TTG C TCT TTC AAC TGT TCC TGG TCT AC CAC CGA AGA ACA CAG ACA CC TCT TCT TGT TCT CAT ACC TAA CTC C AGA CAT TCC TAA AGG TAG AAG CTG GTA TCT GTT TTC TCA TAA GCT CCA G TAT CTT CTG CTC ATC ACA CCT G CTT CCT TGC CAT CCT CTA TAT CG TTC TGT AAG GAG TTG GA GCT TC CAT CAA AGT AAG ACA GCC AGA GT

AGA GTA GCA GGA AGA GGG TT ACC ACA AAC TCA TTC TCA GCA CAG CCT TTC TAC CAC TTC CAG ACA TCG TCA TCC AGT TTG GTG GAT CTC CAC TGA GGC AGT TAT G GTC TGT CAT GGA AGG TGC TC TCT CCA TCC TCT CTT CAC CA TGT CGG AGT ACT GTA GCC A TCA ACG GAC AGG TGA CG AGT TCT CCC AGC AGT TCC T GAA GAA ATC AGG ATA ACA AAG GCA GAC AGG ATG ACA GTG AGC AC GCC AGG AAC AGC TAC CAC AGT GTT TTC TAC CAT CCC TGA G AAG GAA AGA CTG TGG AGA ATG G TGC TCA ACA TGC TAA TAG CCA

Fig. 1. Assessment and validation of RNA quality and integrity. A) RNA extracted from fetal (lanes 1, 3) and maternal (lanes 2, 4) cells after formaldehyde crosslinking and de-crosslinking. Smearing and distinct bands at ∼200 bp indicate RNA fragmentation. B) cDNA prepared from equal amounts of RNA obtained from crude cervical specimen, isolated EVT cells or maternal cells was assayed by qPCR with primers for the indicated RNAs to check for RNA recovery and quality (N = 48; mean ± SEM). The expression of 6 genes known to be markers for C) trophoblast, D) cell invasion/migration and E) epithelial cells were examined by qPCR using equal amounts of cDNA. N = 9 subjects. *p < 0.05; Wilcoxon-Mann-Whitney U test. The boxes represent the 25th to 75th percentiles, and horizontal red lines within the boxes indicate the medians. The whiskers are drawn to indicate 1.5 × Inter Quartile Range (3rd quartile – 1st quartile). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

software (Casava 1.8.2, Illumina). It was then aligned to the human genome build CRCh37.p7/hg19 using TopHat2 ver 2.0.13 (Kim et al., 2013), and to the ribosomal sequences 18s and 28s, using the bioinformatics tool Novoalign (Novocraft, 2010). Novoalign determined unique alignments that were used to generate 1000 reads per coding segment per sample. The reads thus generated were converted into bed.files and imported to the Genomatix mapping station (GMS) (Genomatix Software GmbH). The GMS generated data in the form of Reads Per Kilobase of exon per Million fragments mapped (RPKM) for 25,000 genes in the database, using the GMS RNA-seq analysis suite.

were prepared using 500 pg of fragmented RNA and the ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicenter), following the manufacturer's protocol. Uniquely barcoded adaptors were ligated to each cDNA sample, followed by PCR amplification and library purification. Each uniquely barcoded library was then quantified, and its quality was assessed by bioanalyzer, using the High Sensitivity DNA Chip (Agilent Technologies). The cDNA libraries were combined for sequencing to reduce sequencing lane effects. Paired-end sequencing was performed for 50 cycles using the Illumina HiSeq-2500 sequencer. 2.4. Data alignment & mapping RNA sequencing data was first processed with demultiplexing 4

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listed in Table 3. RNA for each sample was converted to cDNA, using 4 μl iSCRIPT (BioRad) and pre-amplified for 12 cycles in a multiplexed reaction, using all primers and SsoAdvanced PreAmp Supermix (BioRad). Samples were then target-amplified with individual primers and quantified by delta (Bustin et al., 2009) analysis. 3. Results An RNA sequencing-based approach was used to compare the expression patterns of TRIC-isolated EVT cells to the maternal epithelial cells in cervical specimens. The EVT cells were assessed for both RNA quality and expression of trophoblast genes by qPCR. Endocervical specimens collected in PreserCyt (Hologics, Marlborough, MA) fixative were further treated to preserve RNA by crosslinking with formaldehyde. After TRIC, the RNA became fragmented during the reversal of crosslinking and RNA extraction steps, but did not appear to be completely degraded (Fig. 1A). RNA integrity for sequencing was therefore established by assessing expression of 18S rRNA and GAPDH transcripts (Fig. 1B). RNA isolated from the enriched EVT cells demonstrated higher expression of the trophoblast-specific genes HLA-G, KRT7, CDH5 and MMP9 than RNA from maternal cells (Fig. 1C and D). The depleted maternal cells showed higher expression of epithelial markers CDH1 and ITGA6 (Fig. 1E). Fetal and maternal RNA was sequenced, and the data were analyzed using DeSeq2 for identification of differentially expressed genes. A total of 428 differentially expressed transcripts were identified. DESEQ2 analysis for 2.0-fold up or down regulation generated 209 up- and 219 down-regulated transcripts (Fig. 2). Differential expression of transcripts due to GA could not be determined due to the low sample numbers. The sequencing data was validated by assessing the expression of five upregulated and five downregulated genes by qPCR in fetal and maternal RNA (Fig. 3). The differentially expressed genes were subjected to Ingenuity pathway analysis (IPA) to identify the top functional interaction networks. IPA analysis revealed genes involved in interactions characteristic of EVT cells, including invasion and migration, differentiation and proliferation (Fig. 4 and Table 4). Of the top 27 altered genes, 9 (ANGTPLl4, ALOXE3, B3GALNT2, CD83, mir15, mir320, mir-378, MPZL1, PLCG1) are involved in trophoblast cell invasion and related placental development processes, and another 7 (AGTRAP, ACTC1,

Fig. 2. Differential expression of transcripts in TRIC-enriched EVT cells compared to maternal cells. Volcano plot of RNA-seq data represented by log2-fold change (x-axis) versus −log10 p-value. The light colored dots represent significant differentially expressed gene values (p-value < 0.05) in EVT cells compared to maternal cells. Among differentially expressed genes, 5 genes up-regulated (red) and 5 genes down-regulated (blue) in EVT cells are highlighted. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

2.5. Real-time PCR (qPR) Transcripts were assessed by qPCR for GAPDH and 18S rRNA, using validated primers from Qiagen, SYBR green and a BioRad CFX384 thermal cycler. The relative expression of 18S rRNA was calculated by comparison to GAPDH with respect to the number of cell equivalents assayed. RNA sequencing data was validated and trophoblast lineage transcripts were quantified using qPCR with primers (IDT) for genes

Fig. 3. Quantitative validation of select differentially expressed genes identified by RNAseq. Transcript levels of 5 up-regulated (A) and 5 down-regulated (B) genes identified by RNA-Seq and highlighted in Fig. 2 were validated by real-time qPCR. *p < 0.05, analyzed as in Fig. 1C–E using the Wilcoxon-Mann-Whitney U test. 5

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Fig. 4. Protein-protein interaction network. Among the potential regulatory networks identified by Ingenuity pathway analysis of the genes differentially expressed between fetal and maternal cells (p < 0.05), the top protein-protein interaction network is shown. It contains key regulatory genes involved in invasion and migration (blue circles), differentiation (red circles) and proliferation (purple circles). Green symbols indicate downregulated genes, and red symbols indicate upregulated genes. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

adverse pregnancy outcomes such as EPL, PE and IUGR (Fritz et al., 2015b; Bolnick et al., 2016). A picture of cervical EVT cells is emerging that suggests their physiologic status is not highly altered by their dislocation from the placenta. As the placenta grows, EVT cells invade the endometrial glands (Moser et al., 2010, 2015; Weiss et al., 2016b) from which they are apparently transported with secretions into the reproductive tract at the margins of the conceptus (Moser et al., 2018) and may be carried by uterine secretions to the cervical canal without significantly altering their phenotype. It is possible that some changes occur in their RNA expression profiles that could reflect the influence of the local environment. In this study, the transcriptomic signature of cervical EVT cells obtained by TRIC was used to characterize their molecular phenotype. TRIC provides ample RNA from isolated fetal cells for extensive transcriptomic analysis. Statistical analysis of TRIC/RNAseq data revealed that 428 genes differed significantly between cervical EVT and maternal cells, of which trophoblast specific genes were validated by qPCR. EVT RNA expression showed not only higher expression of trophoblast markers, such as HLA-G and KRT7, but also a higher expression of invasion/migration markers, including CDH5 and MMP9. Lower expression of the epithelial markers, CDH1 and ITGA6, was observed, further supporting their EVT phenotype. Interestingly, genes associated with placental disorders were also differentially expressed in cells isolated by TRIC.

APOC2, CLU, CSHL1, mir-342, PRPF4B) are associated with placental disorders. It is interesting to note that the known functions and association of these genes in placental pathologies were determined by studying their expression in either placental tissue or sera from pregnant women (Table 4). 4. Discussion We have provided an overview of cervical EVT cells that migrate from the placenta into the maternal reproductive tract and their potential use for determining both fetal genetic and placental health status. Additionally, evidence was presented validating the utility of cervical EVT cells for investigating pregnancy through global discovery approaches. Further research into the characteristics of these cells combined with longitudinal studies of their molecular profiles during gestation will help fully exploit the potential of TRIC in prenatal medicine. Uteroplacental insufficiency is linked to defects affecting EVT cells (Burton and Jauniaux, 2004; Fisher, 2015; Drewlo and Armant, 2017). The exact origin and biology of EVT-like cells residing in the cervix and their relationship to the human placenta and pregnancy outcomes are unknown. Recently published findings reveal a strong correlation early in gestation between cervical EVT expression patterns of several proteins suspected of involvement in uteroplacental insufficiency and 6

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Table 4 Summary of functions and known pathologies associated with 27 differentially regulated genes included in the network shown in Fig. 4. Gene ID

Gene name

Known to be directly involved in trophoblast and placental development: ANGTPLl4 Angiopoietin Like 4 ALOXE3 Arachidonate Lipoxygenase 3 B3GALNT2 CD83

Beta-1,3-N-Acetylgalactosaminyl-transferase 2 CD83 Molecule

mir15 mir320 mir-378 MPZL1

microRNA 15 microRNA 320 microRNA 378 Myelin Protein Zero Like 1

PLCG1

Phospholipase C Gamma 1

Function

Implicated in trophoblast cell survival, proliferation and migration (Liu et al., 2017) Mutants die shortly after birth and overexpression is associated with growth restriction and altered placental structure (Vierling et al., 2014) Implicated in regulating trophoblast invasion (Liao et al., 2012) Plays a role on trophoblast immune interactions, associated with recurrent miscarriage (Askelund et al., 2004; Qian et al., 2015) Role in (Inhibits) trophoblast cell invasion and angiogenesis (Yang et al., 2016) Known to inhibit trophoblast cell invasion (Gao et al., 2018) Promotes trophoblast differentiation and involved with trophoblast invasion and migration (Luo et al., 2012) Involved in cell migration (Zannettino et al., 2003; Roubelakis et al., 2007) and proposed to be a receptor for primitive Syncytin- like proteins (Denner, 2017) Essential for vascularization in placenta and knockouts die mid-gestation with abnormal placental development (Nakamura et al., 2005)

Known association with placental disorders: AGTRAP Type-1 Angiotensin II Receptor-Associated Protein ACTC1 Actin Alpha Cardiac Muscle 1

Increased hypomethylation associated with PE (Martin et al., 2015) Implicated in fetal cardiac development and differentially methylated in placenta of fetuses with Ventricular septal defect (Bahado-Singh et al., 2018) APOC2 Apolipoprotein C2 Is ratio with negative regulator APOC3 (APOC3/APOC2) is elevated in PE (Flood-Nichols et al., 2011) CLU Clusterin Increased levels (in serum and placenta) associated with PE (Shin et al., 2008) CSHL1 Chorionic Somatomammotropin Hormone Like 1 Reduced placental expression in PE and Small for gestational age cases (Mannik et al., 2010) mir-342 microRNA 342 Elevated levels in maternal serum and placenta of PE women (Wu et al., 2012; Choi et al., 2013) PRPF4B Pre-mRNA Processing Factor 4B Reported to have higher expression in placenta from gestational diabetes cases (Burlina et al., 2019) Known involvement in embryogenesis and cancer related cellular processes: AURKA Aurora kinase A Dysregulation associated with malignant tumors and chromosomal instability (Umene et al., 2015) CHAF1A Chromatin assembly factor 1 subunit A Associated with major features of Down's syndrome (Katsanis and Fisher, 1996) FBXO11 F-box only protein 11 Crucial for germinal-center formation (Schneider et al., 2016) FMNL2 Formin Like 2 Involved in epithelial-mesenchymal transition, regulation of cell morphology and cytoskeletal organization (Li et al., 2010) HNRNPU Heterogeneous nuclear ribonucleoprotein U Reduced expression associated with embryonic lethality (Roshon and Ruley, 2005) LPAR2 Lysophosphatidic Acid Receptor 2 Receptor for lysophosphatidic acid (LPA) known to be involved in vascularization (Beltrame et al., 2342; Teo et al., 2009) MBD2 Methyl-CpG Binding Domain Protein 2 Required for placenta and embryo viability (Itoh et al., 2012) PFKFB3 6-phosphofructo-2-kinase/fructose-2,6Role in angiogenesis and cell proliferation. Also associated with the Warburg effect in cancerous cells (De biphosphatase 3 Bock et al., 2013; Yalcin et al., 2009; Shi et al., 2017) PRR14L Proline Rich 14 Like Promotes myoblast differentiation during skeletal myogenesis (Chase et al., 2019) RNF19A Ring Finger Protein 19A Associated with phenotypes of amyotrophic lateral sclerosis and Parkinson's disease (Sone et al., 2010) Other: COMMD4 COMM Domain Containing 4

real-time placental assessment, and clinical determination of risk for specific adverse pregnancy outcomes. Additionally, it could be used to identify patients at risk for disease as a research tool for evaluating first trimester interventions and novel patient management strategies.

Expression of elevated invasion/migration markers and reduced epithelial markers suggested an epithelial-mesenchymal transition (EMT). During implantation and subsequent placentation, cells of the trophoblast lineage (i.e., cytotrophoblast cells) undergo an EMT that induces trophoblast invasion from the anchoring villi (Vicovac and Aplin, 1996; Aplin et al., 1998; Bischof et al., 2006). EMT results in the loss of cell junctions and polarity of epithelial cells, leading to a reorganization of their cytoskeleton, reprogramming of gene expression, and initiation of increased motility to establish an invasive phenotype (Thiery et al., 2009; Thiery and Sleeman, 2006). Interestingly, bioinformatics analysis of the genes differentially expressed between the maternal and EVT cells generated pathways with highest scores in invasion, migration, and proliferation. Based on the RNA expression of cells isolated by TRIC, we can conclude that the isolated cells are likely EVT cells that have undergone EMT. The transcriptomics analysis demonstrated that cDNA libraries can be constructed from EVT RNA obtained by TRIC and reproducibly sequenced. Furthermore, libraries produced from EVT and maternal cell types showed expected differences. Future studies will be aimed at comparing EVT RNA from normal term pregnancies with those that develop EPL, PE or IUGR to identify changes in their RNA profiles. This translational approach could facilitate development of novel strategies to diagnose and monitor perinatal disease, which will benefit patients who would succumb to obstetrical disorders that arise from uteroplacental insufficiency. TRIC is a novel platform for noninvasive prenatal testing, including comprehensive fetal genetic analysis. It's utility in probing EVT cells from ongoing pregnancies has the potential to be developed as a tool for

Conflicts of interest SD and DRA receive payment for intellectual property that has been licensed by Wayne State University, which has filed patents. SD and DRA have ownership positions in Cradle Genomics, Inc. Funding statement Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and the National Institute on Minority Health and Health Disparities of the National Institutes of Health under Awards Number R43HD092205, R43HD094405 and R43HD097904. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Support was also provided by the W.K. Kellogg Foundation (USA) and PerkinElmer Health Sciences, Inc, Waltham, MA, USA. References Adinolfi, M., Sherlock, J., Soothill, P., Rodeck, C., 1995. Molecular evidence of fetalderived chromosome 21 markers (STRs) in transcervical samples. Prenat. Diagn. 15 (1), 35–39.

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